Figure 5A . Histograms showing the mean distance ( megabases , Mb ) of observed double crossovers ( DCO , red vertical line ) , compared to 2 , 000 matched sets of randomly generated distances ( RAN , black ) . The mean distance of the random sets is shown in by the vertical grey line . B . SNPs / kb values were calculated in physical windows of increasing size ( kb ) around crossover midpoints in wild type ( red ) and msh2 ( blue ) . The same values were calculated for matched randomly chosen positions ( grey ) . Printed above the plot for each window are circles that are coloured green if crossover SNPs / kb values are significantly different to random ( P < 0 . 05 ) , or red if not ( P > 0 . 05 ) ( Bonferroni adjusted t - tests ) . The population analysed ( Col × Ct , Col × Ler , Col × CLC ) and its genome average SNP frequency ( SNPs / kb ) is printed above the plots and the number of crossover positions analysed is printed inset . Beneath are plots showing the significance of SNPs / kb differences around crossovers between the indicated genotypes .

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Figure 5A. Histograms showing the mean distance (megabases, Mb) of observed double crossovers (DCO, red vertical line), compared to 2,000 matched sets of randomly generated distances (RAN, black). The mean distance of the random sets is shown in by the vertical grey line.B. SNPs/kb values were calculated in physical windows of increasing size (kb) around crossover midpoints in wild type (red) and msh2 (blue). The same values were calculated for matched randomly chosen positions (grey). Printed above the plot for each window are circles that are coloured green if crossover SNPs/kb values are significantly different to random (P<0.05), or red if not (P>0.05) (Bonferroni adjusted t-tests). The population analysed (Col×Ct, Col×Ler, Col×CLC) and its genome average SNP frequency (SNPs/kb) is printed above the plots and the number of crossover positions analysed is printed inset. Beneath are plots showing the significance of SNPs/kb differences around crossovers between the indicated genotypes.


Figure 6A . Representative DAPI - stained spreads of pachytene , diakinesis , metaphase - I , dyad and tetrad meiotic stages , for wild type and msh2 , in Col × Ler or Col × CLC hybrid backgrounds . B . Male meiocytes immunostained for MSH2 ( red ) and ASY1 ( green ) , and stained for DAPI ( blue ) in wild type ( Col ) or msh2 . C . As for B , but immunostaining wild type male meiocytes for MSH2 ( red ) , MSH4 ( green ) and staining chromatin with DAPI ( blue ) .

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Figure 6A. Representative DAPI-stained spreads of pachytene, diakinesis, metaphase-I, dyad and tetrad meiotic stages, for wild type and msh2, in Col×Ler or Col×CLC hybrid backgrounds.B. Male meiocytes immunostained for MSH2 (red) and ASY1 (green), and stained for DAPI (blue) in wild type (Col) or msh2.C. As for B, but immunostaining wild type male meiocytes for MSH2 (red), MSH4 (green) and staining chromatin with DAPI (blue).


Figure 7A . Crossover frequency ( black ) and SNP density ( purple ) per 200 kb measured from the Col × Ct F2 population . The position of the centromere ( CEN , vertical dashed line ) and 420 FTL transgenes ( red and green vertical lines ) used to measure crossover frequency are indicated . B . Plots of chromosome 3 showing positions of Col / Col ( black ) or Col / Ct ( purple ) homozygous or heterozygous genotypes at the indicated marker positions ( circles ) . The positions of the 420 FTL transgenes are indicated ( red and green vertical lines ) . C . 420 crossover frequency ( cM ) in the HOM - HOM , HET - HET , HET - HOM , HOM - HET genotypes shown in B , in either wild type or msh2 . Values for replicate individuals are plotted , in addition to the mean ( red ) . D . As for C , but comparing genotypes in wild type or HEI10 - OE backgrounds . Values for replicate individuals are plotted , in addition to the mean ( red ) .

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Figure 7A. Crossover frequency (black) and SNP density (purple) per 200 kb measured from the Col×Ct F2 population. The position of the centromere (CEN, vertical dashed line) and 420 FTL transgenes (red and green vertical lines) used to measure crossover frequency are indicated.B. Plots of chromosome 3 showing positions of Col/Col (black) or Col/Ct (purple) homozygous or heterozygous genotypes at the indicated marker positions (circles). The positions of the 420 FTL transgenes are indicated (red and green vertical lines).C. 420 crossover frequency (cM) in the HOM-HOM, HET-HET, HET-HOM, HOM-HET genotypes shown in B, in either wild type or msh2. Values for replicate individuals are plotted, in addition to the mean (red).D. As for C, but comparing genotypes in wild type or HEI10-OE backgrounds. Values for replicate individuals are plotted, in addition to the mean (red).


Figure 8D . Plots of SNP density per 10 kb along the chromosomes for Arabidopsis Col × Ler and budding yeast S288c × SK1 crosses ( Cooper et al , 2018 ) . Telomere positions are shown by vertical lines , and centromeres by vertical dashed lines . Centromeres are also labeled above the plots in red , as ' CEN ' or ' C ' .

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Figure 8D. Plots of SNP density per 10 kb along the chromosomes for Arabidopsis Col×Ler and budding yeast S288c×SK1 crosses (Cooper et al, 2018). Telomere positions are shown by vertical lines, and centromeres by vertical dashed lines. Centromeres are also labeled above the plots in red, as 'CEN' or 'C'.


Figure 1Cell counting experiments analyzing the response to trastuzumab ( 50 nM , 5 days ) of naïve BT474 as well as the resistant BTRH pool and the BTRH # 10 clone . Western blot analyses of the expression of HER2 , pHER2 , HER3 and pHER3 in BT474 , BTRH and BTRH # 10 cells . HER2 and HER3 were immunoprecipitated from 100 or 500 μg of cell extracts , respectively , and blots probed with anti - HER2 , anti - HER3 or anti - phosphotyrosine . Calnexin was used as a loading control . Numbers below the blots show the quantitation of the signal of each band , referred to BT474 cells ( intensity of band in BT474 cells = 1 ) . Analyses of cell surface levels of HER2 and HER3 in BT474 and BTRH cells by surface immunoprecipitation . Response of BT474 , BTRH and BTRH # 10 to EV20 / MMAF . Cells treated with the indicated doses of EV20 / MMAF for 5 days were counted . Action of the nude EV20 antibody or EV20 / MMAF ( 10 nM each , 5 days ) on BT474 , BTRH and BTRH # 10 cells . Comparison of the effect of MMAF and EV20 / MMAF on the proliferation of BT474 and BTRH cells after five days of treatment with the drugs .

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Figure 1Cell counting experiments analyzing the response to trastuzumab (50 nM, 5 days) of naïve BT474 as well as the resistant BTRH pool and the BTRH#10 clone.Western blot analyses of the expression of HER2, pHER2, HER3 and pHER3 in BT474, BTRH and BTRH#10 cells. HER2 and HER3 were immunoprecipitated from 100 or 500 μg of cell extracts, respectively, and blots probed with anti-HER2, anti-HER3 or anti-phosphotyrosine. Calnexin was used as a loading control. Numbers below the blots show the quantitation of the signal of each band, referred to BT474 cells (intensity of band in BT474 cells=1).Analyses of cell surface levels of HER2 and HER3 in BT474 and BTRH cells by surface immunoprecipitation.Response of BT474, BTRH and BTRH#10 to EV20/MMAF. Cells treated with the indicated doses of EV20/MMAF for 5 days were counted.Action of the nude EV20 antibody or EV20/MMAF (10 nM each, 5 days) on BT474, BTRH and BTRH#10 cells.Comparison of the effect of MMAF and EV20/MMAF on the proliferation of BT474 and BTRH cells after five days of treatment with the drugs.


Figure 2Response to trastuzumab ( 50 nM , 5 days ) of BT474 , BTRH , patient derived cells ( PDX118 ) and their trastuzumab - resistant derivatives TR1 and TR2 . HER2 , HER3 and pHER3 levels in BT474 , BTRH , PDX118 , TR1 and TR2 . pHER3 levels were analyzed by a specific anti - pHER3 antibody . Effect of EV20 / MMAF on BT474 , BTRH , PDX118 , TR1 and TR2 . Cells were counted after 5 days of treatment with the indicated doses of EV20 / MMAF . Effect of trastuzumab ( 50 nM , 5 days ) in HER2 positive breast cancer cell lines ( SKBR3 , BT474 , BTRH , MDA - MB - 361 , HCC1419 , HCC1569 and HCC1954 ) . The blue line indicates the threshold ( established from BTRH ) used to consider a cell line resistant to trastuzumab . Analyses of HER2 , HER3 and pHER3 in the cells used in ( D ) . Numbers below the blots show the quantitation of the signal of each band , referred to SKBR3 cells ( intensity of band in SKBR3 cells = 1 ) . pHER3 levels were analyzed by a specific anti - pHER3 antibody . Effect of EV20 / MMAF on the cell lines used in ( D ) . Cells were counted after 5 days of treatment with the indicated doses of EV20 / MMAF . Knockdown of HER3 in BT474 cells . Control and HER3 - targeted short hairpin sequences were transduced in BT474 cells and HER3 , pHER3 and pAKT levels analyzed by Western . Levels of pHER3 were analyzed by a specific anti - pHER3 antibody . Effect of HER3 knockdown on the antiproliferative action of EV20 / MMAF analyzed by cell counting after 5 days of treatment with the ADC .

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Figure 2Response to trastuzumab (50 nM, 5 days) of BT474, BTRH, patient derived cells (PDX118) and their trastuzumab-resistant derivatives TR1 and TR2.HER2, HER3 and pHER3 levels in BT474, BTRH, PDX118, TR1 and TR2. pHER3 levels were analyzed by a specific anti-pHER3 antibody.Effect of EV20/MMAF on BT474, BTRH, PDX118, TR1 and TR2. Cells were counted after 5 days of treatment with the indicated doses of EV20/MMAF.Effect of trastuzumab (50 nM, 5 days) in HER2 positive breast cancer cell lines (SKBR3, BT474, BTRH, MDA-MB-361, HCC1419, HCC1569 and HCC1954). The blue line indicates the threshold (established from BTRH) used to consider a cell line resistant to trastuzumab.Analyses of HER2, HER3 and pHER3 in the cells used in (D). Numbers below the blots show the quantitation of the signal of each band, referred to SKBR3 cells (intensity of band in SKBR3 cells=1). pHER3 levels were analyzed by a specific anti-pHER3 antibody.Effect of EV20/MMAF on the cell lines used in (D). Cells were counted after 5 days of treatment with the indicated doses of EV20/MMAF.Knockdown of HER3 in BT474 cells. Control and HER3-targeted short hairpin sequences were transduced in BT474 cells and HER3, pHER3 and pAKT levels analyzed by Western. Levels of pHER3 were analyzed by a specific anti-pHER3 antibody.Effect of HER3 knockdown on the antiproliferative action of EV20/MMAF analyzed by cell counting after 5 days of treatment with the ADC.


Figure 3Internalization of EV20 / MMAF ( 10 nM ) at indicated times in BT474 and BTRH cells . Scale bar : 20 µm . HER3 staining : red ; DAPI : blue . Internalization of biotin - S - S - EV20 / MMAF in BT474 and BTRH cells . These cell lines were pulsed with 10 nM of biotin - S - S - EV20 / MMAF for 1 hour at 4 ° C and chased at 37 ° C at the indicated times . Subsequently , surface accessible biotin was cleaved , cells were lysed , and extracts precipitated with streptavidin - sepharose . EV20 / MMAF was analyzed by Western blotting using anti - human - HRP . Quantitative plot of the Western studies shown in ( B ) . Data are presented as the mean + SD of two independent experiments . Co - localization ( yellow ) between EV20 / MMAF and LAMP1 at the indicated incubation times with EV20 / MMAF in BT474 and BTRH cells . Scale bar : 5 µm . HER3 staining : red ; LAMP1 : green , DAPI : blue . Western blot analyses of the effect of EV20 / MMAF ( 10 nM ) on the levels of HER2 and HER3 in BT474 and BTRH cells at the indicated incubation times with the ADC . The same Western was probed for HER2 or HER3 expression using two differently labelled species - specific secondary fluorescent antibodies . Quantitative representation of the Western studies shown in ( E ) .

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Figure 3Internalization of EV20/MMAF (10 nM) at indicated times in BT474 and BTRH cells. Scale bar: 20 µm. HER3 staining: red; DAPI: blue.Internalization of biotin-S-S-EV20/MMAF in BT474 and BTRH cells. These cell lines were pulsed with 10 nM of biotin-S-S-EV20/MMAF for 1 hour at 4°C and chased at 37°C at the indicated times. Subsequently, surface accessible biotin was cleaved, cells were lysed, and extracts precipitated with streptavidin-sepharose. EV20/MMAF was analyzed by Western blotting using anti-human-HRP. Quantitative plot of the Western studies shown in (B). Data are presented as the mean + SD of two independent experiments.Co-localization (yellow) between EV20/MMAF and LAMP1 at the indicated incubation times with EV20/MMAF in BT474 and BTRH cells. Scale bar: 5 µm. HER3 staining: red; LAMP1: green, DAPI: blue.Western blot analyses of the effect of EV20/MMAF (10 nM) on the levels of HER2 and HER3 in BT474 and BTRH cells at the indicated incubation times with the ADC. The same Western was probed for HER2 or HER3 expression using two differently labelled species-specific secondary fluorescent antibodies. Quantitative representation of the Western studies shown in (E).


Figure 4Cell cycle analysis by flow cytometry of BT474 and BTRH cells ( 50 , 000 events ) treated with EV20 / MMAF ( 10 nM ) for the indicated times . BT474 and BTRH cells were treated for the indicated days with EV20 / MMAF ( 10 nM ) , lysed and the amount of different proteins analyzed by Western . Numbers below the blots show the quantitation of the signal of each band , referred to time 0 . Action of EV20 / MMAF on mitotic spindle formation . BT474 and BTRH cells were treated ( 10 nM , 48 hours ) , fixed , stained for β - tubulin ( green ) and DAPI ( blue ) . Scale bar : 7 . 5 μm . Quantitative analysis of mitotic cells with abnormal spindles treated or not with EV20 / MMAF ( 10 nM , 48 hours ) in BT474 and BTRH cells . Bars represent the mean + SD of two independent experiments , calculated as follows : ( number of mitotic cells with abnormal spindles / total number of mitotic cells ) × 100 ( % ) . Cell death induction by EV20 / MMAF ( 10 nM ) analyzed by double annexin V and PI staining in BT474 and BTRH cells ( 50 , 000 events ) . Time - course analysis of the effect of EV20 / MMAF ( 10 nM ) on cleaved - caspase - 3 , analyzed by Western blotting in BT474 and BTRH cells . GAPDH was used as a loading control . Samples with intensity values of 1 were used as reference . Fluorimetric analyses of caspase - 3 activity in BT474 and BTRH cells treated with 10 nM EV20 / MMAF . Data show the mean + SD of triplicates of an experiment that was repeated twice .

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Figure 4Cell cycle analysis by flow cytometry of BT474 and BTRH cells (50,000 events) treated with EV20/MMAF (10 nM) for the indicated times.BT474 and BTRH cells were treated for the indicated days with EV20/MMAF (10 nM), lysed and the amount of different proteins analyzed by Western. Numbers below the blots show the quantitation of the signal of each band, referred to time 0.Action of EV20/MMAF on mitotic spindle formation. BT474 and BTRH cells were treated (10 nM, 48 hours), fixed, stained for β-tubulin (green) and DAPI (blue). Scale bar: 7.5 μm.Quantitative analysis of mitotic cells with abnormal spindles treated or not with EV20/MMAF (10 nM, 48 hours) in BT474 and BTRH cells. Bars represent the mean + SD of two independent experiments, calculated as follows: (number of mitotic cells with abnormal spindles/total number of mitotic cells) ×100 (%).Cell death induction by EV20/MMAF (10 nM) analyzed by double annexin V and PI staining in BT474 and BTRH cells (50,000 events).Time-course analysis of the effect of EV20/MMAF (10 nM) on cleaved-caspase-3, analyzed by Western blotting in BT474 and BTRH cells. GAPDH was used as a loading control. Samples with intensity values of 1 were used as reference.Fluorimetric analyses of caspase-3 activity in BT474 and BTRH cells treated with 10 nM EV20/MMAF. Data show the mean + SD of triplicates of an experiment that was repeated twice.


Figure 5In vivo effect of EV20 ( 10 mg / kg , n = 4 ) , untreated control ( PBS , n = 4 ) and EV20 / MMAF ( 3 . 3 mg / kg , n = 5 ; 10 mg / kg , n = 4 ) on tumor growth in mice injected with BTRH cells . Mice were treated when tumors reached a volume of 500 mm3 . Data are represented as mean + SEM . In vivo effect of single - dose EV20 ( 10 mg / kg , n = 3 ) untreated control ( PBS , n = 4 ) , single - dose EV20 / MMAF ( 10 mg / kg , n = 4 ) or weekly trastuzumab ( 30 mg / kg , n = 4 ) on tumor growth in mice injected with BTRH # 10 cells . Data are represented as mean + SEM . In vivo effect of single - dose EV20 ( 10 mg / kg , n = 4 ) , untreated control ( PBS , n = 4 ) , single - dose EV20 / MMAF ( 10 mg / kg , n = 4 ) or weekly trastuzumab ( 30 mg / kg , n = 4 ) on tumor growth in mice injected with HCC1954 cells . Data are represented as mean + SEM . Analysis of pHistone H3 , HER3 , pHER3 and cleaved caspase - 3 in tumors of mice injected with BTRH and treated ( T ) or not ( C ) with EV20 / MMAF ( 3 . 3 mg / kg for 24 hours or two weeks ) . Numbers below the blots show the quantitation of the signal of each band , referred to that of a control untreated tumor . Long - term monitoring of mice injected with BTRH cells and treated with EV20 / MMAF ( 3 . 3 mg / kg , n = 5 ; 10 mg / kg , n = 4 ) . Where indicated , mice were treated with EV20 / MMAF . Data were represented as mean + SD of the different mice groups .

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Figure 5In vivo effect of EV20 (10 mg/kg, n=4), untreated control (PBS, n=4) and EV20/MMAF (3.3 mg/kg, n=5; 10 mg/kg, n=4) on tumor growth in mice injected with BTRH cells. Mice were treated when tumors reached a volume of 500 mm3. Data are represented as mean + SEM.In vivo effect of single-dose EV20 (10 mg/kg, n=3) untreated control (PBS, n=4), single-dose EV20/MMAF (10 mg/kg, n=4) or weekly trastuzumab (30 mg/kg, n=4) on tumor growth in mice injected with BTRH#10 cells. Data are represented as mean + SEM. In vivo effect of single-dose EV20 (10 mg/kg, n=4), untreated control (PBS, n=4), single-dose EV20/MMAF (10 mg/kg, n=4) or weekly trastuzumab (30 mg/kg, n=4) on tumor growth in mice injected with HCC1954 cells. Data are represented as mean + SEM.Analysis of pHistone H3, HER3, pHER3 and cleaved caspase-3 in tumors of mice injected with BTRH and treated (T) or not (C) with EV20/MMAF (3.3 mg/kg for 24 hours or two weeks). Numbers below the blots show the quantitation of the signal of each band, referred to that of a control untreated tumor.Long-term monitoring of mice injected with BTRH cells and treated with EV20/MMAF (3.3 mg/kg, n=5; 10 mg/kg, n=4). Where indicated, mice were treated with EV20/MMAF. Data were represented as mean + SD of the different mice groups.


Figure 6Dose - response to EV20 / MMAF after 5 days of treatment in BT474 cells resistant to neratinib ( BTRN # 5 and BTRN # 24 ) Levels of HER2 , pHER2 , HER3 , pHER3 , pAKT and calnexin of ( B ) cells resistant to neratinibDose - response to EV20 / MMAF after 5 days of treatment in BT474 cells resistant to lapatinib ( BTRL , BTRL # 109 and BTRL # 3 ) Levels of HER2 , pHER2 , HER3 , pHER3 , pAKT and calnexin of cells resistant to lapatinibDose - response to EV20 / MMAF after 5 days of treatment in BT474 cells resistant to T - DM1 ( BT - TDM1R # 1 and BT - TDM1R # 6 ) . Levels of HER2 , pHER2 , HER3 , pHER3 , pAKT and calnexin of cells resistant to TDM1 .

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Figure 6Dose-response to EV20/MMAF after 5 days of treatment in BT474 cells resistant to neratinib (BTRN#5 and BTRN#24)Levels of HER2, pHER2, HER3, pHER3, pAKT and calnexin of (B) cells resistant to neratinibDose-response to EV20/MMAF after 5 days of treatment in BT474 cells resistant to lapatinib (BTRL, BTRL#109 and BTRL#3)Levels of HER2, pHER2, HER3, pHER3, pAKT and calnexin of cells resistant to lapatinibDose-response to EV20/MMAF after 5 days of treatment in BT474 cells resistant to T-DM1 (BT-TDM1R#1 and BT-TDM1R#6).Levels of HER2, pHER2, HER3, pHER3, pAKT and calnexin of cells resistant to TDM1.


Figure 1Expression of indicated miRNAs in sorted naïve ( CD62L + CD44 - ) CD4 + T cells and following in vitro stimulation with anti - CD3 / anti - CD28 ( 1 - 3 days ) , relative to levels in cells prior to stimulation . Data from 3 independent experiments each using T cells pooled from 4 WT mice . Significance determined by one - way ANOVA . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Expression of indicated miRNAs in purified spleen lymphocytes ( B cells , CD4 + T cells and CD8 + T cells ) from d0 naive ( white ) and day 28 L . donovani - infected ( grey ) mice . Expression of each miRNA normalized to levels in whole naïve spleen ( dotted line ) . Data is mean + SEM of 2 experiments with cells purified from 3 - 5 pooled spleens . Volcano plot of RNAseq gene expression in splenic WT and miR - 132 - / - CD4 + T cells from d28 L . donovani infected mice . Fold change determined as log2 mean FPKM ( miR - 132 - / - / WT ) from 4 WT and 5 miR - 132 - / - mice . Transcripts significantly different between WT and miR - 132 - / - ( p < 0 . 05 ) are shown in red . Dotted box indicates transcripts significantly up - regulated in miR - 132 - / - CD4 + T cells by more than 50 % . STRING network analysis of significantly up - regulated transcripts in CD4 + T cells from spleen of d28 L . donovani infected miR - 132 - / - mice compared to WT cells . Cluster of ribosomal proteins shown in green circle , with coding RP transcripts ( black ) and pseudogenes ( red ) indicated . Secondary clusters are shown in grey . Volcano plot of all RP genes in splenic WT and miR - 132 - / - CD4 + T cells from d28 L . donovani infected mice . RPL genes are shown as circles , RPS genes as triangles , and pseudogenes as squares . Red symbols indicate significant difference between WT and miR - 132 - / - cells ( p < 0 . 05 ) whereas black non - significant . Expression of RP transcripts determined by qPCR from L . donovani infected d28 WT ( blue ) and miR - 132 - / - mice ( red ) . N = 9 for each WT and miR - 132 - / - from 2 independent infection experiments . Box extends from 25 - 75th percentile , whiskers are minimum and maximum values , and horizontal lines indicate median . Significance determined by unpaired t - test . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Fold change of all RP transcripts ( grey ) in Th1 cells compared to naïve CD4 + T cells . Data taken from RNA sequencing experiments described in reference 24 . Fold changes in IL - 10 ( red ) and IFNγ ( blue ) indicated for comparison . The statistical significance of the observed up - regulation of RP transcripts in Th1 cells is determined by Chi - squared test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Expression of indicated miRNAs in sorted naïve (CD62L+ CD44-) CD4+ T cells and following in vitro stimulation with anti-CD3 / anti-CD28 (1-3 days), relative to levels in cells prior to stimulation. Data from 3 independent experiments each using T cells pooled from 4 WT mice. Significance determined by one-way ANOVA. Data information: * p<0.05, ** p<0.01, *** p<0.001.Expression of indicated miRNAs in purified spleen lymphocytes (B cells, CD4+ T cells and CD8+ T cells) from d0 naive (white) and day 28 L. donovani-infected (grey) mice. Expression of each miRNA normalized to levels in whole naïve spleen (dotted line). Data is mean + SEM of 2 experiments with cells purified from 3-5 pooled spleens.Volcano plot of RNAseq gene expression in splenic WT and miR-132-/- CD4+ T cells from d28 L. donovani infected mice. Fold change determined as log2 mean FPKM (miR-132-/-/WT) from 4 WT and 5 miR-132-/- mice. Transcripts significantly different between WT and miR-132-/- (p<0.05) are shown in red. Dotted box indicates transcripts significantly up-regulated in miR-132-/- CD4+ T cells by more than 50%.STRING network analysis of significantly up-regulated transcripts in CD4+ T cells from spleen of d28 L. donovani infected miR-132-/- mice compared to WT cells. Cluster of ribosomal proteins shown in green circle, with coding RP transcripts (black) and pseudogenes (red) indicated. Secondary clusters are shown in grey.Volcano plot of all RP genes in splenic WT and miR-132-/- CD4+ T cells from d28 L. donovani infected mice. RPL genes are shown as circles, RPS genes as triangles, and pseudogenes as squares. Red symbols indicate significant difference between WT and miR-132-/- cells (p<0.05) whereas black non-significant.Expression of RP transcripts determined by qPCR from L. donovani infected d28 WT (blue) and miR-132-/- mice (red). N=9 for each WT and miR-132-/- from 2 independent infection experiments. Box extends from 25-75th percentile, whiskers are minimum and maximum values, and horizontal lines indicate median. Significance determined by unpaired t-test. Data information: * p<0.05, ** p<0.01, *** p<0.001.Fold change of all RP transcripts (grey) in Th1 cells compared to naïve CD4+ T cells. Data taken from RNA sequencing experiments described in reference 24. Fold changes in IL-10 (red) and IFNγ (blue) indicated for comparison. The statistical significance of the observed up-regulation of RP transcripts in Th1 cells is determined by Chi-squared test.


Figure 2A . Volcano plot ( Log2 ( Fold Change ) vs - Log ( P value ) ) of RNA gene expression in purified naïve CD62L + CD44 - WT and miR - 132 - / - CD4 + T cells . Fold change determined as log2 mean FPKM ( miR - 132 - / - / WT ) from 4 WT and 4 miR - 132 - / - mice . Transcripts significantly different between WT and miR - 132 - / - cells ( p < 0 . 05 ) shown in red . B . Volcano plot of RNA gene expression in purified naïve CD62L + CD44 - WT and miR - 132 - / - CD4 + T cells following 18hr in vitro stimulation with anti - CD3 / anti - CD28 under Th1 conditions . Fold change determined as log2 mean FPKM ( miR - 132 - / - / WT ) from 4 WT and 4 miR - 132 - / - mice . Transcripts significantly different between WT and miR - 132 - / - cells ( p < 0 . 05 ) shown in red . C . Volcano plot of transcripts containing a conserved miR - 212 / 132 - 3p target site in naïve CD4 + T cells from WT or miR - 132 - / - mice . D . Volcano plot of transcripts containing a conserved miR - 212 / 132 - 3p target site in in vitro polarised ( Th1 condtions , 18h post stimulation ) CD4 + T cells from WT or miR - 132 - / - mice . E . Volcano plot of transcripts containing a conserved miR - 212 / 132 - 3p target site in spleen CD4 + T cells from d28 L . donovani infected WT or miR - 132 - / - mice . F . BTAF1 transcript levels determined by qRTPCR in WT ( blue ) or miR - 132 - / - ( red ) in naïve ( d0 ) and Th1 polarised for 18h ( d1 ) CD4 + T cells , and CD4 + T cells from d28 L . donovani infected WT or miR - 132 - / - mice . N = 8 - 9 for each WT and miR - 132 - / Data information : Significance in ( F ) determined by unpaired t - test . * p < 0 . 05 , * * p < 0 . 01 . G . Expression of BTAF1 protein in d0 naïve and d1 ( 18hr ) Th1 - polarised WT and miR - 132 - / - CD4 + T cells , as determined by Western blot . Each lane from individual mouse , and representative of two independent experiments . H . Relative luciferase activity in HeLa transfected with plasmid containing WT BTAF1 3 ' UTR ( white ) or BTAF1 3 ' UTR on which the miR - 132 binding site is mutated ( grey ) downstream of renilla luciferase , in the presence of miR - 132 - 3p or miR - 212 - 3p mimics . Error bars indicate SEM from eight replicate treatments . Data information : Significance in ( H ) determined by unpaired t - test . * p < 0 . 05 , * * p < 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Volcano plot (Log2(Fold Change) vs -Log(P value)) of RNA gene expression in purified naïve CD62L+ CD44- WT and miR-132-/- CD4+ T cells. Fold change determined as log2 mean FPKM (miR-132-/-/WT) from 4 WT and 4 miR-132-/- mice. Transcripts significantly different between WT and miR-132-/- cells (p<0.05) shown in red.B. Volcano plot of RNA gene expression in purified naïve CD62L+ CD44- WT and miR-132-/- CD4+ T cells following 18hr in vitro stimulation with anti-CD3/anti-CD28 under Th1 conditions. Fold change determined as log2 mean FPKM (miR-132-/-/WT) from 4 WT and 4 miR-132-/- mice. Transcripts significantly different between WT and miR-132-/- cells (p<0.05) shown in red.C. Volcano plot of transcripts containing a conserved miR-212/132-3p target site in naïve CD4+ T cells from WT or miR-132-/- mice.D. Volcano plot of transcripts containing a conserved miR-212/132-3p target site in in vitro polarised (Th1 condtions, 18h post stimulation) CD4+ T cells from WT or miR-132-/- mice.E. Volcano plot of transcripts containing a conserved miR-212/132-3p target site in spleen CD4+ T cells from d28 L. donovani infected WT or miR-132-/- mice.F. BTAF1 transcript levels determined by qRTPCR in WT (blue) or miR-132-/- (red) in naïve (d0) and Th1 polarised for 18h (d1) CD4+ T cells, and CD4+ T cells from d28 L. donovani infected WT or miR-132-/- mice. N=8-9 for each WT and miR-132-/ Data information: Significance in (F) determined by unpaired t-test. * p<0.05, ** p<0.01.G. Expression of BTAF1 protein in d0 naïve and d1 (18hr) Th1-polarised WT and miR-132-/- CD4+ T cells, as determined by Western blot. Each lane from individual mouse, and representative of two independent experiments.H. Relative luciferase activity in HeLa transfected with plasmid containing WT BTAF1 3'UTR (white) or BTAF1 3'UTR on which the miR-132 binding site is mutated (grey) downstream of renilla luciferase, in the presence of miR-132-3p or miR-212-3p mimics. Error bars indicate SEM from eight replicate treatments. Data information: Significance in (H) determined by unpaired t-test. * p<0.05, ** p<0.01.


Figure 3A . mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with Non - targeting control ( NTC ) mimics ( white ) or miR - 132 - 3p mimics ( grey ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . B . p300 and BTAF1 protein levels in MEF transfected with NTC mimics or miR - 132 - 3p mimics determined by Western blot . GAPDH was used as a loading control . Right panel indicates mean + SEM of 4 experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . C . mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC siRNA ( white ) or p300 siRNA ( grey ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . D . mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC siRNA ( white ) or BTAF1 siRNA ( grey ) . Data information : Statistical significance is determined by unpaired t - testE . mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC or miR - 132 - 3p mimics and NTC siRNA or p300 or BTAF1 siRNAs for 48h . Levels are normalised to cells transfected with NTC siRNA and NTC mimic . Data information : Statistical significance is determined by unpaired t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . F . Puromycin incorporation ( following 10 - minute pulse and 50 - minute chase ) determined by western blot in MEFs transfected with NTC or miR - 132 - 3p or miR - 212 - 3p mimics . Data information : Statistical significance is determined by unpaired t -

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with Non-targeting control (NTC) mimics (white) or miR-132-3p mimics (grey). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.B. p300 and BTAF1 protein levels in MEF transfected with NTC mimics or miR-132-3p mimics determined by Western blot. GAPDH was used as a loading control. Right panel indicates mean + SEM of 4 experiments. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.C. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC siRNA (white) or p300 siRNA (grey). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.D. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC siRNA (white) or BTAF1 siRNA (grey). Data information: Statistical significance is determined by unpaired t-testE. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC or miR-132-3p mimics and NTC siRNA or p300 or BTAF1 siRNAs for 48h. Levels are normalised to cells transfected with NTC siRNA and NTC mimic. Data information: Statistical significance is determined by unpaired t-test * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.F. Puromycin incorporation (following 10-minute pulse and 50-minute chase) determined by western blot in MEFs transfected with NTC or miR-132-3p or miR-212-3p mimics. Data information: Statistical significance is determined by unpaired t-test


Figure 4A . Percentage of IFNγ + live TCRβ + CD4 + cells from L . donovani infected WT ( blue ) or miR - 132 - / - ( red ) mice , determined by intracellular cytokine staining . Data representative of 3 independent experiments with 3 - 5 mice per group . Data information : statistical significance was determined by unpaired t - test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . NS : not significant . B . Percentage of IFNγ + / IL - 10 + live TCRβ + CD4 + cells from L . donovani infected WT ( blue ) or miR - 132 - / - ( red ) mice , determined by intracellular cytokine staining . Data representative of 3 independent experiments with 3 - 5 mice per group . Data information : statistical significance was determined by unpaired t - test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . NS : not significant . C . IL - 10 mRNA levels , determined by RNA - sequencing , in TCRβ + CD4 + cells purified from spleens of L . donovani infected WT ( blue ) or miR - 132 - / - ( red ) mice ( n = 5 per group ) . D . Percentage of IFNγ + WT ( blue ) or miR - 132 - / - ( red ) in vitro polarised Th1 cells ( 6 days ) in the presence or absence of phenylephrine ( PE ) , determined by intracellular cytokine staining . E . Percentage of IL10 + WT ( blue ) or miR - 132 - / - ( red ) in vitro polarised Th1 cells ( 6 days ) in the presence or absence of phenylephrine ( PE ) , determined by intracellular cytokine staining . F . Total cell counts following in vitro Th1 polarisation ( 6 days ) in the presence or absence of phenylephrine ( PE ) . For ( D - E ) , cells were purified from 3 mice per group and 6 replicates performed . Data information : statistical significance was determined with 1 - way ANOVA followed by Bonferroni ' s multiple comparison test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . NS : not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Percentage of IFNγ+ live TCRβ+ CD4+ cells from L. donovani infected WT (blue) or miR-132-/- (red) mice, determined by intracellular cytokine staining. Data representative of 3 independent experiments with 3-5 mice per group. Data information: statistical significance was determined by unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001. NS: not significant.B. Percentage of IFNγ+/IL-10+ live TCRβ+ CD4+ cells from L. donovani infected WT (blue) or miR-132-/- (red) mice, determined by intracellular cytokine staining. Data representative of 3 independent experiments with 3-5 mice per group. Data information: statistical significance was determined by unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001. NS: not significant.C. IL-10 mRNA levels, determined by RNA-sequencing, in TCRβ+ CD4+ cells purified from spleens of L. donovani infected WT (blue) or miR-132-/- (red) mice (n=5 per group).D. Percentage of IFNγ+ WT (blue) or miR-132-/- (red) in vitro polarised Th1 cells (6 days) in the presence or absence of phenylephrine (PE), determined by intracellular cytokine staining. E. Percentage of IL10+ WT (blue) or miR-132-/- (red) in vitro polarised Th1 cells (6 days) in the presence or absence of phenylephrine (PE), determined by intracellular cytokine staining. F. Total cell counts following in vitro Th1 polarisation (6 days) in the presence or absence of phenylephrine (PE). For (D-E), cells were purified from 3 mice per group and 6 replicates performed. Data information: statistical significance was determined with 1-way ANOVA followed by Bonferroni's multiple comparison test. * p<0.05, ** p<0.01, *** p<0.001. NS: not significant.


Figure 5Liver LDU ( Leishman Donovan units ) at day 28 in infected WT mice treated with anti - IL - 10R antibody or isotype control antibody ( left panel , n = 5 mice per group ) , or at day 21 and day 28 from WT ( blue ) , IL - 10 + / - ( open green circles ) and IL - 10 - / - ( filled green circles ) mice ( right panel n = 3 - 6 mice per group ) Data information : Significance determined by unpaired t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Day 28 splenic parasite burdens expressed as LDU with each data point representing an individual mouse in WT ( blue ) and miR - 132 - / - ( miR - 132 - / - ; red ) mice . Data from 4 independent infection experiments . Mean WT and miR - 132 - / - spleen parasite burdens from the 4 independent experiments shown in ( B ) . Lines link individual experiments . Splenic parasite burdens relative to WT group ( WT mean = 1 ) for each of the 4 experiments shown in ( B ) , with each data point representing individual mouse . Data information : Significance determined by unpaired t - test , and in ( C ) by paired t - test of mean values . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Spleen size expressed as % body weight for d0 ( naïve ) or day 28 L . donovani infected WT ( blue ) and miR - 132 - / - ( miR - 132 - / - ; red ) mice . Liver size expressed as % body weight for d0 ( naïve ) or day 28 L . donovani infected WT ( blue ) and miR - 132 - / - ( red ) mice . Data information : Significance determined by unpaired t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Liver LDU (Leishman Donovan units) at day 28 in infected WT mice treated with anti-IL-10R antibody or isotype control antibody (left panel, n=5 mice per group), or at day 21 and day 28 from WT (blue), IL-10+/- (open green circles) and IL-10-/- (filled green circles) mice (right panel n= 3-6 mice per group) Data information: Significance determined by unpaired t-test * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.Day 28 splenic parasite burdens expressed as LDU with each data point representing an individual mouse in WT (blue) and miR-132-/- (miR-132-/-; red) mice. Data from 4 independent infection experiments.   Mean WT and miR-132-/- spleen parasite burdens from the 4 independent experiments shown in (B). Lines link individual experiments.   Splenic parasite burdens relative to WT group (WT mean = 1) for each of the 4 experiments shown in (B), with each data point representing individual mouse.   Data information: Significance determined by unpaired t-test, and in (C) by paired t-test of mean values. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.Spleen size expressed as % body weight for d0 (naïve) or day 28 L. donovani infected WT (blue) and miR-132-/- (miR-132-/-; red) mice.   Liver size expressed as % body weight for d0 (naïve) or day 28 L. donovani infected WT (blue) and miR-132-/- (red) mice.   Data information: Significance determined by unpaired t-test * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.


Figure 1Coq9Q95X mice at 21 postnatal days showing the loss of corporal hair . Representative Western blot images of COQ9protein in kidney homogenate from Coq9 + / + ( n = 4 ) and Coq9Q95Xmice ( n = 4 ) at 3 months of age . Antibody sc - 271892 was used to map the C - terminal region of the COQ9protein and antibody ab - 104189 was used to map the internal sequence of the COQ9protein . High - resolution LC - MS / MS proteomic analysis of kidneymitochondria from Coq9 + / + ( n = 3 ) and Coq9Q95Xmice ( n = 3 ) at 3 months of age . None of the six peptides of the COQ9 protein identified in Coq9 + / + mice was detected in Coq9Q95Xmice .

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Figure 1Coq9Q95X mice at 21 postnatal days showing the loss of corporal hair.Representative Western blot images of COQ9protein in kidney homogenate from Coq9+/+ (n = 4) and Coq9Q95Xmice (n = 4) at 3 months of age. Antibody sc-271892 was used to map the C-terminal region of the COQ9protein and antibody ab-104189 was used to map the internal sequence of the COQ9protein.High-resolution LC-MS/MS proteomic analysis of kidneymitochondria from Coq9+/+ (n = 3) and Coq9Q95Xmice (n = 3) at 3 months of age. None of the six peptides of the COQ9 protein identified in Coq9+/+mice was detected in Coq9Q95Xmice.


Figure 2A - F CoQ9 levels in tissue homogenates from brain ( A ) , cerebellum ( B ) , heart ( C ) , kidney ( D ) , extensor ( E ) and triceps surae ( F ) of male and female Coq9 + / + and Coq9Q95Xmice at 6 and 12 months of age . Data are expressed as mean ± SD . Statistical analysis was performed on 6 - month - old Coq9 + / + mice versus 6 - month - old Coq9Q95Xmice and 12 - month - old Coq9 + / + mice versus 12 - month - old Coq9Q95Xmice . * * P < 0 . 01 ; * * * P < 0 . 001 . Student ' s t - test ( n = 8 for each group ) .

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Figure 2A-F CoQ9 levels in tissue homogenates from brain (A), cerebellum (B), heart (C), kidney (D), extensor (E) and triceps surae (F) of male and female Coq9+/+ and Coq9Q95Xmice at 6 and 12 months of age. Data are expressed as mean ± SD. Statistical analysis was performed on 6-month-old Coq9+/+mice versus 6-month-old Coq9Q95Xmice and 12-month-old Coq9+/+mice versus 12-month-old Coq9Q95Xmice. **P < 0.01; ***P < 0.001. Student's t-test (n = 8 for each group).


Figure 3A - F Residual CoQ9 levels in tissue homogenates from brain ( A ) , cerebellum ( B ) , heart ( C ) , kidney ( D ) , liver ( E ) and skeletal muscle ( F ) of Coq9 + / + , Coq9Q95X and Coq9R239X mice at 1 , 3 and 5 months of age . Data are expressed as mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239Xmice versus Coq9 + / + mice . # P < 0 . 05 ; # # P < 0 . 01 ; # # # P < 0 . 001 ; Coq9Q95X versus Coq9R239Xmice ( one - way ANOVA with a Tukey ' s post hoc test ; n = 8 for each group ; numbers above columns indicate P - values of the one - way ANOVA test ) .

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Figure 3A-F Residual CoQ9 levels in tissue homogenates from brain (A), cerebellum (B), heart (C), kidney (D), liver (E) and skeletal muscle (F) of Coq9+/+, Coq9Q95X and Coq9R239X mice at 1, 3 and 5 months of age. Data are expressed as mean ± SD. **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. #P < 0.05; ##P < 0.01; ###P < 0.001; Coq9Q95X versus Coq9R239Xmice (one-way ANOVA with a Tukey's post hoc test; n = 8 for each group; numbers above columns indicate P-values of the one-way ANOVA test).


Figure 4A - E mRNA expression levels of Coq9 ( A ) , Coq7 ( B ) , Adck3 ( C ) , Coq5 ( D ) and Coq6 ( E ) on cerebrum of Coq9 + / + , Coq9Q95X and Coq9Q95X mice at 3 months of age . * * P < 0 . 01 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239Xmice versus Coq9 + / + mice . # # # P < 0 . 001 ; Coq9Q95X versus Coq9R239Xmice . F - J mRNA expression levels of Coq9 ( F ) , Coq7 ( G ) , Adck3 ( H ) , Coq5 ( I ) and Coq6 ( J ) on kidney of Coq9 + / + , Coq9Q95X and Coq9Q95Xmice at 3 months of age . * * * P < 0 . 001 ; Coq9Q95X and Coq9R239Xmice versus Coq9 + / + mice . # P < 0 . 05 ; # # # P < 0 . 001 ; Coq9Q95X versus Coq9R239Xmice . K - O mRNA expression levels of Coq9 ( K ) , Coq7 ( L ) , Adck3 ( M ) , Coq5 ( N ) and Coq6 ( O ) on triceps surae of Coq9 + / + , Coq9Q95X and Coq9Q95Xmice at 3 months of age . * P < 0 . 05 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239Xmice versus Coq9 + / + mice . # P < 0 . 05 ; Coq9Q95X versus Coq9R239Xmice .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-E mRNA expression levels of Coq9 (A), Coq7 (B), Adck3 (C), Coq5 (D) and Coq6 (E) on cerebrum of Coq9+/+, Coq9Q95X and Coq9Q95X mice at 3 months of age. **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. ###P < 0.001; Coq9Q95X versus Coq9R239Xmice.F-J mRNA expression levels of Coq9 (F), Coq7 (G), Adck3 (H), Coq5 (I) and Coq6 (J) on kidney of Coq9+/+, Coq9Q95X and Coq9Q95Xmice at 3 months of age. ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. #P < 0.05; ###P < 0.001; Coq9Q95X versus Coq9R239Xmice.K-O mRNA expression levels of Coq9 (K), Coq7 (L), Adck3 (M), Coq5 (N) and Coq6 (O) on triceps surae of Coq9+/+, Coq9Q95X and Coq9Q95Xmice at 3 months of age. *P < 0.05; ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. #P < 0.05; Coq9Q95X versus Coq9R239Xmice.


Figure 5A - D Representative Western blot and quantitation of Western blot bands of COQ7 ( A ) , ADCK3 ( B ) , COQ5 ( C ) and COQ6 ( D ) , and VDAC1 as a loading control in the kidneys of 3 - month - old mice . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239X mice versus Coq9 + / + mice . # # P < 0 . 01 ; # # # P < 0 . 001 ; Coq9Q95X versus Coq9R239X mice . One - way ANOVA with a Tukey ' s post hoc test . E - H Representative Western blot and quantitation of Western blot bands of COQ7 ( E ) , ADCK3 ( F ) , COQ5 ( G ) and COQ6 ( H ) , and VDAC1 as a loading control in skeletal muscle of 3 - month - old mice . * * P < 0 . 01 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239X mice versus Coq9 + / + mice . # P < 0 . 05 ; # # P < 0 . 01 ; Coq9Q95X versus Coq9R239X mice . Data information : All values are presented as mean ± SD . One - way ANOVA with a Tukey ' s post hoc test . Numbers above columns indicate P - values of the one - way ANOVA test . Coq9 + / + mice n = 4 ; Coq9Q95X and Coq9R239Xmice n = 5 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-D Representative Western blot and quantitation of Western blot bands of COQ7 (A), ADCK3 (B), COQ5 (C) and COQ6 (D), and VDAC1 as a loading control in the kidneys of 3-month-old mice. *P < 0.05; **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239X mice versus Coq9+/+mice. ##P < 0.01; ###P < 0.001; Coq9Q95X versus Coq9R239X mice. One-way ANOVA with a Tukey's post hoc test.E-H Representative Western blot and quantitation of Western blot bands of COQ7 (E), ADCK3 (F), COQ5 (G) and COQ6 (H), and VDAC1 as a loading control in skeletal muscle of 3-month-old mice. **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239X mice versus Coq9+/+mice. #P < 0.05; ##P < 0.01; Coq9Q95X versus Coq9R239X mice.Data information: All values are presented as mean ± SD. One-way ANOVA with a Tukey's post hoc test. Numbers above columns indicate P-values of the one-way ANOVA test. Coq9+/+mice n = 4; Coq9Q95X and Coq9R239Xmice n = 5.


Figure 6A - C Mitochondrial CoQ9 levels from cerebrum ( A ) , kidney ( B ) and skeletal muscle ( C ) of Coq9 + / + and Coq9Q95X males and females . n = 8 for each group . D - F CI + CIII activity in cerebrum ( D ) , kidney ( E ) and skeletal muscle ( F ) of male and female Coq9 + / + and Coq9Q95X mice . n = 6 for each group . G - I CII + CIII activity in cerebrum ( G ) , kidney ( H ) and skeletal muscle ( I ) of male and female Coq9 + / + and Coq9Q95X mice . n = 6 for each group . J - L Blue - native gel electrophoresis ( BNGE ) followed by immunoblotting analysis of mitochondrial supercomplexes from Coq9 + / + ( n = 3 ) and Coq9Q95X mice ( n = 4 ) at 3 months of age .

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Figure 6A-C Mitochondrial CoQ9 levels from cerebrum (A), kidney (B) and skeletal muscle (C) of Coq9+/+ and Coq9Q95X males and females. n = 8 for each group.D-F CI+CIII activity in cerebrum (D), kidney (E) and skeletal muscle (F) of male and female Coq9+/+ and Coq9Q95X mice. n = 6 for each group.G-I CII+CIII activity in cerebrum (G), kidney (H) and skeletal muscle (I) of male and female Coq9+/+ and Coq9Q95X mice. n = 6 for each group.J-L Blue-native gel electrophoresis (BNGE) followed by immunoblotting analysis of mitochondrial supercomplexes from Coq9+/+ (n = 3) and Coq9Q95X mice (n = 4) at 3 months of age.


Figure 7A - D Measurement of phosphorylating respiration ( represented as State 3o , in the presence of ADP and substrates ) in kidney ( A ) and skeletal muscle ( C ) from male and female Coq9 + / + , Coq9Q95X and Coq9R239X mice at 3 months of age . Representative O2 consumption graphic in kidney ( B ) and skeletal muscle ( D ) from female Coq9 + / + , Coq9Q95X and Coq9R239X mice . Data information : All values are presented as mean ± SD . ( A , C ) * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239Xmice versus Coq9 + / + mice . # P < 0 . 05 ; Coq9Q95X versus Coq9R239Xmice . One - way ANOVA with a Tukey ' s post hoc test . Numbers above columns indicate P - values of the one - way ANOVA test ( n = 3 for each group ) .

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Figure 7A-D Measurement of phosphorylating respiration (represented as State 3o, in the presence of ADP and substrates) in kidney (A) and skeletal muscle (C) from male and female Coq9+/+, Coq9Q95X and Coq9R239X mice at 3 months of age. Representative O2 consumption graphic in kidney (B) and skeletal muscle (D) from female Coq9+/+, Coq9Q95X and Coq9R239X mice.Data information: All values are presented as mean ± SD. (A, C) *P < 0.05; **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. #P < 0.05; Coq9Q95X versus Coq9R239Xmice. One-way ANOVA with a Tukey's post hoc test. Numbers above columns indicate P-values of the one-way ANOVA test (n = 3 for each group).


Figure 8A - H Complex II ( SDH ) and complex IV ( COX ) histochemistry of triceps surae showing a decreased stain in 18 - month - old Coq9Q95X female mice ( D , H ) in contrast to normal SDH and COX activity in 6 - and 18 - month - old Coq9 + / + ( A , C , E , G ) , as well as 6 - month - old Coq9Q95X female mice ( B , F ) . I - L Gomori trichrome stain ( TGM ) of triceps surae showed no differences between 6 - and 18 - month - old Coq9 + / + and Coq9Q95X female mice . M - P Hematoxylin and eosin ( H & amp ; amp ; E ) stains of triceps surae did not reveal any structural abnormality . Data information : Scale bars : 100 μm . n = 3 for each group . Complex IV , cytochrome c oxidase ( COX ) ; complex II , succinate dehydrogenase ( SDH ) .

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Figure 8A-H Complex II (SDH) and complex IV (COX) histochemistry of triceps surae showing a decreased stain in 18-month-old Coq9Q95X female mice (D, H) in contrast to normal SDH and COX activity in 6- and 18-month-old Coq9+/+ (A, C, E, G), as well as 6-month-old Coq9Q95X female mice (B, F).I-L Gomori trichrome stain (TGM) of triceps surae showed no differences between 6- and 18-month-old Coq9+/+ and Coq9Q95X female mice.M-P Hematoxylin and eosin (H&amp;E) stains of triceps surae did not reveal any structural abnormality.Data information: Scale bars: 100 μm. n = 3 for each group. Complex IV, cytochrome c oxidase (COX); complex II, succinate dehydrogenase (SDH).


Figure 9A - C Voluntary wheel running test . Distance traveled on the wheel and average speed during the use of the wheel were decreased in female Coq9Q95X mice at 6 months of age ( B , C ) . D Hanging wire test . Coq9Q95X female mice obtained less reaches score in the ' fall and reaches ' method . E Open - field test . Coq9Q95X mice showed a reduction in the average distance traveled in Coq9Q95X female mice at 6 months of age . F Grip test : Muscle strength was not affected in Coq9Q95X mice at 6 months of age .

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Figure 9A-C Voluntary wheel running test. Distance traveled on the wheel and average speed during the use of the wheel were decreased in female Coq9Q95X mice at 6 months of age (B, C).D Hanging wire test. Coq9Q95X female mice obtained less reaches score in the 'fall and reaches' method.E Open-field test. Coq9Q95X mice showed a reduction in the average distance traveled in Coq9Q95X female mice at 6 months of age.F Grip test: Muscle strength was not affected in Coq9Q95X mice at 6 months of age.


Figure 10A , B KidneyCoQ9 levels in Coq9 + / + , Coq9Q95X and Coq9R239Xmice treated with 2 , 4 - diHB ( + 2 , 4 - diHB ) compared with the non - treated littermate ( vehicle ) . Statistical analysis was performed on + 2 , 4 - diHBCoq9 + / + , Coq9Q95X and Coq9R239Xmice versus vehicle Coq9 + / + , Coq9Q95X and Coq9R239Xmice , respectively ( n = 3 for each group ) . C , D CoQ10 levels in COQ9R244X skinfibroblasts treated with 2 , 4 - diHB ( + 2 , 4 - diHB , blue bars ) compared with the non - treated controls ( vehicle , yellow bars ) . Statistical analysis was performed on + 2 , 4 - diHBCOQ9R244X versus vehicle COQ9R244X ( n = 4 for each group ) .

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Figure 10A, B KidneyCoQ9 levels in Coq9+/+, Coq9Q95X and Coq9R239Xmice treated with 2,4-diHB (+2,4-diHB) compared with the non-treated littermate (vehicle). Statistical analysis was performed on +2,4-diHBCoq9+/+, Coq9Q95X and Coq9R239Xmice versus vehicle Coq9+/+, Coq9Q95X and Coq9R239Xmice, respectively (n = 3 for each group).C, D CoQ10 levels in COQ9R244X skinfibroblasts treated with 2,4-diHB (+2,4-diHB, blue bars) compared with the non-treated controls (vehicle, yellow bars). Statistical analysis was performed on +2,4-diHBCOQ9R244X versus vehicle COQ9R244X (n = 4 for each group).


figf1 ( a ) Immunoblot for the indicated Cx of homogenates ( Hom ) and autophagic vacuoles ( AV ) isolated from fed or 6 h starved ( Stv ) mice . Quantification of enrichment is shown in Supplementary Fig . 1a . ( b ) Immunofluorescence for LC3 and Atg16 in normal rat kidney ( NRK ) cells expressing GFP - Cx43 maintained in the presence or absence of serum for 4 h . Single channels are shown in reverse black and white , and magnification of single and merged images are shown in colour . Arrows : co - localization of Atg16 / Cx43 ( yellow ) or Atg16 / Cx43 / LC3 ( white ) . Quantification is shown in Supplementary Fig . 1c . ( c ) Immunofluorescence for the indicated endogenous proteins in NRK cells maintained in serum - supplemented media without additions ( None ) or treated with chloroquine ( CQ ) for 4 h . Full fields are shown in Supplementary Fig . 1d . ( d ) Maximum projection confocal microscopy 3D reconstruction ( left ) followed by a single - plane time - lapse acquisition of the region outlined by the white square in HeLa cells expressing EBFP2 - Cx43 ( pseudocoloured in red ) and sfGFP - ATG16 . ( e ) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP - Cx43 but knocked down for Eps15 . Single channels are shown in reverse black and white , and merged images in colour . Inset shows higher magnification . Bottom : Quantification of co - localization between Atg16 and Cx43 ( n = 3 wells , 4 independent experiments , > 20 cells per experiment ) . Values are mean + s . e . m . * P 0 . 05 . ( f , g ) Immunoblot for the indicated proteins of immunoprecipitates ( IP ) of endogenous Cx43 and Atg16 from WT MEFs maintained in the presence ( f , g ) or absence of serum ( 4 h ; g ) . I : input ; FT : flow through . ( h ) Immunoblot for the indicated proteins of immunoprecipitates ( IP ) of endogenous Cx43 from untreated WT MEFs , on incubation with 3 - methyladenine ( 3MA ) or in MEFs from Atg5 - null mice ( Atg5 − / − ) . ( i ) Immunoblot for Flag and Cx43 of immunoprecipitates ( IP ) of endogenous Cx43 in WT MEFs transfected with the indicated Flag - tagged constructs of truncated Atg16 .

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figf1(a) Immunoblot for the indicated Cx of homogenates (Hom) and autophagic vacuoles (AV) isolated from fed or 6 h starved (Stv) mice. Quantification of enrichment is shown in Supplementary Fig. 1a.(b) Immunofluorescence for LC3 and Atg16 in normal rat kidney (NRK) cells expressing GFP-Cx43 maintained in the presence or absence of serum for 4 h. Single channels are shown in reverse black and white, and magnification of single and merged images are shown in colour. Arrows: co-localization of Atg16/Cx43 (yellow) or Atg16/Cx43/LC3 (white). Quantification is shown in Supplementary Fig. 1c.(c) Immunofluorescence for the indicated endogenous proteins in NRK cells maintained in serum-supplemented media without additions (None) or treated with chloroquine (CQ) for 4 h. Full fields are shown in Supplementary Fig. 1d.(d) Maximum projection confocal microscopy 3D reconstruction (left) followed by a single-plane time-lapse acquisition of the region outlined by the white square in HeLa cells expressing EBFP2-Cx43 (pseudocoloured in red) and sfGFP-ATG16.(e) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP-Cx43 but knocked down for Eps15. Single channels are shown in reverse black and white, and merged images in colour. Inset shows higher magnification. Bottom: Quantification of co-localization between Atg16 and Cx43 (n = 3 wells, 4 independent experiments, >20 cells per experiment). Values are mean + s.e.m. * P 0.05.(f,g) Immunoblot for the indicated proteins of immunoprecipitates (IP) of endogenous Cx43 and Atg16 from WT MEFs maintained in the presence (f,g) or absence of serum (4 h; g). I: input; FT: flow through.(h) Immunoblot for the indicated proteins of immunoprecipitates (IP) of endogenous Cx43 from untreated WT MEFs, on incubation with 3-methyladenine (3MA) or in MEFs from Atg5-null mice (Atg5−/−).(i) Immunoblot for Flag and Cx43 of immunoprecipitates (IP) of endogenous Cx43 in WT MEFs transfected with the indicated Flag-tagged constructs of truncated Atg16.


figf2 ( a ) Immunostaining for LC3 and GABARAP in MOBs from WT or Cx43 - null mice ( Cx43 − / − ) grown in serum - supplemented media . Reverse black and white channels ( top ) and colour images ( bottom ) . Right : Average number of LC3 and GABARAP puncta per cell ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) . ( b ) Electron micrographs of WT and Cx43 − / − MOBs grown in serum - supplemented media . Yellow arrows : autophagic vacuoles . Insets : autophagic vacuoles ( AV ) at higher magnification . Right : Percentage of cytoplasm area occupied by AV ( top ) and number of AV per area ( bottom ; n = 3 mice , > 10 micrographs per mice ) . ( c ) Immunoblot for LC3 of the same cells grown in serum - supplemented media untreated or treated with lysosomal protease inhibitors ( PI ) for 2 or 4 h . Top : representative immunoblot . GAPDH is shown as loading control . Bottom : LC3 flux ( left ) and increase in LC3 - II during lysosomal inhibition ( right ; n = 3 independent experiments ) . ( d ) Representative images of the same cells transfected with the mCherry - GFP - LC3 tandem reporter and grown in serum - supplemented media . Single - channel images ( inverted black and white ) and merged images ( colour ) . Bottom : Number of vesicles positive for GFP and mCherry ( autophagosomes ; APG ) and only for mCherry ( autophagolysosomes ; APL ; n = 4 wells , 4 independent experiments , > 30 cells per experiment ) . ( e ) Electron micrographs of livers from WT and Cx43 + / − mice . Arrows indicate AV . Right : Percentage of cytosolic area occupied by AVs ( n = 3 mice , > 10 micrographs per mice ) . ( f ) Immunofluorescence for LC3 in Cx43 − / − MOBs expressing GFP - tagged full - length Cx43 . Single channels ( inverted black and white ) and merged channels ( colour ) . Asterisks : transfected ( green ) and untransfected ( red ) cells . ( g ) LC3 - positive puncta per cell in the indicated cells ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) . All values are mean + s . e . m . and differences from WT were significant for * P 0 . 01 . Nuclei are highlighted with DAPI . Scale bars : 5 μm in fluorescence images ; 1 μm and 0 . 1 μm in top panels and insets of b and e . Uncropped images of blots are shown in Supplementary Fig . 9 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(a) Immunostaining for LC3 and GABARAP in MOBs from WT or Cx43-null mice (Cx43−/−) grown in serum-supplemented media. Reverse black and white channels (top) and colour images (bottom). Right: Average number of LC3 and GABARAP puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment).(b) Electron micrographs of WT and Cx43−/− MOBs grown in serum-supplemented media. Yellow arrows: autophagic vacuoles. Insets: autophagic vacuoles (AV) at higher magnification. Right: Percentage of cytoplasm area occupied by AV (top) and number of AV per area (bottom; n = 3 mice, >10 micrographs per mice).(c) Immunoblot for LC3 of the same cells grown in serum-supplemented media untreated or treated with lysosomal protease inhibitors (PI) for 2 or 4 h. Top: representative immunoblot. GAPDH is shown as loading control. Bottom: LC3 flux (left) and increase in LC3-II during lysosomal inhibition (right; n = 3 independent experiments).(d) Representative images of the same cells transfected with the mCherry-GFP-LC3 tandem reporter and grown in serum-supplemented media. Single-channel images (inverted black and white) and merged images (colour). Bottom: Number of vesicles positive for GFP and mCherry (autophagosomes; APG) and only for mCherry (autophagolysosomes; APL; n = 4 wells, 4 independent experiments, >30 cells per experiment).(e) Electron micrographs of livers from WT and Cx43+/− mice. Arrows indicate AV. Right: Percentage of cytosolic area occupied by AVs (n = 3 mice, >10 micrographs per mice).(f) Immunofluorescence for LC3 in Cx43−/− MOBs expressing GFP-tagged full-length Cx43. Single channels (inverted black and white) and merged channels (colour). Asterisks: transfected (green) and untransfected (red) cells. (g) LC3-positive puncta per cell in the indicated cells (n = 3 wells, 3 independent experiments, >50 cells per experiment). All values are mean + s.e.m. and differences from WT were significant for *P 0.01. Nuclei are highlighted with DAPI. Scale bars: 5 μm in fluorescence images; 1 μm and 0.1 μm in top panels and insets of b and e. Uncropped images of blots are shown in Supplementary Fig. 9.


figf3 ( a ) Immunoblot for LC3 in MOBs from WT or Cx43 - null mice ( Cx43 − / − ) treated with vehicle or with 18α glycyrrhetinic acid ( 18α GA ) . Glyceraldehyde - 3 - dehydrogenae ( GPD ) is shown as the loading control . Levels of LC3 - II ( left ) and LC3 - II flux ( right ; n = 3 independent experiments ) . ( b ) Immunofluorescence for LC3 in MOBs treated with vehicle or 18α glycyrrhetinic acid . Where indicated , chloroquine ( CQ ) was added to block lysosomal degradation . Bottom : Quantification of the average number of LC3 - positive puncta per cell ( n = 3 wells , 3 independent experiments , > 30 cells per experiment ) . ( c ) Immunofluorescence for LC3 in WT or Cx43 − / − MOBs treated with the indicated kinase inhibitors for 2 h . Insets : Higher magnification images in inverted black and white . ( d ) Endogenous LC3 puncta in the same cells shown in c ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) . * , differences from WT ; § , differences from untreated . ( e ) LC3 puncta in Cx43 − / − MOBs transfected with constructs coding for different forms of Cx43 ( full length , CT , ΔCT258 or ΔCT245 ) tagged as indicated in the scheme . Left : Representative images . Merged fields ( left ) and LC3 channel in reverse black and white ( right ) are shown ( images of GFP - Cx43 are shown in Fig . 2f ) . Right : Quantification of the average number of LC3 puncta per cell ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) . ( f , g ) Immunoblot for the indicated proteins of the immunoprecipitates ( IP ) of Atg16 from MEFs transfected with GFP - tagged full - length ( FL - ) Cx43 or Flag - tagged C terminus ( CT - ) Cx43 ( f ) or ΔCT258 - Cx43 ( g ) I : input ; FT : flow through . GAPDH and GFP alone were used as negative controls for immunoprecipitation and Atg5 as the positive control . All values are mean + s . e . m . Differences from the control were significant for ( * , § ) P 0 . 01 and ( * * , § § ) P 0 . 001 . Scale bars : 5 μm . Uncropped images of blots are shown in Supplementary Fig . 9 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(a) Immunoblot for LC3 in MOBs from WT or Cx43-null mice (Cx43−/−) treated with vehicle or with 18α glycyrrhetinic acid (18α GA). Glyceraldehyde-3-dehydrogenae (GPD) is shown as the loading control. Levels of LC3-II (left) and LC3-II flux (right; n = 3 independent experiments).(b) Immunofluorescence for LC3 in MOBs treated with vehicle or 18α glycyrrhetinic acid. Where indicated, chloroquine (CQ) was added to block lysosomal degradation. Bottom: Quantification of the average number of LC3-positive puncta per cell (n = 3 wells, 3 independent experiments, >30 cells per experiment).(c) Immunofluorescence for LC3 in WT or Cx43−/− MOBs treated with the indicated kinase inhibitors for 2 h. Insets: Higher magnification images in inverted black and white. (d) Endogenous LC3 puncta in the same cells shown in c (n = 3 wells, 3 independent experiments, >50 cells per experiment). *, differences from WT; §, differences from untreated.(e) LC3 puncta in Cx43−/− MOBs transfected with constructs coding for different forms of Cx43 (full length, CT, ΔCT258 or ΔCT245) tagged as indicated in the scheme. Left: Representative images. Merged fields (left) and LC3 channel in reverse black and white (right) are shown (images of GFP-Cx43 are shown in Fig. 2f). Right: Quantification of the average number of LC3 puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment).(f,g) Immunoblot for the indicated proteins of the immunoprecipitates (IP) of Atg16 from MEFs transfected with GFP-tagged full-length (FL-) Cx43 or Flag-tagged C terminus (CT-) Cx43 (f) or ΔCT258-Cx43 (g) I: input; FT: flow through. GAPDH and GFP alone were used as negative controls for immunoprecipitation and Atg5 as the positive control. All values are mean + s.e.m. Differences from the control were significant for (*, §)P 0.01 and (**, §§)P 0.001. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.


figf4 ( a ) Immunoblots for the indicated proteins in MEFs treated with tamoxifen ( Tx ) or lindane ( Lind ) in the absence or presence of lysosomal protease inhibitors ( PI ) . Right : Changes in LC3 - II levels relative to those detected in untreated cells ( n = 4 independent experiments ) . ( b ) Immunofluorescence for Cx43 and LC3 in NRK cells treated as in a in the presence or absence of chloroquine ( CQ ) for 3 h . Right : Quantification of the average number of LC3 - positive puncta per cell ( n = 3 wells , 3 independent experiments , > 30 cells per experiment ) . ( c ) Time course of changes in the average LC3 - positive puncta per cell after addition of lindane to control NRK cells . ( d ) Fluorescence images of WT or Cx43 − / − MOBs transfected with mCherry - GFP - LC3 and incubated or not with tamoxifen or lindane . Bottom : Quantification of autophagolysosomes ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) . ( e ) Immunostaining for Atg16 and Cx43 in MEFs treated with the indicated compounds . Representative images as merged channels are shown . Right : quantification of the average of cytosolic Atg16 - positive puncta per cell ( n = 3 wells , 3 independent experiments , > 30 cells per experiment ) . ( f ) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP - Cx43 treated with lindane and untreated . Single channel images in inverse black and white and merged colour images are shown . Outlined areas are shown at higher magnification . Arrows : double ( yellow ) and triple ( white ) co - localization . ( g , h ) Immunostaining for Atg16 in Cx43 knocked down ( KD ) ( g ) or knocked out ( − / − ) ( h ) cells . Right : quantification of the average of cytosolic Atg16 positive puncta per cell ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) . ( i ) Immunoblot for Atg16 and Atg14 in immunoprecipitates ( IP ) of endogenous Cx43 from WT MEFs maintained in the absence of serum ( 4 h ) or incubated in the presence of tamoxifen or lindane . I : input ; FT : flow through . All values are mean + s . e . m and differences from untreated cells are significant for * P 0 . 01 . Scale bars : 5 μm . Uncropped images of blots are shown in Supplementary Fig . 9 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(a) Immunoblots for the indicated proteins in MEFs treated with tamoxifen (Tx) or lindane (Lind) in the absence or presence of lysosomal protease inhibitors (PI). Right: Changes in LC3-II levels relative to those detected in untreated cells (n = 4 independent experiments).(b) Immunofluorescence for Cx43 and LC3 in NRK cells treated as in a in the presence or absence of chloroquine (CQ) for 3 h. Right: Quantification of the average number of LC3-positive puncta per cell (n = 3 wells, 3 independent experiments, >30 cells per experiment). (c) Time course of changes in the average LC3-positive puncta per cell after addition of lindane to control NRK cells.(d) Fluorescence images of WT or Cx43−/− MOBs transfected with mCherry-GFP-LC3 and incubated or not with tamoxifen or lindane. Bottom: Quantification of autophagolysosomes (n = 3 wells, 3 independent experiments, >50 cells per experiment).(e) Immunostaining for Atg16 and Cx43 in MEFs treated with the indicated compounds. Representative images as merged channels are shown. Right: quantification of the average of cytosolic Atg16-positive puncta per cell (n = 3 wells, 3 independent experiments, >30 cells per experiment).(f) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP-Cx43 treated with lindane and untreated. Single channel images in inverse black and white and merged colour images are shown. Outlined areas are shown at higher magnification. Arrows: double (yellow) and triple (white) co-localization.(g,h) Immunostaining for Atg16 in Cx43 knocked down (KD) (g) or knocked out (−/−) (h) cells. Right: quantification of the average of cytosolic Atg16 positive puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment).(i) Immunoblot for Atg16 and Atg14 in immunoprecipitates (IP) of endogenous Cx43 from WT MEFs maintained in the absence of serum (4 h) or incubated in the presence of tamoxifen or lindane. I: input; FT: flow through. All values are mean + s.e.m and differences from untreated cells are significant for *P 0.01. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.


figf5 ( a - c ) Immunofluorescence for the indicated endosomal markers in NRK cells expressing GFP - Cx43 maintained in the presence of serum ( a ) , absence of serum ( b ) or treated with lindane ( c ) . Single black and white inverted channels , merged channels and higher magnification insets are shown . ( d ) Immunoblots for indicated proteins in isolated fractions from starved mouse liver . Hom , homogenate ; LE , late endosomes ; Cyt , cytosol ; APG , autophagosomes ; APL , autophagolysosomes . ( e ) Immunoblots for different Cx in homogenates and late endosomes isolated from fed and 6 h starved mice . ( f - h ) Fluorescence images of NRK cells expressing GFP - Cx43 maintained in the presence of serum ( f ) , absence of serum ( g ) or treated with lindane ( h ) and incubated with Alexa594 - transferrin for 15 min before fixation . Top : single and merged channels and outlined area at higher magnification . Bottom : 3D reconstructions and higher magnification details of the outlined areas . Nuclei are highlighted with DAPI . Scale bars : 5 μm . Uncropped images of blots are shown in Supplementary Fig . 9 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a-c) Immunofluorescence for the indicated endosomal markers in NRK cells expressing GFP-Cx43 maintained in the presence of serum (a), absence of serum (b) or treated with lindane (c). Single black and white inverted channels, merged channels and higher magnification insets are shown.(d) Immunoblots for indicated proteins in isolated fractions from starved mouse liver. Hom, homogenate; LE, late endosomes; Cyt, cytosol; APG, autophagosomes; APL, autophagolysosomes.(e) Immunoblots for different Cx in homogenates and late endosomes isolated from fed and 6 h starved mice.(f-h) Fluorescence images of NRK cells expressing GFP-Cx43 maintained in the presence of serum (f), absence of serum (g) or treated with lindane (h) and incubated with Alexa594-transferrin for 15 min before fixation. Top: single and merged channels and outlined area at higher magnification. Bottom: 3D reconstructions and higher magnification details of the outlined areas. Nuclei are highlighted with DAPI. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.


figf6 ( a ) Immunoblot for the indicated proteins in homogenates ( Hom ) and purified PM from fed ( F ) and 24 h starved mice ( Stv ) . Starvation - induced changes in the PM content relative to fed levels were as follows : Vps15 ( 1 . 2 ± 0 . 2 ) , Beclin - 1 ( 0 . 7 ± 0 . 5 ) , Vps34 ( 0 . 9 ± 0 . 1 ) , Atg16 ( 1 . 1 ± 0 . 3 ) , Atg14 ( 2 . 5 ± 0 . 2 ) and only Atg14 were significant for P 0 . 01 . ( b ) Immunoblot of Cx43immunoprecipitates ( IP ) in MEFs maintained in the presence or absence of serum . I , input ; FT , flow through . ( c ) Immunoblot of Cx43 IP in MEFs from WT or Atg5 − / − mice grown in serum - supplemented media . ( d ) Immunoblot of Cx43 IP in WT or Atg16 knockdown ( KD ) MEFs maintained in the presence / absence of serum for 4 h . ( e ) Co - staining for Atg14 and Cx43 in NRK cells maintained in the presence / absence of serum . Merged channels are shown and single channels and broad - field images are shown in Supplementary Fig . 5d . Insets : higher magnification . ( f ) Maximum - intensity confocal microscopy 3D reconstruction in HeLa cells expressing EBFP2 - Cx43 ( pseudocoloured in red ) and EGFP - ATG14 followed by a single - plane time - lapse acquisition of the outlined area ; cells were incubated in serum - deprived medium for 1 h before imaging . ( g ) Immunoblot of Atg14 IP in WT or Atg5 − / − MEFs maintained in the presence of serum . ( h ) Immunoblot in total homogenates and PM purified from WT or Cx43 − / − MOBs . ( i ) Immunofluorescence for e - cadherin and Vps34 in NRK cells WT or knocked down ( KD ) for Cx43 . Single channel and merge ( left ) and z - stack surface reconstruction and higher magnification of the outlined region ( right ) . ( j ) Immunostaining for Cx43 and e - cadherin in NRK cells expressing a tandem FYVE domain ( 2xFYVE ) tagged to GFP and maintained in serum - free media for 4 h . Single channels ( inverted black and white ) and the merged image of the outlined area ( colour ) . Inset : higher magnification image . Scale bars : 5 μm . Uncropped images of blots are shown in Supplementary Fig . 9 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6(a) Immunoblot for the indicated proteins in homogenates (Hom) and purified PM from fed (F) and 24 h starved mice (Stv). Starvation-induced changes in the PM content relative to fed levels were as follows: Vps15 (1.2 ± 0.2), Beclin-1 (0.7 ± 0.5), Vps34 (0.9 ± 0.1), Atg16 (1.1 ± 0.3), Atg14 (2.5 ± 0.2) and only Atg14 were significant for P 0.01.(b) Immunoblot of Cx43immunoprecipitates (IP) in MEFs maintained in the presence or absence of serum. I, input; FT, flow through.(c) Immunoblot of Cx43 IP in MEFs from WT or Atg5−/− mice grown in serum-supplemented media.(d) Immunoblot of Cx43 IP in WT or Atg16 knockdown (KD) MEFs maintained in the presence/absence of serum for 4 h.(e) Co-staining for Atg14 and Cx43 in NRK cells maintained in the presence/absence of serum. Merged channels are shown and single channels and broad-field images are shown in Supplementary Fig. 5d. Insets: higher magnification.(f) Maximum-intensity confocal microscopy 3D reconstruction in HeLa cells expressing EBFP2-Cx43 (pseudocoloured in red) and EGFP-ATG14 followed by a single-plane time-lapse acquisition of the outlined area; cells were incubated in serum-deprived medium for 1 h before imaging.(g) Immunoblot of Atg14 IP in WT or Atg5−/− MEFs maintained in the presence of serum.(h) Immunoblot in total homogenates and PM purified from WT or Cx43−/− MOBs.(i) Immunofluorescence for e-cadherin and Vps34 in NRK cells WT or knocked down (KD) for Cx43. Single channel and merge (left) and z-stack surface reconstruction and higher magnification of the outlined region (right).(j) Immunostaining for Cx43 and e-cadherin in NRK cells expressing a tandem FYVE domain (2xFYVE) tagged to GFP and maintained in serum-free media for 4 h. Single channels (inverted black and white) and the merged image of the outlined area (colour). Inset: higher magnification image. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.


figf7 ( a ) Co - immunostaining for Cx43 and the indicated Atgs in NRK cells maintained in the presence or absence of serum ( top ) . Single and merged channels of the 3D reconstruction of the outlined regions are shown ( bottom ) . Nuclei are highlighted with DAPI . ( b , c ) Immunoblot for the indicated proteins of immunoprecipitates ( IP ) of Cx43 in NRK cells maintained in the presence or absence of serum ( b ) or treated or not with 3 - methyladenine ( c ) . Atg5 is shown as the negative control for immunoprecipitation . ( d ) Co - staining for Atg9 and e - cadherin in control NRK cells and those knocked down for Cx43 . Single and merged channels at higher magnification areas are shown . ( e ) Immunofluorescence for endogenous Atg9 and Cx43 in control NRK cells and those knocked down for Eps15 . ( f , g ) Immunofluorescence for endogenous Cx43 ( green ) in control NRK cells or those knocked down for Atg14 ( f ) maintained in the presence or absence of serum . ( f , g ) Immunofluorescence for endogenous Cx43 ( green ) in control NRK cells or those knocked down for Atg9 ( g ) maintained in the presence or absence of serum . E - cadherin staining ( red ) is shown to highlight PM in g and h . ( h ) Immunofluorescence of endogenous Cx43 in control NRK cells or those knocked down for Atg14 , treated ( or not ) with tamoxifen or lindane ( left ) . E - cadherin staining ( red ) is shown to highlight PM in g and h . Full fields are shown in Supplementary Fig . 6h . Quantification of intracytoplasmic Cx43 - positives vesicles ( right ; n = 3 wells , 3 independent experiments , > 30 cells per experiment ) . Values are mean + s . e . m and significant for * P 0 . 01 and * * P 0 . 001 using analysis of variance + Bonferroni test . Scale bars : 5 μm . Uncropped images of blots are shown in Supplementary Fig . 9 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf7(a) Co-immunostaining for Cx43 and the indicated Atgs in NRK cells maintained in the presence or absence of serum (top). Single and merged channels of the 3D reconstruction of the outlined regions are shown (bottom). Nuclei are highlighted with DAPI.(b,c) Immunoblot for the indicated proteins of immunoprecipitates (IP) of Cx43 in NRK cells maintained in the presence or absence of serum (b) or treated or not with 3-methyladenine (c). Atg5 is shown as the negative control for immunoprecipitation.(d) Co-staining for Atg9 and e-cadherin in control NRK cells and those knocked down for Cx43. Single and merged channels at higher magnification areas are shown.(e) Immunofluorescence for endogenous Atg9 and Cx43 in control NRK cells and those knocked down for Eps15.(f,g) Immunofluorescence for endogenous Cx43 (green) in control NRK cells or those knocked down for Atg14 (f) maintained in the presence or absence of serum.(f,g) Immunofluorescence for endogenous Cx43 (green) in control NRK cells or those knocked down for Atg9 (g) maintained in the presence or absence of serum. E-cadherin staining (red) is shown to highlight PM in g and h.(h) Immunofluorescence of endogenous Cx43 in control NRK cells or those knocked down for Atg14, treated (or not) with tamoxifen or lindane (left). E-cadherin staining (red) is shown to highlight PM in g and h. Full fields are shown in Supplementary Fig. 6h. Quantification of intracytoplasmic Cx43-positives vesicles (right; n = 3 wells, 3 independent experiments, >30 cells per experiment). Values are mean + s.e.m and significant for *P 0.01 and **P 0.001 using analysis of variance + Bonferroni test. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.