B , C . Illustration of the Incucyte readout . Infection of A549 or ACE2 - expressing A549 cells with SARS - CoV - 2 encoding a GFP expression cassette . Infection of cells was monitored microscopically as green fluorescence through live - cell imaging at 24 hours post infection ( hpi ) ( B ) or during the time - course of 48 h and shown as the mean of the GFP - positive area relative to the whole area covered by cells in the same well ( C ) . Error bars represent means ± SD derived from biological triplicates ( N = 3 ) . Scale bar , 500 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, C. Illustration of the Incucyte readout. Infection of A549 or ACE2-expressing A549 cells with SARS-CoV-2 encoding a GFP expression cassette. Infection of cells was monitored microscopically as green fluorescence through live-cell imaging at 24 hours post infection (hpi) (B) or during the time-course of 48 h and shown as the mean of the GFP-positive area relative to the whole area covered by cells in the same well (C). Error bars represent means ± SD derived from biological triplicates (N = 3). Scale bar, 500 µm.


D . A549 - ACE2 cells were treated for 6 hours with different concentrations of indicated inhibitors and then infected with SARS - CoV - 2 - GFP . GFP - fluorescence relative to DMSO - treated control cells was determined 48 hpi as described in ( C ) . Two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction was performed for every indicated concentration compared to DMSO ( 0 µM ) . Asterisks indicate significance compared to DMSO ( N = 3 - 6 ) . Data information : Data are represented as means ± 95 % CI from at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. A549-ACE2 cells were treated for 6 hours with different concentrations of indicated inhibitors and then infected with SARS-CoV-2-GFP. GFP-fluorescence relative to DMSO-treated control cells was determined 48 hpi as described in (C). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed for every indicated concentration compared to DMSO (0 µM). Asterisks indicate significance compared to DMSO (N = 3-6). Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


E . A549 - ACE2 cells were treated for 30 min with PMA at indicated concentrations and then infected with SARS - CoV - 2 - GFP . Infection of cells was monitored as in C . Two - way ANOVA with Tukey ' s post - hoc multiple comparison test . Asterisks indicate significance compared to the DMSO control of each time point ( N = 9 ) . Data information : Data are represented as means ± 95 % CI from at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. A549-ACE2 cells were treated for 30 min with PMA at indicated concentrations and then infected with SARS-CoV-2-GFP. Infection of cells was monitored as in C. Two-way ANOVA with Tukey's post-hoc multiple comparison test. Asterisks indicate significance compared to the DMSO control of each time point (N = 9). Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


F , A549 - ACE2 cells were treated with apratastat , DPC - 333 at indicated concentrations . Experiment was conducted and analyzed as in ( D ) . Two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction was performed for every indicated concentration compared to DMSO ( 0 µM ) . Asterisks indicate significance compared to DMSO ( N = 3 - 6 ) . Data information : Data are represented as means ± 95 % CI from at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, A549-ACE2 cells were treated with apratastat, DPC-333 at indicated concentrations. Experiment was conducted and analyzed as in (D). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed for every indicated concentration compared to DMSO (0 µM). Asterisks indicate significance compared to DMSO (N = 3-6). Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


G . A549 - ACE2 cells were treated with apratastat , DPC - 333 or BB94 at indicated concentrations . Experiment was conducted and analyzed as in ( D ) . Two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction was performed for every indicated concentration compared to DMSO ( 0 µM ) . Asterisks indicate significance compared to DMSO ( N = 3 - 6 ) . Data information : Data are represented as means ± 95 % CI from at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. A549-ACE2 cells were treated with apratastat, DPC-333 or BB94 at indicated concentrations. Experiment was conducted and analyzed as in (D). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed for every indicated concentration compared to DMSO (0 µM). Asterisks indicate significance compared to DMSO (N = 3-6). Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


H , I . A549 - ACE2 cells were treated for 1 h with indicated inhibitors ( 10 µM ) or DMSO before inoculation with GFP - encoding spike pseudoparticles ( VSV pseudotyped with SARS - CoV - 2 spike protein ( VSV - S ) ) ( H ) or as a control LCMV glycoprotein ( VSV - GP ) ( I ) . At 16 hpi , infection was analyzed by counting GFP - positive cells . Data are normalized to the number of infected cells in virus - only control wells without DMSO ( N = 3 - 5 ) . One - way ANOVA with Tukey ' s post - hoc multiple comparison test . Asterisks indicate significance compared to DMSO . Data information : Data are represented as means ± 95 % CI from at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H, I. A549-ACE2 cells were treated for 1 h with indicated inhibitors (10 µM) or DMSO before inoculation with GFP-encoding spike pseudoparticles (VSV pseudotyped with SARS-CoV-2 spike protein (VSV-S)) (H) or as a control LCMV glycoprotein (VSV-GP) (I). At 16 hpi, infection was analyzed by counting GFP-positive cells. Data are normalized to the number of infected cells in virus-only control wells without DMSO (N = 3-5). One-way ANOVA with Tukey's post-hoc multiple comparison test. Asterisks indicate significance compared to DMSO. Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


A , RT - qPCR quantification of viral RNA ( SARS - CoV - 2 N gene relative to RPLP0 ) upon infection of A549 - ACE2 cells with indicated SARS - CoV - 2 strains at 24 hpi . Cells were pretreated with BB94 ( 10 µM ) or DMSO for 6 h . Data are normalized to DMSO ( N = 3 - 7 ) . A two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction was performed . Data information : Data are represented as means ± 95 % CI from at least three ( A , biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, RT-qPCR quantification of viral RNA (SARS-CoV-2 N gene relative to RPLP0) upon infection of A549-ACE2 cells with indicated SARS-CoV-2 strains at 24 hpi. Cells were pretreated with BB94 (10 µM) or DMSO for 6 h. Data are normalized to DMSO (N = 3-7). A two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed. Data information: Data are represented as means ± 95% CI from at least three (A, biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.


B . RT - qPCR quantification of viral RNA ( SARS - CoV - 2 N gene relative to RPLP0 ) upon infection of A549 - ACE2 cells with indicated SARS - CoV - 2 strains at 24 hpi . Cells were pretreated with BB94 ( 10 µM ) , DPC - 333 ( 10 µM ) or DMSO for 6 h . Data are normalized to DMSO ( N = 3 - 7 ) . A two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction was performed . Data information : Data are represented as means ± 95 % CI from at least three , B ) biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. RT-qPCR quantification of viral RNA (SARS-CoV-2 N gene relative to RPLP0) upon infection of A549-ACE2 cells with indicated SARS-CoV-2 strains at 24 hpi. Cells were pretreated with BB94 (10 µM), DPC-333 (10 µM) or DMSO for 6 h. Data are normalized to DMSO (N = 3-7). A two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed. Data information: Data are represented as means ± 95% CI from at least three , B) biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.


C . Normal Human Bronchial Epithelial cells ( NHBE ) from ten donors were infected with SARS - CoV - 2 . NHBE cells were pre - treated with camostat mesylate ( 10µM ) , BB94 ( 1 µM or 10 µM ) or DMSO for 6 hours . RNA was isolated at 24 hpi and levels of SARS - CoV - 2 N relative to RPLP0 from total cellular RNA was measured by RT - qPCR . Data are normalized to the DMSO control ( N = 5 - 10 ) . Two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction . Data information : Data are represented as means ± 95 % CI from at least five / ten biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Normal Human Bronchial Epithelial cells (NHBE) from ten donors were infected with SARS-CoV-2. NHBE cells were pre-treated with camostat mesylate (10µM), BB94 (1 µM or 10 µM) or DMSO for 6 hours. RNA was isolated at 24 hpi and levels of SARS-CoV-2 N relative to RPLP0 from total cellular RNA was measured by RT-qPCR. Data are normalized to the DMSO control (N = 5-10). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI from at least five/ten biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.


D , NHBE cells from five donors were infected with GFP - expressing SARS - CoV - 2 . Infection of cells was monitored microscopically as green fluorescence through live - cell imaging at 72 hours post - infection ( hpi ) and is shown as the mean of the GFP - positive area relative to the whole area covered by cells in the same well . In ( D ) NHBE cells were pre - treated with the indicated drugs for 6 hours before infection Data are normalized to the DMSO control ( N = 5 ) . Two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction . Data information : Data are represented as means ± 95 % CI from at least five / ten biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, NHBE cells from five donors were infected with GFP-expressing SARS-CoV-2. Infection of cells was monitored microscopically as green fluorescence through live-cell imaging at 72 hours post-infection (hpi) and is shown as the mean of the GFP-positive area relative to the whole area covered by cells in the same well. In (D) NHBE cells were pre-treated with the indicated drugs for 6 hours before infection Data are normalized to the DMSO control (N = 5). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI from at least five/ten biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.


E . NHBE cells from five donors were infected with GFP - expressing SARS - CoV - 2 . Infection of cells was monitored microscopically as green fluorescence through live - cell imaging at 72 hours post - infection ( hpi ) and is shown as the mean of the GFP - positive area relative to the whole area covered by cells in the same well . NHBE cells were were treated in ( E ) with the indicated drugs 4 hours after infection . Data are normalized to the DMSO control ( N = 5 ) . Two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction . Data information : Data are represented as means ± 95 % CI from at least five / ten biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. NHBE cells from five donors were infected with GFP-expressing SARS-CoV-2. Infection of cells was monitored microscopically as green fluorescence through live-cell imaging at 72 hours post-infection (hpi) and is shown as the mean of the GFP-positive area relative to the whole area covered by cells in the same well. NHBE cells were were treated in (E) with the indicated drugs 4 hours after infection. Data are normalized to the DMSO control (N = 5). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI from at least five/ten biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.


A . Western Blot ( WB ) analysis of A549 - ACE2 knock - out ( KO ) lines . Two different KO lines were generated using distinct CRISPR guide RNAs for both ADAM10 and ADAM17 . A non - targeting control ( NTC ) guide RNA served as control . Shown are two replicates for each cell line . Mature ( m ) and immature ( im ) forms of ADAM10 and ADAM17 are indicated . ACE2 levels remained unaffected . α - tubulin served as loading control . * : remaining , low amount of immature ADAM10 and ADAM17 . The double KO ( dKO ) was generated from guide 1 of each ADAM10 and ADAM17 . B . Quantification of the abundance of mature ADAM10 and ADAM17 protein forms from ( A ) . Data are normalized to loading control tubulin and to NTC ( N = 3 - 4 ) . The band of mADAM17 runs very close to three unspecific bands ( marked with arrowheads ) - one closely above and two closely below mADAM17 ( A ) . Thus , quantification of remaining mADAM17 in the ADAM10 KO includes also these three unspecific bands . Two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction . Data information : Data are represented as means ± 95 % CI from at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Western Blot (WB) analysis of A549-ACE2 knock-out (KO) lines. Two different KO lines were generated using distinct CRISPR guide RNAs for both ADAM10 and ADAM17. A non-targeting control (NTC) guide RNA served as control. Shown are two replicates for each cell line. Mature (m) and immature (im) forms of ADAM10 and ADAM17 are indicated. ACE2 levels remained unaffected. α-tubulin served as loading control. *: remaining, low amount of immature ADAM10 and ADAM17. The double KO (dKO) was generated from guide 1 of each ADAM10 and ADAM17. B. Quantification of the abundance of mature ADAM10 and ADAM17 protein forms from (A). Data are normalized to loading control tubulin and to NTC (N = 3-4). The band of mADAM17 runs very close to three unspecific bands (marked with arrowheads) - one closely above and two closely below mADAM17 (A). Thus, quantification of remaining mADAM17 in the ADAM10 KO includes also these three unspecific bands. Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


C . RT - qPCR quantification of viral RNA ( SARS - CoV - 2 N gene relative to RPLP0 ) upon infection of A549 - ACE2 NTC or KO cells with SARS - CoV - 2 . Cells were treated with BB94 ( 10 µM ) or DMSO for 6 h prior to infection and RNA was isolated at 24 hpi . Two - way ANOVA with Tukey ' s post - hoc multiple comparison test . Data are normalized to NTC DMSO ( N = 3 ) . Data information : Data are represented as means ± 95 % CI from at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. RT-qPCR quantification of viral RNA (SARS-CoV-2 N gene relative to RPLP0) upon infection of A549-ACE2 NTC or KO cells with SARS-CoV-2. Cells were treated with BB94 (10 µM) or DMSO for 6 h prior to infection and RNA was isolated at 24 hpi. Two-way ANOVA with Tukey's post-hoc multiple comparison test. Data are normalized to NTC DMSO (N = 3). Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


D - G . UMAP of 63 , 103 cells from BAL fluid derived from infected individuals ( D ) Individual cell types are indicated in color . ( E ) Normalized counts of SARS - CoV - 2 reads , ADAM10 , ADAM17 , and TMPRSS2 gene expression levels . ( F ) Dot plot of ADAM10 , ADAM17 , and TMPRSS2 expression , grouped by disease severity . Expression levels are color - coded , the percentage of cells expressing the aforementioned genes is size coded . ( G ) Cells that are infected and additionally express ADAM17 ( ADAM17 + , red ) , ADAM10 ( ADAM10 + , blue ) , both ADAMs ( ADAM10 + ADAM17 + , purple ) , or TMPRSS2 ( TMPRSS2 + , orange ) are indicated .

D-G. UMAP of 63,103 cells from BAL fluid derived from infected individuals (D) Individual cell types are indicated in color. (E) Normalized counts of SARS-CoV-2 reads, ADAM10, ADAM17, and TMPRSS2 gene expression levels. (F) Dot plot of ADAM10, ADAM17, and TMPRSS2 expression, grouped by disease severity. Expression levels are color-coded, the percentage of cells expressing the aforementioned genes is size coded. (G) Cells that are infected and additionally express ADAM17 (ADAM17+, red), ADAM10 (ADAM10+, blue), both ADAMs (ADAM10+ ADAM17+, purple), or TMPRSS2 (TMPRSS2+, orange) are indicated.


B . Representative images of syncytia formation assay . Large syncytia were observed for NTC and ADAM17 KO cells , but were strongly reduced when ADAM10 was knocked - out , either alone or together with ADAM17 or upon inhibition with BB94 ( 10 µM ) . For both ADAMs , the cell lines obtained with gRNA sequence 1 were used . Images show GFP fluorescence and Hoechst staining . Scale bars , 50 µm . C . Quantification of images from fluorescence microscopy ( B ) . Left plot : the area of the fused cells in green ( GFP ) was quantified and normalized to the Hoechst signal ( blue ) of the entire image to account for differences in cell density . Right plot : The nuclei within syncytia were determined by calculating the ratio between the Hoechst signal within syncytia and the total Hoechst signal in the entire image . The data are normalized to NTC DMSO ( N > = 8 ) . Two - way ANOVA with Tukey ' s correction for multiple comparisons . All data are represented as means ± 95 % CI of at least 3 independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Representative images of syncytia formation assay. Large syncytia were observed for NTC and ADAM17 KO cells, but were strongly reduced when ADAM10 was knocked-out, either alone or together with ADAM17 or upon inhibition with BB94 (10 µM). For both ADAMs, the cell lines obtained with gRNA sequence 1 were used. Images show GFP fluorescence and Hoechst staining. Scale bars, 50 µm. C. Quantification of images from fluorescence microscopy (B). Left plot: the area of the fused cells in green (GFP) was quantified and normalized to the Hoechst signal (blue) of the entire image to account for differences in cell density. Right plot: The nuclei within syncytia were determined by calculating the ratio between the Hoechst signal within syncytia and the total Hoechst signal in the entire image. The data are normalized to NTC DMSO (N >= 8). Two-way ANOVA with Tukey's correction for multiple comparisons. All data are represented as means ± 95% CI of at least 3 independent experiments. **p < 0.01, ***p < 0.001.


In vitro cleavage assay of SARS - CoV - 2 spike protein . Recombinant spike protein ( 1 µg ) was digested with ADAM10 , TMPRSS2 ( A ) for 24 hours at a 1 : 1 ratio and analyzed by Western Blot . Camostat mesylate or BB94 ( 10 µM ) were pre - incubated with TMPRSS2 or ADAM proteases , respectively , for 15 min before addition of spike protein . The spike protein and fragments were detected using an antibody against its C - terminal 6xHIS tag .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

In vitro cleavage assay of SARS-CoV-2 spike protein. Recombinant spike protein (1 µg) was digested with ADAM10, TMPRSS2 (A) for 24 hours at a 1:1 ratio and analyzed by Western Blot. Camostat mesylate or BB94 (10 µM) were pre-incubated with TMPRSS2 or ADAM proteases, respectively, for 15 min before addition of spike protein. The spike protein and fragments were detected using an antibody against its C-terminal 6xHIS tag.


B . In vitro cleavage assay of SARS - CoV - 2 spike protein . Recombinant spike protein ( 1 µg ) was digested with ADAM17 ( B ) for 24 hours at a 1 : 1 ratio and analyzed by Western Blot . BB94 ( 10 µM ) were pre - incubated with ADAM proteases , respectively , for 15 min before addition of spike protein . The spike protein and fragments were detected using an antibody against its C - terminal 6xHIS tag .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. In vitro cleavage assay of SARS-CoV-2 spike protein. Recombinant spike protein (1 µg) was digested with ADAM17 (B) for 24 hours at a 1:1 ratio and analyzed by Western Blot. BB94 (10 µM) were pre-incubated with ADAM proteases, respectively, for 15 min before addition of spike protein. The spike protein and fragments were detected using an antibody against its C-terminal 6xHIS tag.


C . Representative images and quantification of a proximity ligation assay ( PLA ) between the SARS - CoV - 2 Spike and ADAM17 in A549 - ACE2 cells . Cells were infected with SARS - CoV - 2 and placed on ice ( 4 ° C ) . After 2 h , cells were shifted to 37 ° C for 15 min or left on ice . Images are presented as overlay between the Nuclei ( Hoechst , blue ) and the PLA ( pseudo - color : red ) channel . Scale bar , 20 µm . The number of PLA foci per cell was determined for the acquired images ( N = 28 - 30 ) . A two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction was performed . Data information : Data are represented as means ± 95 % CI of at least 3 independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Representative images and quantification of a proximity ligation assay (PLA) between the SARS-CoV-2 Spike and ADAM17 in A549-ACE2 cells. Cells were infected with SARS-CoV-2 and placed on ice (4°C). After 2 h, cells were shifted to 37°C for 15 min or left on ice. Images are presented as overlay between the Nuclei (Hoechst, blue) and the PLA (pseudo-color: red) channel. Scale bar, 20 µm. The number of PLA foci per cell was determined for the acquired images (N = 28-30). A two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed. Data information: Data are represented as means ± 95% CI of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


D . A549 - ACE2 cells were treated for 48 h with BB94 ( 10 µM ) , DPC - 333 ( 5 µM ) or DMSO . PMA ( 25 ng / ml ) was added for 3 h before harvest to stimulate ACE2 shedding . Concentration of the soluble ACE2 fragment was determined in the conditioned medium by ELISA and is shown relative to the concentration in the DMSO control ( N = 6 ) . One - way ANOVA with Tukey ' s post - hoc multiple comparison test . Data information : Data are represented as means ± 95 % CI of at least 3 independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. A549-ACE2 cells were treated for 48 h with BB94 (10 µM), DPC-333 (5 µM) or DMSO. PMA (25 ng/ml) was added for 3 h before harvest to stimulate ACE2 shedding. Concentration of the soluble ACE2 fragment was determined in the conditioned medium by ELISA and is shown relative to the concentration in the DMSO control (N = 6). One-way ANOVA with Tukey's post-hoc multiple comparison test. Data information: Data are represented as means ± 95% CI of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


E . GFP - encoding spike pseudoparticles ( VSV pseudotyped with SARS - CoV - 2 spike protein ( VSV - S ) were incubated with indicated amounts of recombinant , soluble ACE2 ( sACE2 ) or bovine serum albumin ( BSA ) as a control prior to infection of A549 - ACE2 cells . At 16 hpi , infection was analyzed by counting GFP - positive cells . Data are normalized to the number of infected cells in virus - only control wells without sACE2 or BSA ( N = 3 ) . Two - sided independent Student ' s t test with Benjamini - Hochberg FDR correction . Data information : Data are represented as means ± 95 % CI of at least 3 independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. GFP-encoding spike pseudoparticles (VSV pseudotyped with SARS-CoV-2 spike protein (VSV-S) were incubated with indicated amounts of recombinant, soluble ACE2 (sACE2) or bovine serum albumin (BSA) as a control prior to infection of A549-ACE2 cells. At 16 hpi, infection was analyzed by counting GFP-positive cells. Data are normalized to the number of infected cells in virus-only control wells without sACE2 or BSA (N = 3). Two-sided independent Student's t test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


( B , C ) Kinetics of the binding between the scFv of FD20 ( scFD20 ) and RBD from SARS - CoV - 2 ( B ) or SARS - CoV ( C ) . Biolayer interferometry ( BLI ) assay was performed with RBD immobilized and scFD20 as analyte at indicated concentrations ( nM ) . Raw data are shown in color and fitted lines are shown in grey . KD values were calculated using 1 : 1 global fitting . Note , although the fitting of the binding curves in D gave a KD of 4 . 8 nM , the accuracy of the kinetic parameters may be compromised owing to the mismatch between the raw data and the fitted curves at the initial dissociation phase . The reason for the abnormal BIL profile is unknown but it may be caused by possible denaturation of the commercial SARS - CoV RBD during storage . It was also noted that the BLI signal in D was ~ 5 times less than that with SARS - CoV - 2 RBD under similar conditions ( C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B, C) Kinetics of the binding between the scFv of FD20 (scFD20) and RBD from SARS-CoV-2 (B) or SARS-CoV (C). Biolayer interferometry (BLI) assay was performed with RBD immobilized and scFD20 as analyte at indicated concentrations (nM). Raw data are shown in color and fitted lines are shown in grey. KD values were calculated using 1:1 global fitting. Note, although the fitting of the binding curves in D gave a KD of 4.8 nM, the accuracy of the kinetic parameters may be compromised owing to the mismatch between the raw data and the fitted curves at the initial dissociation phase. The reason for the abnormal BIL profile is unknown but it may be caused by possible denaturation of the commercial SARS-CoV RBD during storage. It was also noted that the BLI signal in D was ~5 times less than that with SARS-CoV-2 RBD under similar conditions (C).


( D ) Neutralizing assay of scFD20 against SARS - CoV - 2 ( black ) and SARS - CoV ( red ) pseudoviruses .

O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Neutralizing assay of scFD20 against SARS-CoV-2 (black) and SARS-CoV (red) pseudoviruses.


( E ) Neutralization assays using FD20 against pseudoviruses harboring the wildtype SARS - CoV - 2 S ( blue ) , S D614G mutant ( red ) , or SARS - CoV S ( black ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Neutralization assays using FD20 against pseudoviruses harboring the wildtype SARS-CoV-2 S (blue), S D614G mutant (red), or SARS-CoV S (black).


( A ) Neutralization assay of FD20 using SARS - CoV - 2 pp harboring spike derived from four Variants of Concern as indicated . Mean ± s . d . are plotted ( n = 3 biological replicates ) . Numbers in brackets indicate IC50 values in nM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Neutralization assay of FD20 using SARS-CoV-2 pp harboring spike derived from four Variants of Concern as indicated. Mean ± s.d. are plotted (n = 3 biological replicates). Numbers in brackets indicate IC50 values in nM.


( A ) Body weights of hamsters treated with antibodies were measured at indicated days after inoculation with SARS - CoV - 2 . The mean % of starting weight ± s . e . m . ( n = 5 biological replicates ) are plotted .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Body weights of hamsters treated with antibodies were measured at indicated days after inoculation with SARS-CoV-2. The mean % of starting weight ± s.e.m. (n = 5 biological replicates) are plotted.


( B - E ) SARS - CoV - 2 viral RNA ( B and D ) or infectious virus ( C and E ) was detected in the lung ( B and C ) and nasal turbinates ( D and E ) . The mean copy number or the mean infectious titer is shown , error bars represent s . e . m . ( n = 5 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B-E) SARS-CoV-2 viral RNA (B and D) or infectious virus (C and E) was detected in the lung (B and C) and nasal turbinates (D and E). The mean copy number or the mean infectious titer is shown, error bars represent s.e.m. (n = 5 biological replicates).


( D ) Modest perturbation of scFD20 for RBD - ACE2 interaction . A sensor with RBD immobilized was soaked in 200 nM of scFD20 before being further soaked in scFD20 - containing buffer with ( red ) or without ( blue ) 25 nM of ACE2 for BLI signal recording . As a control , the ACE2 - RBD interaction was monitored in the absence of scFD20 ( black ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Modest perturbation of scFD20 for RBD-ACE2 interaction. A sensor with RBD immobilized was soaked in 200 nM of scFD20 before being further soaked in scFD20-containing buffer with (red) or without (blue) 25 nM of ACE2 for BLI signal recording. As a control, the ACE2-RBD interaction was monitored in the absence of scFD20 (black).


( A ) Inhibition of cell - cell fusion by mAbs . Cell fusion was quantified by measuring the luciferase expression induced by full cell membrane fusion and mixing of cytoplasm , and the rate of inhibition by indicated antibodies compared to the condition without antibody is reported . Data depict mean ± s . d . ( n = 4 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Inhibition of cell-cell fusion by mAbs. Cell fusion was quantified by measuring the luciferase expression induced by full cell membrane fusion and mixing of cytoplasm, and the rate of inhibition by indicated antibodies compared to the condition without antibody is reported. Data depict mean ± s.d. (n = 4 biological replicates).


( C ) FD20 pre - incubation with SARS - CoV - 2 pp in the absence of cells strongly impairs entry . To test direct action on particles , SARS - CoV - 2 pp and FD20 were co - incubated for 1 h , then the premixes were used to infect VeroE6 - hACE2 cells for 6 h at 37 ° C . Mean ± s . d . are plotted ( n = 3 biological replicates ) . Numbers in brackets indicate the gained neutralization ( % ) by pre - incubation . As control , cells were incubated at each step with an equal volume of PBS or Y112R . Percentages of primary infection were calculated according to viral titers of PBS control conditions . p < 0 . 0001 ( * * * * ) unless specified otherwise ( 2 - tailed , unpaired student ' s t - test ) . ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) FD20 pre-incubation with SARS-CoV-2 pp in the absence of cells strongly impairs entry. To test direct action on particles, SARS-CoV-2 pp and FD20 were co-incubated for 1 h, then the premixes were used to infect VeroE6-hACE2 cells for 6 h at 37 °C. Mean ± s.d. are plotted (n = 3 biological replicates). Numbers in brackets indicate the gained neutralization (%) by pre-incubation. As control, cells were incubated at each step with an equal volume of PBS or Y112R. Percentages of primary infection were calculated according to viral titers of PBS control conditions. p < 0.0001 (****) unless specified otherwise (2-tailed, unpaired student's t-test). ns, not significant.


( D ) Inhibition of cell surface binding of SARS - CoV - 2 VLP by FD20 . VLP - GFP were preincubated , or not , with FD20 and RBM - targeting mAbs ( CB6 , REGN10933 , REGN10987 ) for 30 min at 37 ° C . Then the VLP premixes were incubated with VeroE6 - hACE2 for 1 h at 37 ° C and the binding detected by flow cytometry . Control binding assays were performed using soluble ACE2 as a competitor . Data are representatives from three experiments .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Inhibition of cell surface binding of SARS-CoV-2 VLP by FD20. VLP-GFP were preincubated, or not, with FD20 and RBM-targeting mAbs (CB6, REGN10933, REGN10987) for 30 min at 37 °C. Then the VLP premixes were incubated with VeroE6-hACE2 for 1 h at 37 °C and the binding detected by flow cytometry. Control binding assays were performed using soluble ACE2 as a competitor. Data are representatives from three experiments.


( B ) FD20 acts irreversibly on particles in the presence or absence of other mAbs . As described in ( A ) , 5 different conditions were used to evaluate the effect of SARS - CoV - 2 pp pre - incubation with mAbs . In some experiments , FD20 was mixed with other mAbs and synergic effects were observed . Inhibition rate was calculated relatively to infection without antibodies at the similar SARS - CoV - 2 pp input ( diluted 20 times or not , Group 2 / 1 in A ) . Mean ± s . d . are plotted ( n = 3 biological replicates ) . p < 0 . 0001 ( * * * * ) unless specified otherwise ( one - way ANOVA with Tukey ' s test ) ; ns , not significant . Source data is available online .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) FD20 acts irreversibly on particles in the presence or absence of other mAbs. As described in (A), 5 different conditions were used to evaluate the effect of SARS-CoV-2 pp pre-incubation with mAbs. In some experiments, FD20 was mixed with other mAbs and synergic effects were observed. Inhibition rate was calculated relatively to infection without antibodies at the similar SARS-CoV-2 pp input (diluted 20 times or not, Group 2/1 in A). Mean ± s.d. are plotted (n = 3 biological replicates). p < 0.0001 (****) unless specified otherwise (one-way ANOVA with Tukey's test); ns, not significant. Source data is available online.


( C - F ) The destruction of SARS - CoV - 2 S by FD20 . ( C ) Negative - stain of FD20 . ( D - F ) Negative - stain image ( i ) and 2D class averages ( ii ) of S alone ( D ) , and S incubated with FD20 ( E ) or 5E1 ( F ) which is an unrelated mAb targeting a protein called Hedgehog ( Maun et al , 2010 ) . Bar = 100 nm . In D and F , red circles indicate typical side views of S . Results are representative of two independent experiments with two different S purification batches .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-F) The destruction of SARS-CoV-2 S by FD20. (C) Negative-stain of FD20. (D-F) Negative-stain image (i) and 2D class averages (ii) of S alone (D), and S incubated with FD20 (E) or 5E1 (F) which is an unrelated mAb targeting a protein called Hedgehog (Maun et al, 2010). Bar = 100 nm. In D and F, red circles indicate typical side views of S. Results are representative of two independent experiments with two different S purification batches.


C Confirmatory screening performed as described above . Schematic distribution of compounds based on Z - score values of cells expressing polyGP - GFP and AUG - RFP ( above ) and on Z - score values of the fluorescent intensity of the two reporters per each compound ( below ) . Data are from four technical replicates , boxes are the Z - score mean value and whiskers represent ± SD . Baseline of Z - score = 0 indicates that Z - score is identical to the mean score . Asterisk ( * ) represents the selected compounds : 1 Forskolin ( FSK ) , 2 Erysolin ( ERY ) , 3 Geldanamycin ( GELD ) , 4 Helenin ( HLN ) and 5 Spironolactone ( SPL ) . Number sign ( # ) represents cellular stress inducers : # 1 Thapsigargin and # 2 Tunicamycin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O

C Confirmatory screening performed as described above. Schematic distribution of compounds based on Z-score values of cells expressing polyGP-GFP and AUG-RFP (above) and on Z-score values of the fluorescent intensity of the two reporters per each compound (below). Data are from four technical replicates, boxes are the Z-score mean value and whiskers represent ±SD. Baseline of Z-score = 0 indicates that Z-score is identical to the mean score. Asterisk (*) represents the selected compounds: 1 Forskolin (FSK), 2 Erysolin (ERY), 3 Geldanamycin (GELD), 4 Helenin (HLN) and 5 Spironolactone (SPL). Number sign (#) represents cellular stress inducers: #1 Thapsigargin and #2 Tunicamycin.


D , E Dose - response analysis of ERY , HLN , SPL , FSK and GELD . Cells were co - transfected with AUG - RFP and polyGP - GFP plasmids and treated with two concentration ranges 0 . 5 , 1 , 5 and 10 µM ( D ) and 20 , 40 and 60 µM ( E ) for 24 h . CHX used only low dosages . Data are mean ± SD from three biological replicates . 1 - way ANOVA followed by Dunnett ' s multiple comparison tests : * P < 0 . 5 ; * * P < 0 . 01 ; * * * P < 0 .

O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E Dose-response analysis of ERY, HLN, SPL, FSK and GELD. Cells were co-transfected with AUG-RFP and polyGP-GFP plasmids and treated with two concentration ranges 0.5, 1, 5 and 10 µM (D) and 20, 40 and 60 µM (E) for 24 h. CHX used only low dosages. Data are mean ± SD from three biological replicates. 1-way ANOVA followed by Dunnett's multiple comparison tests: *P<0.5; **P<0.01; ***P<0.001


B , C HEK293T cells were treated for 24 h and general RNA and protein synthesis were monitored by the incorporation of 5 - ethynyl uridine ( EU ) and O - propargyl - puromycin ( OPP ) , respectively . ActD ( 5 μM ) and CHX ( 350 μM ) were used for 3 h as positive control to block RNA and protein synthesis , respectively . Data are from three biological replicates and represented as mean ± SEM . ( B ) Two - tailed , unpaired t - test ; * * * P < 0 . 001 , * * P = 0 . 0047 . Concentration used : 1 μM GELD , 10 μM ERY , SPL and FSK .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O

B, C HEK293T cells were treated for 24 h and general RNA and protein synthesis were monitored by the incorporation of 5-ethynyl uridine (EU) and O-propargyl-puromycin (OPP), respectively. ActD (5 μM) and CHX (350 μM) were used for 3 h as positive control to block RNA and protein synthesis, respectively. Data are from three biological replicates and represented as mean ± SEM. (B) Two-tailed, unpaired t-test; ***P<0.001, **P = 0.0047. Concentration used: 1 μM GELD, 10 μM ERY, SPL and FSK.


G Luminescence derived from HEK293T transfected with GA - NLuc reporters WT or mutated and treated for 24 h . Mutational analysis of near - cognate CUG start codon from three biological replicates and four technical replicates , n = 12 . Data are mean ± SEM . 2 - way ANOVA followed by Dunnett ' s multiple comparisons test , * * * * P < 0 . 0001 . Concentration used : 1 μM GELD , 10 μM SPL and FSK .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O

G Luminescence derived from HEK293T transfected with GA-NLuc reporters WT or mutated and treated for 24 h. Mutational analysis of near-cognate CUG start codon from three biological replicates and four technical replicates, n = 12. Data are mean ± SEM. 2-way ANOVA followed by Dunnett's multiple comparisons test, ****P<0.0001. Concentration used: 1 μM GELD, 10 μM SPL and FSK.


C Relative expression level of Sqstm1 / p62 , Lc3 , Tfeb , Hspb8 and Bag3 mRNAs in NSC34 cells treated for 24 h . Data are mean ± SEM from three biological replicates . 2 - way ANOVA followed by Dunnett ' s multiple comparisons test , * * * P < 0 . 001 . Dotted lines represent RNA expression in the DMSO control . Concentration used : 1 μM GELD , 10 μM ERY , SPL , FSK .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O

C Relative expression level of Sqstm1/p62, Lc3, Tfeb, Hspb8 and Bag3 mRNAs in NSC34 cells treated for 24 h. Data are mean ± SEM from three biological replicates. 2-way ANOVA followed by Dunnett's multiple comparisons test, ***P<0.001. Dotted lines represent RNA expression in the DMSO control. Concentration used: 1 μM GELD, 10 μM ERY, SPL, FSK.


G Lysates from NSC34 cells transfected with scrambled - GPx100 , treated for 24 h with SPL and / or 10 mM 3MA and immunoblotted for scrambled - GP expression . Quantification from three biological replicates . Data are mean ± SEM . Two - tailed , unpaired t - test , * * * * P < 0 . 0001 for SPL vs DMSO and 3MA vs DMSO , * * * P = 0 . 0008 SPL + 3MA vs SPL . Concentration used : 1 μM GELD , 10 μM

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O

G Lysates from NSC34 cells transfected with scrambled-GPx100, treated for 24 h with SPL and/or 10 mM 3MA and immunoblotted for scrambled-GP expression. Quantification from three biological replicates. Data are mean ± SEM. Two-tailed, unpaired t-test, ****P<0.0001 for SPL vs DMSO and 3MA vs DMSO, ***P = 0.0008 SPL + 3MA vs SPL. Concentration used: 1 μM GELD, 10 μM SPL


B Lysates from HEK293T cells , transfected with 2R or 66R and treated for 24 h , immunoblotted using antibody for poly - GA expression ( HA - tagged ) . Quantification from three biological replicates . Data are mean ± SEM . Two - tailed , unpaired t - test , * * * * P < 0 . 0001 , * * P = 0 . 0027 H89 vs DMSO , * * * P = 0 . 0004 FSK + H89 vs DMSO , * P = 0 . 0234 CLZ vs DMSO , * P = 0 . 0209 FSK + H89 vs FSK . Concentration used : 2 . 5 μM H89 , 10 μM FSK , CLZ .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O

B Lysates from HEK293T cells, transfected with 2R or 66R and treated for 24 h, immunoblotted using antibody for poly-GA expression (HA-tagged). Quantification from three biological replicates. Data are mean ± SEM. Two-tailed, unpaired t-test, ****P<0.0001, **P = 0.0027 H89 vs DMSO, ***P = 0.0004 FSK + H89 vs DMSO, *P = 0.0234 CLZ vs DMSO, *P = 0.0209 FSK + H89 vs FSK. Concentration used: 2.5 μM H89, 10 μM FSK, CLZ.


F Luminescence derived from HEK293T transfected with GA - NLuc reporters WT or mutated and treated with H89 for 24 h . Mutational analysis of near - cognate CUG start codon from three biological replicates and four technical replicates , n = 12 . Data are mean ± SEM . 2 - way ANOVA followed by Dunnett ' s multiple comparisons test , * P = 0 . 0130 WT H89 vs WT DMSO , * * * * P < 0 . 0001 , CUG mutated H89 vs CUG mutated DMSO . Concentration used : 2 . 5 μM H89 ,

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

F Luminescence derived from HEK293T transfected with GA-NLuc reporters WT or mutated and treated with H89 for 24 h. Mutational analysis of near-cognate CUG start codon from three biological replicates and four technical replicates, n = 12. Data are mean ± SEM. 2-way ANOVA followed by Dunnett's multiple comparisons test, *P = 0.0130 WT H89 vs WT DMSO, ****P<0.0001, CUG mutated H89 vs CUG mutated DMSO. Concentration used: 2.5 μM H89,


A , B Lysates from HEK293T cells , transfected with RNAi scrambled or RNAi PRKACA ( 72 h ) and 2R or 66R ( 24 h ) , immunoblotted for PKA - Cα and poly - GA ( HA - tag ) expression . α - Tubulin used as loading control . ( B ) Quantification of PKA - Cα and poly - GA expression from three biological replicates . Data are mean ± SEM . Two - tailed , unpaired t - test . PKA - Cα : ns siScr 66R vs siScr 2R , * * * P = 0 . 0002 siPRKACA 66R vs siScr 2R , * * * P = 0 . 0003 siPRKACA 66R vs siScr 66R . poly - GA expression : * * * * P < 0 . 0001 siScr 66R vs siScr 2R , * * * * P < 0 . 0001 siPRKACA 66R vs siScr 2R , * * P = 0 . 0062 siPRKACA 66R vs siScr 66R . C , D Lysates from HEK293T cells , transfected with RNAi scrambled or RNAi PRKACB ( 72 h ) and 2R or 66R ( 24 h ) , immunoblotted PKA - Cβ and poly - GA ( HA tag ) expression . α - Tubulin used as loading control . ( D ) Quantification of PKA - Cβ and poly - GA expression from three biological replicates . Data are mean ± SEM . Two - tailed , unpaired t - test . PKA - Cβ : ns siScr 66R vs siScr 2R , * * P = 0 . 0049 siPRKACB 66R vs siScr 2R , * * P = 0 . 0091 siPRKACB 66R vs siScr 66R . poly - GA expression : * * * * P < 0 . 0001 siScr 66R vs siScr 2R , * * * * P < 0 . 0001 siPRKACB 66R vs siScr 2R , * * * P = 0 . 0007 siPRKACB 66R vs siScr 66R . E

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B Lysates from HEK293T cells, transfected with RNAi scrambled or RNAi PRKACA (72 h) and 2R or 66R (24 h), immunoblotted for PKA-Cα and poly-GA (HA-tag) expression. α-Tubulin used as loading control. (B) Quantification of PKA-Cα and poly-GA expression from three biological replicates. Data are mean ± SEM. Two-tailed, unpaired t-test. PKA-Cα: ns siScr 66R vs siScr 2R, ***P = 0.0002 siPRKACA 66R vs siScr 2R, ***P = 0.0003 siPRKACA 66R vs siScr 66R. poly-GA expression: ****P<0.0001 siScr 66R vs siScr 2R, ****P<0.0001 siPRKACA 66R vs siScr 2R, **P = 0.0062 siPRKACA 66R vs siScr 66R . C, D Lysates from HEK293T cells, transfected with RNAi scrambled or RNAi PRKACB (72 h) and 2R or 66R (24 h), immunoblotted PKA-Cβ and poly-GA (HA tag) expression. α-Tubulin used as loading control. (D) Quantification of PKA-Cβ and poly-GA expression from three biological replicates. Data are mean ± SEM. Two-tailed, unpaired t-test. PKA-Cβ: ns siScr 66R vs siScr 2R, **P = 0.0049 siPRKACB 66R vs siScr 2R, **P = 0.0091 siPRKACB 66R vs siScr 66R. poly-GA expression: ****P<0.0001 siScr 66R vs siScr 2R, ****P<0.0001 siPRKACB 66R vs siScr 2R, ***P = 0.0007 siPRKACB 66R vs siScr 66R. E


A , B Representative graphs showing the climbing ability flies carrying the UAS - ( G4C2 ) x36 construct in neurons using the Elav - gal4 promoter upon treatment with H89 10 µM diluted with 0 . 1 % of DMSO and 5 % sucrose in PBS . The wild - type ( w1118 ) line was used as control . ( A ) Female flies ( ♀ ) . Data are mean ± SD . 2 - way ANOVA followed by Tukey ' s multiple comparisons , significance is reported for the 6th day of treatment * * * * P < 0 . 0001 ( G4C2 ) x36 vs ( G4C2 ) x36 + H89 . ( B ) Male flies ( ♂ ) . Data are mean ± SD . 2 - way ANOVA followed by Tukey ' s multiple comparisons , significance is reported for the 6th day of treatment . * * * * P < 0 . 0001 ( G4C2 ) x36 vs ( G4C2 ) x36 + H89 . Experiments were performed three times , N = 15 adult flies were used for each genotype , sex and treatment ( N tot = 45 ) . C , D Representative graphs showing climbing activity of flies co - expressing the UAS - Pka - RNAi construct ( 1 , v330111 ) in combination with UAS - ( G4C2 ) x36 in neurons using the Elav - gal4 promoter . The wild - type ( w1118 ) line was used as control . ( C ) Female flies ( ♀ ) . Data are mean ± SD . 2 - way ANOVA followed by Tukey ' s multiple comparisons , significance is reported for the 9th day * * * * P < 0 . 0001 ( G4C2 ) x36 vs Pka - RNAi ( 1 ) ; ( G4C2 ) x36 . ( D ) Male flies ( ♂ ) . Data are mean ± SD . 2 - way ANOVA followed by Tukey ' s multiple comparisons , significance is reported for the 7th day * * * * P < 0 . 0001 ( G4C2 ) x36 vs Pka - RNAi ( 1 ) ; ( G4C2 ) x36 . Experiments were performed twice with different RNA interfering sequence , N = 15 adult flies were used for each genotype and sex ( N tot = 30 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B Representative graphs showing the climbing ability flies carrying the UAS-(G4C2)x36 construct in neurons using the Elav-gal4 promoter upon treatment with H89 10 µM diluted with 0.1 % of DMSO and 5% sucrose in PBS. The wild-type (w1118) line was used as control. (A) Female flies (♀). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 6th day of treatment ****P<0.0001 (G4C2)x36 vs (G4C2)x36 + H89. (B) Male flies (♂). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 6th day of treatment. ****P<0.0001 (G4C2)x36 vs (G4C2)x36 + H89. Experiments were performed three times, N=15 adult flies were used for each genotype, sex and treatment (N tot=45). C, D Representative graphs showing climbing activity of flies co-expressing the UAS-Pka-RNAi construct (1, v330111) in combination with UAS-(G4C2)x36 in neurons using the Elav-gal4 promoter. The wild-type (w1118) line was used as control. (C) Female flies (♀). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 9th day ****P<0.0001 (G4C2)x36 vs Pka-RNAi (1);(G4C2)x36. (D) Male flies (♂). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 7th day ****P<0.0001 (G4C2)x36 vs Pka-RNAi (1);(G4C2)x36. Experiments were performed twice with different RNA interfering sequence, N=15 adult flies were used for each genotype and sex (N tot=30).


A , B Three independent C9 patient iPSC - MN lines ( Becker S6 , M211R2 , C90202 ) were treated with GELD , SPL , FSK , H89 or DMSO vehicle control and levels of poly - GA ( A ) and poly - GP ( B ) measured after 7 days . Values are displayed as percentages of mean DMSO signal . No significant changes in either DPR were observed ( linear mixed effects model , all not significant , P > 0 . 05 ; P = 0 . 1 DMSO vs FSK ) . Treatments were performed in four technical replicates .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

A, B Three independent C9 patient iPSC-MN lines (Becker S6, M211R2, C90202) were treated with GELD, SPL, FSK, H89 or DMSO vehicle control and levels of poly-GA (A) and poly-GP (B) measured after 7 days. Values are displayed as percentages of mean DMSO signal. No significant changes in either DPR were observed (linear mixed effects model, all not significant, P>0.05; P = 0.1 DMSO vs FSK). Treatments were performed in four technical replicates.


C , D Three independent C9 patient iPSC - MN lines were treated with 10 µM H89 and levels of poly - GA ( C ) and poly - GP ( D ) were measured after 14 days . Values are displayed as percentages of mean DMSO signal . H89 significantly reduced poly - GA * P = 0 . 039 and poly - GP levels * P = 0 . 018 ( linear mixed effects model ) . Treatments were performed in four technical replicates .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D Three independent C9 patient iPSC-MN lines were treated with 10 µM H89 and levels of poly-GA (C) and poly-GP (D) were measured after 14 days. Values are displayed as percentages of mean DMSO signal. H89 significantly reduced poly-GA *P = 0.039 and poly-GP levels *P = 0.018 (linear mixed effects model). Treatments were performed in four technical replicates.


E No increased LDH release was observed after 14 - day 10 µM H89 treatment compared to vehicle treatment in two independent C9 patient iPSC - MN lines ( unpaired t - tests , Becker S6 : significant decrease in LDH , * * P = 0 . 0064 ; M211R2 : not significant , P > 0 . 05 ) . In Becker S6 , mean LDH release was 22 % lower after H89 treatment compared to vehicle . Treatments were performed in eight technical replicates . Data information : Error bars reported as ± SD . Not significant = P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E No increased LDH release was observed after 14-day 10 µM H89 treatment compared to vehicle treatment in two independent C9 patient iPSC-MN lines (unpaired t-tests, Becker S6: significant decrease in LDH, **P = 0.0064; M211R2: not significant, P>0.05). In Becker S6, mean LDH release was 22% lower after H89 treatment compared to vehicle. Treatments were performed in eight technical replicates. Data information: Error bars reported as ± SD. Not significant = P>0.05, *P<0.05, **P<0.01.


F Relative fold changes of C9orf72 variant RNA levels normalised to RPL13A , after 14 - day 10 µM H89 treatment vs vehicle treatment ( N = 1 C9 patient line , four technical replicates ) . No significant changes were observed between H89 and vehicle for any of the isoforms ( 2 - way ANOVA with Šídák ' s multiple comparisons test , all not significant , P > 0 . 05 ) . Data information : Error bars reported as ± SD . Not significant = P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 . T

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Relative fold changes of C9orf72 variant RNA levels normalised to RPL13A, after 14-day 10 µM H89 treatment vs vehicle treatment (N=1 C9 patient line, four technical replicates). No significant changes were observed between H89 and vehicle for any of the isoforms (2-way ANOVA with Šídák's multiple comparisons test, all not significant, P>0.05). Data information: Error bars reported as ± SD. Not significant = P>0.05, *P<0.05, **P<0.01. T


, Maximum intensity projections of confocal z - stacks taken from the apical cochlear region of control ( A ) mice at postnatal day 11 . Inner hair cells ( IHCs ) were stained with antibodies against Kir2 . 1 ( green ) and the hair cell marker MYO7a ( blue ) . At least 3 mice for each genotype were used .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

, Maximum intensity projections of confocal z-stacks taken from the apical cochlear region of control (A) mice at postnatal day 11. Inner hair cells (IHCs) were stained with antibodies against Kir2.1 (green) and the hair cell marker MYO7a (blue). At least 3 mice for each genotype were used.


C , D , Currents from IHCs of control ( C , P9 ) and Kir2 . 1 - OE ( D , P10 ) pre - hearing mice . Currents were elicited by using depolarizing and hyperpolarizing voltage steps , with a nominal increment of 10 mV , from a holding potential of − 84 mV . Test potentials are shown next to some of the traces . Note that the large inward rectifier Kir2 . 1 current is only present in the IHC of the Kir2 . 1 - OE mouse ( D ) . The outward current is primarily carried by a delayed rectifier current IK . IK1 identifies the small inwardly rectifying K + current normally expressed in IHCs . E , Steady - state current - voltage curves obtained from IHCs of control ( P9 - P11 ) and Kir2 . 1 - OE ( P9 - P11 ) mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D, Currents from IHCs of control (C, P9) and Kir2.1-OE (D, P10) pre-hearing mice. Currents were elicited by using depolarizing and hyperpolarizing voltage steps, with a nominal increment of 10 mV, from a holding potential of −84 mV. Test potentials are shown next to some of the traces. Note that the large inward rectifier Kir2.1 current is only present in the IHC of the Kir2.1-OE mouse (D). The outward current is primarily carried by a delayed rectifier current IK. IK1 identifies the small inwardly rectifying K+ current normally expressed in IHCs. E, Steady-state current-voltage curves obtained from IHCs of control (P9-P11) and Kir2.1-OE (P9-P11) mice.


F , G , Representative ∆ F / F0 traces from the IHCs and GER of P7 - P9 control ( F ) and Kir2 . 1 - OE ( G ) mice in the presence of 0 . 3 mM Ca2 + . Spontaneous ATP - dependent Ca2 + waves from the GER ( green traces ) were eliciting coordinated Ca2 + signals in the IHCs from both controls and Kir2 . 1 - OE mice . For each genotype , two separate sets of recordings from 2 mice are shown ( top and bottom right ) , with the top traces being linked to the images on the left : before [ 1 ] , during [ 2 ] and after [ 3 ] ) the generation of a large Ca2 + wave from the GER . For details about the frequency and duration of the Ca2 + waves , and the number of mice and recordings see Fig EV3 . All recordings were obtained at body temperature . Traces are computed as pixel averages of regions of interest centred on IHCs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, G, Representative ∆F/F0 traces from the IHCs and GER of P7-P9 control (F) and Kir2.1-OE (G) mice in the presence of 0.3 mM Ca2+. Spontaneous ATP-dependent Ca2+ waves from the GER (green traces) were eliciting coordinated Ca2+ signals in the IHCs from both controls and Kir2.1-OE mice. For each genotype, two separate sets of recordings from 2 mice are shown (top and bottom right), with the top traces being linked to the images on the left: before [1], during [2] and after [3]) the generation of a large Ca2+ wave from the GER. For details about the frequency and duration of the Ca2+ waves, and the number of mice and recordings see Fig EV3. All recordings were obtained at body temperature. Traces are computed as pixel averages of regions of interest centred on IHCs.


C , Peak - to - peak MET current - voltage curves from P6 - P7 apical - coil IHCs of 7 control ( 12 IHCs ) and 3 littermates Kir2 . 1 - OE mice ( 7 IHCs ) . Recordings were obtained by mechanically stimulating the hair bundles of IHCs at the same time as stepping their membrane potential from − 124 mV to + 96 mV in 20 mV increments . The two sets of data are not significantly different : P = 0 . 6320 , 2 - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, Peak-to-peak MET current-voltage curves from P6-P7 apical-coil IHCs of 7 control (12 IHCs) and 3 littermates Kir2.1-OE mice (7 IHCs). Recordings were obtained by mechanically stimulating the hair bundles of IHCs at the same time as stepping their membrane potential from −124 mV to +96 mV in 20 mV increments. The two sets of data are not significantly different: P = 0.6320, 2-way ANOVA.


C , Peak - to - peak MET current - voltage curves from P8 - P9 apical - coil IHCs of 12 control ( 17 IHCs ) and 13 littermates Kir2 . 1 - OE mice ( 24 IHCs ) . The two sets of data are significantly different : P < 0 . 0001 , 2 - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, Peak-to-peak MET current-voltage curves from P8-P9 apical-coil IHCs of 12 control (17 IHCs) and 13 littermates Kir2.1-OE mice (24 IHCs). The two sets of data are significantly different: P < 0.0001, 2-way ANOVA.


J , Example of FM1 - 43 uptake by IHCs from P11 control ( top ) and Kir2 . 1 - OE ( bottom ) mice , showing the lack of fluorescence labelling in the latter , which is an indication of the lack of MET channels open at rest at this stage in the IHCs overexpressing the Kir2 . 1 channels . At least 3 mice for each genotype were used .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J, Example of FM1-43 uptake by IHCs from P11 control (top) and Kir2.1-OE (bottom) mice, showing the lack of fluorescence labelling in the latter, which is an indication of the lack of MET channels open at rest at this stage in the IHCs overexpressing the Kir2.1 channels. At least 3 mice for each genotype were used.


A , B , Scanning electron microscope ( SEM ) images showing the IHC hair bundle structure in the apical coil of the cochlea of P11 control ( A ) and P8 - P11 Kir2 . 1 - OE ( B ) mice . Control IHCs and the large majority of P8 IHCs from Kir2 . 1 - OE mice show a normal hair bundle structure composed of three rows of stereocilia : tall , intermediate and short ( arrows ) . From about P9 in Kir2 . 1 - OE mice , some IHCs start to lose the third row of stereocilia ( arrowheads ) and some already exhibit some fusion of the stereocilia ( asterisk ) . These changes in hair - bundle structure became more prominent at P11 . At least 3 mice for each genotype were used . In these panels and those below , asterisks are used to define some of the abnormal hair bundles .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B, Scanning electron microscope (SEM) images showing the IHC hair bundle structure in the apical coil of the cochlea of P11 control (A) and P8-P11 Kir2.1-OE (B) mice. Control IHCs and the large majority of P8 IHCs from Kir2.1-OE mice show a normal hair bundle structure composed of three rows of stereocilia: tall, intermediate and short (arrows). From about P9 in Kir2.1-OE mice, some IHCs start to lose the third row of stereocilia (arrowheads) and some already exhibit some fusion of the stereocilia (asterisk). These changes in hair-bundle structure became more prominent at P11. At least 3 mice for each genotype were used. In these panels and those below, asterisks are used to define some of the abnormal hair bundles.


C , D , SEM images of both IHCs and OHCs from the cochlea of P26 control ( C , upper panel ) and P26 Kir2 . 1 - OE ( D , upper panel ) mice . Lower panels show a higher - magnification view of the hair bundle of IHCs from both genotypes , highlighting the profound disruption of the stereocilia in IHCs overexpressing Kir2 . 1 channels . At least 3 mice for each genotype were used .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D, SEM images of both IHCs and OHCs from the cochlea of P26 control (C, upper panel) and P26 Kir2.1-OE (D, upper panel) mice. Lower panels show a higher-magnification view of the hair bundle of IHCs from both genotypes, highlighting the profound disruption of the stereocilia in IHCs overexpressing Kir2.1 channels. At least 3 mice for each genotype were used.


A , B , Maximum intensity projections of confocal z - stacks showing images of the hair bundles from apical - coil IHCs of P6 and P11 control ( A ) and Kir2 . 1 - OE ( B ) mice immunostained with antibodies against MYOSIN VI ( blue ) and EPS8 ( magenta ) . At least 3 mice for each genotype were used . In all panels ( A - D ) , stereocilia are labelled with phalloidin ( green ) . Note that despite the disrupted hair - bundle structure in the IHCs overexpressing Kir2 . 1 channels ; the stereocilia retained a normal distribution of these bundle proteins . At least 3 mice for each genotype were used .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A,B, Maximum intensity projections of confocal z-stacks showing images of the hair bundles from apical-coil IHCs of P6 and P11 control (A) and Kir2.1-OE (B) mice immunostained with antibodies against MYOSIN VI (blue) and EPS8 (magenta). At least 3 mice for each genotype were used. In all panels (A-D), stereocilia are labelled with phalloidin (green). Note that despite the disrupted hair-bundle structure in the IHCs overexpressing Kir2.1 channels; the stereocilia retained a normal distribution of these bundle proteins. At least 3 mice for each genotype were used.


C , D , Confocal images of the hair bundles of P11 IHCs from control ( C ) and Kir2 . 1 - OE ( D ) mice immunostained with antibodies against MYOSIN XV isoform 1 ( blue ) and WHIRLIN ( magenta ) . In all panels ( A - D ) , stereocilia are labelled with phalloidin ( green ) . Note that despite the disrupted hair - bundle structure in the IHCs overexpressing Kir2 . 1 channels ; the stereocilia retained a normal distribution of these bundle proteins . At least 3 mice for each genotype were used .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C,D, Confocal images of the hair bundles of P11 IHCs from control (C) and Kir2.1-OE (D) mice immunostained with antibodies against MYOSIN XV isoform 1 (blue) and WHIRLIN (magenta). In all panels (A-D), stereocilia are labelled with phalloidin (green). Note that despite the disrupted hair-bundle structure in the IHCs overexpressing Kir2.1 channels; the stereocilia retained a normal distribution of these bundle proteins. At least 3 mice for each genotype were used.


A , B , Maximum intensity projections of confocal z - stacks showing the IHCs of the apical cochlear region from control ( A ) and Kir2 . 1 - OE ( B ) pups with the females being in the continuous presence of DOX in the drinking water from conception up to when the pups were P5 ( upper panels ) . Middle and bottom panels show IHCs at P7 and P14 following the removal of DOX at P5 for both control ( A ) and Kir2 . 1 - OE mice ( B ) . IHCs were stained with antibodies against the K + channel Kir2 . 1 ( green ) and Myosin 7a ( Myo7a , blue : cell marker ) . Note that after 2 days following the removal of DOX , Kir2 . 1overexpression was already largely downregulated . At least 3 mice for each genotype were used .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A,B, Maximum intensity projections of confocal z-stacks showing the IHCs of the apical cochlear region from control (A) and Kir2.1-OE (B) pups with the females being in the continuous presence of DOX in the drinking water from conception up to when the pups were P5 (upper panels). Middle and bottom panels show IHCs at P7 and P14 following the removal of DOX at P5 for both control (A) and Kir2.1-OE mice (B). IHCs were stained with antibodies against the K+ channel Kir2.1 (green) and Myosin 7a (Myo7a, blue: cell marker). Note that after 2 days following the removal of DOX, Kir2.1overexpression was already largely downregulated. At least 3 mice for each genotype were used.


H , I , Maximum intensity projections of confocal z - stacks showing images of the hair bundles from apical - coil IHCs of P14 control ( H ) and Kir2 . 1 - OE ( I ) mice stained with phalloidin . DOX was removed from the mother ' s drinking water when the pups were P5 . At least 3 mice for each genotype were used .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H,I, Maximum intensity projections of confocal z-stacks showing images of the hair bundles from apical-coil IHCs of P14 control (H) and Kir2.1-OE (I) mice stained with phalloidin. DOX was removed from the mother's drinking water when the pups were P5. At least 3 mice for each genotype were used.


J , K , SEM images showing the normal structure of the hair bundles of the IHCs in the apical coil of the cochlea of P14 control ( J ) and P14 Kir2 . 1 - OE ( K ) mice . DOX was removed from the mother ' s drinking water when the pups were P5 . Note that the morphological profile of the hair bundles in IHCs is comparable between control and Kir2 . 1 - OE mice , indicating that the removal of the intrinsic Ca2 + action potentials prior to the second postnatal week has no effect on the mechanoelectrical transduction apparatus . At least 3 mice for each genotype were used .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J,K, SEM images showing the normal structure of the hair bundles of the IHCs in the apical coil of the cochlea of P14 control (J) and P14 Kir2.1-OE (K) mice. DOX was removed from the mother's drinking water when the pups were P5. Note that the morphological profile of the hair bundles in IHCs is comparable between control and Kir2.1-OE mice, indicating that the removal of the intrinsic Ca2+ action potentials prior to the second postnatal week has no effect on the mechanoelectrical transduction apparatus. At least 3 mice for each genotype were used.


D , Heatmap of the counts for each RNA - library for the 118 genes associated with microtubule or cytoskeletal processes . Each row is z - scored . 118 genes were all differentially expressed as per previous analysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, Heatmap of the counts for each RNA-library for the 118 genes associated with microtubule or cytoskeletal processes. Each row is z-scored. 118 genes were all differentially expressed as per previous analysis.


A ) Representative size - exclusion chromatography ( SEC ) chromatogram with absorbance measured at nanometers ( nm ) 260 or 280 ( for this chromatogram and the following ones , absorbances are in units of 1000 - 1 ( mAU ) ) of reconstituted K . lactis COMA - Nkp1 - Nkp2 , and image of Coomassie Blue - stained SDS - PAGE gel with fractions from principal SEC peaks . Molecular masses of protein standards ( Mm . S . ) are in kiloDalton ( kDa ) ; L : sample loaded on column . † : Escherichia Coli DnaK ; ╪ : proteolysed Nkp1 - Nkp2 ; Nkp1 - Nkp2 is less prone to spontaneous proteolysis when associated with COMA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) Representative size-exclusion chromatography (SEC) chromatogram with absorbance measured at nanometers (nm) 260 or 280 (for this chromatogram and the following ones, absorbances are in units of 1000-1 (mAU)) of reconstituted K. lactis COMA-Nkp1-Nkp2, and image of Coomassie Blue-stained SDS-PAGE gel with fractions from principal SEC peaks. Molecular masses of protein standards (Mm. S.) are in kiloDalton (kDa); L: sample loaded on column. †: Escherichia Coli DnaK; ╪: proteolysed Nkp1-Nkp2; Nkp1-Nkp2 is less prone to spontaneous proteolysis when associated with COMA.


B , C ) Nanoflow mass - spectra of COMA - Nkp1 - Nkp2 ( B ) acquired with different injected concentrations , showing an increase in COMA - Nkp1 - Nkp2 dimers or COMA dimers with increasing injectant concentration .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B,C) Nanoflow mass-spectra of COMA-Nkp1-Nkp2 (B) acquired with different injected concentrations, showing an increase in COMA-Nkp1-Nkp2 dimers or COMA dimers with increasing injectant concentration.


B , C ) Nanoflow mass - spectra of COMA ( C ) , acquired with different injected concentrations , showing an increase in COMA - Nkp1 - Nkp2 dimers or COMA dimers with increasing injectant concentration .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B,C) Nanoflow mass-spectra of COMA (C), acquired with different injected concentrations, showing an increase in COMA-Nkp1-Nkp2 dimers or COMA dimers with increasing injectant concentration.


D ) Graph with measured differential refractive index ( relative scale ) and molar mass calculated with multi - angle light scattering ( MALS ) data of COMA eluting from SEC ( SEC - MALS ) . We injected COMA at a concentration of ~ 43 μM ( COMA dimer ) on the column . Dashed lines indicate expected molar masses for monomeric COMA or dimeric COMA : 142337 g mol − 1 or 284674 g mol − 1 ; our experimentally determined molar mass from the principal SEC elution peak : 299100 g mol − 1 ± 5683 g mol − 1 ( mean ± standard deviation from a single SEC - MALS experiment ) ; max . dimeric COMA concentration , from refractive index measurement , in principal peak : 2 . 4 μM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Graph with measured differential refractive index (relative scale) and molar mass calculated with multi-angle light scattering (MALS) data of COMA eluting from SEC (SEC-MALS). We injected COMA at a concentration of ~ 43 μM (COMA dimer) on the column. Dashed lines indicate expected molar masses for monomeric COMA or dimeric COMA: 142337 g mol−1 or 284674 g mol−1; our experimentally determined molar mass from the principal SEC elution peak: 299100 g mol−1 ± 5683 g mol−1 (mean ± standard deviation from a single SEC-MALS experiment); max. dimeric COMA concentration, from refractive index measurement, in principal peak: 2.4 μM.


A ) Excerpt from a representative nanoflow mass - spectrum of COMA ( full spectrum and enlarged spectrum areas are in Appendix Fig S2A ) ; B ) mass - spectrum from tandem mass - spectrometry of the isolated 28 + charge state of COMA ( from spectrum shown in A ) , after its collision - induced dissociation in the gas phase . Charge states of assigned masses are indicated above peaks . For this spectrum and our other spectra of this type , charge states of assigned masses are indicated above peaks . Inset table shows identified proteins or protein assemblies , their measured mass values as mean ± standard deviation from multiple peaks in a peak series assigned to the same molecular species ; and their respective expected mass .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) Excerpt from a representative nanoflow mass-spectrum of COMA (full spectrum and enlarged spectrum areas are in Appendix Fig S2A); B) mass-spectrum from tandem mass-spectrometry of the isolated 28+ charge state of COMA (from spectrum shown in A), after its collision-induced dissociation in the gas phase. Charge states of assigned masses are indicated above peaks. For this spectrum and our other spectra of this type, charge states of assigned masses are indicated above peaks. Inset table shows identified proteins or protein assemblies, their measured mass values as mean ± standard deviation from multiple peaks in a peak series assigned to the same molecular species; and their respective expected mass.


A , B ) Plots showing hydrogen - deuterium exchanged peptides , after 10 sec of deuterium exchange of Okp1 ( A ) each in COMA or COMA - Nkp1 - Nkp2 . For this representation , and all following of this type , detected peptides are colour - coded according to their measured deuterium - exchange in % of the maximum measured exchange ( 1 - 25 % : dark blue ; 26 - 50 % : light blue ; 51 - 75 % : yellow ; 76 - 100 % : red ) . Peptides are represented as bars . Each bar is plotted row - wise corresponding to its position in the amino - acid sequence of the protein it derives from . Amino - acid sequence scheme is illustrated at the top with thin or thick lines ; thin line : flexible element : thick line : structured segment . Positions of predicted coiled coils are shown as helices . Plots for full time courses are in Appendix Fig S3C , E .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A,B) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Okp1 (A) each in COMA or COMA-Nkp1-Nkp2. For this representation, and all following of this type, detected peptides are colour-coded according to their measured deuterium-exchange in % of the maximum measured exchange (1-25 %: dark blue; 26-50 %: light blue; 51-75 %: yellow; 76-100 %: red). Peptides are represented as bars. Each bar is plotted row-wise corresponding to its position in the amino-acid sequence of the protein it derives from. Amino-acid sequence scheme is illustrated at the top with thin or thick lines; thin line: flexible element: thick line: structured segment. Positions of predicted coiled coils are shown as helices. Plots for full time courses are in Appendix Fig S3C,E.


A , B ) Plots showing hydrogen - deuterium exchanged peptides , after 10 sec of deuterium exchange of Ame1 ( B ) , each in COMA or COMA - Nkp1 - Nkp2 . For this representation , and all following of this type , detected peptides are colour - coded according to their measured deuterium - exchange in % of the maximum measured exchange ( 1 - 25 % : dark blue ; 26 - 50 % : light blue ; 51 - 75 % : yellow ; 76 - 100 % : red ) . Peptides are represented as bars . Each bar is plotted row - wise corresponding to its position in the amino - acid sequence of the protein it derives from . Amino - acid sequence scheme is illustrated at the top with thin or thick lines ; thin line : flexible element : thick line : structured segment . Positions of predicted coiled coils are shown as helices . Plots for full time courses are in Appendix Fig S3C , E .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A,B) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Ame1 (B), each in COMA or COMA-Nkp1-Nkp2. For this representation, and all following of this type, detected peptides are colour-coded according to their measured deuterium-exchange in % of the maximum measured exchange (1-25 %: dark blue; 26-50 %: light blue; 51-75 %: yellow; 76-100 %: red). Peptides are represented as bars. Each bar is plotted row-wise corresponding to its position in the amino-acid sequence of the protein it derives from. Amino-acid sequence scheme is illustrated at the top with thin or thick lines; thin line: flexible element: thick line: structured segment. Positions of predicted coiled coils are shown as helices. Plots for full time courses are in Appendix Fig S3C,E.


B , C ) Representative images of Coomassie Blue - stained SDS - PAGE gels with Ni2 + - affinity purification eluates . B ) of full - length COMA ( lane 11 ) with full - length polyhistidine - tagged Mcm21 ( His - Mcm21 ) and Ctf19 , or COMA variants with polyhistidine - tagged Mcm21 D - RWD domain ( His - Mcm21 _ D - RWD ; K . lactis Mcm21 residues 108 - 293 ) and Ctf19 D - RWD domain ( Ctf19 _ D - RWD ; K . lactis Ctf19 residues 107 - 270 ) We show one - letter identifiers for protein versions in A . Lanes between lanes 5 and 6 are not relevant .

B,C) Representative images of Coomassie Blue-stained SDS-PAGE gels with Ni2+-affinity purification eluates. B) of full-length COMA (lane 11) with full-length polyhistidine-tagged Mcm21 (His-Mcm21) and Ctf19, or COMA variants with polyhistidine-tagged Mcm21 D-RWD domain (His-Mcm21_D-RWD; K. lactis Mcm21 residues 108-293) and Ctf19 D-RWD domain (Ctf19_D-RWD; K. lactis Ctf19 residues 107-270) We show one-letter identifiers for protein versions in A. Lanes between lanes 5 and 6 are not relevant.


B , C ) Representative images of Coomassie Blue - stained SDS - PAGE gels with Ni2 + - affinity purification eluates . C ) of Ame1 - Okp1 variants with polyhistidine - tagged Okp1 variants ( His - Okp1 variants ) . We show one - letter identifiers for protein versions in A . Lanes between lanes 5 and 6 are not relevant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B,C) Representative images of Coomassie Blue-stained SDS-PAGE gels with Ni2+-affinity purification eluates. C) of Ame1-Okp1 variants with polyhistidine-tagged Okp1 variants (His-Okp1 variants). We show one-letter identifiers for protein versions in A. Lanes between lanes 5 and 6 are not relevant.


D ) Image of SDS - PAGE gel with fractions of two purified Ctf19 - Mcm21 - Okp1 variants ( after SEC ) ; expected migrating position of non - proteolysed Okp1 variant ( compare with its position in SDS - PAGE images of COMA or Ame1 - Okp1 variants in B or C ) is marked ; 2 and 3 : SEC - purified fractions of samples from different elution fractions from ion - exchange chromatography . When we co - expressed full - length Okp1 with Ctf19 and Mcm21 , which has a coding regions for an N - terminal polyhistidine - tag , we did not co - purify substantial amounts of full - length Okp1 with Ctf19 - Mcm21 , probably because Okp1 was prone to proteolysis ( in the absence of Ame1 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Image of SDS-PAGE gel with fractions of two purified Ctf19-Mcm21-Okp1 variants (after SEC); expected migrating position of non-proteolysed Okp1 variant (compare with its position in SDS-PAGE images of COMA or Ame1-Okp1 variants in B or C) is marked; 2 and 3: SEC-purified fractions of samples from different elution fractions from ion-exchange chromatography. When we co-expressed full-length Okp1 with Ctf19 and Mcm21, which has a coding regions for an N-terminal polyhistidine-tag, we did not co-purify substantial amounts of full-length Okp1 with Ctf19-Mcm21, probably because Okp1 was prone to proteolysis (in the absence of Ame1).


A , B ) Overlayed chromatograms showing absorbance at 280 nm from SEC of Ctf19 - Mcm21 - Okp1 variants without Okp1 segment 3 ( A ) or with Okp1 segment 3 ( B ) ; Ctf19 - Mcm21 - Okp1 variants : CMO , Nkp1 - Nkp2 : NN , Ctf19 - Mcm21 - Okp1 variants combined with Nkp1 - Nkp2 : CMO - NN ; and images of SDS - PAGE gels with equivalent elution fractions from five SEC experiments ; L : loaded on column . The elution - volume range that we analysed fractions of on SDS - PAGE is indicated below the chromatograms .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A,B) Overlayed chromatograms showing absorbance at 280 nm from SEC of Ctf19-Mcm21-Okp1 variants without Okp1 segment 3 (A) or with Okp1 segment 3 (B); Ctf19-Mcm21-Okp1 variants: CMO, Nkp1-Nkp2: NN, Ctf19-Mcm21-Okp1 variants combined with Nkp1-Nkp2: CMO-NN; and images of SDS-PAGE gels with equivalent elution fractions from five SEC experiments; L: loaded on column. The elution-volume range that we analysed fractions of on SDS-PAGE is indicated below the chromatograms.


C ) Representative microscopy - images of living haploid S . cerevisiae cells with Okp1 _ fl ( integrated full - length Okp1 ) or our Okp1 version that lacks the coding region for segment 3 - Okp1 _ nnΔ , with Nkp1 - GFP or Nkp2 - GFP . Fluorescence images are maximum intensity projected , and we show those from Okp1 _ fl or Okp1 _ nnΔ with the same fluorophor - fusion kinetochore protein on the same intensity scale ; scale bar : 2 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Representative microscopy-images of living haploid S. cerevisiae cells with Okp1_fl (integrated full-length Okp1) or our Okp1 version that lacks the coding region for segment 3-Okp1_nnΔ, with Nkp1-GFP or Nkp2-GFP. Fluorescence images are maximum intensity projected, and we show those from Okp1_fl or Okp1_nnΔ with the same fluorophor-fusion kinetochore protein on the same intensity scale; scale bar: 2 μm.


D , E ) Plots showing hydrogen - deuterium exchanged peptides , after 10 sec of deuterium exchange of Nkp1 ( D ) each in Nkp1 - Nkp2 or COMA - Nkp1 - Nkp2 . Nkp1 has an N - terminal SNA residual . Plots for full time courses are in Appendix Fig S5A , B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D,E) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Nkp1 (D) each in Nkp1-Nkp2 or COMA-Nkp1-Nkp2. Nkp1 has an N-terminal SNA residual. Plots for full time courses are in Appendix Fig S5A,B.


D , E ) Plots showing hydrogen - deuterium exchanged peptides , after 10 sec of deuterium exchange of Nkp2 ( E ) , each in Nkp1 - Nkp2 or COMA - Nkp1 - Nkp2 . Nkp1 has an N - terminal SNA residual . Plots for full time courses are in Appendix Fig S5A , B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D,E) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Nkp2 (E), each in Nkp1-Nkp2 or COMA-Nkp1-Nkp2. Nkp1 has an N-terminal SNA residual. Plots for full time courses are in Appendix Fig S5A,B.


A ) Plots showing deuterium - exchanged peptides , after deuterium exchange of 10 sec of Okp1 in Ctf19D - RWD - Mcm21D - RWD - Okp1229 - 336 , in full - length COMA , or in Ame11 - 260 - Okp1123 - 336 . Plots for full time course are in Appendix Fig S6C .

A) Plots showing deuterium-exchanged peptides, after deuterium exchange of 10 sec of Okp1 in Ctf19D-RWD-Mcm21D-RWD-Okp1229-336, in full-length COMA, or in Ame11-260-Okp1123-336. Plots for full time course are in Appendix Fig S6C.


C ) Semi - transparent surface representation of our Ctf19D - RWD - Mcm21D - RWD - Okp1295 - 360 crystal structure , with secondary structure cartoon - diagram underneath ; left : in a similar orientation as Ctf19 - Mcm21 in B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Semi-transparent surface representation of our Ctf19D-RWD-Mcm21D-RWD-Okp1295-360 crystal structure, with secondary structure cartoon-diagram underneath; left: in a similar orientation as Ctf19-Mcm21 in B.


D ) Close - up view of the Okp1 binding site of Ctf19 - Mcm21 with Ctf19 - Mcm21 binding motif of Okp1 ; Okp1 residues , and some Ctf19 or Mcm21 sidechains , are shown as sticks ( nitrogen : blue , oxygen : red ) ; Ctf19 - Mcm21 shown as secondary structure cartoon - diagram .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

D) Close-up view of the Okp1 binding site of Ctf19-Mcm21 with Ctf19-Mcm21 binding motif of Okp1; Okp1 residues, and some Ctf19 or Mcm21 sidechains, are shown as sticks (nitrogen: blue, oxygen: red); Ctf19-Mcm21 shown as secondary structure cartoon-diagram.