. Adult flies were fed with regular sucrose diet ( RS ) or high sucrose diet ( HS ) , and survival percentage of adult flies of the indicated genotypes were examined . Kaplan - Meier survival analysis , n ≥ 40 for each group .

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.Adult flies were fed with regular sucrose diet (RS) or high sucrose diet (HS), and survival percentage of adult flies of the indicated genotypes were examined. Kaplan-Meier survival analysis, n ≥ 40 for each group.


CFTR splicing assay was performed in cells with knockdown of either OGT , TDP - 43 , or combination . ACTB levels served as loading controls ( top panel ) . The indicated protein levels were examined by immunoblotting ( bottom panel ) .

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CFTR splicing assay was performed in cells with knockdown of either OGT, TDP-43, or combination. ACTB levels served as loading controls (top panel). The indicated protein levels were examined by immunoblotting (bottom panel).


CFTR splicing assay was examined in siTDP - 43 cells overexpressing the WT or mutant OGT by PCR analysis . ACTB levels served as loading controls ( top panel ) . Various protein levels were examined by immunoblotting ( bottom panel ) .

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CFTR splicing assay was examined in siTDP-43 cells overexpressing the WT or mutant OGT by PCR analysis. ACTB levels served as loading controls (top panel). Various protein levels were examined by immunoblotting (bottom panel).


Representative PCR analysis to examine expression levels of the spliced mRNA isoform containing exon 2a of STMN2 in SH - SY5Y cells with the indicated constructs ( top panel ) . TDP - 43 protein levels were examined by immunoblotting ( bottom panel ) .

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Representative PCR analysis to examine expression levels of the spliced mRNA isoform containing exon 2a of STMN2 in SH-SY5Y cells with the indicated constructs (top panel). TDP-43 protein levels were examined by immunoblotting (bottom panel).


( B ) Representative images of mCherry - optoYAP in HEK293T cells imaged with only 561 nm laser following the protocol in ( A ) . Scale bars , 10 μm . ( B ' ) Fold - change in nuclear localisation of mCherry - optoYAP ( n = 50 cells from 3 independent experiments ) .

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(B) Representative images of mCherry-optoYAP in HEK293T cells imaged with only 561 nm laser following the protocol in (A). Scale bars, 10 μm. (B') Fold-change in nuclear localisation of mCherry-optoYAP (n=50 cells from 3 independent experiments).


( C ) Representative images of the same cells in ( B ) exposed to both 488 and 561 nm following the protocol shown in ( A ) . Scale bars , 10 μm . ( C ' ) Fold - change in nuclear localisation of the mCherry - optoYAP ( n = 50 cells from 3 independent experiments ) .

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(C) Representative images of the same cells in (B) exposed to both 488 and 561 nm following the protocol shown in (A). Scale bars, 10 μm. (C') Fold-change in nuclear localisation of the mCherry-optoYAP (n=50 cells from 3 independent experiments).


( D ) HEK293T cells transfected with optoYAP were subjected to three cycles of activation protocol and recovery in the dark . ( Top ) The ratio of mCherry - optoYAP signal in the nucleus to the cytoplasmic mCherry - optoYAP signal ( n = 8 cells from 2 independent experiments ) . ( Bottom ) Representative images of the mCherry - optoYAP signal from the same HEK293T cells at each cycle . Scale bars , 10 μm .

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(D) HEK293T cells transfected with optoYAP were subjected to three cycles of activation protocol and recovery in the dark. (Top) The ratio of mCherry-optoYAP signal in the nucleus to the cytoplasmic mCherry-optoYAP signal (n=8 cells from 2 independent experiments). (Bottom) Representative images of the mCherry-optoYAP signal from the same HEK293T cells at each cycle. Scale bars, 10 μm.


( B ) Example images of embryos expressing optofYap ( mCherry - tagged ) in the EVL and deep cells kept in the dark or subjected to the activation protocol at 6 hpf . Scale bars , 10 μm . ( B ' ) Fold - change in nuclear localisation of mCherry - optofYap ( n = 20 cells from 3 independent experiments ) .

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(B) Example images of embryos expressing optofYap (mCherry-tagged) in the EVL and deep cells kept in the dark or subjected to the activation protocol at 6 hpf. Scale bars, 10 μm. (B') Fold-change in nuclear localisation of mCherry-optofYap (n=20 cells from 3 independent experiments).


Soft agar colony formation assay for HFF ( A - B ) cells respectively grown on soft agar for one week under different light conditions as described in Fig . 1A . Scale bars , 1 mm . Representative images of colony growth in HFF cells respectively under different activation conditions . Diameter of individual colonies formed under different light conditions and genetic backgrounds . Colonies were counted and measured from 3 biological replicates .

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Soft agar colony formation assay for HFF (A-B) cells respectively grown on soft agar for one week under different light conditions as described in Fig. 1A. Scale bars, 1 mm. Representative images of colony growth in HFF cells respectively under different activation conditions. Diameter of individual colonies formed under different light conditions and genetic backgrounds. Colonies were counted and measured from 3 biological replicates.


Soft agar colony formation assay for MKN28 ( C - D ) cells respectively grown on soft agar for one week under different light conditions as described in Fig . 1A . Scale bars , 1 mm . Representative images of colony growth in MKN28 cells respectively under different activation conditions . Diameter of individual colonies formed under different light conditions and genetic backgrounds . Colonies were counted and measured from 3 biological replicates .

Soft agar colony formation assay for MKN28 (C-D) cells respectively grown on soft agar for one week under different light conditions as described in Fig. 1A. Scale bars, 1 mm. Representative images of colony growth in MKN28 cells respectively under different activation conditions. Diameter of individual colonies formed under different light conditions and genetic backgrounds. Colonies were counted and measured from 3 biological replicates.


( B ) Example images of brightfield and fluorescently labelled HPG ( Alexa Fluor 647 ) of wild type cells ( PN1 ) assayed for global cellular translation . Scale bars represent 5 µm . ( C ) Change in HPG labelling signal with different durations of HPG incubation ( in PN1 ) , measured by flow cytometry . Population medians of at least 200 , 000 cells are shown . The red line is the ordinary least square ( OLS ) linear regression fitted on the medians between 0 and 5 minutes . (

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(B) Example images of brightfield and fluorescently labelled HPG (Alexa Fluor 647) of wild type cells (PN1) assayed for global cellular translation. Scale bars represent 5 µm. (C) Change in HPG labelling signal with different durations of HPG incubation (in PN1), measured by flow cytometry. Population medians of at least 200,000 cells are shown. The red line is the ordinary least square (OLS) linear regression fitted on the medians between 0 and 5 minutes. (


( E ) Example images of brightfield and fluorescently labelled EU ( Alexa Fluor 488 ) of hENT1 and hsvTK cells ( PN10567 ) assayed for global cellular transcription . Scale bars represent 5 µm . ( F ) Change in EU labelling signal with different lengths of EU incubations , measured by flow cytometry ( in PN10597 ) . The mean and standard deviation ( SD ) of the population medians of at least 200 , 000 cells in experimental triplicates are shown in black . The dark green line is the OLS linear regression fitted on the mean data between 2 and 12 minutes .

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(E) Example images of brightfield and fluorescently labelled EU (Alexa Fluor 488) of hENT1 and hsvTK cells (PN10567) assayed for global cellular transcription. Scale bars represent 5 µm. (F) Change in EU labelling signal with different lengths of EU incubations, measured by flow cytometry (in PN10597). The mean and standard deviation (SD) of the population medians of at least 200,000 cells in experimental triplicates are shown in black. The dark green line is the OLS linear regression fitted on the mean data between 2 and 12 minutes.


( H ) Medians of global translation ( solid black line ) and IQR ( shaded area ) of cdc25 - 22 ( PN143 ) cells grown at 30 ° C and grouped in length bins of 2 µm . Bins containing more than 30 cells are shown . The solid red lines represent a locally estimated scatterplot smoothing ( LOESS ) function fitted on the single - cell data .

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(H) Medians of global translation (solid black line) and IQR (shaded area) of cdc25-22 (PN143) cells grown at 30 °C and grouped in length bins of 2 µm. Bins containing more than 30 cells are shown. The solid red lines represent a locally estimated scatterplot smoothing (LOESS) function fitted on the single-cell data.


( I ) Global cellular translation of cells shown in ( H ) divided by their cell length . The solid black line represents the LOESS function fitted in ( H ) .

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(I) Global cellular translation of cells shown in (H) divided by their cell length. The solid black line represents the LOESS function fitted in (H).


( D ) For each of the 5 length bins boxed in ( C ) , both 1C and 2C medians are normalised to their respective 1C global cellular translation values . The normalised values are represented as dots , each dot corresponding to one of the 5 length bins , the mean and SD of the normalised values are shown for each DNA content . For each DNA content , the normalised values ( dots ) are in the same order ( left to right ) as their corresponding length bins in ( C ) . The p - value is calculated using a Welch ' s unequal variances t - test .

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(D) For each of the 5 length bins boxed in (C), both 1C and 2C medians are normalised to their respective 1C global cellular translation values. The normalised values are represented as dots, each dot corresponding to one of the 5 length bins, the mean and SD of the normalised values are shown for each DNA content. For each DNA content, the normalised values (dots) are in the same order (left to right) as their corresponding length bins in (C). The p-value is calculated using a Welch's unequal variances t-test.


( F ) Example images of brightfield and EGFP - PCNA fluorescence in CCP ∆ EGFP - pcn1 cells ( PN6001 ) . The dashed lines in the EGFP - PCNA channel delimit the cell masks generated from the brightfield image . Cells with visible foci in the EGFP - PCNA channel are highlighted in yellow and marked with arrows . Scale bars represent 5 µm .

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(F) Example images of brightfield and EGFP-PCNA fluorescence in CCP∆ EGFP-pcn1 cells (PN6001). The dashed lines in the EGFP-PCNA channel delimit the cell masks generated from the brightfield image. Cells with visible foci in the EGFP-PCNA channel are highlighted in yellow and marked with arrows. Scale bars represent 5 µm.


( K ) For each of the 4 length bins boxed in ( J ) , G1 , S , and G2 / M medians are normalised to their respective G1 global cellular translation value . The normalised values are represented as dots , each dot corresponding to one of the 4 length bins , the mean and SD of the normalised values are shown for each cell cycle stage . For each cell cycle stage , the normalised values ( dots ) are in the same order ( left to right ) as their corresponding length bins in ( J ) . The p - value is calculated using a Welch ' s unequal variances t - test .

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(K) For each of the 4 length bins boxed in (J), G1, S, and G2/M medians are normalised to their respective G1 global cellular translation value. The normalised values are represented as dots, each dot corresponding to one of the 4 length bins, the mean and SD of the normalised values are shown for each cell cycle stage. For each cell cycle stage, the normalised values (dots) are in the same order (left to right) as their corresponding length bins in (J). The p-value is calculated using a Welch's unequal variances t-test.


( C ) Cells shown in ( A ) and ( B ) are grouped in length bins of 1 µm . Medians of global cellular transcription ( solid lines ) and IQR ( shaded areas ) are shown for G1 ( blue ) , S ( yellow ) , and G2 / M ( red ) subpopulations . The dashed line box marks the length bins which have G1 , S , and G2 / M median global transcription values . Bins containing more than 75 cells are shown .

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(C) Cells shown in (A) and (B) are grouped in length bins of 1 µm. Medians of global cellular transcription (solid lines) and IQR (shaded areas) are shown for G1 (blue), S (yellow), and G2/M (red) subpopulations. The dashed line box marks the length bins which have G1, S, and G2/M median global transcription values. Bins containing more than 75 cells are shown.


( D ) For each of the 3 length bins boxed in ( C ) , G1 , S , and G2 / M medians are normalised to their respective G1 global transcription value . The normalised values are represented as dots , each dot corresponding to one of the 3 length bins , the mean and SD of the normalised values are shown for each cell cycle stage . For each cell cycle stage , the normalised values ( dots ) are in the same order ( left to right ) as their corresponding length bins in ( C ) . The p - value is calculated using a Welch ' s unequal variances t - test .

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(D) For each of the 3 length bins boxed in (C), G1, S, and G2/M medians are normalised to their respective G1 global transcription value. The normalised values are represented as dots, each dot corresponding to one of the 3 length bins, the mean and SD of the normalised values are shown for each cell cycle stage. For each cell cycle stage, the normalised values (dots) are in the same order (left to right) as their corresponding length bins in (C). The p-value is calculated using a Welch's unequal variances t-test.


( E ) For each of the 3 length bins boxed in ( D ) , the medians of each subpopulation are normalised to their respective " uninucleate , low nuclear synCut3 - mCherry " global cellular translation value . The normalised values are represented as dots , each dot corresponding to one of the 3 length bins , the mean and SD of the normalised values are shown for each cell cycle stage . For each mitotic stage , the normalised values ( dots ) are in the same order ( left to right ) as their corresponding length bins in ( D ) . The p - value is calculated using a Welch ' s unequal variances t - test . For visual clarity , synCut3 - mCherry is shortened to synCut3 in the figure .

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(E) For each of the 3 length bins boxed in (D), the medians of each subpopulation are normalised to their respective "uninucleate, low nuclear synCut3-mCherry" global cellular translation value. The normalised values are represented as dots, each dot corresponding to one of the 3 length bins, the mean and SD of the normalised values are shown for each cell cycle stage. For each mitotic stage, the normalised values (dots) are in the same order (left to right) as their corresponding length bins in (D). The p-value is calculated using a Welch's unequal variances t-test. For visual clarity, synCut3-mCherry is shortened to synCut3 in the figure.


( G ) Cells shown in ( F ) are grouped in length bins of 1 µm . Medians of global cellular transcription ( solid lines ) and IQR ( shaded areas ) are shown for uninucleates ( blue ) and binucleates ( red ) , and with low ( light ) and high ( dark ) nuclear synCut3 - mCherry signal subpopulations . Bins containing more than 25 cells are shown .

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(G) Cells shown in (F) are grouped in length bins of 1 µm. Medians of global cellular transcription (solid lines) and IQR (shaded areas) are shown for uninucleates (blue) and binucleates (red), and with low (light) and high (dark) nuclear synCut3-mCherry signal subpopulations. Bins containing more than 25 cells are shown.


Mean ΔF images of Rhod - FF and iGluSnFR signals in response to a voltage ramp and a 50 - ms - long step depolarization , respectively . The synapse marked with * on the overlaid image is further analyzed in the following panels . ( P : pillar side , M : modiolar side ; Scale bar : 2 μm )

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Mean ΔF images of Rhod-FF and iGluSnFR signals in response to a voltage ramp and a 50-ms-long step depolarization, respectively. The synapse marked with * on the overlaid image is further analyzed in the following panels. (P: pillar side, M: modiolar side; Scale bar: 2 μm)


Voltage command ( top ) , corresponding whole - cell Ca2 + - influx ( middle ) and the functional fluorescence responses ( bottom ) ( bandstop filtered at 33 . 3 Hz ) from Rhod - FF and iGluSnFR , respectively . A modified Boltzmann function ( see Materials and Methods , R2 = 0 . 81 ) was fitted to a Rhod - FF fluorescence trace in response to a voltage ramp ( C - C ' ) . iGluSnFR - AUC was calculated per depolarization voltage and used for a Boltzmann fit ( D ' , R2 = 0 . 92 ) . The voltage of half - maximal activation ( V1 / 2 ) and the dynamic range ( 10 - 90 % ) of synaptic Ca2 + - influx and glutamate release were calculated from the fits and depicted as circle and bar in C ' and D ' , respectively . The obtained fits from a Ca2 + " hotspot " ( C ' ) and from glutamate release ( D ' ) were plotted against each other in a voltage range from - 57 mV to - 17 mV in 1 mV increments . A power function was fitted to the 25 % of the maximum iGluSnFR - AUC ( R2 = 0 . 99 ) to obtain the m - estimate . ( ruptured patch - clamp , 10 mM intracellular EGTA , 5 mM [ Ca2 + ] e ) ( See also Fig EV4 )

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Voltage command (top), corresponding whole-cell Ca2+-influx (middle) and the functional fluorescence responses (bottom) (bandstop filtered at 33.3 Hz) from Rhod-FF and iGluSnFR, respectively. A modified Boltzmann function (see Materials and Methods, R2 = 0.81) was fitted to a Rhod-FF fluorescence trace in response to a voltage ramp (C-C'). iGluSnFR-AUC was calculated per depolarization voltage and used for a Boltzmann fit (D', R2 = 0.92). The voltage of half-maximal activation (V1/2) and the dynamic range (10-90%) of synaptic Ca2+-influx and glutamate release were calculated from the fits and depicted as circle and bar in C' and D', respectively. The obtained fits from a Ca2+ "hotspot" (C') and from glutamate release (D') were plotted against each other in a voltage range from -57 mV to -17 mV in 1 mV increments. A power function was fitted to the 25% of the maximum iGluSnFR-AUC (R2 = 0.99) to obtain the m-estimate. (ruptured patch-clamp, 10 mM intracellular EGTA, 5 mM [Ca2+]e) (See also Fig EV4)


A voltage ramp was applied to obtain ΔF of Rhod - FF as a proxy synaptic Ca2 + influx . The V1 / 2 of synaptic Ca2 + - influx was calculated from a modified Boltzman function ( see Materials and Methods ) fitted to ΔF / F0 . ( mean ± SD , n = 55 synapses ; individual synapses are color coded ) ( see Appendix Fig S7 for individual fits ) .

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A voltage ramp was applied to obtain ΔF of Rhod-FF as a proxy synaptic Ca2+ influx. The V1/2 of synaptic Ca2+-influx was calculated from a modified Boltzman function (see Materials and Methods) fitted to ΔF/F0. (mean ± SD, n = 55 synapses; individual synapses are color coded) (see Appendix Fig S7 for individual fits).


Normalized iGluSnFR - AUC , in response to 50 - ms - long step depolarizations , same as A ( see Appendix Fig S8 for individual fits ) .

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Normalized iGluSnFR-AUC, in response to 50-ms-long step depolarizations, same as A (see Appendix Fig S8 for individual fits).


Left panel shows the linear regression analysis ( solid lines ) of V1 / 2 of synaptic Ca2 + - influx ( B ) , glutamate release ( C ) , threshold ( D ) and dynamic range of release ( E ) as a function of position along the pillar - modiolar axis . Shaded areas depict the associated 95 % confidence intervals . Significance of correlation coefficients is reported by a two - tailed p - value . Right panel shows box and whisker plots of these properties of synapses grouped into pillar and modiolar halves of the IHCs . Box plots indicate first quartile ( 25th percentile ) , median and third quartile ( 75th percentile ) with whiskers reaching from 10 - 90 % . For comparison of pillar and modiolar synapses , either student ' s t - test ( for normally distributed data ) or Mann - Whitney - U test ( for non - normally distributed data ) was applied . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 .

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Left panel shows the linear regression analysis (solid lines) of V1/2 of synaptic Ca2+-influx (B), glutamate release (C), threshold (D) and dynamic range of release (E) as a function of position along the pillar-modiolar axis. Shaded areas depict the associated 95% confidence intervals. Significance of correlation coefficients is reported by a two-tailed p-value. Right panel shows box and whisker plots of these properties of synapses grouped into pillar and modiolar halves of the IHCs. Box plots indicate first quartile (25th percentile), median and third quartile (75th percentile) with whiskers reaching from 10-90%. For comparison of pillar and modiolar synapses, either student's t-test (for normally distributed data) or Mann-Whitney-U test (for non-normally distributed data) was applied. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001


Single IHC exhibits different modes of Ca2 + - control of release . The overlaid ΔF image of Rhod - FF ( red ) and iGluSnFR ( green ) shows synaptic Ca2 + - influx and glutamate release at three neighboring modiolar synapses ( individual synapses are color coded based on their clusters ) . E ' The relation between Ca2 + - influx and glutamate release of given synapses in the voltage range of - 57 to - 17 mV plotted with 1 mV increments . A power function was fitted until the 25 % of normalized iGluSnFR AUC ( ΔF / F0 ) . Synapses showed different Ca2 + - dependencies ( m = 2 . 0 , 1 . 3 , 7 . 4 ; green , purple , orange ) .

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Single IHC exhibits different modes of Ca2+-control of release. The overlaid ΔF image of Rhod-FF (red) and iGluSnFR (green) shows synaptic Ca2+-influx and glutamate release at three neighboring modiolar synapses (individual synapses are color coded based on their clusters). E' The relation between Ca2+-influx and glutamate release of given synapses in the voltage range of -57 to -17 mV plotted with 1 mV increments. A power function was fitted until the 25% of normalized iGluSnFR AUC(ΔF/F0). Synapses showed different Ca2+-dependencies (m = 2.0, 1.3, 7.4; green, purple, orange).


Single IHC exhibits different modes of Ca2 + - control of release . The overlaid ΔF image of Rhod - FF ( red ) and iGluSnFR ( green ) shows synaptic Ca2 + - influx and glutamate release at three neighboring modiolar synapses ( individual synapses are color coded based on their clusters ) .

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Single IHC exhibits different modes of Ca2+-control of release. The overlaid ΔF image of Rhod-FF (red) and iGluSnFR (green) shows synaptic Ca2+-influx and glutamate release at three neighboring modiolar synapses (individual synapses are color coded based on their clusters).


Mean glutamate release ( iGluSnFR - AUC ) as a function of depolarization voltage in the three identified clusters ( mean ± SEM , Cluster 1 : n = 27 synapses , Cluster 2 : n = 22 synapses , Cluster 3 : n = 6 synapses ) .

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Mean glutamate release (iGluSnFR-AUC) as a function of depolarization voltage in the three identified clusters (mean ± SEM, Cluster 1: n = 27 synapses, Cluster 2: n = 22 synapses, Cluster 3: n = 6 synapses).


( A ) Following transfection of given siRNAs and synchronization in G2 - phase , Hek293 cells were DRB - or mock - treated and then subjected to IFM using the indicated antibodies . Lower panels display a 3 - fold magnification of the boxed area shown above . Scales bars correspond to 5 and 1 μm ,

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(A) Following transfection of given siRNAs and synchronization in G2-phase, Hek293 cells were DRB- or mock-treated and then subjected to IFM using the indicated antibodies. Lower panels display a 3-fold magnification of the boxed area shown above. Scales bars correspond to 5 and 1 μm, respectively


( B ) Hek293 cells were thymidine - arrested for 20h , mock - or OHT - treated to induce DSBs by nuclear accumulation of ER - AsiSI and then subjected to ChIP - multiplex PCR

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(B) Hek293 cells were thymidine-arrested for 20h, mock- or OHT-treated to induce DSBs by nuclear accumulation of ER-AsiSI and then subjected to ChIP-multiplex PCR


( C ) ChIP samples from ( B ) were analysed by qPCR . Shown are averages ( bars ) of 3 independent experiments ( dots )

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(C) ChIP samples from (B) were analysed by qPCR. Shown are averages (bars) of 3 independent experiments (dots)


Separase interacts with γH2AX in DSB - containing G2 but not G1 cells . Transgenic Hek293 cells treated with Dox to induce expression of Myc - separase - WT and with OHT to induce nuclear accumulation of ER - AsiSI and infliction of DSBs were synchronized in G1 - or G2 - phase and analysed by IP -

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Separase interacts with γH2AX in DSB-containing G2 but not G1 cells. Transgenic Hek293 cells treated with Dox to induce expression of Myc-separase-WT and with OHT to induce nuclear accumulation of ER-AsiSI and infliction of DSBs were synchronized in G1- or G2-phase and analysed by IP-Western


Separase interacts with γH2AX in DSB - containing G2 but not G1 cells . Transgenic Hek293 cells treated with Dox to induce expression of Myc - separase - WT and with OHT to induce nuclear accumulation of ER - AsiSI and infliction of DSBs were synchronized in G1 - or G2 - phase and analysed b IFM for γH2AX - and Myc - separase - positive foc The quantification of the IFM in ( E ) shows averages ( bars ) of 3 independent experiments ( dots ) counting ≥ 100 cells

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Separase interacts with γH2AX in DSB-containing G2 but not G1 cells. Transgenic Hek293 cells treated with Dox to induce expression of Myc-separase-WT and with OHT to induce nuclear accumulation of ER-AsiSI and infliction of DSBs were synchronized in G1- or G2-phase and analysed b IFM for γH2AX- and Myc-separase-positive foc The quantification of the IFM in (E) shows averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each


Separase is required for proper HDR but dispensable for NHEJ . U2OS DR - GFP ( HDR reporter ) and U2OS EJ5 - GFP ( NHEJ reporter ) cells were separase - depleted by RNAi ( SEP - 1 ) or control - treated with GL2 siRNA , transfected to express HA - tagged ER - I - SceI , and then supplemented with OHT in G2 - phase to induce nuclear accumulation of the homing endonuclease . Ethanol - supplemented samples served as negative controls . Two days later , cells were subjected to immunoblotting (

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

Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls. Two days later, cells were subjected to immunoblotting (A


Separase is required for proper HDR but dispensable for NHEJ . U2OS DR - GFP ( HDR reporter ) and U2OS EJ5 - GFP ( NHEJ reporter ) cells were separase - depleted by RNAi ( SEP - 1 ) or control - treated with GL2 siRNA , transfected to express HA - tagged ER - I - SceI , and then supplemented with OHT in G2 - phase to induce nuclear accumulation of the homing endonuclease . Ethanol - supplemented samples served as negative controls . Two days later , cells were subjected t flow cytometry to quantify the percentage of GFP positive cells ( B The GFP quantification in ( B ) displays averages ( bars ) of 3 independent experiments ( dots )

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Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls. Two days later, cells were subjected t flow cytometry to quantify the percentage of GFP positive cells (B The GFP quantification in (B) displays averages (bars) of 3 independent experiments (dots)


Separase is required for proper HDR but dispensable for NHEJ . U2OS DR - GFP ( HDR reporter ) and U2OS EJ5 - GFP ( NHEJ reporter ) cells were separase - depleted by RNAi ( SEP - 1 ) or control - treated with GL2 siRNA , transfected to express HA - tagged ER - I - SceI , and then supplemented with OHT in G2 - phase to induce nuclear accumulation of the homing endonuclease . Ethanol - supplemented samples served as negative controls Two days later PI - stained cellular DNA ( C )

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Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls Two days later PI-stained cellular DNA (C)


Co - depletion of Wapl and separase has a synergistic effect on HDR . U2OS DR - GFP cells were transfected with the indicated siRNAs , treated as in ( A + B ) and analysed by GFP - flow cytometry ( D Shown in ( D ) are averages ( bars ) of 2 - 3 independent experiments ( dots )

Co-depletion of Wapl and separase has a synergistic effect on HDR. U2OS DR-GFP cells were transfected with the indicated siRNAs, treated as in (A+B) and analysed by GFP-flow cytometry (D Shown in (D) are averages (bars) of 2-3 independent experiments (dots)


Co - depletio of Wapl and separase has a synergistic effect on HDR . U2OS DR - GFP cells were transfected with the indicated siRNAs , treated as in ( A + B ) and analysed b immunoblotting ( E )

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Co-depletio of Wapl and separase has a synergistic effect on HDR. U2OS DR-GFP cells were transfected with the indicated siRNAs, treated as in (A+B) and analysed b immunoblotting (E)


( A ) Transgenic Hek293 cells constitutively expressing a separase sensor ( cartoon below ) and inducibly expressing Myc - separase were transiently transfected to express Flag - AsiSI - ER , Dox - and / or OHT - treated in G2 - phase and analysed by ( IP - )

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(A) Transgenic Hek293 cells constitutively expressing a separase sensor (cartoon below) and inducibly expressing Myc-separase were transiently transfected to express Flag-AsiSI-ER, Dox- and/or OHT-treated in G2-phase and analysed by (IP-)Western


( B ) Cells from ( A ) were analysed by IP - Western for cleavage of endogenous Rad21 . As a control , in vitro - translated ( IVT ) Rad21 was incubated with hyperactive separase - SA or a protease - dead ( PD ) varian

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(B) Cells from (A) were analysed by IP-Western for cleavage of endogenous Rad21. As a control, in vitro-translated (IVT) Rad21 was incubated with hyperactive separase-SA or a protease-dead (PD) varian


( C ) Sensor expressing cells were treated in G2 - phase with DRB and nocodazole for 6 and 2 h , respectively , prior to chromosome spreading and IFM using Hec1 - and γH2AX - antibodies . The separase sensor was detected based on autofluorescence of eGFP and mCherry , while Hec1 - and γH2AX - antibodies were detected with corresponding Cy5 - and marina blue - labeled secondary antibodies , respectively . Note that sizes of spread chromosomes vary greatly with buffer conditions , which is why no scale bar is shown

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(C) Sensor expressing cells were treated in G2-phase with DRB and nocodazole for 6 and 2 h, respectively, prior to chromosome spreading and IFM using Hec1- and γH2AX-antibodies. The separase sensor was detected based on autofluorescence of eGFP and mCherry, while Hec1- and γH2AX-antibodies were detected with corresponding Cy5- and marina blue-labeled secondary antibodies, respectively. Note that sizes of spread chromosomes vary greatly with buffer conditions, which is why no scale bar is shown


Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX . Hek293 cells were arrested in G2 phase by sequential thymidine - and RO - 3306 treatment , DRB - ( + ) or mock treated ( - ) , and then analysed as indicated Myc - separase - WT or - S1660A expressing cells were subjected to ( IP - ) Western using , amongst others , a pan - specific antibody against phosphorylated serine ( A , pan -

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Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX. Hek293 cells were arrested in G2 phase by sequential thymidine- and RO-3306 treatment, DRB- (+) or mock treated (-), and then analysed as indicated Myc-separase-WT or -S1660A expressing cells were subjected to (IP-)Western using, amongst others, a pan-specific antibody against phosphorylated serine (A, pan-pS


Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX . Hek293 cells were arrested in G2 phase by sequential thymidine - and RO - 3306 treatment , DRB - ( + ) or mock treated ( - ) , and then analysed as indicated Myc - separase - WT or - S1660A expressing cells were subjected to ( IP - ) Western using , amongst other a separase antibody specific for phosphorylated Ser1660 ( B , pS1660 )

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Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX. Hek293 cells were arrested in G2 phase by sequential thymidine- and RO-3306 treatment, DRB- (+) or mock treated (-), and then analysed as indicated Myc-separase-WT or -S1660A expressing cells were subjected to (IP-)Western using, amongst other a separase antibody specific for phosphorylated Ser1660 (B, pS1660)


Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX . Hek293 cells were arrested in G2 phase by sequential thymidine - and RO - 3306 treatment , DRB - ( + ) or mock treated ( - ) , and then analysed as indicated ( C ) DNA damage - induced Ser1660 phosphorylation of endogenous separase is largely blocked by ATM inhibition . G2 - enriched Hek293 cells were treated with KU - 55933 ( 0 . 3 μM ) and / or DRB and analyzed by ( IP - ) Western 12 h thereafter using the indicated

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Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX. Hek293 cells were arrested in G2 phase by sequential thymidine- and RO-3306 treatment, DRB- (+) or mock treated (-), and then analysed as indicated (C) DNA damage-induced Ser1660 phosphorylation of endogenous separase is largely blocked by ATM inhibition. G2-enriched Hek293 cells were treated with KU-55933 (0.3 μM) and/or DRB and analyzed by (IP-)Western 12 h thereafter using the indicated antibodies


( D ) Preventing NES phosphorylation spoils nuclear localization of separase in response to DSBs . HeLaK cells expressing N - terminally NLS - eGFP - tagged separase variants were treated with DRB or carrier solvent ( − DRB ) for 4 h and then subjected to IFM using anti - γH2AX and anti - Nup153 to visualize sites of DNA damage and nuclear pore complexes , respectively . Transgenic separase was detected based on the eGFP autofluorescence . Note that due to their relatively high nuclear concentration co - localization of separase - WT and - S1660D with γH2AX - foci is not discernable . Scale bar = 5 μm

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(D) Preventing NES phosphorylation spoils nuclear localization of separase in response to DSBs. HeLaK cells expressing N-terminally NLS-eGFP-tagged separase variants were treated with DRB or carrier solvent (− DRB) for 4 h and then subjected to IFM using anti-γH2AX and anti-Nup153 to visualize sites of DNA damage and nuclear pore complexes, respectively. Transgenic separase was detected based on the eGFP autofluorescence. Note that due to their relatively high nuclear concentration co-localization of separase-WT and -S1660D with γH2AX-foci is not discernable. Scale bar = 5 μm


( E ) In vitro phosphorylation of separase on Ser1660 by ATM kinase . Incubation of GST - p53 ( amino acids 9 - 22 ) , GST , separase - WT , or separase - S1660 with recombinant ATM - WT , ATM - KD ( kinase dead ) , and / or KU - 55933 in presence of [ γ - 33P ] - ATP was followed by immunoblotting and

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(E) In vitro phosphorylation of separase on Ser1660 by ATM kinase. Incubation of GST-p53 (amino acids 9-22), GST, separase-WT, or separase-S1660 with recombinant ATM-WT, ATM-KD (kinase dead), and/or KU-55933 in presence of [γ-33P]-ATP was followed by immunoblotting and autoradiography


( F ) Arg - methylation of RG - repeats mediates recruitment of separase to DSB - containing chromatin . Myc - separase - WT or - KG expressing cells were treated with DRB as indicated and analysed by ( IP - ) Western and Coomassie staining

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(F) Arg-methylation of RG-repeats mediates recruitment of separase to DSB-containing chromatin. Myc-separase-WT or -KG expressing cells were treated with DRB as indicated and analysed by (IP-)Western and Coomassie staining


( G ) In vitro Arg - methylation of separase ' s RG - repeats by PRMT1 . Incubation of histone H4 , separase - WT , or separase - KG with recombinant PRMT1 or reference buffer in presence of S - adenosyl - L - [ methyl - 3H ] - methionine was followed by Coomassie staining and autoradiography

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(G) In vitro Arg-methylation of separase's RG-repeats by PRMT1. Incubation of histone H4, separase-WT, or separase-KG with recombinant PRMT1 or reference buffer in presence of S-adenosyl-L-[methyl-3H]-methionine was followed by Coomassie staining and autoradiography


( A ) Persistence of γH2AX - and MDC1 - positive foci in absence of NES - phosphorylation or RG - methylation of separase . Transfected Hek293T cells were siRNA - and Dox - treated to deplete endogenous separase and induce expression of the indicated , siRNA resistant Myc - tagged separase variants , respectively . Then , they were constitutively ( − washout ) or transiently exposed to DRB ( + washout ) and finally quantitatively assessed by IFM for γH2AX - and MDC1 - positive foci . Shown are averages ( bars ) of 3 independent experiments ( dots ) counting ≥ 100 cells each . Scale bar = 5

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(A) Persistence of γH2AX- and MDC1-positive foci in absence of NES-phosphorylation or RG-methylation of separase. Transfected Hek293T cells were siRNA- and Dox-treated to deplete endogenous separase and induce expression of the indicated, siRNA resistant Myc-tagged separase variants, respectively. Then, they were constitutively (− washout) or transiently exposed to DRB (+ washout) and finally quantitatively assessed by IFM for γH2AX- and MDC1-positive foci. Shown are averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each. Scale bar = 5 μm


( C ) Recruitment of separase - WT and - S1660D but not separase - superNES , - S1660A , and - KG to DSBs . Cells from ( A ) were treated as indicated and analysed by IFM for co - localization of Myc - separase with γH2AX - foci . Scale bar = 5 μm

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(C) Recruitment of separase-WT and -S1660D but not separase-superNES, -S1660A, and -KG to DSBs. Cells from (A) were treated as indicated and analysed by IFM for co-localization of Myc-separase with γH2AX-foci. Scale bar = 5 μm


( D ) Interaction of separase - WT and - S1660D but not separase - superNES , - S1660A , and - KG with γH2AX . Cells from ( A ) were subjected to ( IP - ) Western analysis as indicated . Lanes that illustrate cellular levels of cyclin A2 and γH2AX after DRB - washout and those that analyse interaction of Myc - separase variants with γH2AX in presence of DRB are labeled by red arrows and blue arrow heads ,

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(D) Interaction of separase-WT and -S1660D but not separase-superNES, -S1660A, and -KG with γH2AX. Cells from (A) were subjected to (IP-)Western analysis as indicated. Lanes that illustrate cellular levels of cyclin A2 and γH2AX after DRB-washout and those that analyse interaction of Myc-separase variants with γH2AX in presence of DRB are labeled by red arrows and blue arrow heads, respectively


Separase is sumoylated in response to DSBs . Hek293T cells transfected to express His6 - separase ( A were treated with DRB or carrier solvent ( − DRB ) and then subjected to denaturing IMAC followed by immunoblotting of input samples and eluates using the indicated

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Separase is sumoylated in response to DSBs. Hek293T cells transfected to express His6-separase (A were treated with DRB or carrier solvent (− DRB) and then subjected to denaturing IMAC followed by immunoblotting of input samples and eluates using the indicated antibodies


Separase is sumoylated in response to DSBs . Hek293T cells transfected to expres His6 - Sumo2 ( B ) were treated with DRB or carrier solvent ( − DRB ) and then subjected to denaturing IMAC followed by immunoblotting of input samples and eluates using the indicated

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Separase is sumoylated in response to DSBs. Hek293T cells transfected to expres His6-Sumo2 (B) were treated with DRB or carrier solvent (− DRB) and then subjected to denaturing IMAC followed by immunoblotting of input samples and eluates using the indicated antibodies


Lys - 1034 is a major target of DSB - induced sumoylation of separase . Hek293T cells expressing His6 - Sumo2 ( C or - K1034R ( D ) were DRB - or mock - treated and subjected to denaturing IMA Input samples and eluates were immunoblotted using the indicated

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Lys-1034 is a major target of DSB-induced sumoylation of separase. Hek293T cells expressing His6-Sumo2 (C or -K1034R (D) were DRB- or mock-treated and subjected to denaturing IMA Input samples and eluates were immunoblotted using the indicated antibodies


Lys - 1034 is a major target of DSB - induced sumoylation of separase . Hek293T cells expressin Myc - separase - WT or - K1034R ( D ) were DRB - or mock - treated and subjected t Myc - I Input samples and eluates were immunoblotted using the indicated

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Lys-1034 is a major target of DSB-induced sumoylation of separase. Hek293T cells expressin Myc-separase-WT or -K1034R (D) were DRB- or mock-treated and subjected t Myc-I Input samples and eluates were immunoblotted using the indicated antibodies


( E ) Mms21 - dependent in vitro sumoylation of separase at Lys - 1034 . Myc beads were loaded with separase - WT or - K1034R or left empty ( last lane ) , combined with recombinant Ubc9 and / or Mms21 , as indicated , and incubated in presence of His6 - Sumo2 , Sae1 - Sae2 and ATP . Supernatant and washed beads were analysed by Coomassie staining ( lower panels ) and immunoblotting ( upper panels ) , respectively

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(E) Mms21-dependent in vitro sumoylation of separase at Lys-1034. Myc beads were loaded with separase-WT or -K1034R or left empty (last lane), combined with recombinant Ubc9 and/or Mms21, as indicated, and incubated in presence of His6-Sumo2, Sae1-Sae2 and ATP. Supernatant and washed beads were analysed by Coomassie staining (lower panels) and immunoblotting (upper panels), respectively


( F ) Cells expressing the indicated separase variants were DRB - or mock - treated and then lysed . Lysates and chromatin pelleted therefrom were assessed by immunoblotting and Coomassie staining , as indicated . Note that the lanes shown in the upper panels , although not directly juxtaposed , nevertheless originate from the same gel

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(F) Cells expressing the indicated separase variants were DRB- or mock-treated and then lysed. Lysates and chromatin pelleted therefrom were assessed by immunoblotting and Coomassie staining, as indicated. Note that the lanes shown in the upper panels, although not directly juxtaposed, nevertheless originate from the same gel


( G ) Preventing sumoylation of Lys - 1034 compromises separase ' s ability to support DSB repair . The indicated variants were probed for their ability to functionally replace endogenous separase in HDR as described in figures 6A and 6B . Shown are averages ( bars ) of 3 independent experiments ( dots ) counting ≥ 100 cells each

(G) Preventing sumoylation of Lys-1034 compromises separase's ability to support DSB repair. The indicated variants were probed for their ability to functionally replace endogenous separase in HDR as described in figures 6A and 6B. Shown are averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each


( A , B ) SEPARASE + / + and SEPARASE + / - MEFs were constitutively ( − washout ) or transiently exposed to DRB ( + washout ) and then analysed by IF for cells with γH2AX - and 53BP1 - positive foci . Shown are averages ( bars ) of 3 independent experiments ( dots ) counting ≥ 100 cells each . Scale bar = 5 μm . ( C ) Cell lysates from one experiment in ( A ) were analysed by immunoblotting using the indicated antibodies

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(A, B) SEPARASE+/+ and SEPARASE+/- MEFs were constitutively (− washout) or transiently exposed to DRB (+ washout) and then analysed by IF for cells with γH2AX- and 53BP1-positive foci. Shown are averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each. Scale bar = 5 μm. (C) Cell lysates from one experiment in (A) were analysed by immunoblotting using the indicated antibodies


SEPARASE + / + and SEPARASE + / - MEFs were treated with DRB ( + ) or carrier solvent ( − ) for the indicated times and then analysed by immunoblotting of total cell lysates (

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SEPARASE+/+ and SEPARASE+/- MEFs were treated with DRB (+) or carrier solvent (−) for the indicated times and then analysed by immunoblotting of total cell lysates (D


SEPARASE + / + and SEPARASE + / - MEFs were treated with DRB ( + ) or carrier solvent ( − ) for the indicated times and then analysed b IPs using anti - Rad21 or unspecific IgG ( E ) . The lower right two lanes show in vitro - expressed mouse Rad21 treated with hyperactive ( SA ) separase or a protease - dead ( PD )

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SEPARASE+/+ and SEPARASE+/- MEFs were treated with DRB (+) or carrier solvent (−) for the indicated times and then analysed b IPs using anti-Rad21 or unspecific IgG (E). The lower right two lanes show in vitro-expressed mouse Rad21 treated with hyperactive (SA) separase or a protease-dead (PD) variant


( A ) Transformation susceptibility of primary MEFs . SEPARASE + / + versus SEPARASE + / - MEFs were infected to express E1A and hRasV12 and then subjected to a colony formation assay . Shown are representative images ( left ) and the graphical representation of the number of transformed foci ( right ) from two independent experiments ( bars = standard deviations )

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(A) Transformation susceptibility of primary MEFs. SEPARASE+/+ versus SEPARASE+/- MEFs were infected to express E1A and hRasV12 and then subjected to a colony formation assay. Shown are representative images (left) and the graphical representation of the number of transformed foci (right) from two independent experiments (bars = standard deviations)


( B ) Two - stage DMBA ( initiation ) plus TPA ( promotion ) skin carcinogenesis . The number and size of skin papillomas are plotted against time for each genotype : SEPARASE + / + ( n = 8 ) versus SEPARASE + / - ( n = 11 )

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(B) Two-stage DMBA (initiation) plus TPA (promotion) skin carcinogenesis. The number and size of skin papillomas are plotted against time for each genotype: SEPARASE+/+ (n=8) versus SEPARASE+/- (n= 11)


( C ) Representative hematoxylin - and eosin - stained sections from SEPARASE + / - biopsies . ( i ) glandular hyperplasia ; ( ii ) benign papilloma ; ( iii ) squamous cell carcinoma . Scale bars are 200 µm in ( i ) and 500 µm in ( ii ) and ( iii ) . ( D ) Quantitative histological assessment of the DMBA - TPA skin cancerogenesis assay . Given are numbers of total , and corresponding percentage in brackets

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(C) Representative hematoxylin- and eosin-stained sections from SEPARASE+/- biopsies. (i) glandular hyperplasia; (ii) benign papilloma; (iii) squamous cell carcinoma. Scale bars are 200 µm in (i) and 500 µm in (ii) and (iii). (D) Quantitative histological assessment of the DMBA-TPA skin cancerogenesis assay. Given are numbers of total, and corresponding percentage in brackets


C Western blots of total protein of CHO cells transfected with ApCatSper 2 or 3 , non ‐ transfected control cells ( ‐ ) , and A . punctulata sperm ( Sp ) . The Western blots were probed with anti ‐ HA , anti ‐ ApCatSper 2 , or anti ‐ ApCatSper 3 antibodies . Arrows indicate bands representing ApCatSper 2 and 3 .

C Western blots of total protein of CHO cells transfected with ApCatSper 2 or 3, non‐transfected control cells (‐), and A. punctulata sperm (Sp). The Western blots were probed with anti‐HA, anti‐ApCatSper 2, or anti‐ApCatSper 3 antibodies. Arrows indicate bands representing ApCatSper 2 and 3.


D Immunocytochemistry analysis of sperm stained with anti ‐ ApCatSper 3 , anti ‐ GC , or anti ‐ CNGK antibodies ; superposition of images obtained by fluorescence microscopy and bright ‐ field light microscopy ; scale bar = 10 μm . The DNA was stained with DAPI ( blue ) .

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D Immunocytochemistry analysis of sperm stained with anti‐ApCatSper 3, anti‐GC, or anti‐CNGK antibodies; superposition of images obtained by fluorescence microscopy and bright‐field light microscopy; scale bar = 10 μm. The DNA was stained with DAPI (blue).


E Western blot analysis of co ‐ immunoprecipitation ( IP ) of A . punctulata sperm proteins . The input ( I ) , flow through ( FT ) , washes ( W1 ‐ 5 ) , and the eluate ( E ) of the IP using the anti ‐ ApCatSper 2 antibody were probed with the anti ‐ ApCatSper 3 antibody ( upper panel ) and vice versa ( lower panel ) . ApCatSper 3 and ApCatSper 2 were co ‐ immunoprecipitated with the anti ‐ ApCatSper 2 ( upper panel ) and anti ‐ ApCatSper 3 antibody ( lower panel ) , respectively .

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E Western blot analysis of co‐immunoprecipitation (IP) of A. punctulata sperm proteins. The input (I), flow through (FT), washes (W1‐5), and the eluate (E) of the IP using the anti‐ApCatSper 2 antibody were probed with the anti‐ApCatSper 3 antibody (upper panel) and vice versa (lower panel). ApCatSper 3 and ApCatSper 2 were co‐immunoprecipitated with the anti‐ApCatSper 2 (upper panel) and anti‐ApCatSper 3 antibody (lower panel), respectively.


F Analysis by mass spectrometry of immunoprecipitated proteins . ApCatSper 1 ‐ 4 , β , γ , and δ were identified in the immunoprecipitates obtained with both the anti ‐ ApCatSper 2 and anti ‐ ApCatSper 3 antibodies . The number of tryptic peptides identified and the respective sequence coverage are given .

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F Analysis by mass spectrometry of immunoprecipitated proteins. ApCatSper 1‐4, β, γ, and δ were identified in the immunoprecipitates obtained with both the anti‐ApCatSper 2 and anti‐ApCatSper 3 antibodies. The number of tryptic peptides identified and the respective sequence coverage are given.


A Alkaline ‐ evoked Ca2 + signals in sperm mixed with NH4Cl ; sperm were loaded with the Ca2 + indicator Fluo ‐ 4 . ΔF / F ( % ) indicates the change in Fluo ‐ 4 fluorescence ( ΔF ) with respect to the basal fluorescence ( F , mean of the first 3 - 5 data points ) . B Ca2 + signals evoked by mixing of sperm simultaneously with NH4Cl ( 30 mM ) and the CatSper inhibitor MDL12330A . C Dose - response relation of inhibition of the Ca2 + signals shown in panel ( B ) , Ki = 20 μM , amplitudes were determined at t = 4 s .

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A Alkaline‐evoked Ca2+ signals in sperm mixed with NH4Cl; sperm were loaded with the Ca2+ indicator Fluo‐4. ΔF/F (%) indicates the change in Fluo‐4 fluorescence (ΔF) with respect to the basal fluorescence (F, mean of the first 3-5 data points).B Ca2+ signals evoked by mixing of sperm simultaneously with NH4Cl (30 mM) and the CatSper inhibitor MDL12330A.C Dose-response relation of inhibition of the Ca2+ signals shown in panel (B), Ki = 20 μM, amplitudes were determined at t = 4 s.


A Changes in pHi evoked by mixing with pHi ‐ clamp solutions ( see explanation in the text ) ; sperm were loaded with the pHi indicator BCECF . ΔR / R ( % ) indicates the change in the BCECF fluorescence emission ratio ( ΔR = F494 / F540 ) with respect to the basal ratio ( R , mean of the first 3 - 5 data points ) . B Steady ‐ state change ( at t = 14 s ) of BCECF fluorescence for the pHi signals shown in ( A ) . The intercept of the fitted straight line with the x ‐ axis yields the resting pHi ; the slope of the straight line yields the ΔR / R ( % ) × ΔpH − 1 . Inset : calibrated changes in pHi evoked by various pHi ‐ clamp solutions .

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A Changes in pHi evoked by mixing with pHi‐clamp solutions (see explanation in the text); sperm were loaded with the pHi indicator BCECF. ΔR/R (%) indicates the change in the BCECF fluorescence emission ratio (ΔR = F494/F540) with respect to the basal ratio (R, mean of the first 3-5 data points).B Steady‐state change (at t = 14 s) of BCECF fluorescence for the pHi signals shown in (A). The intercept of the fitted straight line with the x‐axis yields the resting pHi; the slope of the straight line yields the ΔR/R (%) × ΔpH−1. Inset: calibrated changes in pHi evoked by various pHi‐clamp solutions.


C Ca2 + signals evoked by mixing of sperm with pHi ‐ clamp solutions .

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C Ca2+ signals evoked by mixing of sperm with pHi‐clamp solutions.


D Dose - response relation for the Ca2 + signals shown in ( C ) .

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D Dose-response relation for the Ca2+ signals shown in (C).


E Calibrated pHi increase ( red ) and respective Ca2 + response ( black ) evoked by mixing of sperm with a pHi 7 . 6 ‐ clamp solution ; depicted are , on an extended time scale , the first 500 ms of the respective pHi increase and Ca2 + signal shown in ( B , inset ) and ( C ) , respectively . The threshold pHi for CatSper activation was deduced from the latency of the Ca2 + signal . F Threshold pHi and latency of Ca2 + signals evoked by various pHi ‐ clamp solutions ( mean ± SD ; n ≥ 3 ) .

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E Calibrated pHi increase (red) and respective Ca2+ response (black) evoked by mixing of sperm with a pHi 7.6‐clamp solution; depicted are, on an extended time scale, the first 500 ms of the respective pHi increase and Ca2+ signal shown in (B, inset) and (C), respectively. The threshold pHi for CatSper activation was deduced from the latency of the Ca2+ signal.F Threshold pHi and latency of Ca2+ signals evoked by various pHi‐clamp solutions (mean ± SD; n ≥ 3).


A Depolarization ‐ evoked Ca2 + signals in sperm mixed with ASW containing high KCl concentrations .

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A Depolarization‐evoked Ca2+ signals in sperm mixed with ASW containing high KCl concentrations.


B Ca2 + signals evoked by mixing of sperm with 80 mM KCl and the CatSper inhibitor MDL12330A .

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B Ca2+ signals evoked by mixing of sperm with 80 mM KCl and the CatSper inhibitor MDL12330A.


C Ca2 + signals evoked by mixing of sperm with 160 mM KCl and MDL12330A .

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C Ca2+ signals evoked by mixing of sperm with 160 mM KCl and MDL12330A.


D Dose - response relation for the Ca2 + signals shown in ( B , C ) at t = 1 - 2 s .

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D Dose-response relation for the Ca2+ signals shown in (B, C) at t = 1-2 s.


E Threshold pHi for Ca2 + signals evoked by pHi ‐ clamp solutions in sperm bathed in ASW containing low ( 3 mM ) , high ( 191 mM ) , and normal ( 9 mM ) KCl ( mean ± SD ; n ≥ 3 ) ; data for 9 mM KCl are from Fig F . F Resting pHi and resting Vm in sperm bathed in ASW containing low ( 3 mM ) , high ( 191 mM ) , and normal ( 9 mM ) KCl ( black ) ( mean ± SD ; n ≥ 3 ) . Mean threshold pHi for CatSper activation at different membrane potentials ( red ) ; mean threshold pHi was derived from data shown in ( E ) .

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E Threshold pHi for Ca2+ signals evoked by pHi‐clamp solutions in sperm bathed in ASW containing low (3 mM), high (191 mM), and normal (9 mM) KCl (mean ± SD; n ≥ 3); data for 9 mM KCl are from Fig F.F Resting pHi and resting Vm in sperm bathed in ASW containing low (3 mM), high (191 mM), and normal (9 mM) KCl (black) (mean ± SD; n ≥ 3). Mean threshold pHi for CatSper activation at different membrane potentials (red); mean threshold pHi was derived from data shown in (E).


A Ca2 + signals in sperm evoked by photorelease ( at t = 0 ) of resact from caged resact in the presence of the CatSper inhibitor MDL12330A .

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A Ca2+ signals in sperm evoked by photorelease (at t = 0) of resact from caged resact in the presence of the CatSper inhibitor MDL12330A.


B Ca2 + signals evoked by intracellular photorelease ( at t = 0 ) of cGMP in sperm loaded with caged cGMP in the presence of the CatSper inhibitor MDL12330A .

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B Ca2+ signals evoked by intracellular photorelease (at t = 0) of cGMP in sperm loaded with caged cGMP in the presence of the CatSper inhibitor MDL12330A.


C Normalized dose - response relation for inhibition of the resact ‐ and cGMP ‐ induced Ca2 + signals shown in ( A , B ) ( Ki = 6 . 2 and 4 . 3 μM , respectively ) .

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C Normalized dose-response relation for inhibition of the resact‐ and cGMP‐induced Ca2+ signals shown in (A, B) (Ki = 6.2 and 4.3 μM, respectively).


D Normalized dose ‐ response relation for inhibition of the resact ‐ and cGMP ‐ induced Ca2 + signals shown in Supplementary Fig S4 by the CatSper inhibitor mibefradil ( Ki = 7 . 7 and 20 . 9 μM , respectively ) .

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D Normalized dose‐response relation for inhibition of the resact‐ and cGMP‐induced Ca2+ signals shown in Supplementary Fig S4 by the CatSper inhibitor mibefradil (Ki = 7.7 and 20.9 μM, respectively).


A Calibrated Vm ( left ) and pHi ( right ) changes and Ca2 + signals ( middle ) evoked by resact .

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A Calibrated Vm (left) and pHi (right) changes and Ca2+ signals (middle) evoked by resact.


B Normalized Vm , pHi , and Ca2 + signals evoked by 10 pM resact ; the first 600 ms after mixing are shown . The hyperpolarization precedes the pHi increase , whereas the pHi increase precedes the Ca2 + increase .

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B Normalized Vm, pHi, and Ca2+ signals evoked by 10 pM resact; the first 600 ms after mixing are shown. The hyperpolarization precedes the pHi increase, whereas the pHi increase precedes the Ca2+ increase.


C Latency of the Vm , the pHi , and the Ca2 + signals evoked by various resact concentrations ( mean ± SD ; n = 3 ) .

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C Latency of the Vm, the pHi, and the Ca2+ signals evoked by various resact concentrations (mean ± SD; n = 3).


D Normalized pHi and Ca2 + signals evoked by photorelease of cGMP in sperm loaded with caged cGMP .

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D Normalized pHi and Ca2+ signals evoked by photorelease of cGMP in sperm loaded with caged cGMP.


E Calibrated Vm and Ca2 + signals evoked by 1 nM resact ; the threshold voltage ( Vthr ) for the Ca2 + influx was deduced from the latency of the Ca2 + signal .

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E Calibrated Vm and Ca2+ signals evoked by 1 nM resact; the threshold voltage (Vthr) for the Ca2+ influx was deduced from the latency of the Ca2+ signal.


F Calibrated pHi and Ca2 + signals evoked by 1 nM resact ; the threshold pHi ( pHthr ) at which the Ca2 + influx commences was deduced from the latency of the Ca2 + signal .

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F Calibrated pHi and Ca2+ signals evoked by 1 nM resact; the threshold pHi (pHthr) at which the Ca2+ influx commences was deduced from the latency of the Ca2+ signal.


G Linear relationship between pHthr and Vthr for activation of Ca2 + influx by various resact concentrations ( data derived from Supplementary Fig S6 ; mean ± SD ; n ≥ 3 ) .

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G Linear relationship between pHthr and Vthr for activation of Ca2+ influx by various resact concentrations (data derived from Supplementary Fig S6; mean ± SD; n ≥ 3).


A Dark ‐ field light microscopy images of a sperm suspension before ( top ) and after ( bottom ) photorelease of a resact gradient ( middle ) in the absence ( control ; left ) or presence of the CatSper inhibitor MDL12330A ( 10 μM ; right ) . MDL12330A abolishes resact ‐ induced sperm accumulation . B Relative change of the sperm dispersion in the field of view evoked by photorelease of resact ( t = 0 , flash ) in the absence ( control ; blue ) or presence of MDL12330A ( red ) ; a decrease in dispersion indicates sperm accumulation in the irradiated area ( mean ± SD ; n = 4 ) .

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A Dark‐field light microscopy images of a sperm suspension before (top) and after (bottom) photorelease of a resact gradient (middle) in the absence (control; left) or presence of the CatSper inhibitor MDL12330A (10 μM; right). MDL12330A abolishes resact‐induced sperm accumulation.B Relative change of the sperm dispersion in the field of view evoked by photorelease of resact (t = 0, flash) in the absence (control; blue) or presence of MDL12330A (red); a decrease in dispersion indicates sperm accumulation in the irradiated area (mean ± SD; n = 4).


C Changes in pHi evoked by repetitive release ( arrows ) of cGMP from caged cGMP . The first UV flash was delivered at t = 0 .

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C Changes in pHi evoked by repetitive release (arrows) of cGMP from caged cGMP. The first UV flash was delivered at t = 0.


For SPR measurements , IL - 27Rα or GP130 were immobilized on the chip surface by biotin - streptavidin interaction and IL - 27 were flowed across the chip in solution . SPR data are representative of three biological replicates . Kinetic charts for IL - 27Rα ( A ) and GP130 ( B ) . Concentrations used are shown on the curves . Data traces were fitted using a 1 : 1 interaction model ( black ) to quantify the kinetics ( kon , koff ) and binding affinity ( KD ) of the interactions .

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For SPR measurements, IL-27Rα or GP130 were immobilized on the chip surface by biotin-streptavidin interaction and IL-27 were flowed across the chip in solution. SPR data are representative of three biological replicates. Kinetic charts for IL-27Rα (A) and GP130 (B). Concentrations used are shown on the curves. Data traces were fitted using a 1:1 interaction model (black) to quantify the kinetics (kon, koff) and binding affinity (KD) of the interactions.


For SPR measurements , IL - 27Rα were immobilized on the chip surface by biotin - streptavidin interaction and p28 were flowed across the chip in solution . SPR data are representative of three biological replicates . ( C , Equilibrium chart for p28 binding to IL - 27Rα ( C , left panel ) and curve - fitting to data points generated at various concentrations of p28 ( C , right panel ) .

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For SPR measurements, IL-27Rα were immobilized on the chip surface by biotin-streptavidin interaction and p28 were flowed across the chip in solution. SPR data are representative of three biological replicates. (C, Equilibrium chart for p28 binding to IL-27Rα (C, left panel) and curve-fitting to data points generated at various concentrations of p28 (C, right panel).


For SPR measurements , GP130 were immobilized on the chip surface by biotin - streptavidin interaction and p28 were flowed across the chip in solution . SPR data are representative of three biological replicates . ( D ) Equilibrium chart for p28 binding GP130 .

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For SPR measurements, GP130 were immobilized on the chip surface by biotin-streptavidin interaction and p28 were flowed across the chip in solution. SPR data are representative of three biological replicates. (D) Equilibrium chart for p28 binding GP130.


( F , G ) Dose response curves for pSTAT1 ( F ) and pSTAT3 ( G ) in resting mouse CD8 + T cells . Cells were stimulated with mIL - 27 or p28 for 15 minutes with the indicated doses . Data shown are the mean of four biological replicates with error bars depicting standard error of the mean .

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(F,G) Dose response curves for pSTAT1 (F) and pSTAT3 (G) in resting mouse CD8+ T cells. Cells were stimulated with mIL-27 or p28 for 15 minutes with the indicated doses. Data shown are the mean of four biological replicates with error bars depicting standard error of the mean.


Quantification of IL - 1β concentration ( pg / ml ) in the supernatant of primary macrophage cultures from COVID - 19 patients ( n = 44 ; red bars ) or SC - naïve individuals ( n = 24 ; blue bars ) stimulated with LPS or S - protein ( 0 . 1 µg / ml ) . To activate IL - 1β secretion nigericin was added for 2h . For statistical analysis , two - way ANOVA with tukey post hoc test was used . TNF - α ( pg / ml ) secreted from macrophages derived from SC - naïve and COVID - 19 patients as described above . Cells were stimulated with LPS ( SC - naïve / COVID - 19 n = 4 ) , LPS with nigericin ( SC - naïve n = 4 ; COVID - 19 n = 6 ) , S - protein ( SC - naïve n = 5 ; COVID - 19 n = 4 ) , S - protein with nigericin ( SC - naïve n = 3 ; COVID - 19 n = 4 ) or left unstimulated ( SC - naïve n = 3 ; COVID - 19 n = 4 ) . Significances shown in the figure are always in comparison to the unstimulated control .

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Quantification of IL-1β concentration (pg/ml) in the supernatant of primary macrophage cultures from COVID-19 patients (n = 44; red bars) or SC-naïve individuals (n = 24; blue bars) stimulated with LPS or S-protein (0.1 µg/ml). To activate IL-1β secretion nigericin was added for 2h. For statistical analysis, two-way ANOVA with tukey post hoc test was used. TNF-α (pg/ml) secreted from macrophages derived from SC-naïve and COVID-19 patients as described above. Cells were stimulated with LPS (SC-naïve/COVID-19 n = 4), LPS with nigericin (SC-naïve n = 4; COVID-19 n = 6), S-protein (SC-naïve n = 5; COVID-19 n = 4), S-protein with nigericin (SC-naïve n = 3; COVID-19 n = 4) or left unstimulated (SC-naïve n = 3; COVID-19 n = 4). Significances shown in the figure are always in comparison to the unstimulated control.


Representative confocal microscope pictures of macrophages from SC - naïve ( top ) and COVID - 19 patients ( bottom ) . Nuclei are stained with DAPI ( blue ) and ASC speck are labeled by immunofluorescence staining ( green ) . Macrophages were stimulated with LPS ( left ) and S - Protein ( right ) for 4h followed by stimulation with nigericin in both cases . Pictures were taken with a 60x objective using the same microscope settings .

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Representative confocal microscope pictures of macrophages from SC-naïve (top) and COVID-19 patients (bottom). Nuclei are stained with DAPI (blue) and ASC speck are labeled by immunofluorescence staining (green). Macrophages were stimulated with LPS (left) and S-Protein (right) for 4h followed by stimulation with nigericin in both cases. Pictures were taken with a 60x objective using the same microscope settings.


Subgroup analysis of IL - 1β secretion of macrophages after stimulation with S - protein and nigericin from COVID - 19 patients with severe ( red / black ; n = 23 ) or mild ( white / red ; n = 21 ) disease . Student ' s t - test with Welsh corrections was used to calculate statistical differences . Graph shows mean ± SEM .

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Subgroup analysis of IL-1β secretion of macrophages after stimulation with S-protein and nigericin from COVID-19 patients with severe (red/black; n=23) or mild (white/red; n=21) disease. Student's t-test with Welsh corrections was used to calculate statistical differences. Graph shows mean ± SEM.


CD14 + cells of two convalescent individuals were isolated sequentially at d18 , d26 and d56 after confirmation of SARS CoV2 infection by PCR . Following M - CSF differentiation , macrophages were stimulated with LPS ( red triangle ) or S - Protein ( red circles ) and IL - 1β concentration ( pg / ml ) was quantified upon incubation with nigericin . Blue dashed line indicates mean IL - 1β level of SC - naïve individuals stimulated with S - protein and nigericin .

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CD14+ cells of two convalescent individuals were isolated sequentially at d18, d26 and d56 after confirmation of SARS CoV2 infection by PCR. Following M-CSF differentiation, macrophages were stimulated with LPS (red triangle) or S-Protein (red circles) and IL-1β concentration (pg/ml) was quantified upon incubation with nigericin. Blue dashed line indicates mean IL-1β level of SC-naïve individuals stimulated with S-protein and nigericin.


Volcano plot showing DEGs in S - protein stimulated macrophages from convalescent COVID - 19 patients ( SC - conv ) compared to unstimulated cells . Negative log10 adjusted p - values are plotted against the log2 fold - change . Select genes are labeled . P - values are calculated using Wald test and p - adjusted values using the FDR / Benjamini - Hochberg approach .

Volcano plot showing DEGs in S-protein stimulated macrophages from convalescent COVID-19 patients (SC-conv) compared to unstimulated cells. Negative log10 adjusted p-values are plotted against the log2 fold-change. Select genes are labeled. P-values are calculated using Wald test and p-adjusted values using the FDR/Benjamini-Hochberg approach.


IL - 1β concentration ( pg / ml ) in supernatants of COVID - 19 patient derived macrophages pretreated with DMSO ( n = 44 ) , KINK - 1 ( n = 11 ) and MMG - 11 ( n = 19 ) and subsequent stimulation with LPS and nigericin . For statistical analysis , one - way ANOVA was used .

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IL-1β concentration (pg/ml) in supernatants of COVID-19 patient derived macrophages pretreated with DMSO (n = 44), KINK-1 (n = 11) and MMG-11 (n = 19) and subsequent stimulation with LPS and nigericin. For statistical analysis, one-way ANOVA was used.


IL - 1β concentration ( pg / ml ) in supernatants from macrophages of SC naïve ( blue ; n = 7 ) and COVID - 19 patients ( red ; n = 11 ) . Macrophages were pre - treated with DMSO ( SC - naïve n = 7 ; COVID - 19 n = 11 ) , KINK - 1 ( SC - naïve n = 6 ; COVID - 19 n = 7 ) and MMG - 11 ( SC - naïve n = 4 ; COVID - 19 n = 4 ) for 2h and then stimulated with zymosan ( 4h ) and nigericin ( 2h ) as indicated . For statistical analysis , two - way ANOVA with tukey post hoc test was used .

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IL-1β concentration (pg/ml) in supernatants from macrophages of SC naïve (blue; n = 7) and COVID-19 patients (red; n = 11). Macrophages were pre-treated with DMSO (SC-naïve n = 7; COVID-19 n = 11), KINK-1 (SC-naïve n = 6; COVID-19 n = 7) and MMG-11 (SC-naïve n = 4; COVID-19 n = 4) for 2h and then stimulated with zymosan (4h) and nigericin (2h) as indicated. For statistical analysis, two-way ANOVA with tukey post hoc test was used.


IL - 1β concentration ( pg / ml ) in supernatants from COVID - 19 patients which were stimulated for 2h with a blocking anti - TLR2 monoclonal antibody in different concentrations as indicated . Subsequently , macrophages were stimulated with LPS ( 1 ng / ml : n = 6 ; 10 and 100 ng / ml : n = 7 ) and S - Protein ( 1 ng / ml : n = 12 ; 10 and 100 ng / ml : n = 13 ) for 4h and both groups for 2h with nigericin . For statistical analysis , one - way ANOVA was

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IL-1β concentration (pg/ml) in supernatants from COVID-19 patients which were stimulated for 2h with a blocking anti-TLR2 monoclonal antibody in different concentrations as indicated. Subsequently, macrophages were stimulated with LPS (1 ng/ml: n = 6; 10 and 100 ng/ml: n = 7) and S-Protein (1 ng/ml: n = 12; 10 and 100 ng/ml: n = 13) for 4h and both groups for 2h with nigericin. For statistical analysis, one-way ANOVA was used


Volcano plot showing DEGs in unstimulated COVID - 19 patient - derived macrophages ( SC - conv ) compared to macrophages from SARS - CoV - 2 naïve individuals ( SC - naïve ) . Negative log10 padj values are plotted against the log2 fold - change . Selected genes of interest are labeled .

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Volcano plot showing DEGs in unstimulated COVID-19 patient-derived macrophages (SC-conv) compared to macrophages from SARS-CoV-2 naïve individuals (SC-naïve). Negative log10 padj values are plotted against the log2 fold-change. Selected genes of interest are labeled.


IL - 1β concentration ( pg / ml ) in supernatants of SC - naïve individuals ( n = 7 ) which were pre - treated for 24h with FCCP ( 1µg / ml ) and H2O2 ( 10µg / ml ) . Macrophages were then incubated with LPS or S - Protein ( 4h ) and nigericin ( 2h ) as indicated in the figure . Control cells were left unstimulated or stimulated with LPS or S - protein without pre - treatment with FCSP or H2O2 ( n = 6 ) . For statistical analysis , Brown - Forsythe and Welsh ANOVA test was used .

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IL-1β concentration (pg/ml) in supernatants of SC-naïve individuals (n = 7) which were pre-treated for 24h with FCCP (1µg/ml) and H2O2 (10µg/ml). Macrophages were then incubated with LPS or S-Protein (4h) and nigericin (2h) as indicated in the figure. Control cells were left unstimulated or stimulated with LPS or S-protein without pre-treatment with FCSP or H2O2 (n = 6). For statistical analysis, Brown-Forsythe and Welsh ANOVA test was used.


Representative gene loci ( TLR2 ( top ) and S100A7A / 8 / 9 / 12 ( bottom ) H3K27ac peaks ) comparing SC - naïve ( blue ) and SC - conv ( red ) monocytes . Red peaks reach higher values than blue peaks ( representative examples of four samples for each condition ) .

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Representative gene loci (TLR2 (top) and S100A7A/8/9/12 (bottom) H3K27ac peaks) comparing SC-naïve (blue) and SC-conv (red) monocytes. Red peaks reach higher values than blue peaks (representative examples of four samples for each condition).


Quantification of IL - 1β concentrations ( pg / ml ) in the supernatant of primary macrophage cultures from patients with active / untreated tuberculosis ( n = 6 ; red / black bars ) or healthy / SARS - CoV - 2 naïve individuals ( n = 3 ; blue bars ) or the same patients after 6 months of anti - tuberculous treatment ; n = 6 ; white / red bars ) stimulated with LPS or S - protein ( 0 . 1 µg / ml ) for 4h and subsequent incubation with nigericin for 2h . For statistical analysis , two - way ANOVA with tukey post hoc test was used .

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Quantification of IL-1β concentrations (pg/ml) in the supernatant of primary macrophage cultures from patients with active/untreated tuberculosis (n = 6; red/black bars) or healthy/SARS-CoV-2 naïve individuals (n = 3; blue bars) or the same patients after 6 months of anti-tuberculous treatment; n = 6; white/red bars) stimulated with LPS or S-protein (0.1 µg/ml) for 4h and subsequent incubation with nigericin for 2h. For statistical analysis, two-way ANOVA with tukey post hoc test was used.


A . Immunoblot analysis of the levels of the indicated proteins in lysates from non - infected ( ni ) and infected wild - type iBMDMs for 60 or 120 min .

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A. Immunoblot analysis of the levels of the indicated proteins in lysates from non-infected (ni) and infected wild-type iBMDMs for 60 or 120 min.


C . Immunoblot analysis of Klf4 and tubulin levels in lysates from non - infected ( ni ) and infected wild - type iBMDMs for 60 or 120 min .

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C. Immunoblot analysis of Klf4 and tubulin levels in lysates from non-infected (ni) and infected wild-type iBMDMs for 60 or 120 min.


H . Immunoblot analysis of Arg1 and tubulin levels in lysates from non - infected ( ni ) and infected wild - type and stat6 - / - iBMDMs for 60 or 120 min .

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H. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type and stat6-/- iBMDMs for 60 or 120 min.


D . Immunofluorescence confocal microscopy of the colocalization of Kp52145 harbouring pFPV25 . 1Cm , and cresyl violet dye in wild - type ( WT ) and stat6 - / - macrophages . The images were taken 90 min post infection . Images are representative of duplicate coverslips of three independent experiments . E . Percentage of Kp52145 harbouring pFPV25 . 1Cm co - localization with cresyl violet over a time course . Values are given as mean percentage of Kp52145 co - localizing with the marker ± SEM . The number of infected cells counted per time in three independent experiments are indicated in the figure .

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D. Immunofluorescence confocal microscopy of the colocalization of Kp52145 harbouring pFPV25.1Cm, and cresyl violet dye in wild-type (WT) and stat6-/- macrophages. The images were taken 90 min post infection. Images are representative of duplicate coverslips of three independent experiments. E. Percentage of Kp52145 harbouring pFPV25.1Cm co-localization with cresyl violet over a time course. Values are given as mean percentage of Kp52145 co-localizing with the marker ± SEM. The number of infected cells counted per time in three independent experiments are indicated in the figure.


A . Immunoblot analysis of phospho - STAT6 ( pSTAT6 ) and tubulin levels in lysates from non - infected ( ni ) and infected wild - type ( WT ) , tlr2 - / - , tlr4 - / - and tlr2 / 4 - / - iBMDMs for 60 or 120 min . B . Immunoblot analysis of Arg1and tubulin levels in lysates from non - infected ( ni ) and infected wild - type ( WT ) , tlr2 - / - , tlr4 - / - and tlr2 / 4 - / - iBMDMs for 60 or 120 min .

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A. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), tlr2-/-, tlr4-/- and tlr2/4-/- iBMDMs for 60 or 120 min. B. Immunoblot analysis of Arg1and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), tlr2-/-, tlr4-/- and tlr2/4-/- iBMDMs for 60 or 120 min.


F . Immunoblot analysis of phospho - STAT6 ( pSTAT6 ) and tubulin levels in lysates from non - infected ( ni ) and infected wild - type ( WT ) , myd88 - / - , tram / trif - / - for 60 or 120 min . G . Immunoblot analysis of Arg1 and tubulin levels in lysates from non - infected ( ni ) and infected wild - type ( WT ) , myd88 - / - , tram / trif - / - for 60 or 120 min .

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F. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), myd88-/-, tram/trif-/- for 60 or 120 min. G. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), myd88-/-, tram/trif-/- for 60 or 120 min.


A . Immunoblot analysis of phospho - STAT6 ( pSTAT6 ) and tubulin levels in lysates from non - infected ( ni ) and infected wild - type ( WT ) , or ifnar1 - / - for 60 or 120 min . B . Immunoblot analysis of Arg1 and tubulin levels in lysates from non - infected ( ni ) and infected wild - type ( WT ) , or ifnar1 - / - for 60 or 120 min .

A. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or ifnar1-/- for 60 or 120 min. B. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or ifnar1-/- for 60 or 120 min.


K . Immunoblot analysis of phospho - STAT6 ( pSTAT6 ) and tubulin levels in lysates from non - infected ( ni ) and infected wild - type ( WT ) , or irf3 - / - for 60 or 120 min . L Immunoblot analysis of Arg1 and tubulin levels in lysates from non - infected ( ni ) and infected wild - type ( WT ) , or irf31 - / - for 60 or 120 min .

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K. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or irf3-/- for 60 or 120 min. L Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or irf31-/- for 60 or 120 min.


A . Immunoblot analysis of phospho - CREB ( pCREB ) and tubulin levels in lysates from non - infected ( ni ) and infected wild - type ( WT ) , or tlr4 - / - for 60 or 120 min . B . Immunoblot analysis of phospho - CREB ( pCREB ) and tubulin levels in lysates from non - infected ( ni ) and infected wild - type ( WT ) , or myd88 - / - for 60 or 120 min .

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A. Immunoblot analysis of phospho-CREB (pCREB) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or tlr4-/- for 60 or 120 min. B. Immunoblot analysis of phospho-CREB (pCREB) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or myd88-/- for 60 or 120 min.


D . Immunoblot analysis of phospho - STAT6 ( pSTAT6 ) and tubulin levels in lysates from non - infected ( ni ) and infected wild - type ( WT ) , or il10 - / - for 60 or 120 min .

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D. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or il10-/- for 60 or 120 min.


F . Immunoblot analysis of Arg1 and tubulin levels in lysates from non - infected ( ni ) and infected wild - type ( WT ) , or il10 - / - for 60 or 120 min .

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F. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or il10-/- for 60 or 120 min.