D . Full thickness skin reconstitution assay using 3C cells from passage 1 ( p1 ) and passage 15 ( p15 ) show that cells cultured in 3C maintain their multipotency and self - renewal capacity in long - term cultures . A representative recipient of 4 mice / condition is shown . See also Appendix figure S2 .

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D. Full thickness skin reconstitution assay using 3C cells from passage 1 (p1) and passage 15 (p15) show that cells cultured in 3C maintain their multipotency and self-renewal capacity in long-term cultures. A representative recipient of 4 mice/condition is shown. See also Appendix figure S2.


B . Heatmap and Euclidian distance dendrogram of quantified transcripts from RNAseq data generated as shown in panel A . 3C cultures cluster with purified CD34 + α6 + HFSCs ( n = 3 biological replicates ) .

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B. Heatmap and Euclidian distance dendrogram of quantified transcripts from RNAseq data generated as shown in panel A. 3C cultures cluster with purified CD34+α6+ HFSCs (n=3 biological replicates).


E . Heatmap of selected genes differentially expressed in 3C - CD34 + α6 + and 3C - CD34 ¯ α6 + cells . Note upregulation of multiple key HFSC lineage identity transcription factors and bulge markers ( triangles ) in 3C - CD34 + α6 + cells , and upregulation of genes implicated in lineage progression in 3C - CD34 ¯ α6 + cells ( circles ) .

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E. Heatmap of selected genes differentially expressed in 3C-CD34+α6+ and 3C-CD34¯α6+ cells. Note upregulation of multiple key HFSC lineage identity transcription factors and bulge markers (triangles) in 3C-CD34+α6+ cells, and upregulation of genes implicated in lineage progression in 3C-CD34¯α6+ cells (circles).


G . RT - qPCR analyses of FACS - purified CD34 + α6 + and CD34 - α6 + cells from either 3C cultures or from freshly isolated epidermis show upregulation of HFSC identity in both freshly isolated and 3C - CD34 + α6 + cells compared to freshly isolated CD34 - α6 + progenitors . 3C - CD34 - α6 + cells show intermediated expression ( mean ± SEM ; n = 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) .

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G. RT-qPCR analyses of FACS-purified CD34+α6+ and CD34-α6+ cells from either 3C cultures or from freshly isolated epidermis show upregulation of HFSC identity in both freshly isolated and 3C-CD34+α6+ cells compared to freshly isolated CD34-α6+ progenitors. 3C- CD34-α6+ cells show intermediated expression (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis).


H . Both 3C - CD34 + α6 + and 3C - CD34 - α6 + cells show upregulation of a panel of cell cycle genes and genes shown to be enriched in HFSC progeny ( mean ± SEM ; n = 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) . See also Appendix figure S3 and Tables S1 - S3 .

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H. Both 3C-CD34+α6+ and 3C-CD34-α6+ cells show upregulation of a panel of cell cycle genes and genes shown to be enriched in HFSC progeny (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis). See also Appendix figure S3 and Tables S1-S3.


A . FACS analysis of CD34 + α6 + cells over time shows that 3C cultures reach a ~ 50 : 50 population equilibrium after 10 days of culture ( mean ± SEM ; n = 3 ) .

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A. FACS analysis of CD34+α6+ cells over time shows that 3C cultures reach a ~50:50 population equilibrium after 10 days of culture (mean ±SEM; n=3).


B . Cells were EdU - labeled for 24 h ( day 10 - 11 ) prior to analysis by flow cytometry to quantify proliferating cells . Percentage of EdU + cells is indicated ( n = 4 ; mean ± SD ; * p ≤ 0 . 05 , Mann - Whitney U - test ) .

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B. Cells were EdU-labeled for 24 h (day 10-11) prior to analysis by flow cytometry to quantify proliferating cells. Percentage of EdU+ cells is indicated (n=4; mean ±SD; *p ≤ 0.05, Mann-Whitney U-test).


D . Simulated population proportions of CD34 + α6 + HFSCs and CD34 ¯ α6 + non - HFSCs derived from the mathematical model describing steady state cultures . Experimental data from ( A ) are shown as reference ( in grey ) . See also Appendix figure S4 and Dataset EV4 .

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D. Simulated population proportions of CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs derived from the mathematical model describing steady state cultures. Experimental data from (A) are shown as reference (in grey). See also Appendix figure S4 and Dataset EV4.


A . CD34 + α6 + HFSCs and CD34 ¯ α6 + non - HFSCs were FACS - purified from total epidermis ( d0 ) to ≥ 98 % purity post sorting ( d0 post - sort ) , and grown 14 days in 3C cultures ( d14 3C ) . A significant population of CD34 + α6 + HFSCs and CD34 ¯ α6 + non - HFSCs is generated from both purified populations after 14 days . A representative of 3 independent experiments is shown ( mean ± SD ) .

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A. CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs were FACS-purified from total epidermis (d0) to ≥98% purity post sorting (d0 post-sort), and grown 14 days in 3C cultures (d14 3C). A significant population of CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs is generated from both purified populations after 14 days. A representative of 3 independent experiments is shown (mean ±SD).


B - C . Time course analyses of 3C cultures established from FACS - purified populations of CD34 + α6 + HFSCs ( B ) or CD34 ¯ α6 + non - HFSCs ( C ) from 3C cultures . Both cultures reach population equilibrium between day 6 and 10 ( n = 4 ; mean ± SD ) .

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B-C. Time course analyses of 3C cultures established from FACS-purified populations of CD34+α6+ HFSCs (B) or CD34¯α6+ non-HFSCs (C) from 3C cultures. Both cultures reach population equilibrium between day 6 and 10 (n=4; mean ±SD).


D - E . Simulated population proportions of CD34 + α6 + HFSCs ( D ) and CD34 ¯ α6 + non - HFSCs ( E ) from the mathematical model describing behavior of pure cell populations . The model parameters were estimated from the experimental data in B - C ( shown in grey as reference ) .

D-E. Simulated population proportions of CD34+α6+ HFSCs (D) and CD34¯α6+ non-HFSCs (E) from the mathematical model describing behavior of pure cell populations. The model parameters were estimated from the experimental data in B-C (shown in grey as reference).


G . Lineage tracing of FACS - purified CD34 + α6 + HFSCs and CD34 ¯ α6 + non - HFSCs from EGFP ¯ and EGFP + 3C cultures , respectively . CD34 + α6 + and CD34 ¯ α6 + were mixed at the indicated ratios at d0 and EGFP expression was used to trace the cells of origin over time by flow cytometry . Presence of CD34 + α6 + EGFP + cells in cultures at d10 indicates non - HFSC to HFSC interconversion .

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G. Lineage tracing of FACS-purified CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs from EGFP¯ and EGFP+ 3C cultures, respectively. CD34+α6+ and CD34¯α6+ were mixed at the indicated ratios at d0 and EGFP expression was used to trace the cells of origin over time by flow cytometry. Presence of CD34+α6+ EGFP+ cells in cultures at d10 indicates non-HFSC to HFSC interconversion.


H . Lineage tracing as in ( F ) shows that 3C cultures reach equilibrium between day 6 and 10 independent of the initial proportions of HFSCs and non - HFSCs .

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H. Lineage tracing as in (F) shows that 3C cultures reach equilibrium between day 6 and 10 independent of the initial proportions of HFSCs and non-HFSCs.


I . Quantification of the proportion of CD34 + α6 + cells that maintain their fate over time ( experiments with both EGFP + or EGFP ¯ labeled cells are indicated ) .

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I. Quantification of the proportion of CD34+α6+ cells that maintain their fate over time (experiments with both EGFP+ or EGFP¯ labeled cells are indicated).


J . Quantification of the proportion of CD34 ¯ α6 + cells that maintain their fate over time ( experiments with both EGFP + or EGFP ¯ labeled cells are indicated ) . Data in panels G - J show n = 4 ; mean ± SD . See also Appendix figure S5 .

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J. Quantification of the proportion of CD34¯α6+ cells that maintain their fate over time (experiments with both EGFP+ or EGFP¯ labeled cells are indicated). Data in panels G-J show n=4; mean ±SD. See also Appendix figure S5.


A - B . Inhibitor treatment scheme ( A ) and FACS plots ( B ) of 3C cultures treated with the Shh inhibitor cyclopamine or the BMP inhibitor dorsomorphin ( mean ± SD ; n = 3 ) . C . Quantification of experiments as in ( B ) shows enrichment of HFSCs upon cyclopamine ( Cyclo ) treatment , whereas dorsomorphin ( Dorso ) treatment leads to depletion of HFSCs ( mean ± SD ; n = 3 ; * p ≤ 0 . 05 , Kruskal - Wallis ) .

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A-B. Inhibitor treatment scheme (A) and FACS plots (B) of 3C cultures treated with the Shh inhibitor cyclopamine or the BMP inhibitor dorsomorphin (mean ±SD; n=3). C. Quantification of experiments as in (B) shows enrichment of HFSCs upon cyclopamine (Cyclo) treatment, whereas dorsomorphin (Dorso) treatment leads to depletion of HFSCs (mean ±SD; n=3; *p ≤ 0.05, Kruskal-Wallis).


D . Heatmap of significantly ( padj value < 0 . 05 ) differentially expressed genes from RNAseq analysis of 3C cultures treated with Cyclo or Dorso for 48h . HFSC identity genes ( triangles ) and genes implicated in HFSC lineage progression ( circles ) are indicated .

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D. Heatmap of significantly (padj value<0.05) differentially expressed genes from RNAseq analysis of 3C cultures treated with Cyclo or Dorso for 48h. HFSC identity genes (triangles) and genes implicated in HFSC lineage progression (circles) are indicated.


E . RT - qPCR analysis of 3C cultures treated as in ( D ) confirms that Id 1 , 2 and 3 are significantly upregulated in Cyclo treated cells ( mean ± SEM ; n = 4 ; * p ≤ 0 . 05 , Mann - Whitney U test ) .

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E. RT-qPCR analysis of 3C cultures treated as in (D) confirms that Id 1,2 and 3 are significantly upregulated in Cyclo treated cells (mean ±SEM; n=4; *p ≤ 0.05, Mann-Whitney U test).


F . RT - qPCR analysis of 3C cultures treated as in ( A ) shows upregulation of HFSC identity genes upon cyclopamine treatment whereas regulators of HFSC quiescence are downregulated upon dorsomorphin treatment ( mean ± SEM ; n = 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) .

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F. RT-qPCR analysis of 3C cultures treated as in (A) shows upregulation of HFSC identity genes upon cyclopamine treatment whereas regulators of HFSC quiescence are downregulated upon dorsomorphin treatment (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis).


H . Quantification of experiments as in E ( mean ± SD ; n = 3 - 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) . See also Appendix figure S6 and Dataset EV5 .

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H. Quantification of experiments as in E (mean ±SD; n=3-4; * p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis). See also Appendix figure S6 and Dataset EV5.


( e ) Binding of monoclonal antibodies to 2019 - nCoV RBD determined by ELISA .

(e) Binding of monoclonal antibodies to 2019-nCoV RBD determined by ELISA.


( f ) Binding profiles of 2019 - nCoV RBD to ACE2 and

(f) Binding profiles of 2019-nCoV RBD to ACE2 and antibodies


( g ) competition of CR3022 and ACE2 with 2019 - nCoV RBD measured by BLI in OctetRED96 . Binding kinetics was evaluated using a 1 : 1 Langmuir binding model by ForteBio Data Analysis 7 . 0 software .

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(g) competition of CR3022 and ACE2 with 2019-nCoV RBD measured by BLI in OctetRED96. Binding kinetics was evaluated using a 1:1 Langmuir binding model by ForteBio Data Analysis 7.0 software.


B , Immunofluorescence staining of single nuclei for H3K79me3 or H3K79me2 ( green ) and the pericentromeric heterochromatin marker HMGA1 ( red ) . Upper panels , prometaphase ; lower panels , interphase . DNA is stained with DAPI ( blue ) . Scale bar , 2µm .

B, Immunofluorescence staining of single nuclei for H3K79me3 or H3K79me2 (green) and the pericentromeric heterochromatin marker HMGA1 (red). Upper panels, prometaphase; lower panels, interphase. DNA is stained with DAPI (blue). Scale bar, 2µm.


H , H3K79me2 and H3K79me3 ChIP - seq enrichment relative to input at selected repetitive element sequences .

H, H3K79me2 and H3K79me3 ChIP-seq enrichment relative to input at selected repetitive element sequences.


K , Immunofluorescence staining for H3K79me3 in control and Dot1l KO nuclei . Scale bar , 2µm .

K, Immunofluorescence staining for H3K79me3 in control and Dot1l KO nuclei. Scale bar, 2µm.


M , DNA Fluorescence in situ hybridization ( DNA FISH ) for the mouse major satellite element with immunofluorescence staining for H3K79me3 ( top ) or H3K79me2 ( bottom ) in single mESC nuclei . Scale bar , 5µm . Plot shows Pearson ' s correlation values between FISH and H3K79me2 or H3K79me3 signal , each point representing an individual nucleus . * * * p < 0 . 0001 , two - tailed unpaired t - test .

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M, DNA Fluorescence in situ hybridization (DNA FISH) for the mouse major satellite element with immunofluorescence staining for H3K79me3 (top) or H3K79me2 (bottom) in single mESC nuclei. Scale bar, 5µm. Plot shows Pearson's correlation values between FISH and H3K79me2 or H3K79me3 signal, each point representing an individual nucleus. ***p<0.0001, two-tailed unpaired t-test.


B , Metagene plots showing mean ChIP signal for H3K79me2 and H3K79me3 at downregulated and upregulated genes identified in ( A ) .

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B, Metagene plots showing mean ChIP signal for H3K79me2 and H3K79me3 at downregulated and upregulated genes identified in (A).


F , RNA FISH for major satellite transcripts and simultaneous staining for H3K79me3 in control and Dot1l KO mESCs . Scale bar , 5µm . Below left , quantitation of fluorescence intensity along a line bisecting a representative nucleus . Bisecting lines used to generate the plots are shown in the merged images . Below right , quantitation of total FISH fluorescence signal for n = 46 cells of each genotype . Line summaries show mean ± standard deviation . * * * p < 0 . 0001 , unpaired Welch ' s t - test .

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F, RNA FISH for major satellite transcripts and simultaneous staining for H3K79me3 in control and Dot1l KO mESCs. Scale bar, 5µm. Below left, quantitation of fluorescence intensity along a line bisecting a representative nucleus. Bisecting lines used to generate the plots are shown in the merged images. Below right, quantitation of total FISH fluorescence signal for n=46 cells of each genotype. Line summaries show mean ± standard deviation. ***p<0.0001, unpaired Welch's t-test.


A , ChIP - qPCR for HP1β at selected repeat elements in Dot1l KO and wild type mESCs . Bars represent mean of n = 2 biological replicates . Neg . control indicates an intergenic locus ( mm10 chr19 : 51758385 - 58758494 ) not strongly associated with either euchromatin or heterochromatin .

A, ChIP-qPCR for HP1β at selected repeat elements in Dot1l KO and wild type mESCs. Bars represent mean of n=2 biological replicates. Neg. control indicates an intergenic locus (mm10 chr19:51758385-58758494) not strongly associated with either euchromatin or heterochromatin.


B , Immunofluorescence staining of HP1β ( green ) in wild type and Dot1l KO mESCs . DNA is stained with DAPI ( blue ) . Quantification of signal is shown at right . Each point represents one nucleus ( Dot1l KO n = 83 and control n = 104 nuclei ) . Scale bar , 10µm . * * * p < 0 . 0001 , Mann - Whitney U test .

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B, Immunofluorescence staining of HP1β (green) in wild type and Dot1l KO mESCs. DNA is stained with DAPI (blue). Quantification of signal is shown at right. Each point represents one nucleus (Dot1l KO n=83 and control n=104 nuclei). Scale bar, 10µm. ***p<0.0001, Mann-Whitney U test.


G , Western blot for co - immunoprecipitation of DOT1L and SMARCA5 in mESCs . AF10 is a positive control and GAPDH is a loading control .

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G, Western blot for co-immunoprecipitation of DOT1L and SMARCA5 in mESCs. AF10 is a positive control and GAPDH is a loading control.


H , Immunofluorescence staining of SMARCA5 ( green ) and HMGA1 ( red ) in mESCs , indicating localization of SMARCA5 to heterochromatic regions . Arrowheads indicate an example of SMARCA5 enrichment at PCH . DNA is stained with DAPI ( blue ) . Scale bar , 4µm . Bottom , quantitation of fluorescence intensity in each channel along a line bisecting the nucleus .

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H, Immunofluorescence staining of SMARCA5 (green) and HMGA1 (red) in mESCs, indicating localization of SMARCA5 to heterochromatic regions. Arrowheads indicate an example of SMARCA5 enrichment at PCH. DNA is stained with DAPI (blue). Scale bar, 4µm. Bottom, quantitation of fluorescence intensity in each channel along a line bisecting the nucleus.


J , H3K79me3 enrichment at major satellite sequences in Smarca5 knockdown ( KD ) relative to scrambled shRNA control mESCs measured by ChIP - qPCR . Bars represent mean of n = 3 biological replicates and error bars represent ± SD . p = 0 . 0876 for the major satellite element ( one - sample t - test compared to expected fold change of 1 , shown as a dashed line ) .

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J, H3K79me3 enrichment at major satellite sequences in Smarca5 knockdown (KD) relative to scrambled shRNA control mESCs measured by ChIP-qPCR. Bars represent mean of n=3 biological replicates and error bars represent ± SD. p=0.0876 for the major satellite element (one-sample t-test compared to expected fold change of 1, shown as a dashed line).


L , Immunofluorescence staining of H3K79me3 ( red ) in mouse 3T3 fibroblasts transduced with a vector expressing GFP and a short hairpin against Smarca5 ( shSmarca5 ) compared to untransduced control . Arrowheads indicate DAPI - dense chromocenters . Merge includes GFP channel for transduced cells . Scale bar , 10µm .

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L, Immunofluorescence staining of H3K79me3 (red) in mouse 3T3 fibroblasts transduced with a vector expressing GFP and a short hairpin against Smarca5 (shSmarca5) compared to untransduced control. Arrowheads indicate DAPI-dense chromocenters. Merge includes GFP channel for transduced cells. Scale bar, 10µm.


A , Immunofluorescence staining of H3K79me3 ( green ) and H3S10P ( red ) in a single nucleus of control and Dot1l KO mESCs . DNA is stained with DAPI ( blue ) . Scale bar , 5 µm .

A, Immunofluorescence staining of H3K79me3 (green) and H3S10P (red) in a single nucleus of control and Dot1l KO mESCs. DNA is stained with DAPI (blue). Scale bar, 5 µm.


B , Example metaphase spreads in wild type and Dot1l KO mESCs showing examples of a chromosome break , premature centromere separation , and chromosome fusion ( yellow boxes ) . Scale bar , 5 µm .

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B, Example metaphase spreads in wild type and Dot1l KO mESCs showing examples of a chromosome break, premature centromere separation, and chromosome fusion (yellow boxes). Scale bar, 5 µm.


F , Western blot for the DNA damage marker γH2A . X in control and Dot1l KO mESCs . Quantitation of signal on the blot for n = 2 biological replicates is shown .

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F, Western blot for the DNA damage marker γH2A.X in control and Dot1l KO mESCs. Quantitation of signal on the blot for n=2 biological replicates is shown.


A , Immunofluorescence staining of H3K79me3 ( green ) and H3K9me3 ( red ) in wild type mouse embryos during the first 48 hours of preimplantation development . Bottom images show an embryo treated with DOT1L inhibitor at 48 hours post fertilization ( hpf ) . DNA is stained with DAPI ( blue ) . Scale bars , 10 µm ( main image ) or 4 µm ( inset ) . Quantitation of pixel - by - pixel fluorescence intensity for H3K9me3 and H3K79me3 for a representative embryo is shown at right . AU , arbitrary units .

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A, Immunofluorescence staining of H3K79me3 (green) and H3K9me3 (red) in wild type mouse embryos during the first 48 hours of preimplantation development. Bottom images show an embryo treated with DOT1L inhibitor at 48 hours post fertilization (hpf). DNA is stained with DAPI (blue). Scale bars, 10 µm (main image) or 4 µm (inset). Quantitation of pixel-by-pixel fluorescence intensity for H3K9me3 and H3K79me3 for a representative embryo is shown at right. AU, arbitrary units.


B , Quantitation of numbers of total , H3K9me3 - positive , and H3K79me3 - positive chromocenters in control and inhibitor - treated embryos at 48 hpf . Chromocenters were defined as DAPI - dense foci . Each point represents one nucleus . N = 69 ( control ) or n = 20 nuclei ( inhibitor treated ) in n = 19 ( control ) or n = 5 ( inhibitor treated ) embryos * p < 0 . 05 , * * * p < 0 . 001 , two - tailed unpaired t - test .

B, Quantitation of numbers of total, H3K9me3-positive, and H3K79me3-positive chromocenters in control and inhibitor-treated embryos at 48 hpf. Chromocenters were defined as DAPI-dense foci. Each point represents one nucleus. N=69 (control) or n=20 nuclei (inhibitor treated) in n=19 (control) or n=5 (inhibitor treated) embryos *p<0.05, ***p<0.001, two-tailed unpaired t-test.


A , Volcano plot showing differentially expressed single - copy genes in DOT1L inhibitor - treated vs control embryos at 56hpf ( 8 cell stage ) . Each dot signifies one gene . Differentially expressed genes ( adjusted p - value ≤ 0 . 05 ; no threshold for log2 fold change ) are shown in red . P - values were corrected for multiple hypothesis testing using the Benjamini - Hochberg method .

A, Volcano plot showing differentially expressed single-copy genes in DOT1L inhibitor-treated vs control embryos at 56hpf (8 cell stage). Each dot signifies one gene. Differentially expressed genes (adjusted p-value ≤ 0.05; no threshold for log2 fold change) are shown in red. P-values were corrected for multiple hypothesis testing using the Benjamini-Hochberg method.


Antibacterial competition assay . E . coli K12 recipient cells ( W3110 gfp + , kanR ) were mixed with the indicated EAEC attacker cells ( 1 : 4 ratio ) : WT , ∆ T6SS - 1 and ∆ tle1 - tli1 carrying pBAD18 and pBAD33 vectors , or producing the indicated proteins , and spotted on SIM 0 . 02 % arabinose agar plates for 4 - h at 37 ° C . The image of a representative bacterial spot is shown . The number of recovered prey cells is indicated in the upper graph ( in log of colony - forming units ( cfu ) ) . The black , dark grey and light grey circles indicate values obtained from three different spots , and the average is indicated by the bar . Western blot analysis of the production of Tle1V and Tle1 ∆ 1 - 26 V is shown in the inset . The experiment was performed in triplicate and a representative result is shown .

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Antibacterial competition assay. E. coli K12 recipient cells (W3110 gfp+, kanR) were mixed with the indicated EAEC attacker cells (1:4 ratio): WT, ∆T6SS-1 and ∆tle1-tli1 carrying pBAD18 and pBAD33 vectors, or producing the indicated proteins, and spotted on SIM 0.02 % arabinose agar plates for 4-h at 37°C. The image of a representative bacterial spot is shown. The number of recovered prey cells is indicated in the upper graph (in log of colony-forming units (cfu)). The black, dark grey and light grey circles indicate values obtained from three different spots, and the average is indicated by the bar. Western blot analysis of the production of Tle1V and Tle1∆1-26 V is shown in the inset. The experiment was performed in triplicate and a representative result is shown.


Co - immunoprecipitation assay . Lysates from E . coli K - 12 W3110 cells producing VSVG - tagged Tle1 ( Tle1V ) or VSVG - tagged Tle1 ∆ 1 - 26 truncated variant ( Tle1 ∆ 1 - 26 V ) were mixed with lysates from W3110 cells producing FLAG - tagged VgrG ( VgrGFL ) or not ( - , empty vector ) and subjected to immunoprecipitation on anti - FLAG - coupled beads . The mixed soluble lysates ( Input ) and the immunoprecipitated material ( IP ) were subjected to 12 . 5 % SDS - PAGE and immunodetected with anti - FLAG ( upper panel ) and anti - VSVG ( lower panel ) monoclonal antibodies . Molecular weight markers ( in kDa ) are indicated on the left . This experiment was performed in triplicate and a representative result is shown .

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

Co-immunoprecipitation assay. Lysates from E. coli K-12 W3110 cells producing VSVG-tagged Tle1 (Tle1V) or VSVG-tagged Tle1∆1-26 truncated variant (Tle1∆1-26 V) were mixed with lysates from W3110 cells producing FLAG-tagged VgrG (VgrGFL) or not (-, empty vector) and subjected to immunoprecipitation on anti-FLAG-coupled beads. The mixed soluble lysates (Input) and the immunoprecipitated material (IP) were subjected to 12.5% SDS-PAGE and immunodetected with anti-FLAG (upper panel) and anti-VSVG (lower panel) monoclonal antibodies. Molecular weight markers (in kDa) are indicated on the left. This experiment was performed in triplicate and a representative result is shown.


Specific phospholipase A1 ( PLA1 ) activity measurements of Tle1H , SVgrG - Tle1H complex , and isolated SVgrG . Thermomyces lanuginosus lipase ( TLL ) was used as positive standard for PLA1 activity . Mean values and standard deviation from at least three independent assays are shown . Statistical analysis relative to the Tle1 activity is indicated . * * * P < 0 . 001 ( P = 0 , 0001998 ) , unpaired two - sample Fisher - Pitman permutation test .

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

Specific phospholipase A1 (PLA1) activity measurements of Tle1H, SVgrG-Tle1H complex, and isolated SVgrG. Thermomyces lanuginosus lipase (TLL) was used as positive standard for PLA1 activity. Mean values and standard deviation from at least three independent assays are shown. Statistical analysis relative to the Tle1 activity is indicated. ***P<0.001 (P=0,0001998), unpaired two-sample Fisher-Pitman permutation test.


VgrG inhibition of Tle1H . Specific phospholipase A1 ( PLA1 ) activity measurements of purified Tle1H in the presence of 1 : 10 molar ratio of lysozyme from chicken egg white ( Lyso ) and increasing molar ratio ( 0 , 1 , 5 , and 10 ) of SVgrG or SVgrG1 - 490 . Inhibition experiments with SVgrG were repeated three times with independent protein preparations , each measured in triplicate . Inhibition experiments with SVgrG1 - 490 were repeated twice with two independent protein preparations , each measured in triplicate . Mean values and standard deviation are shown . Statistical analysis relative to the Tle1 activity is indicated . ns , non - significant ( P > 0 . 05 ) , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , unpaired two - sample Fisher - Pitman permutation test . ( P values : Lyso , 0 . 7006 ; VgrG 1 : 1 , 0 . 004946 ; VgrG 1 : 5 , 1 . 448e - 07 ; VgrG1 : 10 , 1 . 448e - 07 ; VgrG1 - 490 1 : 1 , 0 . 5627 ; VgrG1 - 490 1 : 5 , 0 . 2977 ; VgrG1 - 490 1 : 10 , 0 . 002631 ) .

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

VgrG inhibition of Tle1H. Specific phospholipase A1 (PLA1) activity measurements of purified Tle1H in the presence of 1:10 molar ratio of lysozyme from chicken egg white (Lyso) and increasing molar ratio (0, 1, 5, and 10) of SVgrG or SVgrG1-490. Inhibition experiments with SVgrG were repeated three times with independent protein preparations, each measured in triplicate. Inhibition experiments with SVgrG1-490 were repeated twice with two independent protein preparations, each measured in triplicate. Mean values and standard deviation are shown. Statistical analysis relative to the Tle1 activity is indicated. ns, non-significant (P>0.05), *P<0.05, **P<0.01, ***P<0.001, unpaired two-sample Fisher-Pitman permutation test. (P values: Lyso, 0.7006; VgrG 1:1, 0.004946; VgrG 1:5, 1.448e-07; VgrG1:10, 1.448e-07; VgrG1-490 1:1, 0.5627; VgrG1-490 1:5, 0.2977; VgrG1-490 1:10, 0.002631).


Representative SDS - PAGE / Coomassie blue staining of the Tle1H , SVgrG and SVgrG1 - 490 purified proteins used in B ( 5 μg / well ) .

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Representative SDS-PAGE / Coomassie blue staining of the Tle1H, SVgrG and SVgrG1-490 purified proteins used in B (5 μg/well).


( A , B ) PTPRN2 ( A ) and PLCB1 ( B ) expression levels were determined by qRT - PCR .

(A,B) PTPRN2 (A) and PLCB1 (B) expression levels were determined by qRT-PCR.


( C ) Bioluminescence imaging quantification of lung colonization by 40 , 000 LM2 breast cancer cells transduced with shRNAs targeting PTPRN2 or a control hairpin . For shCntrl , sh1PTPRN2 , N = 5 mice / group . For sh2PTPRN2 N = 6 mice . Right , H & amp ; amp ; E staining of representative lung sections .

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(C) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 breast cancer cells transduced with shRNAs targeting PTPRN2 or a control hairpin. For shCntrl, sh1PTPRN2, N = 5 mice/group. For sh2PTPRN2 N = 6 mice. Right, H&amp;E staining of representative lung sections.


( D ) Bioluminescence imaging quantification of lung colonization by 40 , 000 LM2 cells transfected with siRNA targeting PLCβ1 or a control siRNA . For siCntrl , N = 5 mice . For si1PLCβ1 , si2PLCβ1 , N = 6 mice / group . Right , H & amp ; amp ; E staining of representative lung sections .

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(D) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells transfected with siRNA targeting PLCβ1 or a control siRNA. For siCntrl, N = 5 mice. For si1PLCβ1, si2PLCβ1, N = 6 mice/group. Right, H&amp;E staining of representative lung sections.


( E , F ) PTPRN2 ( E ) and PLCB1 ( F ) levels were analyzed in human breast cancers ( stages I - IV ) and normal breast tissue from TissueScan qPCR Array Breast Cancer Panels II and III ( Origene , N = 97 ) . Expression levels were normalized to levels in normal tissue for each gene .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,F) PTPRN2 (E) and PLCB1 (F) levels were analyzed in human breast cancers (stages I-IV) and normal breast tissue from TissueScan qPCR Array Breast Cancer Panels II and III (Origene, N = 97). Expression levels were normalized to levels in normal tissue for each gene.


( G ) Kaplan - Meier curve representing overall survival of a cohort of breast cancer patients ( N = 528 ) as a function of their primary tumor ' s PTPRN2 and PLCB1 expression levels ( data from the TCGA Research Network ( Cancer Genome Atlas , 2012 ) ) . Patients whose primary tumors ' PTPRN2 and PLCB1 expression levels were higher or lower than the median of the population were classified as low ( blue ) or high ( red ) expression .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kaplan-Meier curve representing overall survival of a cohort of breast cancer patients (N = 528) as a function of their primary tumor's PTPRN2 and PLCB1 expression levels (data from the TCGA Research Network (Cancer Genome Atlas, 2012)). Patients whose primary tumors' PTPRN2 and PLCB1 expression levels were higher or lower than the median of the population were classified as low (blue) or high (red) expression.


( H ) Kaplan - Meier curve representing distal metastasis - free survival of a cohort of breast cancer patients ( N = 1609 ) as a function of their primary tumor ' s PTPRN2 and PLCB1 expression levels ( Data from KMPlot ( Gyorffy et al . , 2010 ) ) . Patients ' primary tumors ' PTPRN2 and PLCB1 expression levels were classified as low ( blue ) or high ( red ) expression .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor's PTPRN2 and PLCB1 expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients' primary tumors' PTPRN2 and PLCB1 expression levels were classified as low (blue) or high (red) expression.


( A ) Matrigel invasion by 50 , 000 LM2 cells transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA . Data normalized to control values . N = 6 inserts / group .

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(A) Matrigel invasion by 50,000 LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. Data normalized to control values. N = 6 inserts/group.


( B ) Migration assay by 100 , 000 LM2 cells transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA . Data normalized to control values . N = 6 inserts / group . Right , representative images of the migration assay . Scale bar , 100 μm .

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(B) Migration assay by 100,000 LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. Data normalized to control values. N = 6 inserts/group. Right, representative images of the migration assay. Scale bar, 100 μm.


( C ) Quantification of area covered by cells 24 h after a scratch was made through confluent cells transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA . N = 5 wells / group . Right , representative images of the scratch assay .

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(C) Quantification of area covered by cells 24 h after a scratch was made through confluent cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. N = 5 wells/group. Right, representative images of the scratch assay.


( D , E ) MDA - MB - 231 cells transduced with PTPRN2 , PTPRN2C945A , or control vector were subjected to the Matrigel invasion ( D ) and migration assays ( E ) . N = 5 inserts / group .

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(D,E) MDA-MB-231 cells transduced with PTPRN2, PTPRN2C945A, or control vector were subjected to the Matrigel invasion (D) and migration assays (E). N = 5 inserts/group.


( F ) Bioluminescence imaging quantification of lung colonization by 40 , 000 MDA - MB - 231 cells overexpressing PTPRN2 , PTPRN2C945A , or control vector . N = 5 mice / group . Right , H & amp ; amp ; E staining of representative lung sections .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bioluminescence imaging quantification of lung colonization by 40,000 MDA-MB-231 cells overexpressing PTPRN2, PTPRN2C945A, or control vector. N = 5 mice/group. Right, H&amp;E staining of representative lung sections.


( G , H ) MDA - MB - 231 cells transduced with PLCβ1 , PLCβ1H331Q , or control vector were subjected to the Matrigel invasion ( G ) and migration assays ( H ) . N = 5 inserts / group .

(G,H) MDA-MB-231 cells transduced with PLCβ1, PLCβ1H331Q, or control vector were subjected to the Matrigel invasion (G) and migration assays (H). N = 5 inserts/group.


( I ) Bioluminescence imaging quantification of lung colonization by 40 , 000 MDA - MB - 231 cells overexpressing PLCβ1 , PLCβ1H331Q , or control vector . For Cntrl and PLCβ1H331Q OE , N = 6 mice / group . For PLCβ1 OE , N = 5 mice . Right , H & amp ; amp ; E staining of representative lung sections .

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

(I) Bioluminescence imaging quantification of lung colonization by 40,000 MDA-MB-231 cells overexpressing PLCβ1, PLCβ1H331Q, or control vector. For Cntrl and PLCβ1H331Q OE, N = 6 mice/group. For PLCβ1 OE, N = 5 mice. Right, H&amp;E staining of representative lung sections.


( A ) Mean fluorescence intensity of membrane PI ( 4 , 5 ) P2 was analyzed in MDA - MB - 231 and LM2 cells immunostained with PI ( 4 , 5 ) P2 antibody using fluorescence microscopy . N = 50 cells / group . Scale bar , 10 μm .

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

(A) Mean fluorescence intensity of membrane PI(4,5)P2 was analyzed in MDA-MB-231 and LM2 cells immunostained with PI(4,5)P2 antibody using fluorescence microscopy. N = 50 cells/group. Scale bar, 10 μm.


( B ) Bioluminescence imaging quantification of lung colonization by 40 , 000 LM2 cells treated with carrier incubated with PI ( 4 , 5 ) P2 or carrier alone for one hour and then immediately injected . Right , H & amp ; amp ; E staining of representative lung sections . N = 6 mice / group .

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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-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

(B) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells treated with carrier incubated with PI(4,5)P2 or carrier alone for one hour and then immediately injected. Right, H&amp;E staining of representative lung sections. N=6 mice/group.


( C , D ) MDA - MB - 231 cells overexpressing PTPRN2 , PLCβ1 , or a control vector ( C ) or LM2 cells transfected with siRNA targeting PTPRN2 , PLCβ1 , or a control siRNA ( D ) were immunostained for PI ( 4 , 5 ) P2 levels and analyzed by fluorescence microscopy . Mean fluorescence intensity of plasma membrane levels of the lipid were quantified . N = 50 cells / group . Left , representative immunofluorescence images of cells stained with anti - PI ( 4 , 5 ) P2 antibody ( red ) and 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI , blue ) . Scale bar , 10 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-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

(C,D) MDA-MB-231 cells overexpressing PTPRN2, PLCβ1, or a control vector (C) or LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or a control siRNA (D) were immunostained for PI(4,5)P2 levels and analyzed by fluorescence microscopy. Mean fluorescence intensity of plasma membrane levels of the lipid were quantified. N = 50 cells/group. Left, representative immunofluorescence images of cells stained with anti-PI(4,5)P2 antibody (red) and 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 10 μm.


( A , B ) LM2 cells transfected with siRNA targeting PTPRN2 ( A ) , PLCβ1 ( B ) or a control siRNA were transfected with Lyn11 - FRB and INPP5E - FKBP , treated with either DMSO or 100 nm rapamycin and subjected to the migration assay . N = 5 inserts / group .

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(A,B) LM2 cells transfected with siRNA targeting PTPRN2 (A), PLCβ1 (B) or a control siRNA were transfected with Lyn11-FRB and INPP5E-FKBP, treated with either DMSO or 100 nm rapamycin and subjected to the migration assay. N = 5 inserts/group.


( C , D ) MDA - MB - 231 cells overexpressing PTPRN2 ( C ) , PLCβ1 ( D ) or control vector were treated with carrier alone or carrier incubated with PI ( 4 , 5 ) P2 for one hour and then immediately subjected to the migration assay . N = 5 inserts / group .

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

(C,D) MDA-MB-231 cells overexpressing PTPRN2 (C), PLCβ1 (D) or control vector were treated with carrier alone or carrier incubated with PI(4,5)P2 for one hour and then immediately subjected to the migration assay. N = 5 inserts/group.


( E ) Kaplan - Meier curve representing distal metastasis - free survival of a cohort of breast cancer patients ( N = 1609 ) as a function of their primary tumor ' s mean PIP5K1A , PIP5KB , and PIP5KC expression levels ( Data from KMPlot ( Gyorffy et al . , 2010 ) ) . Patients ' primary tumors ' combined PIP5K expression levels were classified as low ( blue ) or high ( red ) expression .

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(E) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor's mean PIP5K1A, PIP5KB, and PIP5KC expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients' primary tumors' combined PIP5K expression levels were classified as low (blue) or high (red) expression.


( F , G ) Migration ( F ) and Matrigel invasion ( G ) of LM2 cells transduced with a retroviral vector overexpressing PIP5K1A or control vector . Data normalized to control values . N = 5 inserts / group .

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(F,G) Migration (F) and Matrigel invasion (G) of LM2 cells transduced with a retroviral vector overexpressing PIP5K1A or control vector. Data normalized to control values. N = 5 inserts/group.


( A ) Kaplan - Meier curve representing distal metastasis - free survival of a cohort of breast cancer patients ( N = 1609 ) as a function of their primary tumor ' s CFL1 expression levels ( Data from KMPlot ( Gyorffy et al . , 2010 ) ) . Patients ' primary tumors ' CFL1 expression levels were classified as low ( blue ) or high ( red ) expression .

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(A) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor's CFL1 expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients' primary tumors' CFL1 expression levels were classified as low (blue) or high (red) expression.


( B , C ) Membrane and membrane - associated proteins were purified from cells transfected with siRNA targeting PTPRN2 ( B ) or PLCβ1 ( C ) or control siRNA . Fractions were subjected to Western blot analysis for CFL1 and EGFR levels . Right , densitometry analysis of CFL1 levels normalized to EGFR levels .

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(B,C) Membrane and membrane-associated proteins were purified from cells transfected with siRNA targeting PTPRN2 (B) or PLCβ1 (C) or control siRNA. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels.


( D , E ) Membrane and membrane - associated proteins were purified from cells overexpressing PTPRN2 ( D ) , PLCβ1 ( E ) or a control vector . Fractions were subjected to Western blot analysis for CFL1 and EGFR levels . Right , densitometry analysis of CFL1 levels normalized to EGFR levels .

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(D,E) Membrane and membrane-associated proteins were purified from cells overexpressing PTPRN2 (D), PLCβ1 (E) or a control vector. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels.


( F ) LM2 cells transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA were partially permeabilized and incubated with biotin - actin monomers . Cells were stained for incorporated biotin - actin monomers using Streptavidin - 555 ( red ) and DAPI ( blue ) . Right , quantification of mean fluorescence intensity of incorporated biotin - actin monomers . N = 100 cells / group . Scale bar , 20 μm .

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(F) LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA were partially permeabilized and incubated with biotin-actin monomers. Cells were stained for incorporated biotin-actin monomers using Streptavidin-555 (red) and DAPI (blue). Right, quantification of mean fluorescence intensity of incorporated biotin-actin monomers. N = 100 cells/group. Scale bar, 20 μm.


( A ) LM2 cells transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA were stained with phalloidin ( red ) and DAPI ( blue ) and analyzed using fluorescence microscopy . Right , mean fluorescence intensity quantification of whole cell phalloidin signal . N = 40 cells / group . Scale bar , 10 μm .

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(A) LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA were stained with phalloidin (red) and DAPI (blue) and analyzed using fluorescence microscopy. Right, mean fluorescence intensity quantification of whole cell phalloidin signal. N = 40 cells/group. Scale bar, 10 μm.


( B ) Mean fluorescence intensity quantification of whole cell phalloidin signal in MDA - MB - 231 cells overexpressing PTPRN2 , PLCβ1 , or control vector . N = 40 cells / group .

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(B) Mean fluorescence intensity quantification of whole cell phalloidin signal in MDA-MB-231 cells overexpressing PTPRN2, PLCβ1, or control vector. N = 40 cells/group.


( C , D ) MDA - MB - 231 cells were transfected with siRNAs targeting CFL1 or a control siRNA in the setting of control or PTPRN2 overexpression and subjected to the migration ( C ) or invasion ( D ) assays . N = 5 inserts / group .

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(C,D) MDA-MB-231 cells were transfected with siRNAs targeting CFL1 or a control siRNA in the setting of control or PTPRN2 overexpression and subjected to the migration (C) or invasion (D) assays. N = 5 inserts/group.


( E ) MDA - MB - 231 cells were transfected with siRNAs targeting CFL1 or a control siRNA in the setting of control or PLCβ1 overexpression were subjected to the migration assay . N = 5 inserts / group .

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(E) MDA-MB-231 cells were transfected with siRNAs targeting CFL1 or a control siRNA in the setting of control or PLCβ1 overexpression were subjected to the migration assay. N = 5 inserts/group.


( F ) Bioluminescence imaging quantification of 40 , 000 MDA - MB - 231 cells overexpressing PTPRN2 or control vector and transfected with control siRNA or siRNA targeting CFL1 . Right , H & amp ; amp ; E staining of representative lung sections . N = 5 mice / group .

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(F) Bioluminescence imaging quantification of 40,000 MDA-MB-231 cells overexpressing PTPRN2 or control vector and transfected with control siRNA or siRNA targeting CFL1. Right, H&amp;E staining of representative lung sections. N = 5 mice/group.


( G , H ) MDA - MB - 231 cells overexpressing PTPRN2 ( G ) or PLCβ1 ( H ) were transfected with siRNA targeting the 3 ' UTR of CFL1 to deplete endogenous CFL1 . Cells were further transfected with plasmids encoding either GFP - CFL1 - WT or GFP - CFL1 - Lck and subjected to the migration assay . N = 5 inserts / group .

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(G,H) MDA-MB-231 cells overexpressing PTPRN2 (G) or PLCβ1 (H) were transfected with siRNA targeting the 3'UTR of CFL1 to deplete endogenous CFL1. Cells were further transfected with plasmids encoding either GFP-CFL1-WT or GFP-CFL1-Lck and subjected to the migration assay. N = 5 inserts/group.


( A , B ) ELISA screen of the lectin - Fc library against full - length trimeric SARS - CoV - 2 Spike ( A ) or monomeric RBD ( B ) . Results are shown as mean OD values of 2 replicates normalized against a BSA control and ranked by value . Lectin - Fc fusion proteins with a normalized OD > 0 . 5 in either ( A ) or ( B ) are indicated in both panels .

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(A,B) ELISA screen of the lectin-Fc library against full-length trimeric SARS-CoV-2 Spike (A) or monomeric RBD (B). Results are shown as mean OD values of 2 replicates normalized against a BSA control and ranked by value. Lectin-Fc fusion proteins with a normalized OD > 0.5 in either (A) or (B) are indicated in both panels.


( C ) Lectin - Fc and human ACE2 - mIgG1 Fc - fusion protein ( hACE2 ) binding to untreated or heat - denatured full - length SARS - CoV - 2 Spike by ELISA . hACE2 - mIgG1 was used as control for complete denaturation of Spike protein . Results are shown as mean OD values ± SD normalized to the BSA control ( technical replicates , N = 3 ) .

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(C) Lectin-Fc and human ACE2-mIgG1 Fc-fusion protein (hACE2) binding to untreated or heat-denatured full-length SARS-CoV-2 Spike by ELISA. hACE2-mIgG1 was used as control for complete denaturation of Spike protein. Results are shown as mean OD values ± SD normalized to the BSA control (technical replicates, N=3).


( D ) Lectin - Fc binding to full - length SARS - CoV - 2 Spike with or without de - N - glycosylation by PNGase F . " PNGase F only " denotes wells that were not coated with the Spike protein . Results are shown as mean OD values ± SD normalized to BSA controls ( technical replicates , N = 3 ) .

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(D) Lectin-Fc binding to full-length SARS-CoV-2 Spike with or without de-N-glycosylation by PNGase F. "PNGase F only" denotes wells that were not coated with the Spike protein. Results are shown as mean OD values ± SD normalized to BSA controls (technical replicates, N=3).


( E ) High speed AFM of single trimeric Spike visualizing the real - time interaction dynamics with lectins . Top panel shows 5 frames of trimeric Spike alone imaged on mica . Middle and bottom panels show 5 sequential frames of trimeric Spike / Clec4g and trimeric Spike / CD209c complexes , acquired at a rate of 153 . 6 and 303 ms / frame , respectively . Association and dissociation events between lectin and Spike are indicated by white and red arrows , respectively . The blue dotted ellipses display the core of the complexes showing low conformational mobility . Color schemes indicate height of the molecules in nanometers ( nm ) . Volumes of single trimeric Spike , trimeric Spike / Clec4g and trimeric Spike / CD209c complexes are indicated , as well as numbers of lectins bound to trimeric Spike , averaged over the experimental recording period .

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(E) High speed AFM of single trimeric Spike visualizing the real-time interaction dynamics with lectins. Top panel shows 5 frames of trimeric Spike alone imaged on mica. Middle and bottom panels show 5 sequential frames of trimeric Spike/Clec4g and trimeric Spike/CD209c complexes, acquired at a rate of 153.6 and 303 ms/frame, respectively. Association and dissociation events between lectin and Spike are indicated by white and red arrows, respectively. The blue dotted ellipses display the core of the complexes showing low conformational mobility. Color schemes indicate height of the molecules in nanometers (nm). Volumes of single trimeric Spike, trimeric Spike/Clec4g and trimeric Spike/CD209c complexes are indicated, as well as numbers of lectins bound to trimeric Spike, averaged over the experimental recording period.


( A ) ELISA analyses of human lectin - hIgG1 Fc - fusion protein ( hCLEC4g , hCD209 , hCD299 ) binding to untreated or heat - denatured full - length SARS - CoV - 2 Spike . A human ACE2 - hIgG1 - Fc fusion protein ( hACE2 ) was used to control for the complete denaturation of Spike . Results are shown as mean OD values ± SD normalized to a BSA control ( technical replicates , N = 3 ) .

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(A) ELISA analyses of human lectin-hIgG1 Fc-fusion protein (hCLEC4g, hCD209, hCD299) binding to untreated or heat-denatured full-length SARS-CoV-2 Spike. A human ACE2-hIgG1-Fc fusion protein (hACE2) was used to control for the complete denaturation of Spike. Results are shown as mean OD values ± SD normalized to a BSA control (technical replicates, N=3).


( B ) hCLEC4g , hCD209 , and hCD299 binding to full - length SARS - CoV - 2 Spike with or without de - N - glycosylation by PNGase F . " PNGase F only " denotes wells that were not coated with the Spike protein . Results are shown as mean OD values ± SD normalized against the BSA control ( technical replicates , N = 3 ) .

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(B) hCLEC4g, hCD209, and hCD299 binding to full-length SARS-CoV-2 Spike with or without de-N-glycosylation by PNGase F. "PNGase F only" denotes wells that were not coated with the Spike protein. Results are shown as mean OD values ± SD normalized against the BSA control (technical replicates, N=3).


( C , D ) SPR analysis with immobilized full - length trimeric Spike , probed with various concentrations of hCLEC4G ( C ) and hCD209 ( D ) .

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(C, D) SPR analysis with immobilized full-length trimeric Spike, probed with various concentrations of hCLEC4G (C) and hCD209 (D).


( C ) Infectivity of mouse Clec4g , CD209c , Asgr1 and hCLEC4G pre - treated SARS - CoV - 2 virus in Vero E6 cells . Data represent two to three independent experiments for Clec4g , CD209c and hCLEC4G lectins ( N = 5 - 9 ) and , one experiment for Asgr1 ( N = 2 - 4 ) . ( D ) Infectivity of mouse Clec4g , CD209c , Asgr1 and hCLEC4G pre - treated SARS - CoV - 2 virus in human airway epithelial Calu3 cells measured in triplicates and compared to non - treated SARS - CoV - 2 infected Calu3 cells measured in quadruplicates ( technical replicates ) . : ( C ) and ( D ) Viral RNA was measured with qRT - PCR 15 hours after infection with 0 . 02 MOI ( 103 PFUs ) of SARS - CoV - 2 . Data are presented as fold changes of viral loads over mock ( only SARS - CoV - 2 was added ) treated controls ( mean log10 values ± SD ) . All no virus controls were negative for SARS - CoV - 2 .

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(C) Infectivity of mouse Clec4g, CD209c, Asgr1 and hCLEC4G pre-treated SARS-CoV-2 virus in Vero E6 cells. Data represent two to three independent experiments for Clec4g, CD209c and hCLEC4G lectins (N=5-9) and, one experiment for Asgr1 (N=2-4). (D) Infectivity of mouse Clec4g, CD209c, Asgr1 and hCLEC4G pre-treated SARS-CoV-2 virus in human airway epithelial Calu3 cells measured in triplicates and compared to non-treated SARS-CoV-2 infected Calu3 cells measured in quadruplicates (technical replicates). : (C) and (D) Viral RNA was measured with qRT-PCR 15 hours after infection with 0.02 MOI (103 PFUs) of SARS-CoV-2. Data are presented as fold changes of viral loads over mock (only SARS-CoV-2 was added) treated controls (mean log10 values ± SD). All no virus controls were negative for SARS-CoV-2.


Survival curve of N . furzeri in the FLI facility . Recording of deaths starts at age of 5 wph , which corresponds to sexual maturity , and the colored dashed lines indicate the three age groups analysed in this study ( 5 animals / group ) , namely 5 weeks post hatching ( wph , young , sexual maturity ) , 12 wph ( adult ) and 39 wph ( old , past median lifespan ) of a wild - derived strain that exhibits a median lifespan of 7 - 8 months .

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Survival curve of N. furzeri in the FLI facility. Recording of deaths starts at age of 5 wph, which corresponds to sexual maturity, and the colored dashed lines indicate the three age groups analysed in this study (5 animals/group), namely 5 weeks post hatching (wph, young, sexual maturity), 12 wph (adult) and 39 wph (old, past median lifespan) of a wild-derived strain that exhibits a median lifespan of 7-8 months.


Global protein - transcript correlation for each sample , grouped by age . RPKM and iBAQ values were used to estimate transcript and protein levels from matched RNA - seq and TMT - based proteomics data obtained from the same animal . An ANOVA test was performed to evaluate significance among the age groups ( mean correlation at 5 wph : 0 . 48 ; at 12 wph : 0 . 43 ; and at 39 wph : 0 . 33 ; p = 3 . 05e - 07 , n = 5 per age group ) . In boxplots , the horizontal line represents the median , the bottom and top of the box the 25th and 75th percentile , respectively , and the whiskers extend 1 . 5 folds the interquartile range .

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Global protein-transcript correlation for each sample, grouped by age. RPKM and iBAQ values were used to estimate transcript and protein levels from matched RNA-seq and TMT-based proteomics data obtained from the same animal. An ANOVA test was performed to evaluate significance among the age groups (mean correlation at 5 wph: 0.48; at 12 wph: 0.43; and at 39 wph: 0.33; p = 3.05e-07, n=5 per age group). In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.


Scatter plot of log2 fold changes for genes differentially expressed both at transcript and protein levels ( adj . p < 0 . 05 ) . The color gradients indicate gene density in the regions where individual points overlap . Numbers of genes in each quadrant and the value of Pearson ' s coefficient of correlation , r , are reported for each graph . Solid lines represent a spline fit ( r = 0 . 505 for genes significantly affected at both transcript and protein levels , p < 2 . 2 x 10 - 16 , D ) ( r = 0 . 126 , p = 0 . 007 , E ) .

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Scatter plot of log2 fold changes for genes differentially expressed both at transcript and protein levels (adj. p < 0.05). The color gradients indicate gene density in the regions where individual points overlap. Numbers of genes in each quadrant and the value of Pearson's coefficient of correlation, r, are reported for each graph. Solid lines represent a spline fit (r = 0.505 for genes significantly affected at both transcript and protein levels, p < 2.2 x 10-16, D) (r = 0.126, p = 0.007, E).


Mechanisms affecting protein abundance during aging . Bar plots are based on all the proteins affected in either one of the age comparisons ( adj . p < 0 . 05 ) . Proteins were divided in the following five groups : ( i ) proteins and transcripts with significant and consistent changes ( dark brown ) , ( ii ) proteins with significant changes , and with consistent changes of the transcripts ( light brown ) , ( iii ) proteins with no transcripts detected ( dark gray ) , ( iv ) proteins with transcripts whose translation is potentially regulated by miRNAs ( light gray ) , as assessed by the workflow displayed in ( Figure EV2D ) , ( v ) all the remaining proteins that we classified as regulated by other post - transcriptional mechanisms ( violet ) . pgs = protein groups .

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Mechanisms affecting protein abundance during aging. Bar plots are based on all the proteins affected in either one of the age comparisons (adj. p < 0.05). Proteins were divided in the following five groups: (i) proteins and transcripts with significant and consistent changes (dark brown), (ii) proteins with significant changes, and with consistent changes of the transcripts (light brown), (iii) proteins with no transcripts detected (dark gray), (iv) proteins with transcripts whose translation is potentially regulated by miRNAs (light gray), as assessed by the workflow displayed in (Figure EV2D), (v) all the remaining proteins that we classified as regulated by other post-transcriptional mechanisms (violet). pgs = protein groups.


Abundance changes of ribosomal proteins and their transcripts during aging . Cytoplasmic ribosomal proteins of large and small subunits are displayed separately and changes are shown for both the age comparisons as box plots . Transcripts are displayed as light blue and proteins as dark blue boxes . Changes of individual proteins are displayed as dots ; orange dots identify significant cases ( adj . p < 0 . 05 , n = 4 per age group for proteome and n = 5 per age group for transcriptome ) . In boxplots , the horizontal line represents the median , the bottom and top of the box the 25th and 75th percentile , respectively , and the whiskers extend 1 . 5 folds the interquartile range .

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Abundance changes of ribosomal proteins and their transcripts during aging. Cytoplasmic ribosomal proteins of large and small subunits are displayed separately and changes are shown for both the age comparisons as box plots. Transcripts are displayed as light blue and proteins as dark blue boxes. Changes of individual proteins are displayed as dots; orange dots identify significant cases (adj. p < 0.05, n=4 per age group for proteome and n=5 per age group for transcriptome). In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.


Brains from young ( 5 wph ) and old ( 39 wph ) were homogenized and clarified lysates separated by size - exclusion chromatography ( SEC ) . For each age group , two pools of brains were processed separately . For each experiment ( 4 in total ) , 39 fractions were collected along the chromatogram , digested into peptides and analysed by Data Independent Acquisition ( DIA ) quantitative mass spectrometry .

Brains from young (5 wph) and old (39 wph) were homogenized and clarified lysates separated by size-exclusion chromatography (SEC). For each age group, two pools of brains were processed separately. For each experiment (4 in total), 39 fractions were collected along the chromatogram, digested into peptides and analysed by Data Independent Acquisition (DIA) quantitative mass spectrometry.


Co - elution profiles for selected protein complexes . For each complex , the median abundance of all the quantified subunits was used to generate the complex profile across fractions ( Dataset EV5 ) . All the complex profiles are scaled to the max value ( set to 1 ) to make profiles comparable across experiments . The estimated molecular weight of the displayed complexes is indicated in brackets .

Co-elution profiles for selected protein complexes. For each complex, the median abundance of all the quantified subunits was used to generate the complex profile across fractions (Dataset EV5). All the complex profiles are scaled to the max value (set to 1) to make profiles comparable across experiments. The estimated molecular weight of the displayed complexes is indicated in brackets.


Statistics of protein complexes undergoing stoichiometry changes with aging . Only protein complexes that had at least 5 members quantified were considered for each comparison . Complexes were considered affected if at least two members showed significant stoichiometry change ( adj . p < 0 . 05 and absolute log2 fold change > 0 . 5 ) . The complete list of stoichiometry changes is available in Dataset EV6 .

Statistics of protein complexes undergoing stoichiometry changes with aging. Only protein complexes that had at least 5 members quantified were considered for each comparison. Complexes were considered affected if at least two members showed significant stoichiometry change (adj. p < 0.05 and absolute log2 fold change > 0.5). The complete list of stoichiometry changes is available in Dataset EV6.


Violin plots depicting interquartile ranges ( IQRs ) of individual members of protein complexes during aging . The IQR for each protein complex considered in G was calculated using transcript ( total RNA dataset , light blue ) or protein ( dark blue ) log2 fold changes between two age groups ( n = 4 per age group for proteome and n = 5 per age group for transcriptome ) . * p < 0 . 05 , Wilcoxon Rank Sum test . The central line of the violin plots indicates the median value .

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Violin plots depicting interquartile ranges (IQRs) of individual members of protein complexes during aging. The IQR for each protein complex considered in G was calculated using transcript (total RNA dataset, light blue) or protein (dark blue) log2 fold changes between two age groups (n=4 per age group for proteome and n=5 per age group for transcriptome). * p < 0.05, Wilcoxon Rank Sum test. The central line of the violin plots indicates the median value.


Heat map showing relative protein fold changes for members of selected complexes affected by aging . Names of significantly affected members in the 39 wph vs . 12 wph comparison ( adj . p < 0 . 05 and absolute log2 fold change > 0 . 5 ) are highlighted in bold with a star . Related to Figure EV3 and Dataset EV5 and EV6 .

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Heat map showing relative protein fold changes for members of selected complexes affected by aging. Names of significantly affected members in the 39 wph vs. 12 wph comparison (adj. p < 0.05 and absolute log2 fold change > 0.5) are highlighted in bold with a star. Related to Figure EV3 and Dataset EV5 and EV6.


Representative Coomassie - stained SDS - PAGE gel showing the isolation of SDS insoluble aggregates from mouse brain lysates . TH = total homogenate , SUP = supernatant , W1 = SDS - soluble fraction , Pellet = formic - acid soluble fraction ( see Figure EV4A ) . Quantification of the yield of SDS - insoluble aggregates from young and old brain lysates was based on densitometry analysis of Coomassie - stained gel bands obtained from different animals , n = 5 per age group ( Figure EV4B ) ; * * p < 0 . 01 , unpaired t - test . In boxplots , the horizontal line represents the median , the bottom and top of the box the 25th and 75th percentile , respectively , and the whiskers extend 1 . 5 folds the interquartile range .

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Representative Coomassie-stained SDS-PAGE gel showing the isolation of SDS insoluble aggregates from mouse brain lysates. TH = total homogenate, SUP = supernatant, W1 = SDS-soluble fraction, Pellet = formic-acid soluble fraction (see Figure EV4A). Quantification of the yield of SDS-insoluble aggregates from young and old brain lysates was based on densitometry analysis of Coomassie-stained gel bands obtained from different animals, n=5 per age group (Figure EV4B); ** p < 0.01, unpaired t-test. In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.


Volcano plot based on protein quantification by label - free mass spectrometry depicting the enrichment of specific proteins in protein aggregates . The x - axis indicates the log2 ratio between protein abundance in aggregates ( Pellet ) and starting total homogenate ( TH ) . The horizontal dashed line indicates a p value cut - off of 0 . 05 and vertical lines a log2 fold change cut off of ± 0 . 5 . Selected proteins are highlighted as colored dots as indicated in the figure legend . Protein quantification was based on samples obtained from 3 independent isolations .

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Volcano plot based on protein quantification by label-free mass spectrometry depicting the enrichment of specific proteins in protein aggregates. The x-axis indicates the log2 ratio between protein abundance in aggregates (Pellet) and starting total homogenate (TH). The horizontal dashed line indicates a p value cut-off of 0.05 and vertical lines a log2 fold change cut off of ± 0.5. Selected proteins are highlighted as colored dots as indicated in the figure legend. Protein quantification was based on samples obtained from 3 independent isolations.


Double - labelling of telencephalic sections of N . furzeri with anti - RPS6 ( green ) as ribosomal marker and Proteostat as a marker for aggregated proteins ( red ) . Nuclear counterstaining was performed with DAPI ( blue ) . Scale bar = 10 μm .

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Double-labelling of telencephalic sections of N. furzeri with anti-RPS6 (green) as ribosomal marker and Proteostat as a marker for aggregated proteins (red). Nuclear counterstaining was performed with DAPI (blue). Scale bar = 10 μm.


Abundance changes of proteasome proteins and their transcripts during aging . Members of the 19S and 20S complex are displayed separately and changes are shown for both the age comparisons as box plots . Transcripts are displayed as light blue and proteins as dark blue boxes . Changes of individual proteins are displayed as dots , orange dots represent significant cases ( adj . p < 0 . 05 , n = 4 per age group for proteome and n = 5 per age group for transcriptome ) . In boxplots , the horizontal line represents the median , the bottom and top of the box the 25th and 75th percentile , respectively , and the whiskers extend 1 . 5 folds the interquartile range .

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Abundance changes of proteasome proteins and their transcripts during aging. Members of the 19S and 20S complex are displayed separately and changes are shown for both the age comparisons as box plots. Transcripts are displayed as light blue and proteins as dark blue boxes. Changes of individual proteins are displayed as dots, orange dots represent significant cases (adj. p < 0.05, n=4 per age group for proteome and n=5 per age group for transcriptome). In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.


Heat map showing transcript and protein fold changes for members of the 26 proteasome ( 19S and 12S complexes ) . Genes are annotated according to significance of their changes at the level of transcript ( adj . p < 0 . 05 : black , adj . p > 0 . 05 : white ) or protein ( adj . p < 0 . 05 : green , adj . p > 0 . 05 : white ) .

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Heat map showing transcript and protein fold changes for members of the 26 proteasome (19S and 12S complexes). Genes are annotated according to significance of their changes at the level of transcript (adj. p < 0.05: black, adj. p > 0.05: white) or protein (adj. p < 0.05: green, adj. p > 0.05: white).


In - gel proteasome assay following native gel electrophoresis ( top ) and immunoblotting of proteasome complexes ( 30S , 26S and 20S ) ( bottom ) in young ( 5wph ) , adult ( 12 wph ) and old ( 39 wph ) killifish brains . For additional samples and low exposure pictures see Figure EV5B and EV5C .

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In-gel proteasome assay following native gel electrophoresis (top) and immunoblotting of proteasome complexes (30S, 26S and 20S) (bottom) in young (5wph), adult (12 wph) and old (39 wph) killifish brains. For additional samples and low exposure pictures see Figure EV5B and EV5C.


Bar plots depicting the quantification of chymotrypsin - like ( CT - L ) activity from native gels calculated for doubly capped ( 30S ) or singly capped ( 26S ) proteasomes . n > = 5 per sample group ; error bars indicate standard error of the mean . * p < 0 . 05 , * * p < 0 . 005 , * * * p = 0 . 0001 , * * * * p < 0 . 001 , one - way ANOVA , Holm - Sidak ' s multiple comparison test . For each sample group , the mean value of activity in young samples ( 5 wph ) was set to 100 % .

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Bar plots depicting the quantification of chymotrypsin-like (CT-L) activity from native gels calculated for doubly capped (30S) or singly capped (26S) proteasomes. n >=5 per sample group; error bars indicate standard error of the mean. * p < 0.05, ** p < 0.005, *** p=0.0001, **** p < 0.001, one-way ANOVA, Holm-Sidak's multiple comparison test. For each sample group, the mean value of activity in young samples (5 wph) was set to 100%.


Percentage ( % ) of chymotrypsin - like ( CT - L ) , trypsin - like ( T - L ) and peptidylglutamyl peptide hydrolysing or caspase - like ( PGPH ) proteasome activities in brain extracts of killifish of different ages . n > = 6 per sample group ; error bars indicate standard error of the mean . * * p < 0 . 005 , * * * p = 0 . 0001 , * * * * p < 0 . 001 , one - way ANOVA , Holm - Sidak ' s multiple comparison test . For each sample group , the mean value of each activity in young samples ( 5 wph ) was set to 100 % .

Percentage (%) of chymotrypsin-like (CT-L), trypsin-like (T-L) and peptidylglutamyl peptide hydrolysing or caspase-like (PGPH) proteasome activities in brain extracts of killifish of different ages. n >=6 per sample group; error bars indicate standard error of the mean. ** p < 0.005, *** p=0.0001, **** p < 0.001, one-way ANOVA, Holm-Sidak's multiple comparison test. For each sample group, the mean value of each activity in young samples (5 wph) was set to 100%.


Age - related changes of proteins involved in the ubiquitin cycle . All the displayed proteins showed significant protein level changes in the 39 wph vs . 12 wph comparison ( adj . p < 0 . 05 ) .

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Age-related changes of proteins involved in the ubiquitin cycle. All the displayed proteins showed significant protein level changes in the 39 wph vs. 12 wph comparison (adj. p < 0.05).


Chymotrypsin - like ( CT - L ) proteasome activity in brain extracts of killifish treated with different proteasome inhibitors . The activity was measured at day 4 after the beginning of treatment . n = 8 per sample group ; error bars indicate standard deviation . * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA followed by Holm - Sidak ' s multiple comparisons test .

Chymotrypsin-like (CT-L) proteasome activity in brain extracts of killifish treated with different proteasome inhibitors. The activity was measured at day 4 after the beginning of treatment. n=8 per sample group; error bars indicate standard deviation. ** p < 0.01, **** p < 0.0001, one-way ANOVA followed by Holm-Sidak's multiple comparisons test.


Proteasome inhibition induces proteasome activators ( PSME4 ) , assembly factors ( POMP ) ubiquitin ligases ( UBE3C ) , mediators of autophagosome formation ( ATG2B ) , heat shock proteins and chaperones in killifish brain . Protein abundances were quantified by DIA mass spectrometry and they are shown relative to the mean value of vehicle control samples ( DMSO ) set to 1 , n = 10 per sample group . Adj . p < 0 . 05 for all the displayed proteins . Error bars indicate mean + / - SD .

B-CONTROLLED_VAR 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

Proteasome inhibition induces proteasome activators (PSME4), assembly factors (POMP) ubiquitin ligases (UBE3C), mediators of autophagosome formation (ATG2B), heat shock proteins and chaperones in killifish brain. Protein abundances were quantified by DIA mass spectrometry and they are shown relative to the mean value of vehicle control samples (DMSO) set to 1, n=10 per sample group. Adj. p < 0.05 for all the displayed proteins. Error bars indicate mean +/-SD.


Bortezomib treatment affects the stoichiometry of a subset of protein complexes . The number of affected proteins ( adj . p < 0 . 25 ) for each protein complex is indicated . Only protein complexes that had at least two members affected are shown .

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

Bortezomib treatment affects the stoichiometry of a subset of protein complexes. The number of affected proteins (adj. p < 0.25) for each protein complex is indicated. Only protein complexes that had at least two members affected are shown.


Members of the mitochondrial and cytoplasmic ribosomes affected by Bortezomib treatment . Relative protein abundances ( normalized to the mean of the protein complex to which they belong ) are shown . The mean value of vehicle control samples ( DMSO ) was set to 1 . n = 10 per sample group . Adj . p < 0 . 25 for all the displayed proteins . Error bars indicate mean + / - SD .

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

Members of the mitochondrial and cytoplasmic ribosomes affected by Bortezomib treatment. Relative protein abundances (normalized to the mean of the protein complex to which they belong) are shown. The mean value of vehicle control samples (DMSO) was set to 1. n=10 per sample group. Adj. p < 0.25 for all the displayed proteins. Error bars indicate mean +/-SD.


Distribution of change in expression for proteasome transcripts across the entire cohort of 159 fish . Lifespan was compared among fish that showed extreme changes in proteasome levels between 10 and 20 wph ( 32 fish showing the most pronounced decreases , shown in black vs . 32 fish showing the most pronounce increases ) . * * * p < 0 . 001 Log - rank test .

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Distribution of change in expression for proteasome transcripts across the entire cohort of 159 fish. Lifespan was compared among fish that showed extreme changes in proteasome levels between 10 and 20 wph (32 fish showing the most pronounced decreases, shown in black vs. 32 fish showing the most pronounce increases). *** p<0.001 Log-rank test.


Percentage of 2 - cell - like cells obtained after hyperosmotic treatments for 24h ( B ) Shown are the mean ± s . d . from at least 2 replicates . Individual dots indicate measurement of an independent biological replicate by FACS . Unpaired two - tailed t - tests were performed . Data information : Stars indicate significant differences obtained using indicated statistical tests with * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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Percentage of 2-cell-like cells obtained after hyperosmotic treatments for 24h (B) Shown are the mean ± s.d. from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.


Percentage of 2 - cell - like cells obtained after hyperosmotic treatments the indicated time ( C - D ) . Shown are the mean ± s . d . from at least 2 replicates . Individual dots indicate measurement of an independent biological replicate by FACS . Unpaired two - tailed t - tests were performed . Data information : Stars indicate significant differences obtained using indicated statistical tests with * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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Percentage of 2-cell-like cells obtained after hyperosmotic treatments the indicated time (C-D). Shown are the mean ± s.d. from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.


H - I . Protein levels assessed by Western blotting in the indicated conditions . Shown is one representative image from 2 independent replicates .

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H-I. Protein levels assessed by Western blotting in the indicated conditions. Shown is one representative image from 2 independent replicates.


A . ( Left ) Representative single confocal z - section of CellROX - DeepRed fluorescence of ESC treated with 0 . 2M sorbitol for the indicated time from 2 biological replicates . Scale bar : 10µm . ( Right ) Quantification of CellROX - DeepRed fluorescence intensity from single z - sections from 2 biological replicates . Two - tailed Mann - Whitney tests were performed . Boxes indicate the range between the first and third quartile , the line indicates the median and the whiskers display the spread from 5 to 95 % of the data . Data information : Stars indicate significant differences obtained using indicated statistical tests with * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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

A. (Left) Representative single confocal z-section of CellROX-DeepRed fluorescence of ESC treated with 0.2M sorbitol for the indicated time from 2 biological replicates. Scale bar: 10µm. (Right) Quantification of CellROX-DeepRed fluorescence intensity from single z-sections from 2 biological replicates. Two-tailed Mann-Whitney tests were performed. Boxes indicate the range between the first and third quartile, the line indicates the median and the whiskers display the spread from 5 to 95% of the data. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.


B . Percentage of 2CLCs obtained by FACS after the indicated sorbitol treatment with addition of ROS scavenger N - AcetylCysteine ( NAC ) during either the sorbitol treatment or recovery time . Shown are the mean ± s . d . from at least 2 replicates . Individual dots indicate measurement of an independent biological replicate by FACS . Unpaired two - tailed t - tests were performed . Data information : Stars indicate significant differences obtained using indicated statistical tests with * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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

B. Percentage of 2CLCs obtained by FACS after the indicated sorbitol treatment with addition of ROS scavenger N-AcetylCysteine (NAC) during either the sorbitol treatment or recovery time. Shown are the mean ± s.d. from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.


C . Protein levels assessed by Western blotting in the indicated conditions . Shown is one representative image from 3 independent replicates .

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C. Protein levels assessed by Western blotting in the indicated conditions. Shown is one representative image from 3 independent replicates.


D . Percentage of 2CLCs obtained by FACS after the indicated hyperosmotic treatment with addition of ATR inhibitor during either the hyperosmotic stress or recovery time . Shown are the mean ± s . d . from at least 2 replicates . Individual dots indicate measurement of an independent biological replicate by FACS . Same untreated dots are shown . Two - tail t - tests were performed . Data information : Stars indicate significant differences obtained using indicated statistical tests with * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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D. Percentage of 2CLCs obtained by FACS after the indicated hyperosmotic treatment with addition of ATR inhibitor during either the hyperosmotic stress or recovery time. Shown are the mean ± s.d. from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Same untreated dots are shown. Two-tail t-tests were performed. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.


A . Percentage of 2CLCs obtained by FACS after the indicated sorbitol treatment with addition of ROS scavenger N - AcetylCysteine ( NAC ) or ATR inhibitor . Shown are the mean ± s . d from 5 replicates . Individual dots indicate measurement of an independent biological replicate by FACS . Unpaired two - tailed t - tests were performed . B . Percentage of 2CLCs obtained by FACS after the indicated NaCl treatment with the addition of ROS scavenger N - AcetylCysteine ( NAC ) or ATR inhibitor . Shown are the mean ± s . d . from at least 2 replicates . Individual dots indicate measurement of an independent biological replicate by FACS . Unpaired two - tailed t - tests were performed . Data information : Stars indicate significant differences obtained using indicated statistical tests with * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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

A. Percentage of 2CLCs obtained by FACS after the indicated sorbitol treatment with addition of ROS scavenger N-AcetylCysteine (NAC) or ATR inhibitor. Shown are the mean ± s.d from 5 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. B. Percentage of 2CLCs obtained by FACS after the indicated NaCl treatment with the addition of ROS scavenger N-AcetylCysteine (NAC) or ATR inhibitor. Shown are the mean ± s.d. from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.


C . Percentage of 2CLCs obtained by FACS after the indicated treatments . Shown are the mean ± s . d from at least 2 replicates . Individual dots indicate measurement of an independent biological replicate by FACS . Individual and combinatorial treatments performed with 0 . 2M of sorbitol , 24mM of sodium acetate , 2 . 5nM of PlaB spliceosome inhibitor and 0 . 53µM of retinoic acid . Unpaired two - tailed t - tests were performed . Unpaired two - tailed t - tests were performed against each individual treatment in the combinatorial experiments . Data information : Stars indicate significant differences obtained using indicated statistical tests with * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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

C. Percentage of 2CLCs obtained by FACS after the indicated treatments. Shown are the mean ± s.d from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Individual and combinatorial treatments performed with 0.2M of sorbitol, 24mM of sodium acetate, 2.5nM of PlaB spliceosome inhibitor and 0.53µM of retinoic acid. Unpaired two-tailed t-tests were performed. Unpaired two-tailed t-tests were performed against each individual treatment in the combinatorial experiments. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.


D . Time course of pyrene actin polymerization showing the influence of different SPIN90 constructs in reactions containing 50 nM Bos taurus Arp2 / 3 complex and 3 μM 15 % pyrene labeled actin . E . Plot of maximum polymerization rate versus concentration of SPIN90 for reactions shown in ( D )

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D. Time course of pyrene actin polymerization showing the influence of different SPIN90 constructs in reactions containing 50 nM Bos taurus Arp2/3 complex and 3 μM 15% pyrene labeled actin. E. Plot of maximum polymerization rate versus concentration of SPIN90 for reactions shown in (D)


F . Plot of maximum polymerization rate versus concentration of SPIN90 for pyrene actin polymerization assays containing 2 . 5 μM SPIN90 , 500 nM Arp2 / 3 complex , 3 μM actin and the indicated concentration of the SPIN90 ARM domain construct

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F. Plot of maximum polymerization rate versus concentration of SPIN90 for pyrene actin polymerization assays containing 2.5 μM SPIN90, 500 nM Arp2/3 complex, 3 μM actin and the indicated concentration of the SPIN90 ARM domain construct


G . Time courses of pyrene actin polymerization as described in panel ( D ) , except the influence of the SPIN90 middle segment is tested on its own or in the presence of the 351 - 722 construct

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G. Time courses of pyrene actin polymerization as described in panel (D), except the influence of the SPIN90 middle segment is tested on its own or in the presence of the 351-722 construct