EMBO/SourceData huggingface dataset:
Figure 1(A) Representative images of H&E staining of colon from 3m C57Bl (n=4) and mdx (n=4) mice. High magnification (scale bar: 20 μm) and low magnification (scale bar: 200 μm).(B) Mucus layer, area between yellow dash lines; crypt length, yellow headed arrow. Scale bar: 100 μm. (C) Mucus layer thickness and crypt length were quantified for n=4 mice per group (with pooled samples of n=60 for mucus layer thickness and n=80 for crypt length).(D) SCFA faecal quantification of 3m C57Bl (n=3) and mdx (n=4-5) mice. Data information: Data are presented as mean ± SD (*p<0.05; **p<0.01, ***p<0.001, ****p<0.0001; Student t-test).(E) Colon images captured with the iMScope TRIO described altered pattern of expression of different Phosphatidylcholines (PC) and Lysophosphatidylcholines (LysoPC) (as indicated by m/z values) in 3m mdx mice (n=3). Scale bar: 50 μm. For each lipid, the mean intensities measured at 12 positions throughout colon images are shown on the right side where bars are mean ± SEM (n = 3). Data information: Data are presented as mean ± SD (*p<0.05; **p<0.01, ***p<0.001, ****p<0.0001; Student t-test).(F) Cropped images of representative WB showing the expression of proteins involved in inflammation and fibrosis in colon tissues of 3m C57Bl (n=3) and 3m mdx (n=5). Densitometric analyses of protein expression was shown as ratio to actin. Data information: Data are presented as mean ± SD (*p<0.05; **p<0.01, ***p<0.001, ****p<0.0001; Student t-test).
Figure 4(E) Faecal content quantification of SCFAs in 3m mdx and 3m mdx+ABX mice (n=5 per group). Data equal to 0. Data are presented as mean ± SD (**p< 0.01, ***p< 0.001; Student t-test).
Figure 5(A-D) FACS analysis of spleen and muscle homogenates from 3m C57Bl (n=4), mdx (n=5) and mdx+ABX (n=7) mice demonstrates no significant alteration of CD45+CD11b+CD4-CD8- myeloid cells (A and C) and few differences in CD4+ or CD8+ naïve (CD62L+ CD44-), central memory (CD62L+ CD44+) and effector (CD62L- CD44+) T cells (B and D). Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 ordinary one-way ANOVA, Tukey's multiple comparison test.(E) FACS analysis of spleen of 3m C57Bl (n=7), mdx (n=9) and GFmdx (n=5) mice revealed similar proportions of CD4+ and CD8+ T cells but reduced activated CD44+ T cells in GFmdx mice. Representative plots are depicted. Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 ordinary one-way ANOVA, Tukey's multiple comparison test.(F) Graphs show cumulative frequencies of CD4+ and CD8+ T cells on live cells of 3m C57Bl (n=7), mdx (n=4) and GFmdx (n=5) mice. Representative dot plots and cumulative frequencies of splenic CD4+GITR+CD25+ Treg. Frequencies of effector CD44+ T cells were significantly decreased in spleen of GFmdx mice. Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 ordinary one-way ANOVA, Tukey's multiple comparison test.(G) Representative dot-plots showing the proportion of muscle-infiltrating CD45+ cells of 3m C57Bl (n=6), mdx (n=6) and GFmdx (n=5) mice. Cumulative frequencies of muscle-infiltrating CD45+ cells are shown. Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 ordinary one-way ANOVA, Tukey's multiple comparison test.(H) Representative images of TA muscles from 3m mdx and GFmdx mice stained for CD45 (in green), isolectin (in red) and phalloidin (in purple). Nuclei were counterstained with DAPI (in blue). Scale bar: 10 μm.(I) Absolute number of CD3+ inflammatory cells (white arrows) were quantified in n=12 images of TA of 3m C57Bl, 3m mdx and 3m GFmdx mice (n=6 each). CD3 staining is shown in green and DAPI in blue. Scale bars: 50 μm. Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 The comparisons among the averages of CD3+ cells were evaluated using unpaired t-test.
Figure 6(D) RT-qPCR analysis of TA muscles of two independent experiments with 3m mdx (n=4), mdx+ABX (n=4) and GFmdx (n=5) mice determined the expression of myogenic markers. Data information: Data are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, ordinary one-way ANOVA and non-parametric test followed by Kruskal-Wallis test for RT-qPCR).(E) Representative Gomori modified staining and quantification of myofiber area and relative frequency of the myofiber cross-sectional area (CSA) expressed as the frequency distribution of the TA muscles of 3m C57Bl (n=4), 3m mdx (n=4), mdx+ABX (n=4) and GFmdx mice (n=5). Pooled samples for each group with n=6240 for 3m C57Bl; n=6001 for 3m mdx; n=10556 for 3m mdx+ABX; n=23059 for GFmdx. For morphometric analysis, images were quantified with Image J software for each mouse. Scale bars: 50 μm. Data information: Data are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, ordinary one-way ANOVA(G) Representative images of skeletal muscle showed the distribution and composition of the myosin heavy chain (MyHC) isoforms (Type IIa, Type IIx and Type IIb). (H, I) Graph portrays (H) the percentage of myofibers expressing different MyHC isoforms and (I) myofibers area per type of MyHC in TAs of 3m mdx (n=4), mdx+ABX (n=4) and GFmdx (n=5) mice (n=12 images per animal). Data information: Data are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, ordinary one-way ANOVA(J) Representative SDH staining and quantification of percentage of SDH+ myofibers of TAs from 3m mdx (n=4), mdx+ABX (n=4) and GFmdx mice (n=5) (n=12 images per animal). Scale bars: 50 μm.
Figure 7(D, E) Representative H&E staining (D) and quantification of myofiber area with Image J software (E) of TA muscles from 3m C57Bl (n=5), mdx (n=5) and ABX-mdxFMT_C57Bl (n=6). Scale bars for H&E: 200 μm. Data information: Data for staining quantification are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, one-way ANOVA, Tukey's multiple comparison test).(H) Representative images of skeletal muscle showed the distribution and composition of MyHC isoforms (Type IIa, Type IIx and Type IIb). Scale bar: 50 μm. (I) Graph portrays the percentage of myofibers expressing different MyHC isoforms. n=12 images were analysed for each mouse.(J-K) Representative SDH staining and quantification of percentage of SDH+ myofibers of TA muscles from mdx (n=5) and ABX-mdxFMT_C57Bl (n=6) (n=12 images per mouse). Scale bar: 200 μm. Data information: Data for staining quantification are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, one-way ANOVA, Tukey's multiple comparison test).(L-M) Representative image of CD31 (in cyan), α-SMA (in green) and isolectin (in red) staining and their quantification in TA muscles from mdx (n=5) and ABX-mdxFMT_C57Bl (n=6) mice. Scale bar: 500 μm. Data information: Data for staining quantification are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, one-way ANOVA, Tukey's multiple comparison test).
Figure 1(C) pS65-Ub (left) and total Ub (right) immunoblots of mitochondria-enriched fractions from wild-type flies treated 3 days with either paraquat or vehicle (sucrose).(D) Immunoblots for the indicated antibodies following subcellular fractionation of flies treated with paraquat for 3 days. T, total lysate; N, nuclear-enriched fraction; M, mitochondria-enriched fraction; S, post-mitochondrial supernatant.(E) Immunoblots for the indicated antibodies of (T) total lysates or (M) mitochondria-enriched fractions from fly heads (with proboscises removed) and bodies (thoraces and abdomens).
Figure 2(C) pS65-Ub immunoblot of mitochondria-enriched fractions following the paraquat pulse-chase assay in wild-type and park-/- flies. UT, untreated; PQ, 1-day paraquat treatment; Recovery, flies removed from paraquat and returned to normal food. Asterisk (*) denotes non-specific band. (D) pS65-Ub lane densitometry analysis of n = 3 independent replicates from (C), expressed relative to the most intense lane signal in each blot.
Figure 3(A-B) pS65-Ub immunoblot of whole-animal lysates from untreated flies of the indicated genotypes. park RNAi was induced by da-GAL4 (ubiquitous), Mef2-GAL4 (muscle) or nSyb-GAL4 (neurons). (C) pS65-Ub immunoblot of lysates from bodies or heads (without proboscises), as indicated, from park-/- animals.(E) Higher magnification image of pS65-Ub positive structures in flight muscle counterstained for ATP5A. Scale bar = 5 μm. Chart shows quantification of ATP5A levels of pS65-Ub-positive (+ve) versus -negative (-ve) mitochondrial structures from 3 biological replicates (4-7 images per animal; average per animal shown in open circles).
Figure 5(A) pS65-Ub immunoblot of whole-animal lysates from wild-type, Atg5- and park-/- animals harvested at the indicated ages.(B) pS65-Ub immunoblot of whole-animal lysates from young animals of the following genotypes: wild-type, Pink1-, park-/-, park RNAi (daG4>UAS-park RNAi), Atg1 RNAi (daG4>UAS-Atg1 RNAi), Atg8a-.
Figure 6(A-G) Representative images of pS65-Ub immunostaining false-coloured for intensity (Fire LUT) in muscle segments 6/7 from wandering L3 larvae of the following genotypes: (A) wild type, (B) Pink1-, (C) park-/-, (D) Atg5-, (E) Atg8a-, (F) Atg5-; park-/-, (G) Atg8a-; park-/-. (H) Quantification of number of pS65-Ub puncta from A-G displaying mean +/- SEM from the indicated number of animals (n = 3-5 as indicated). (I) Puncta volume from C-G expressed as mean +/- SEM (n = 4-5 animals).
Figure 7(A) pS65-Ub immunoblot of whole-animal lysates following paraquat pulse-chase assay of Atg5- flies with parkin overexpression (OE) (Atg55cc5 daG4>UAS-park) compared with Atg5- flies overexpressing mitochondrially targeted GFP (Atg55cc5 daG4>UAS-mito-HA-GFP). UT, untreated flies; PQ, 1-day paraquat treatment; Recovery, flies removed from paraquat and returned to normal food.(B) pS65-Ub lane densitometry, normalised to total protein (RevertTM stain), of n = 3 independent replicates of (A), expressed as pS65-Ub intensity relative to the most intense band in each blot.
Fig 1(B) Locomotor behavior, measured by thrashing rates in liquid medium, in the C. elegans strains with neuronal expression of human WT SOD1 or ALS-linked mutant SOD1G85R, in the presence (M1/M1) or absence (+/+) of the suppressor mutation (n = 16).(C) Northern (top panel) and western (middle, bottom panels) blot analyses of total RNA and protein from SOD1G85R strains, with (M1/M1) or without (+/+) suppressor mutations, demonstrating that the levels of SOD1G85R mRNA and total protein are not changed by the suppressor mutation (M1/M1). The western blot lanes are from the same gel and exposure.(E) C. elegans SPR-5 and UFD-2 and their mammalian homologs lysine-specific demethylase 1 (LSD1) and ubiquitination factor E4 B (UBE4B) share all major protein domains. These include the Swi3-Rsc8-Moira (SWIRM), amine oxidase-like (AOL), and TOWER domains in LSD1/SPR-5 and the Ufd2 Core and U-box domains in UBE4B/UFD-2. Positions of the missense, nonsense, and deletion mutations in mutant C. elegans are indicated.(F) Locomotor behavior, measured by thrashing rates, of the C. elegans carrying the SOD1G85R transgene on the normal background (WT), with the null mutation of either ufd-2(tm1380) or spr-5(by134), the double mutation ufd-2(tm1380);spr-5(by134), or the M1 suppressor ufd-2(W824X);spr-5(R646Q) (n = 16).(G) Western blotting of the supernatant (S) and pellet (P) protein fractions from the C. elegans carrying the SOD1G85R transgene with the double mutation ufd-2(tm1380);spr-5(by134) compared with controls. While SOD1G85R protein levels are unchanged in the S fraction, those in the P fraction are decreased in the double suppressor mutant. The P fraction represents only about 1.7% of total SOD1G85R in the WT sample; therefore, a higher ratio of the P fraction relative to the S fraction (approximately 2:1) is used for the western analysis.(H) The overexpression (OE) of WT UFD-2 or SPR-5 in the C. elegans nervous system blocks the protection conferred by the double mutation ufd-2(tm1380);spr-5(by134) (n = 16). Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
Fig 2(A) Top: schematic drawing depicts pan-neuronal expression of YFP in head and ventral neurons in the context of the C. elegans body plan. Left: micrographs show the SOD1G85R-YFP proteins expressed in WT or the spr-5(by134);ufd-2(tm1380) mutant background. The double-mutant worms show a marked decrease in protein aggregation in neurons. Enlarged sections of headneurons (red framed) and ventral cord neurons (white framed) are shown. Middle: quantification of locomotion in the spr-5(by134);ufd-2(tm1380) and the WT C. elegans with neuronal expression of SOD1G85R-YFP (n = 30). Right: a decrease in the protein levels of SOD1G85R-YFP in the presence of spr-5(by134);ufd-2(tm1380) is shown by western blots of the supernatant (S) and the pellet (P) fractions. (B and C) Analyses of the spr-5(by134);ufd-2(tm1380) and the WT C. elegans with neuronal expression of TDP-43-c25-YFP (n = 30) or PolyQ-YFP (n = 12) as in (A).(D) Neurodegenerative rough-eye phenotype in adults is alleviated by the knockdown of the Drosophila homologs of ufd-2/UBE4B and spr-5/LSD1-CG9934 and Su(Var)3-3, respectively, compared to the control (CTRL). Eye-specific expression of TDP-43M337V, FUSR521C, and RNA interference (RNAi) was driven by GMR-Gal4.(E) Drosophila eye pigment quantitation. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
Fig 3(A) Western blots of cell lysates derived from mock (CTRL), single UBE4B or LSD1, or double UBE4B and LSD1 knockdowns. Supernatant (S) and pellet (P) fractions were probed with indicated antibodies. While the LSD1 or UBE4B single-knockdown reduces the SOD1G85R aggregates in both supernatant and pellet fractions, the combined knockdown produces the strongest reduction in the aggregates. (B) Quantification of SOD1G85R protein levels by western blotting (A). n = 3 (supernatant); n = 8 (pellet).(C) Western blots of a representative cycloheximide chase experiment to determine SOD1 protein half-lives in the double UBE4B and LSD1 knockdown cells versus controls. (D) Quantification of SOD1G85R clearance, as analyzed by western blotting in (C). The graph indicates the relative band intensity of SOD1G85R at each chase time point. n = 5; Overall p = 0.02 (paired t test, CTRL versus UBE4B and LSD1 double knockdown). Individual p = 0.03 (3 h), p = 0.003 (6 h), p = 0.06 (9 h), and p = 0.004 (12-21 h).(E) The half-life of SOD1G85R is reduced from 8.5 h to 5 h upon knockdown of UBE4B and LSD1. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
Fig 4(B) The volcano scatter plot indicates fold changes in the levels of gene transcripts affected differentially by the UBE4B and LSD1 double knockdown versus control shRNA. Gray spots represent 22,148 annotated transcripts. Red spots are predicted p53-activated targets, and blue spots are predicted p53-inhibited targets. The enrichment of red spots in the up-regulated genes (right upper quadrant, >1.2-fold change, p ≤ 0.04) and blue spots in the down-regulated genes (left upper quadrant) indicates that the p53-mediated transcription is activated by the UBE4B and LSD1 double knockdown.C) RT-qPCR validation of the expression levels of representative p53 target genes in the UBE4B and LSD1 double-knockdown samples. n = (2 to 6), p ≤ 0.04.(D) The p53 protein level is significantly increased by UBE4B and LSD1 double knockdown. Left: representative western blots showing p53 levels, the knockdown of UBE4B and LSD1, and the actin control in HEK293T cells. Right: quantification of the p53 protein levels normalized against actin (n = 3).E) The p53-mediated transcriptional activity is measured by a luciferase reporter under the control of a p53-response element promoter, which was transfected into HEK293T cells 72-96 h after the initiation of the UBE4B and LSD1 single or double knockdowns (n = 6).(F) Increased interaction of p53 with its activating partner 53BP1 in response to UBE4B and LSD1 knockdown. Left: representative western blots of 53BP1 co-immunoprecipitation. Right: quantification of the 53BP1 co-immunoprecipitation (n = 2). Data represent means ± SEM.(G) RT-qPCR validation of the expression levels of FOXO3a, FOXO4, and PSMD11 (n = 6).(H) The FOXO3a-mediated transcriptional activity was measured with a luciferase reporter under the control of a FOXO-response element promoter. HEK293T cells were transfected with shRNAs for control, LSD1, UBE4B, or both LSD1 and UBE4B, followed by transfection of the luciferase reporter together with a constitutively active form of FOXO3a (TM) 72-96 h later (n = 6). Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
Fig 5(A) Increased protein levels of proteasome subunits upon the UBE4B and LSD1 double knockdown in HEK293T cells. (B) The UBE4B and LSD1 single or double knockdowns increase the proteasomal degradation of a reporter substrate, Suc-LLVY-Luciferin, whose degradation is measured in a luciferase release assay. The double knockdown shows synergistic proteasomal activation when compared with the individual knockdowns (n = 6).(C) The autophagy activity is significantly increased in cells with the UBE4B and LSD1 double knockdown. The in vivo activity of the ATG4B protease, which cleaves LC3 precursors, was quantified via a Gaussia luciferase release assay (see S5D Fig., S5E Fig., and Materials and Methods for details). The HCT116 cells were analyzed 48 h after the initiation of the knockdown.(D) Quantification of LC3-II levels in HEK293T cells with the UBE4B and LSD1 double knockdown or the mock control. The cells were treated with or without 10 mM 3-methyladenine (3-MA) for 48h before lysis. Western blots indicate an increase in LC3 levels (top panels) in the double UBE4B and LSD1 knockdown cells (middle panels), while the actin control is unchanged (bottom panels). The graph shows the quantification of LC3-II levels as normalized to actin levels (n = 3). A one-way ANOVA test with matched multiple comparisons and Tukey correction was used for statistics. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
Fig 6(A) p53 small molecule activators Tenovin-1 and CP-31398 reduce the levels of misfolded SOD1 proteins, as determined by the SOD1G85R solubility assay in HEK293 cells. Increasing concentrations of the p53 activators significantly decrease the levels of SOD1G85R but not the endogenous WT SOD1 proteins in western blots of both supernatant and pellet fractions.(B) A decrease in p53 as the result of shRNA knockdown increases the levels of SOD1G85R but not WT SOD1 proteins in the SOD1G85R aggregation assay, as shown by western blots of both supernatant (S) (n = 2) and pellet (P) (n = 3) fractions.(C) A complete absence of p53 increases the accumulation of SOD1G85R mutant proteins in p53-/- HCT116 cells when compared with controls. Representative western blots (left panels) and quantification of SOD1G85R levels in the supernatant lysates are shown. The middle graph indicates the ratio of G85R to WT SOD1 proteins in the presence or absence of p53 with varying amounts of transfected mutant SOD1. The right graph panel shows the same data as shown in the middle panel, but normalized to the average SOD1G85R level for each amount of the transfected plasmid. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
Fig 7(A) Left: p53 knockdown reverses the enhanced clearance of SOD1G85R proteins conferred by the UBE4B and LSD1 double knockdown. Total amounts of shRNAs were adjusted to be equal with nontargeting CTRL shRNAs. Right: quantification of insoluble aggregated SOD1G85R in pellet fractions from HEK293T cells transfected with control, double (UBE4B/LSD1), or triple (UBE4B/LSD1/p53) shRNAs (n = 2).(B) The degenerative TDP-43M337V eye phenotype is exacerbated by the knockdown of the Drosophila homolog of p53 (p53 RNAi), or by the overexpression of a dominant-negative p53 mutant (p53.R155H). Expression of p53 RNAi, p53.R155H, and TDP-43M337V are driven by GMR-Gal4.(C) The p53-activating drug Tenovin-1 (TEN1) protects spinal cord motor neurons from SOD1G85R-induced proteotoxicity. Rat spinal cord cultures were grown as described in Materials and Methods and treated with 0.8 μM TEN1 or vehicle (VEH) DMSO, followed by infection of HSV-SOD1G85R or the HSV-LacZ control after 24 h. At day 5 post-infection, cells were fixed and stained with a motor neuron-specific anti-neurofilament H (NF-H) antibody. Left: representative images of motor neurons in each condition. Scale bar = 50 μm. Right: quantification of motor neuron survival (one-way ANOVA with multiple comparisons and Tukey correction). Data represent means ± SEM.
Figure 1(A) Top hit selected candidates are listed. The plot shows relative S9.6 nuclear intensity values for cells transformed with each of the indicated pool of siRNAs.(B) Representative images of HeLa cells immunostained with S9.6 and nucleolin antibodies after transfection with at least a pool of two siRNAs of each original pool.(C) Relative S9.6 signal intensity per nucleus after nucleolus signal removal in HeLa cells transfected with the indicated siRNAs. More than 250 total cells from three independent experiments were considered. The median of each population is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. ***p<0.001 (Mann-Whitney U test).
Figure 2(A) Relative DRIP-qPCR signal values at RPL13A, APOE, MIB2 and RHOT2 genes in HeLa cells transfected with the indicated siRNAs and treated in vitro with RNase H pre-immunoprecipitation where indicated. The mean ± SEM from at least three independent experiments is shown. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). Black stars denote significant increases whereas red stars denote significant decreases.(B) Representative images of immunostaining with S9.6 and anti-nucleolin antibodies in HeLa cells transfected with the indicated siRNAs(C) Relative S9.6 signal intensity per nucleus after nucleolus signal removal in HeLa cells after cytoplasm pre-extraction (CE) and treated in vitro with RNaseIII and RNase H where indicated. More than 500 total cells from three independent experiments were considered. The median of each population is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. **p<0.01, ***p<0.001 (Mann-Whitney U test).
Figure 3(A) Representative images of single-cell alkaline gel electrophoresis (comet assay) of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+).(B) Comet tail moment from single-cell alkaline gel electrophoresis (comet assay) of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). More than 250 total cells were considered. The mean ± SEM of the median from at least three independent experiments is shown, except for RAD1, RAD17 and UBE2B (n=2). *p<0.05, **p<0.01, ***p<0.001 (one-tailed unpaired t-test). Black stars denote significant increases whereas red stars denote significant decreases.
Figure 4(A) Representative images and percentage of HeLa cells with more than 5 H3S10-P foci after transfection with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). Mitotic cells were excluded for the analysis by DAPI staining. More than 300 total cells were considered. Data represent mean ± SEM from three independent experiments. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). Black stars denote significant increases whereas red stars denote significant decreases.(B) Representative images and H3K9me2 nuclear signal intensity of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). At least three experiments were performed. The median of each population in a representative experiment with at least 100 cells per condition is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). Black stars denote significant increases whereas red stars denote significant decreases.