( C ) Immunoblot analysis of sTREM2 in conditioned media from BMDM upon treatment with 4D9 and isotype antibodies . BMDM from TREM2 knockout mice were included to show the specificity of the anti - TREM2 antibody . Soluble APP served as a loading control . Note that heavy and light chains of the antibodies used for treatment are also detected and annotated as follows : Ab hc = antibody heavy chain ; Ab lc = antibody light chain . Levels of sTREM2 were quantified by MSD ELISA . TREM2 antibody clone 5F4 was used for detection . Data represent the mean ± SEM ( n = 6 ) . Unpaired t - test ( two - tailed ) with Welch ' s correction ; p < 0 . 0001 . No gender - specific differences could be observed .

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(C) Immunoblot analysis of sTREM2 in conditioned media from BMDM upon treatment with 4D9 and isotype antibodies. BMDM from TREM2 knockout mice were included to show the specificity of the anti-TREM2 antibody. Soluble APP served as a loading control. Note that heavy and light chains of the antibodies used for treatment are also detected and annotated as follows: Ab hc= antibody heavy chain; Ab lc= antibody light chain. Levels of sTREM2 were quantified by MSD ELISA. TREM2 antibody clone 5F4 was used for detection. Data represent the mean ± SEM (n=6). Unpaired t-test (two-tailed) with Welch's correction; p<0.0001. No gender-specific differences could be observed.


( D ) Wildtype , Trem2 + / - , and Trem2 - / - BMDMs were plated in low concentration of M - CSF on 4D9 or isotype coated wells for 5 days and cellular ATP levels were measured by luminescence detection to indicate cell viability . 4D9 and isotype data for each genotype represent the mean ± SEM ( n = 2 and n = 1 , respectively ) .

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(D) Wildtype, Trem2+/-, and Trem2-/- BMDMs were plated in low concentration of M-CSF on 4D9 or isotype coated wells for 5 days and cellular ATP levels were measured by luminescence detection to indicate cell viability. 4D9 and isotype data for each genotype represent the mean ± SEM (n=2 and n=1, respectively).


( A ) Flow cytometry detection of cell surface 4D9 ( red ) binding and isotype control ( blue ) on primary wild type and Trem2 - / - mouse microglia . Data are shown as MFI ( mean fluorescent intensity ) .

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(A) Flow cytometry detection of cell surface 4D9 (red) binding and isotype control (blue) on primary wild type and Trem2-/- mouse microglia. Data are shown as MFI (mean fluorescent intensity).


( B ) Uptake assay for fluorescently labelled myelin , Aβ ( 1 - 42 ) and inactivated E . coli particles . The top row shows that myelin and Aβ ( pseudocoloured in white ) accumulate within the plasma membrane of primary microglia ( labeled with dylight - 649 isolectin and pseudocoloured in red ) . Overnight pre - treatment with antibody 4D9 significantly increased the percentage of substrate positive cells ( middle row ) . Pre - treatment with the isotope control did not affect the uptake rate ( bottom row ) . No changes in the uptake of E . coli particles were observed . Substrate uptake in the absence of antibody is shown in the top row . Hoechst 33342 was used to counter stain the nuclei ( in cyan ) and to assess cell density . Scale bar = 20µm .

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(B) Uptake assay for fluorescently labelled myelin, Aβ(1-42) and inactivated E.coli particles. The top row shows that myelin and Aβ (pseudocoloured in white) accumulate within the plasma membrane of primary microglia (labeled with dylight-649 isolectin and pseudocoloured in red). Overnight pre-treatment with antibody 4D9 significantly increased the percentage of substrate positive cells (middle row). Pre-treatment with the isotope control did not affect the uptake rate (bottom row). No changes in the uptake of E.coli particles were observed. Substrate uptake in the absence of antibody is shown in the top row. Hoechst 33342 was used to counter stain the nuclei (in cyan) and to assess cell density. Scale bar= 20µm.


( A ) 4D9 demonstrates standard IgG pharmacokinetics in vivo . Peripheral clearance rates of 4D9 on a human IgG effectorless backbone compared to an isotype control were determined in wild type mice by hIgG ELISA detection of plasma antibody concentrations 1 and 24 hours , 4 and 7 days after 10 mg / kg intravenous injection of antibody .

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(A) 4D9 demonstrates standard IgG pharmacokinetics in vivo. Peripheral clearance rates of 4D9 on a human IgG effectorless backbone compared to an isotype control were determined in wild type mice by hIgG ELISA detection of plasma antibody concentrations 1 and 24 hours, 4 and 7 days after 10 mg/kg intravenous injection of antibody.


( B ) Brain antibody concentration was measured 24hrs post intravenous dosing of isotype hIgG at 100mg / kg and 4D9 - hIgG dosed intravenously at 100 , 50 and 10mg / kg . Detection of hIgG levels by ELISA demonstrated dose dependent brain concentrations of 4D9 in the single digit nM range . Animals were perfused to minimize IgG contribution from the plasma . Data represent the mean ± SEM ( n = 5 ) .

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(B) Brain antibody concentration was measured 24hrs post intravenous dosing of isotype hIgG at 100mg/kg and 4D9-hIgG dosed intravenously at 100, 50 and 10mg/kg. Detection of hIgG levels by ELISA demonstrated dose dependent brain concentrations of 4D9 in the single digit nM range. Animals were perfused to minimize IgG contribution from the plasma. Data represent the mean ± SEM (n=5).


( D ) Target engagement timecourse demonstrated near 100 % 4D9 - bound sTREM2 in CSF of wildtype mice at 24 hrs post dose . Over a 10 day timecourse with timepoints at day 1 , 2 , 4 , 7 and study termination at day 10 , the bound / total sTREM2 reduces gradually to reach ~ 50 % by day 10 . Animals were dosed intravenously with 50mg / kg of isotype and 4D9 antibodies . Data represent the mean ± SEM ( n = 5 ) .

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(D) Target engagement timecourse demonstrated near 100% 4D9-bound sTREM2 in CSF of wildtype mice at 24 hrs post dose. Over a 10 day timecourse with timepoints at day 1, 2, 4, 7 and study termination at day 10, the bound/total sTREM2 reduces gradually to reach ~50% by day 10. Animals were dosed intravenously with 50mg/kg of isotype and 4D9 antibodies. Data represent the mean ± SEM (n=5).


( E ) Target engagement dose response demonstrated saturated bound sTREM2 at 50 and 10mg / kg with > 50 % bound sTREM2 at 1mg / kg of antibody . 4D9 - bound sTREM2 was undetectable at 0 . 1mg / kg and lower . 4D9 was IV - dosed at 50 , 10 , 1 , 0 . 1 , 0 . 01 , 0 . 001 , 0 . 0001 mg / kg , and isotype dosed at 50mg / kg . CSF bound : total sTREM2 in wildtype mice was measured at 24hrs . Data represent the mean ± SEM ( n = 5 ) .

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(E) Target engagement dose response demonstrated saturated bound sTREM2 at 50 and 10mg/kg with >50% bound sTREM2 at 1mg/kg of antibody. 4D9-bound sTREM2 was undetectable at 0.1mg/kg and lower. 4D9 was IV-dosed at 50, 10, 1, 0.1, 0.01, 0.001, 0.0001 mg/kg, and isotype dosed at 50mg/kg. CSF bound: total sTREM2 in wildtype mice was measured at 24hrs. Data represent the mean ± SEM (n=5).


( F ) 4D9 antibody demonstrates dose dependent increase of total brain TREM2 levels . Quantification of total TREM2 in brain lysates from wildtype mice dosed with 4D9 or isotype control was performed by a MSD - platform based ELISA . Data represent the mean ± SEM ( n = 5 ) and are shown as pg TREM2 per ug of total protein . One - way ANOVA , Dunnett ' s post hoc test , p ( isotype vs 4D9 ( 100mg / kg ) ) < 0 . 0001 ; p ( isotype vs 4D9 ( 50mg / kg ) ) = 0 . 0007 .

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(F) 4D9 antibody demonstrates dose dependent increase of total brain TREM2 levels. Quantification of total TREM2 in brain lysates from wildtype mice dosed with 4D9 or isotype control was performed by a MSD-platform based ELISA. Data represent the mean ± SEM (n=5) and are shown as pg TREM2 per ug of total protein. One-way ANOVA, Dunnett's post hoc test, p (isotype vs 4D9 (100mg/kg)) < 0.0001; p (isotype vs 4D9 (50mg/kg)) = 0.0007.


( B ) Immunohistochemical costainings of TREM2 ( magenta ) and IBA1 ( green ) microglia in the cortex of isotype control and 4D9 injected WT and APP - NL - G - F mice . Side panel shows images from each staining at a larger magnification indicated by the dotted white boxes . Scale bar = 10µm ; scale bar ( inset ) = 2 . 5µm .

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(B) Immunohistochemical costainings of TREM2 (magenta) and IBA1 (green) microglia in the cortex of isotype control and 4D9 injected WT and APP-NL-G-F mice. Side panel shows images from each staining at a larger magnification indicated by the dotted white boxes. Scale bar = 10µm; scale bar (inset) = 2.5µm.


( C ) Quantification of cortical TREM2 stainings shown in B . Two - way ANOVA , Tukey ' s multiple comparison test ( Genotype effect : F1 , 23 = 70 . 63 , p = < 0 . 0001 ; Treatment effect : F1 , 23 = 14 . 33 , p = 0 . 0010 ; Genotype x Treatment effect : F1 , 23 = 14 . 51 , p = 0 . 0009 ) ; p ( WT isotype vs WT 4D9 ) > 0 . 9999 ; p ( WT isotype vs APP isotype ) = 0 . 02 ; p ( WT 4D9 vs APP 4D9 ) < 0 . 0001 ; p ( APP isotype vs APP 4D9 ) = 0 . 0001 ; n . s . , not significant .

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(C) Quantification of cortical TREM2 stainings shown in B. Two-way ANOVA, Tukey's multiple comparison test (Genotype effect: F1,23=70.63, p=<0.0001; Treatment effect: F1,23=14.33, p=0.0010; Genotype x Treatment effect: F1,23=14.51, p=0.0009); p (WT isotype vs WT 4D9) > 0.9999; p (WT isotype vs APP isotype) = 0.02; p (WT 4D9 vs APP 4D9) < 0.0001; p (APP isotype vs APP 4D9) = 0.0001; n.s., not significant.


( D ) Quantification of cortical IBA1 staining shown in B . Two - way ANOVA , Tukey ' s multiple comparison test ( Genotype effect : F1 , 21 = 215 . 4 , p = < 0 . 0001 ; Treatment effect : F1 , 21 = 0 . 5994 , p = 0 . 4475 ; Genotype x Treatment effect : F1 , 21 = 0 . 2992 , p = 0 . 5902 ) ; p ( WT isotype vs APP isotype ) < 0 . 0001 ; p ( WT 4D9 vs APP 4D9 ) < 0 . 0001 ; p ( APP isotype vs APP 4D9 ) = 0 . 9986 ; n . s . , not significant .

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(D) Quantification of cortical IBA1 staining shown in B. Two-way ANOVA, Tukey's multiple comparison test (Genotype effect: F1,21=215.4, p=<0.0001; Treatment effect: F1,21=0.5994, p=0.4475; Genotype x Treatment effect: F1,21=0.2992, p=0.5902); p (WT isotype vs APP isotype) < 0.0001; p (WT 4D9 vs APP 4D9) < 0.0001; p (APP isotype vs APP 4D9) = 0.9986; n.s., not significant.


( E ) Confocal images of P2RY12 ( red ) , IBA1 ( green ) , and Aβ ( gray ) costainings from cortex . Top panel : Dotted white boxes indicate the area in P2RY12 and IBA1 costainings that are magnified as inset . Bottom panel : Insets show P2RY12 , IBA1 and Aβ null at a larger magnification . Of note , we did not observe a complete co - localization of P2RY12 and null suggesting different microglial populations . Scale bar = 10µm ; scale bar ( insets ) = 5µm .

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(E) Confocal images of P2RY12 (red), IBA1 (green), and Aβ (gray) costainings from cortex. Top panel: Dotted white boxes indicate the area in P2RY12 and IBA1 costainings that are magnified as inset. Bottom panel: Insets show P2RY12, IBA1 and Aβ null at a larger magnification. Of note, we did not observe a complete co-localization of P2RY12 and null suggesting different microglial populations. Scale bar= 10µm; scale bar (insets) = 5µm.


( F ) Quantification of P2RY12 stained cells in the cortex shown in E . Two - way ANOVA , Tukey ' s multiple comparison test ( Genotype effect : F1 , 23 = 71 . 99 , p = < 0 . 0001 ; Treatment effect : F1 , 23 = 9 . 029 , p = 0 . 0063 ; Genotype x Treatment effect : F1 , 23 = 5 . 93 , p = 0 . 0231 ) ; p ( WT isotype vs APP isotype ) = 0 . 0018 ; p ( WT null vs APP 4D9 ) < 0 . 0001 ; p ( APP isotype vs APP 4D9 ) = 0 . 0051 .

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(F) Quantification of P2RY12 stained cells in the cortex shown in E. Two-way ANOVA, Tukey's multiple comparison test (Genotype effect: F1,23=71.99, p=<0.0001; Treatment effect: F1,23=9.029, p=0.0063; Genotype x Treatment effect: F1,23=5.93, p=0.0231); p (WT isotype vs APP isotype) = 0.0018; p (WT null vs APP 4D9) < 0.0001; p (APP isotype vs APP 4D9) = 0.0051.


( A , B ) Overview of the cortex immunohistochemically stained for Aβ plaques in 6 months old APP - NL - G - F mice treated with isotype control ( A ) and 4D9 antibody ( B ) . Scale bar = 100µm .

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(A, B) Overview of the cortex immunohistochemically stained for Aβ plaques in 6 months old APP-NL-G-F mice treated with isotype control (A) and 4D9 antibody (B). Scale bar= 100µm.


( C ) Higher magnification images of cortical Aβ plaques in APP isotype and 4D9 injected mice . Scale bar = 10µm . ( D ) Quantification of cortical plaque area from stainings shown in C . Mann - Whitney U test , p = 0 . 0082 . ( E ) Quantification of total plaque coverage of Aβ stainings in the cortex shown in C . Mann - Whitney U test , p = 0 . 014 .

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(C) Higher magnification images of cortical Aβ plaques in APP isotype and 4D9 injected mice. Scale bar= 10µm. (D) Quantification of cortical plaque area from stainings shown in C. Mann-Whitney U test, p=0.0082. (E) Quantification of total plaque coverage of Aβ stainings in the cortex shown in C. Mann-Whitney U test, p=0.014.


( F ) Immunoblot analysis of soluble ( DEA fraction , top ) and insoluble Aβ levels ( FA fraction , bottom ) in brains of APP - NL - G - F mice treated with either isotype control or 4D9 antibody . While a reduction in levels of soluble Aβ is evident from the DEA fractions no change in levels of insoluble Aβ could be detected in the FA fractions . Note that the higher background in the DEA immunoblot is due to a much longer exposure time , which was needed to visualize soluble Aβ .

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(F) Immunoblot analysis of soluble (DEA fraction, top) and insoluble Aβ levels (FA fraction, bottom) in brains of APP-NL-G-F mice treated with either isotype control or 4D9 antibody. While a reduction in levels of soluble Aβ is evident from the DEA fractions no change in levels of insoluble Aβ could be detected in the FA fractions. Note that the higher background in the DEA immunoblot is due to a much longer exposure time, which was needed to visualize soluble Aβ.


( G ) MSD ELISA quantification of soluble Aβ42 in DEA extracts as shown in ( F ) . Mann - Whitney U test , p = 0 . 026 . ( H ) MSD ELISA quantification of insoluble Aβ42 in FA extracts as shown in ( F ) . Unpaired t - test ( two - tailed ) with Welch ' s correction ; p = 0 . 0877 .

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(G) MSD ELISA quantification of soluble Aβ42 in DEA extracts as shown in (F). Mann-Whitney U test, p=0.026. (H) MSD ELISA quantification of insoluble Aβ42 in FA extracts as shown in (F). Unpaired t-test (two-tailed) with Welch's correction; p=0.0877.


Volcano plot representation of AFG3L2E408Q - FLAG interaction partners . Mitochondria isolated from Flp - In HEK293T - REx cells expressing AFG3L2E408Q - FLAG were subjected to immunoprecipitation . Co - purifying proteins were identified by quantitative mass spectrometry ( MS ) ( n = 5 independent experiments ) followed by an unpaired two - sided t - test and permutation - based FDR estimation to correct for multiple testing ( FDR < 0 . 05 . Significantly enriched MitoCarta 3 . 0 proteins are colored in red , gene names indicate previously verified AFG3L2 interactors and TMBIM5 ( GHITM , MICS1 ) .

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Volcano plot representation of AFG3L2E408Q-FLAG interaction partners. Mitochondria isolated from Flp-In HEK293T-REx cells expressing AFG3L2E408Q-FLAG were subjected to immunoprecipitation. Co-purifying proteins were identified by quantitative mass spectrometry (MS) (n=5 independent experiments) followed by an unpaired two-sided t-test and permutation-based FDR estimation to correct for multiple testing (FDR<0.05. Significantly enriched MitoCarta 3.0 proteins are colored in red, gene names indicate previously verified AFG3L2 interactors and TMBIM5 (GHITM, MICS1).


Representative immunoblot of HeLa wildtype ( WT ) cells transfected with scrambled siRNA ( control ) or siRNA targeting TMBIM5 and treated with the indicated drugs for 16 h . Control ( 0 . 1 % DMSO ) , actinomycin D ( 1 µM ) , venetoclax ( 1 µM ) , A - 1155463 ( 1 µM ) , staurosporine ( 1 µM ) , thapsigargin ( 2 µM ) . n = 3 independent experiments .

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Representative immunoblot of HeLa wildtype (WT) cells transfected with scrambled siRNA (control) or siRNA targeting TMBIM5 and treated with the indicated drugs for 16 h. Control (0.1% DMSO), actinomycin D (1 µM), venetoclax (1 µM), A-1155463 (1 µM), staurosporine (1 µM), thapsigargin (2 µM). n=3 independent experiments.


Representative immunoblot of HeLa WT and TMBIM5 - / - cells transfected with scrambled siRNA ( control ) or siRNA targeting MCU for 72 h . Where indicated samples were treated with staurosporine ( STS ; 1 µM ) for 16 h . ( n = 3 independent experiments ) .

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Representative immunoblot of HeLa WT and TMBIM5-/- cells transfected with scrambled siRNA (control) or siRNA targeting MCU for 72 h. Where indicated samples were treated with staurosporine (STS; 1 µM) for 16 h. (n=3 independent experiments).


Viability of WT and TMBIM5 - / - cells ( depleted of MCU when indicated ) after incubation with staurosporine ( STS ; 0 . 1 µM ) for 16 h . Cell viability was assessed cytofluorimetrically monitoring NucView488 and RedDot2 staining and expressed as percentage of all cells . n = 3 independent experiments ; two - tailed t - test ; mean ± SD . A p - value of < 0 . 05 was considered statistically significant .

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Viability of WT and TMBIM5-/- cells (depleted of MCU when indicated) after incubation with staurosporine (STS; 0.1 µM) for 16 h. Cell viability was assessed cytofluorimetrically monitoring NucView488 and RedDot2 staining and expressed as percentage of all cells. n=3 independent experiments; two-tailed t-test; mean ±SD. A p-value of <0.05 was considered statistically significant.


WT and TMBIM5 - / - HeLa cells were transfected with the indicated scrambled siRNA ( control ) and siRNA targeting MCU for 48 h and incubated with staurosporine ( 0 . 1 µM ) for 16 h in the presence of Z - VAD - FMK ( 50 µM ) and epoxomicin ( 1 µM ) to prevent apoptosis . Cells were subjected to immunofluorescence analysis with antibodies directed against cytochrome c ( green ) and TOMM20 ( magenta ) . * Cells with cytosolic cytochrome c . ( n = 3 independent experiments ; number of cells > 150 each experiment ) .

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WT and TMBIM5-/- HeLa cells were transfected with the indicated scrambled siRNA (control) and siRNA targeting MCU for 48 h and incubated with staurosporine (0.1 µM) for 16 h in the presence of Z-VAD-FMK (50 µM) and epoxomicin (1 µM) to prevent apoptosis. Cells were subjected to immunofluorescence analysis with antibodies directed against cytochrome c (green) and TOMM20 (magenta). *Cells with cytosolic cytochrome c. (n=3 independent experiments; number of cells > 150 each experiment).


Calcium retention capacity ( CRC ) was assessed in isolated mitochondria of HEK293T WT cells transfected with the indicated siRNA . The experiment was performed in sucrose - based medium containing respiratory substrates and the membrane - impermeable Ca2 + sensor Ca2 + Green - 5N . Ca2 + Green - 5N fluorescence was monitored following repeated addition of Ca2 + pulses ( 10 µM ) . Data show representative traces ( 2 independent experiments run in triplicate each condition ) .

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Calcium retention capacity (CRC) was assessed in isolated mitochondria of HEK293T WT cells transfected with the indicated siRNA. The experiment was performed in sucrose-based medium containing respiratory substrates and the membrane-impermeable Ca2+ sensor Ca2+ Green-5N. Ca2+ Green-5N fluorescence was monitored following repeated addition of Ca2+ pulses (10 µM). Data show representative traces (2 independent experiments run in triplicate each condition).


Fluorescence ( F ) F / F0 ratio quantifications , where F0 is the average of the first ten seconds and F is the fluorescence peak measured after thapsigargin ( 2 µM ) administration ( left panel ) . The right shows mtRED - GECO ratio traces of HeLa cells transfected with scrambled siRNA ( control ) and siRNA targeting TMBIM5 . Each measurement was performed in at least 10 cells from 6 different preparations . Mean ± SD ; two - tailed t - test . A p - value of < 0 . 05 was considered statistically significant .

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Fluorescence (F) F/F0 ratio quantifications, where F0 is the average of the first ten seconds and F is the fluorescence peak measured after thapsigargin (2 µM) administration (left panel). The right shows mtRED-GECO ratio traces of HeLa cells transfected with scrambled siRNA (control) and siRNA targeting TMBIM5. Each measurement was performed in at least 10 cells from 6 different preparations. Mean ±SD; two-tailed t-test. A p-value of <0.05 was considered statistically significant.


Matrix pH was measured with Sypher3smito in wildtye ( WT ) HeLa cells transfected with scrambled siRNA ( control ) or siRNA targeting TMBIM5 for 72 h . The probe was calibrated in KRB buffer by the stepwise addition / removal of permeant weak acid . Each measurement was performed in at least 80 cells from 7 different preparations . Mean ± SD ; two - tailed t - test . A p - value of < 0 . 05 was considered statistically significant .

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

Matrix pH was measured with Sypher3smito in wildtye (WT) HeLa cells transfected with scrambled siRNA (control) or siRNA targeting TMBIM5 for 72 h. The probe was calibrated in KRB buffer by the stepwise addition/removal of permeant weak acid. Each measurement was performed in at least 80 cells from 7 different preparations. Mean ±SD; two-tailed t-test. A p-value of <0.05 was considered statistically significant.


Mitochondrial membrane potential was monitored with TMRM in WT and TMBIM5 - / - HeLa cells . Fluorescent intensity was calculated as ratio to the value upon CCCP addition ( 15 µM ) . Values are expressed as mean ± SD relative to control . Each measurement was performed in triplicate from 4 different preparations .

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

Mitochondrial membrane potential was monitored with TMRM in WT and TMBIM5-/- HeLa cells. Fluorescent intensity was calculated as ratio to the value upon CCCP addition (15 µM). Values are expressed as mean ±SD relative to control. Each measurement was performed in triplicate from 4 different preparations.


Matrix pH was measured with Sypher3smito in WT HeLa cells transiently expressing TMBIM5 or TMBIM5D294K D325K for 24 h . The ratio of absorbance at 488 nm and 405 nm is shown in arbitrary units ( a . u . ) . Each measurement was performed in at least 20 cells from 3 different preparations . Representative immunoblot of WT HeLa cells transfected with TMBIM5 or TMBIM5D294K D325K for 24 h ( right panel ) . Mean ± SD ; two - tailed t - test . A p - value of < 0 . 05 was considered statistically significant .

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

Matrix pH was measured with Sypher3smito in WT HeLa cells transiently expressing TMBIM5 or TMBIM5D294K D325K for 24 h. The ratio of absorbance at 488 nm and 405 nm is shown in arbitrary units (a.u.). Each measurement was performed in at least 20 cells from 3 different preparations. Representative immunoblot of WT HeLa cells transfected with TMBIM5 or TMBIM5D294K D325K for 24 h (right panel). Mean ±SD; two-tailed t-test. A p-value of <0.05 was considered statistically significant.


Liposome assay . Recombinant TMBIM5 was reconstituted in liposomes containing 100 µM Fura - 2 . CaCl2 was added later at the concentration of 100 µM . Rmax was obtained at the end of the experiment after the addition of ionomycin ( 10 µM ) . Data are expressed as mean of R / Rmax , where R is the peak after CaCl2 addition . Where indicated GdCl3 ( 100 µM ) was added 10 min before the experiment . n = 3 independent experiments , shown as black , white and grey dots . Mean ± SD ; two - tailed t - test . A p - value of < 0 . 05 was considered statistically significant . Liposome assay ( as in E ) . Slope was calculated in the 100 s after 100 µM CaCl2 addition as expressed as mean ± SD . n = 3 independent experiments , shown as black , white and grey dots . Two - tailed t - test . A p - value of < 0 . 05 was considered statistically significant .

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

Liposome assay. Recombinant TMBIM5 was reconstituted in liposomes containing 100 µM Fura-2. CaCl2 was added later at the concentration of 100 µM. Rmax was obtained at the end of the experiment after the addition of ionomycin (10 µM). Data are expressed as mean of R/Rmax, where R is the peak after CaCl2 addition. Where indicated GdCl3 (100 µM) was added 10 min before the experiment. n=3 independent experiments, shown as black, white and grey dots. Mean ±SD; two-tailed t-test. A p-value of <0.05 was considered statistically significant. Liposome assay (as in E). Slope was calculated in the 100 s after 100 µM CaCl2 addition as expressed as mean ±SD. n=3 independent experiments, shown as black, white and grey dots. Two-tailed t-test. A p-value of <0.05 was considered statistically significant.


Alterations in mitochondrial proteome in WT and TMBIM5 - / - HeLa cells upon depletion of AFG3L2 . WT and TMBIM5 - / - HeLa cells were transfected with scrambled siRNA ( control ) or siRNA targeting AFG3L2 for 48 h . Proteins , which were downregulated in the absence of TMBIM5 and whose steady state levels was altered upon depletion of AFG3L2 , were identified using a two - way ANOVA . An interaction p - value of 0 . 01 was used as a cutoff . Log2 - transformed LFQ intensities of MitoCarta 3 . 0 proteins were Z - Score normalized and visualized in a hierarchical cluster analysis ( Euclidean distance , complete method ) . The dendrogram is omitted . Proteins accumulating upon depletion of AFG3L2 ( cluster 1 , C1 ) and proteins whose steady state levels are decreased upon depletion of AFG3L2 ( cluster 2 , C2 ) are shown .

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

Alterations in mitochondrial proteome in WT and TMBIM5-/- HeLa cells upon depletion of AFG3L2. WT and TMBIM5-/- HeLa cells were transfected with scrambled siRNA (control) or siRNA targeting AFG3L2 for 48 h. Proteins, which were downregulated in the absence of TMBIM5 and whose steady state levels was altered upon depletion of AFG3L2, were identified using a two-way ANOVA. An interaction p-value of 0.01 was used as a cutoff. Log2-transformed LFQ intensities of MitoCarta 3.0 proteins were Z-Score normalized and visualized in a hierarchical cluster analysis (Euclidean distance, complete method). The dendrogram is omitted. Proteins accumulating upon depletion of AFG3L2 (cluster 1, C1) and proteins whose steady state levels are decreased upon depletion of AFG3L2 (cluster 2, C2) are shown.


Representative immunoblot of TMBIM5 - / - HeLa cells transfected with scrambled siRNA ( control ) or siRNA targeting AFG3L2 for 48 h . Samples were treated with the protein synthesis inhibitor cycloheximide ( CHX ; 10 μg / ml ) and collected at the indicated time points ( n = 3 independent experiments ) .

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

Representative immunoblot of TMBIM5-/- HeLa cells transfected with scrambled siRNA (control) or siRNA targeting AFG3L2 for 48 h. Samples were treated with the protein synthesis inhibitor cycloheximide (CHX; 10 μg/ml) and collected at the indicated time points (n=3 independent experiments).


Volcano plot of mitochondrial protein ( MitoCarta 3 . 0 ) changes in WT HeLa cells treated with oligomycin ( 10 μM for 16 h ) when compared to untreated cells . Before MitoCarta 3 . 0 - filtering , significantly altered proteins were identified using a two - sided t - test followed by permutation - based FDR estimation to 0 . 05 and are colored in blue ( n = 5 independent experiments ) .

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

Volcano plot of mitochondrial protein (MitoCarta 3.0) changes in WT HeLa cells treated with oligomycin (10 μM for 16 h) when compared to untreated cells. Before MitoCarta 3.0-filtering, significantly altered proteins were identified using a two-sided t-test followed by permutation-based FDR estimation to 0.05 and are colored in blue (n=5 independent experiments).


Representative immunoblot of WT HeLa cells treated with the indicated drugs for 16 h . Antimycin A ( AA ; 10 μM ) , piericidin A ( 10 μM ) and oligomycin ( 10 μM ) .

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

Representative immunoblot of WT HeLa cells treated with the indicated drugs for 16 h. Antimycin A (AA; 10 μM), piericidin A (10 μM) and oligomycin (10 μM).


Representative immunoblot of WT HeLa cells transfected with scrambled siRNA ( control ) and siRNA targeting AFG3L2 for 48 h , treated when indicated with oligomycin ( 10 μM ) for 16 h . Quantification of TMBIM5 levels is shown on the right panel . TMBIM5 levels in untreated WT cells was set to 1 ( n = 3 independent experiments ; mean ± SD ) .

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

Representative immunoblot of WT HeLa cells transfected with scrambled siRNA (control) and siRNA targeting AFG3L2 for 48 h, treated when indicated with oligomycin (10 μM) for 16 h. Quantification of TMBIM5 levels is shown on the right panel. TMBIM5 levels in untreated WT cells was set to 1 (n=3 independent experiments; mean ±SD).


TMBIM5 abundance determined by mass spectrometry in WT HeLa cells treated with oligomycin ( 10 µM ; 16 h ) and depleted of AFG3L2 when indicated . n = 5 independent experiments . P - value was calculated using a two - sided unpaired t - test . The central band of each box is the median value , and the box defines the 25th ( lower ) and 75th ( higher ) quantile . The whiskers represent the minimum and maximum value in the data excluding outliers ( greater than 1 . 5 * inter quantile range distance to median - no outliers found here ) .

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

TMBIM5 abundance determined by mass spectrometry in WT HeLa cells treated with oligomycin (10 µM; 16 h) and depleted of AFG3L2 when indicated. n=5 independent experiments. P-value was calculated using a two-sided unpaired t-test. The central band of each box is the median value, and the box defines the 25th (lower) and 75th (higher) quantile. The whiskers represent the minimum and maximum value in the data excluding outliers (greater than 1.5 * inter quantile range distance to median - no outliers found here).


Zoom in to selected clusters of hierarchical clustering of Z - Score normalized log2 LFQ intensities . Proteins that were significantly ( FDR < 0 . 05 ) accumulated between HeLa WT and TMBIM5 - / - cells after oligomycin treatment ( 10 µM ) for 16 h were clustered using Euclidean distance and the complete method on protein features . The figure shows identified cluster C4 and C5 with gene names and MitoCarta 3 . 0 localization annotations . The row dendrogram is omitted .

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

Zoom in to selected clusters of hierarchical clustering of Z-Score normalized log2 LFQ intensities. Proteins that were significantly (FDR<0.05) accumulated between HeLa WT and TMBIM5-/- cells after oligomycin treatment (10 µM) for 16 h were clustered using Euclidean distance and the complete method on protein features. The figure shows identified cluster C4 and C5 with gene names and MitoCarta 3.0 localization annotations. The row dendrogram is omitted.


C - E . Luciferase reporter assay in HeLa cells using the synthetic miR - Control or miR - 182 - 3p in combination with the wild type ( C ) or the mutant 3 ' UTR of TRF2 construct ( D ) or the wild type 3 ' UTR of TRF1 ( E ) . Data information : data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by Mann - Whitney test .

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

C-E. Luciferase reporter assay in HeLa cells using the synthetic miR-Control or miR-182-3p in combination with the wild type (C) or the mutant 3'UTR of TRF2 construct (D) or the wild type 3'UTR of TRF1 (E). Data information: data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by Mann-Whitney test.


F , G . Western blotting for TRF2 expression in Telomerase Positive ( HeLa , HCT116 , MDA - MB - 231 , MDA - MB - 436 ) and ALT positive ( U2 - OS , Saos - 2 ) cells transiently transfected with miR - Control or miR - 182 - 3p . Upper panel shows the quantification of TRF2 expression . Bottom panel , representative images are shown , actin was used as loading control . Data information : data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by Mann - Whitney test .

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

F,G. Western blotting for TRF2 expression in Telomerase Positive (HeLa, HCT116, MDA-MB-231, MDA-MB-436) and ALT positive (U2-OS, Saos-2) cells transiently transfected with miR-Control or miR-182-3p. Upper panel shows the quantification of TRF2 expression. Bottom panel, representative images are shown, actin was used as loading control. Data information: data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by Mann-Whitney test.


H . U2 - OS cells transiently transfected with the miR - Control , miR - 182 - 3p or miR - 182 - 3p inhibitor were assayed by quantitative immunofluorescence for TRF2 three days post - transfection . Left panel , representative images . Scale bar : 10µm . Right panel , quantification of TRF2 fluorescence intensity . a . f . u . arbitrary fluorescence units . N = number of analysed nuclei . Red bar indicates mean value . Data information : Three independent experiments were performed ( n = 3 ) . P values are determined by Student ' s t - test ; for ( H )

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

H. U2-OS cells transiently transfected with the miR-Control, miR-182-3p or miR-182-3p inhibitor were assayed by quantitative immunofluorescence for TRF2 three days post-transfection. Left panel, representative images. Scale bar: 10µm. Right panel, quantification of TRF2 fluorescence intensity. a.f.u. arbitrary fluorescence units. N=number of analysed nuclei. Red bar indicates mean value. Data information: Three independent experiments were performed (n=3). P values are determined by Student's t-test; for (H)


I . U2 - OS cells transfected as described in ( H ) were assayed by immuno - fluorescence combined with telomeric FISH . Left panel , representative images of co - localizations between TRF2 and telomeres ( white arrowheads ) . Scale bar : 10µm . Right panel , co - localizations were analysed using imageJ software . N = number of analysed nuclei . Data information : For I , data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by Mann - Whitney test .

I. U2-OS cells transfected as described in (H) were assayed by immuno-fluorescence combined with telomeric FISH. Left panel, representative images of co-localizations between TRF2 and telomeres (white arrowheads). Scale bar: 10µm. Right panel, co-localizations were analysed using imageJ software. N=number of analysed nuclei. Data information: For I, data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by Mann-Whitney test.


A . Immunofluorescence analysis of γH2AX combined with telomeric FISH ( TIFs ) was performed in MDA - MB - 231 cells transfected with the indicated miRNAs or siRNAs . The mean number of TIFs per nucleus was analysed . B . Representative images and enlargements of co - localizations ( white arrowheads ) relative to the experiment described in A . Scale bar : 10µm . Data information : For A data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test . At least 60 nuclei were analysed for each experimental condition . All the experiments were performed three days post - transfection with the indicated miRNAs or siRNAs .

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

A. Immunofluorescence analysis of γH2AX combined with telomeric FISH (TIFs) was performed in MDA-MB-231 cells transfected with the indicated miRNAs or siRNAs. The mean number of TIFs per nucleus was analysed. B. Representative images and enlargements of co-localizations (white arrowheads) relative to the experiment described in A. Scale bar: 10µm. Data information: For A data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test. At least 60 nuclei were analysed for each experimental condition. All the experiments were performed three days post-transfection with the indicated miRNAs or siRNAs.


C . Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe ( PIFs ) was performed in MDA - MB - 231 cells transfected with the indicated miRNAs or siRNAs . The γH2AX positive cells with ≥ 1 PIFs per nucleus were analysed . D . Representative images of co - localizations ( white arrowheads ) relative to the experiment described in C . Scale bar : 10µm . Data information : For C , data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test . At least 60 nuclei were analysed for each experimental condition . All the experiments were performed three days post - transfection with the indicated miRNAs or siRNAs .

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

C. Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in MDA-MB-231 cells transfected with the indicated miRNAs or siRNAs. The γH2AX positive cells with ≥1 PIFs per nucleus were analysed. D. Representative images of co-localizations (white arrowheads) relative to the experiment described in C. Scale bar: 10µm. Data information: For C, data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test. At least 60 nuclei were analysed for each experimental condition. All the experiments were performed three days post-transfection with the indicated miRNAs or siRNAs.


E . Quantification of TIFs in MDA - MB - 231 cells over - expressing TRF2 or an empty vector ( pBabe ) , transfected with indicated miRNAs . The mean number of TIFs per nucleus was quantified . F . Representative images and enlargements relative to the experiment described in E . White arrowheads indicate co - localizations events . Scale bar : 10µm . Data information : data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test . At least 60 nuclei were analysed for each experimental condition . All the experiments were performed three days post - transfection with the indicated

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

E. Quantification of TIFs in MDA-MB-231 cells over-expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The mean number of TIFs per nucleus was quantified. F. Representative images and enlargements relative to the experiment described in E. White arrowheads indicate co-localizations events. Scale bar: 10µm. Data information: data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test. At least 60 nuclei were analysed for each experimental condition. All the experiments were performed three days post-transfection with the indicated miRNAs


G . Quantification of PIFs in MDA - MB - 231 cells over - expressing TRF2 or an empty vector ( pBabe ) , transfected with indicated miRNAs . The γH2AX positive cells with ≥ 1 PIFs per nucleus were analysed . H . Representative images relative to the experiment described in G . White arrowheads indicate co - localizations events . Scale bar : 10µm . Data information : For G data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test . At least 60 nuclei were analysed for each experimental condition . All the experiments were performed three days post - transfection with the indicated

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

G. Quantification of PIFs in MDA-MB-231 cells over-expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The γH2AX positive cells with ≥1 PIFs per nucleus were analysed. H. Representative images relative to the experiment described in G. White arrowheads indicate co-localizations events. Scale bar: 10µm. Data information: For G data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test. At least 60 nuclei were analysed for each experimental condition. All the experiments were performed three days post-transfection with the indicated miRNAs


A , B . MDA - MB - 436 and MDA - MB - 231 cells underwent two rounds of transfection with miR - Control , miR - 182 - 3p or miR - 182 - 3p inhibitor . Starting from the day of the second transfection , cell confluence was monitored by Incucyte every 24h up to a maximum of 3 days . The percentage of cell confluence was analysed . Data information : For A , B data are shown as mean ± SEM . three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test .

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

A,B. MDA-MB-436 and MDA-MB-231 cells underwent two rounds of transfection with miR-Control, miR-182-3p or miR-182-3p inhibitor. Starting from the day of the second transfection, cell confluence was monitored by Incucyte every 24h up to a maximum of 3 days. The percentage of cell confluence was analysed. Data information: For A,B data are shown as mean ± SEM. three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test.


C , D . Cell number of MDA - MB - 436 ( C ) and MDA - MB - 231 ( D ) cells and TRF2 expression were analysed by automatic cell count and by western blotting at the end of the experiment described in A and B . Actin was used as loading control . Data information : For C , D data are shown as mean ± SD . three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test .

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

C,D. Cell number of MDA-MB-436 (C) and MDA-MB-231 (D) cells and TRF2 expression were analysed by automatic cell count and by western blotting at the end of the experiment described in A and B. Actin was used as loading control. Data information: For C,D data are shown as mean ± SD. three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test.


E . Two - dimensional scatter plots of Annexin V analysis performed in MDA - MB - 436 at the end of the second cycle of transfection with miR - Control , miR - 182 - 3p or miR - 182 - 3p inhibitor . Red boxes indicate early and late apoptotic cells . F . Quantification of Annexin V positive cells ( % ) of experiment described in E . G . Two - dimensional scatter plots of Annexin V analysis performed in MDA - MB - 231 as described in E . H . Quantification of Annexin V positive cells ( % ) of experiment described in G . Data information : For F , H data are shown as mean ± SD . P values are determined by unpaired two - tailed t test . For F , H two different biological replicates were performed .

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

E. Two-dimensional scatter plots of Annexin V analysis performed in MDA-MB-436 at the end of the second cycle of transfection with miR-Control, miR-182-3p or miR-182-3p inhibitor. Red boxes indicate early and late apoptotic cells. F. Quantification of Annexin V positive cells (%) of experiment described in E. G. Two-dimensional scatter plots of Annexin V analysis performed in MDA-MB-231 as described in E. H. Quantification of Annexin V positive cells (%) of experiment described in G. Data information: For F,H data are shown as mean ± SD. P values are determined by unpaired two-tailed t test. For F, H two different biological replicates were performed.


I , J . MDA - MB - 436 cells over - expressing TRF2 or an empty vector ( pBabe ) were transiently transfected with indicated miRNAs and cell count ( I ) or apoptosis ( J ) analysis was performed 72h post - transfection . Data information : For , I , J , data are shown as mean ± SD . For I , three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test . For J , two different biological replicates were performed .

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

I,J. MDA-MB-436 cells over-expressing TRF2 or an empty vector (pBabe) were transiently transfected with indicated miRNAs and cell count (I) or apoptosis (J) analysis was performed 72h post-transfection. Data information: For ,I,J, data are shown as mean ± SD. For I, three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test. For J, two different biological replicates were performed.


A . Western blotting for TRF2 expression in BJ cells transiently transfected with miR - Control or miR - 182 - 3p . The graph represents the quantification of three independent experiments . Representative images are shown , actin was used as loading control . Unspecific bands are indicated with ( * ) .

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A. Western blotting for TRF2 expression in BJ cells transiently transfected with miR-Control or miR-182-3p. The graph represents the quantification of three independent experiments. Representative images are shown, actin was used as loading control. Unspecific bands are indicated with (*).


B , C . Mean of γH2AX foci per nucleus was analysed in BJ cells 72h post - transfection with the indicated miRNAs . Representative images of γH2AX foci are shown in C . Data information : student t test was used to calculate statistical significance . Scale bars ( 10µm ) . P values are indicated .

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B, C. Mean of γH2AX foci per nucleus was analysed in BJ cells 72h post-transfection with the indicated miRNAs. Representative images of γH2AX foci are shown in C. Data information: student t test was used to calculate statistical significance. Scale bars (10µm). P values are indicated.


D . Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe ( TIFs ) was performed in BJ cells 72h post - transfection with the indicated miRNAs . Left panel : the mean number of TIFs per nucleus was analysed . Right panel : representative images and enlargements of co - localizations . Data information : a student t test was used to calculate statistical significance . Scale bars ( 10µm ) . P values are indicated .

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D. Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in BJ cells 72h post-transfection with the indicated miRNAs. Left panel: the mean number of TIFs per nucleus was analysed. Right panel: representative images and enlargements of co-localizations. Data information: a student t test was used to calculate statistical significance. Scale bars (10µm). P values are indicated.


E . Cells number of BJ cells was analysed by automatic cell count at the end of the second round of transfection with miR - Control or miR - 182 - 3p . Data information : a student t test was used to calculate statistical significance . P values are indicated .

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E. Cells number of BJ cells was analysed by automatic cell count at the end of the second round of transfection with miR-Control or miR-182-3p. Data information: a student t test was used to calculate statistical significance. P values are indicated.


F . FACS analysis to evaluate cell cycle progression by Propidium Iodide ( PI ) staining in BJ cells treated as indicated in E .

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F. FACS analysis to evaluate cell cycle progression by Propidium Iodide (PI) staining in BJ cells treated as indicated in E.


G . β - Galactosidase assay in BJ cells after two rounds of transfection with mimic miR - Control or miR - 182 - 3p . Left panel : analysis of β - Galactosidase positive cells . Right panel : representative images . Data information : a student t test was used to calculate statistical significance . P values are indicated .

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G. β-Galactosidase assay in BJ cells after two rounds of transfection with mimic miR-Control or miR-182-3p. Left panel: analysis of β-Galactosidase positive cells. Right panel: representative images. Data information: a student t test was used to calculate statistical significance. P values are indicated.


H - J . IL - 6 ( H ) , CXCL1 ( I ) , IL - 8 ( J ) factors were analysed by ELISA to evaluated the Senescence - associated secretory phenotype ( SASP ) in BJ cells treated as indicated in G . Data information : a student t test was used to calculate statistical significance . P values are indicated .

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H-J. IL-6 (H), CXCL1 (I), IL-8 (J) factors were analysed by ELISA to evaluated the Senescence-associated secretory phenotype (SASP) in BJ cells treated as indicated in G. Data information: a student t test was used to calculate statistical significance. P values are indicated.


A , B . MDA - MB - 231 ( A ) and MDA - MB - 436 ( B ) tumor xenografts were treated with LNPs - empty , LNPs - miR - Control or by LNPs - miR - 182 - 3p when the tumors became palpable . Mice were treated 6 times by intravenous tail vein injections with 20µg of LNPs - miR - Control , LNPs - miR - 182 - 3p or equivalent volume of LNPs - empty as indicated in the scheduling . The mean of tumor volumes ( n = 5 per group ) is shown . Data information : For A , B data are shown as mean ± SD . P values are determined by unpaired two - tailed t

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A,B. MDA-MB-231 (A) and MDA-MB-436 (B) tumor xenografts were treated with LNPs-empty, LNPs-miR-Control or by LNPs-miR-182-3p when the tumors became palpable. Mice were treated 6 times by intravenous tail vein injections with 20µg of LNPs-miR-Control, LNPs-miR-182-3p or equivalent volume of LNPs-empty as indicated in the scheduling. The mean of tumor volumes (n=5 per group) is shown. Data information: For A, B data are shown as mean ± SD. P values are determined by unpaired two-tailed t test


C , D . Tumors from mice treated in A and B were processed to measure miR - 182 - 3p expression by TaqMan qPCR . Data information : For C , D the line in the middle of the box plot denote a median value , the limits of box represent the interquartile range ( 25th to 75th percentiles ) , while , the whiskers denote the minimum to maximum values . , P values are determined by unpaired two - tailed t test ;

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C,D. Tumors from mice treated in A and B were processed to measure miR-182-3p expression by TaqMan qPCR. Data information: For C, D the line in the middle of the box plot denote a median value, the limits of box represent the interquartile range (25th to 75th percentiles), while, the whiskers denote the minimum to maximum values. , P values are determined by unpaired two-tailed t test;


E . Representative images of IHC analysis of the indicated markers on tumor samples from mice bearing MDA - MB - 231 human breast cancer xenografts . Scale bar : 50μm . F . The histograms show : the expression of TRF2 , calculated as immunoreactivity score ( IRS ) by IHC , and the count of positive cells to γH2AX , Tunel or CD31 staining . The analyses were performed on 3 mice per group , the points represent the number of field analysed for each condition . Data information : For F , data are shown as mean ± SD . ( F ) , P values are determined by Mann - Whitney test .

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E. Representative images of IHC analysis of the indicated markers on tumor samples from mice bearing MDA-MB-231 human breast cancer xenografts. Scale bar: 50μm. F. The histograms show: the expression of TRF2, calculated as immunoreactivity score (IRS) by IHC, and the count of positive cells to γH2AX, Tunel or CD31 staining. The analyses were performed on 3 mice per group, the points represent the number of field analysed for each condition. Data information: For F, data are shown as mean ± SD. (F), P values are determined by Mann-Whitney test.


G , H . Luminescent MDA - MB - 436 cells were injected into the brain and monitored by IVIS imaging system . After one week from implant , treatment with LNPs - miR - Control and LNPs - miR - 182 - 3p was performed as indicated in A and B . Representative images from in vivo ( upper panel ) or ex - vivo ( bottom panel ) brain tumors are shown in G . Boxplots ( H ) show the measurement of photons for each brain tumor ( n = 5 per group ) acquired at the indicated times . Data information : For H , the line in the middle of the box plot denote a median value , the limits of box represent the interquartile range ( 25th to 75th percentiles ) , while , the whiskers denote the minimum to maximum values . For H , P values are determined by unpaired two - tailed t test ;

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G, H. Luminescent MDA-MB-436 cells were injected into the brain and monitored by IVIS imaging system. After one week from implant, treatment with LNPs-miR-Control and LNPs-miR-182-3p was performed as indicated in A and B. Representative images from in vivo (upper panel) or ex-vivo (bottom panel) brain tumors are shown in G. Boxplots (H) show the measurement of photons for each brain tumor (n=5 per group) acquired at the indicated times. Data information: For H, the line in the middle of the box plot denote a median value, the limits of box represent the interquartile range (25th to 75th percentiles), while, the whiskers denote the minimum to maximum values. For H, P values are determined by unpaired two-tailed t test;


A , B . PDTCs # 1 and # 2 underwent two rounds of transfection with miR - Control or miR - 182 - 3p . Three days after the second transfection , miR - 182 - 3p and TRF2 expression were analysed by TaqMan qPCR and western blotting , respectively . Actin was used as loading control . Data information : data are shown as mean ± SD . P values are determined by unpaired two - tailed t test For the experiments showed in A , B two or three biological replicates were performed , respectively .

A,B. PDTCs #1 and #2 underwent two rounds of transfection with miR-Control or miR-182-3p. Three days after the second transfection, miR-182-3p and TRF2 expression were analysed by TaqMan qPCR and western blotting, respectively. Actin was used as loading control. Data information: data are shown as mean ± SD. P values are determined by unpaired two-tailed t test For the experiments showed in A, B two or three biological replicates were performed, respectively.


C , D . Left panel , area of each PDTCs was measured by ImageJ . Right panel , representative images are shown . Scale bar : 50μm . At least 85 3D cells were analysed for each experimental condition . Data information : data are shown as mean ± SD . P values are determined by unpaired two - tailed t test For the experiments showed in C , D two or three biological replicates were performed , respectively .

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C,D. Left panel, area of each PDTCs was measured by ImageJ. Right panel, representative images are shown. Scale bar: 50μm. At least 85 3D cells were analysed for each experimental condition. Data information: data are shown as mean ± SD. P values are determined by unpaired two-tailed t test For the experiments showed in C, D two or three biological replicates were performed, respectively.


E . NSG mice implanted with breast PDTX ( # 2 ) were treated with LNPs - empty , LNPs - miR - Control or LNPs - miR - 182 - 3p as indicated in the scheduling . Caliper measurement of tumors were taken at the indicated days . The mean of tumor volumes ( n = 5 per group ) is shown . Data information : data are shown as mean ± SD . P values are determined by unpaired two - tailed t test ;

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E. NSG mice implanted with breast PDTX (#2) were treated with LNPs-empty, LNPs-miR-Control or LNPs-miR-182-3p as indicated in the scheduling. Caliper measurement of tumors were taken at the indicated days. The mean of tumor volumes (n=5 per group) is shown. Data information: data are shown as mean ± SD. P values are determined by unpaired two-tailed t test;


F . miR - 182 - 3p expression of tumors from mice treated in ( E ) was assayed by TaqMan qPCR . Data information : data are shown as mean ± SD . P values are determined by unpaired two - tailed t

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F. miR-182-3p expression of tumors from mice treated in (E) was assayed by TaqMan qPCR. Data information: data are shown as mean ± SD. P values are determined by unpaired two-tailed t test


G . Representative images of IHC analysis of the indicated markers from tumors of the experiment showed in E . Scale bar : 50μm . H . The histograms show : the expression levels of TRF2 measured as immunoreactivity score ( IRS ) , the percentage of positive cells to γH2AX and Tunel . The analysis was performed on 3 mice per group , the points represent the number of field analysed for each condition . Data information : For H , data are shown as mean ± SD . For ( H ) , P values are determined by Mann - Whitney test .

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G. Representative images of IHC analysis of the indicated markers from tumors of the experiment showed in E. Scale bar: 50μm. H. The histograms show: the expression levels of TRF2 measured as immunoreactivity score (IRS), the percentage of positive cells to γH2AX and Tunel. The analysis was performed on 3 mice per group, the points represent the number of field analysed for each condition. Data information: For H, data are shown as mean ± SD. For (H), P values are determined by Mann-Whitney test.


A . Representative images of intestine sections from mice previously treated with LNPs - Empty or LNPs - miR - 182 - 3p . H & E staining ( scale bar : 200μm ) and IHC analysis with TRF2 or γH2AX antibodies are shown ( scale bar : 50μm ) . B , C . Quantification of TRF2 expression as immunoreactivity score ( IRS ) ( B ) and of γH2AX positive cells ( % ) ( C ) on intestine samples . D . Representative H & E ( scale bar : 200μm ) , TRF2 and γH2AX images of skin samples corresponding to LNPs - Empty or LNPs - miR - 182 - 3p treated animals ( scale bar : 50μm ) . E , F . Quantification of TRF2 expression as immunoreactivity score ( IRS ) ( E ) and of γH2AX positive cells ( % ) ( F ) on skin samples . G . Representative H & E ( scale bar : 200μm ) , TRF2 and γH2AX images of bone marrow samples corresponding to LNPs - Empty or LNPs - miR - 182 - 3p treated animals ( scale bar : 50μm ) . H , I . Quantification of TRF2 expression as immunoreactivity score ( IRS ) ( H ) and of γH2AX positive cells ( % ) ( I ) on bone marrow samples . Data information : For B , C , E , F , H , I , data are shown as mean ± SD . A Mann - Whitney test t test was used to calculate statistical significance . Four mice per group were analysed the points represent the number of field analysed for each condition .

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A. Representative images of intestine sections from mice previously treated with LNPs-Empty or LNPs-miR-182-3p. H&E staining (scale bar: 200μm) and IHC analysis with TRF2 or γH2AX antibodies are shown (scale bar: 50μm). B,C. Quantification of TRF2 expression as immunoreactivity score (IRS) (B) and of γH2AX positive cells (%) (C) on intestine samples. D. Representative H&E (scale bar: 200μm), TRF2 and γH2AX images of skin samples corresponding to LNPs-Empty or LNPs-miR-182-3p treated animals (scale bar: 50μm). E,F. Quantification of TRF2 expression as immunoreactivity score (IRS) (E) and of γH2AX positive cells (%) (F) on skin samples. G. Representative H&E (scale bar: 200μm), TRF2 and γH2AX images of bone marrow samples corresponding to LNPs-Empty or LNPs-miR-182-3p treated animals (scale bar: 50μm). H,I. Quantification of TRF2 expression as immunoreactivity score (IRS) (H) and of γH2AX positive cells (%) (I) on bone marrow samples. Data information: For B,C,E,F,H,I, data are shown as mean ± SD. A Mann-Whitney test t test was used to calculate statistical significance. Four mice per group were analysed the points represent the number of field analysed for each condition.


( a ) UVRAG interaction with C - Vps . 293T cells were co - transfected with Flag - UVRAG and HA - Vps16 , HA - Vps11 , HA - Vps18 , HA - Vps33 or HA - Vps39 . Whole - cell lysates ( WCLs ) were used for immunoprecipitation ( IP ) with an anti - Flag antibody , followed by immunoblotting ( IB ) with an anti - HA antibody . 1 % WCL was used as the input .

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(a) UVRAG interaction with C-Vps. 293T cells were co-transfected with Flag-UVRAG and HA-Vps16, HA-Vps11, HA-Vps18, HA-Vps33 or HA-Vps39. Whole-cell lysates (WCLs) were used for immunoprecipitation (IP) with an anti-Flag antibody, followed by immunoblotting (IB) with an anti-HA antibody. 1% WCL was used as the input.


( b ) Interaction between endogenous UVRAG and C - Vps subunits . WCLs of 293T cells were used for IP with control serum ( control ) or an anti - UVRAG antibody , followed by IB with the indicated antibodies . The right panel shows endogenous protein expression .

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(b) Interaction between endogenous UVRAG and C-Vps subunits. WCLs of 293T cells were used for IP with control serum (control) or an anti-UVRAG antibody, followed by IB with the indicated antibodies. The right panel shows endogenous protein expression.


( c ) Confocal microscopy analysis of the colocalization of endogenous UVRAG and Vps16 or Vps18 in HeLa cells . Insets highlight colocalization .

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(c) Confocal microscopy analysis of the colocalization of endogenous UVRAG and Vps16 or Vps18 in HeLa cells. Insets highlight colocalization.


( d ) Confocal microscopy analysis of the colocalization of UVRAG with C - Vps subunits in HeLa cells transfected with Flag - UVRAG , and epitope - tagged C - Vps subunits as indicated .

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(d) Confocal microscopy analysis of the colocalization of UVRAG with C-Vps subunits in HeLa cells transfected with Flag-UVRAG, and epitope-tagged C-Vps subunits as indicated.


( e ) Colocalization of UVRAG - C - Vps in early endosomes . HeLa cells were co - transfected with EGFP - Vps16 and Flag - UVRAG , stained with antibodies to LAMP1 , LAMP2 , EEA1 and Rab5 , followed by confocal microscopy . All images are representative of at least three independent experiments . Scale bars , 5 μm . The raw data for a and b are shown in Supplementary Information , Fig .

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(e) Colocalization of UVRAG-C-Vps in early endosomes. HeLa cells were co-transfected with EGFP-Vps16 and Flag-UVRAG, stained with antibodies to LAMP1, LAMP2, EEA1 and Rab5, followed by confocal microscopy. All images are representative of at least three independent experiments. Scale bars, 5 μm. The raw data for a and b are shown in Supplementary Information, Fig. S6


( a ) The N - terminal C2 domain or the C - terminal region of UVRAG interacts individually with Vps16 . 293T cells were co - transfected with HA - Vps16 , together with vector , Flag - UVRAG or Flag - UVRAG mutants . WCLs were immunoprecipitated with anti - Flag followed by immunoblotting with an anti - HA antibody .

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(a) The N-terminal C2 domain or the C-terminal region of UVRAG interacts individually with Vps16. 293T cells were co-transfected with HA-Vps16, together with vector, Flag-UVRAG or Flag-UVRAG mutants. WCLs were immunoprecipitated with anti-Flag followed by immunoblotting with an anti-HA antibody.


( b ) HeLa cells were transfected with EGFP - Vps16 together with Flag - UVRAG mutants as indicated and stained with anti - Flag ( red ) , followed by confocal microscopy . Scale bars , 5 μm .

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(b) HeLa cells were transfected with EGFP-Vps16 together with Flag-UVRAG mutants as indicated and stained with anti-Flag (red), followed by confocal microscopy. Scale bars, 5 μm.


( c ) Interactions of the UVRAG C - terminal truncated mutants with Vps16 . At 48 h post - transfection with HA - Vps16 and Flag - UVRAG C - terminal mutants , 293T WCLs were immunoprecipitated with anti - Flag , followed by immunoblotting with anti - HA .

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(c) Interactions of the UVRAG C-terminal truncated mutants with Vps16. At 48 h post-transfection with HA-Vps16 and Flag-UVRAG C-terminal mutants, 293T WCLs were immunoprecipitated with anti-Flag, followed by immunoblotting with anti-HA.


( d ) UVRAG C2 domain or C - terminal region deletion abolishes Vps16 binding . At 48 h post - transfection with HA - Vps16 together with Flag - UVRAG or its mutants , 293T WCLs were immunoprecipitated with anti - Flag followed by immunoblotting with an anti - HA antibody .

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(d) UVRAG C2 domain or C-terminal region deletion abolishes Vps16 binding. At 48 h post-transfection with HA-Vps16 together with Flag-UVRAG or its mutants, 293T WCLs were immunoprecipitated with anti-Flag followed by immunoblotting with an anti-HA antibody.


( a ) HeLa . Vec and HeLa . UVRAG cells transfected with HA - Vps16 and GFP - LC3 were treated with 2 μM rapamycin for 2 h , and processed for confocal microscopy ( left panel ; scale bars , 5 μm ) . The percentage of GFP - LC3 punctae positive for HA - Vps16 staining was quantified ( right panel ; data are mean ± s . e . m . , n = 60 , * P 0 . 01 ) .

(a) HeLa.Vec and HeLa.UVRAG cells transfected with HA-Vps16 and GFP-LC3 were treated with 2 μM rapamycin for 2 h, and processed for confocal microscopy (left panel; scale bars, 5 μm). The percentage of GFP-LC3 punctae positive for HA-Vps16 staining was quantified (right panel; data are mean ± s.e.m., n = 60, *P 0.01).


( b ) Effects of Vps16 on UVRAG - mediated autophagosome formation . Light microscopic quantification of autophagosomes in HeLa . Vec and HeLa . UVRAG cells ( upper left panel ) or in HCT116 . Vec and HCT116 . UVRAG cells ( bottom left panel ) when transfected with GFP - LC3 together with control siRNA or Vps16 siRNA ( data are mean ± s . e . m . , n = 60 for HeLa cells ; n = 450 for HCT116 cells , * P 0 . 01 ; * * P 0 . 001 ) . Immunoblotting of Vps16 and tubulin are shown in the right panel .

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(b) Effects of Vps16 on UVRAG-mediated autophagosome formation. Light microscopic quantification of autophagosomes in HeLa.Vec and HeLa.UVRAG cells (upper left panel) or in HCT116.Vec and HCT116.UVRAG cells (bottom left panel) when transfected with GFP-LC3 together with control siRNA or Vps16 siRNA (data are mean ± s.e.m., n = 60 for HeLa cells; n = 450 for HCT116 cells, *P 0.01; **P 0.001). Immunoblotting of Vps16 and tubulin are shown in the right panel.


( a ) UVRAG enhances the colocalization efficiency of GFP - LC3 with LAMP1 . HeLa . Vec and HeLa . UVRAG cells transfected with GFP - LC3 were either untreated or treated with rapamycin ( 2 μM ) in the absence or presence of bafilomycin A1 ( 0 . 1 μM ) and stained for LAMP1 . Insets highlight the colocalization . The percentage of LAMP1 - positive autophagosomes was calculated in each setting ( data are mean ± s . e . m . , n = 200 ; 5 independent experiments , * * P 0 . 01 ; * P 0 . 05 ) .

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(a) UVRAG enhances the colocalization efficiency of GFP-LC3 with LAMP1. HeLa.Vec and HeLa.UVRAG cells transfected with GFP-LC3 were either untreated or treated with rapamycin (2 μM) in the absence or presence of bafilomycin A1 (0.1 μM) and stained for LAMP1. Insets highlight the colocalization. The percentage of LAMP1-positive autophagosomes was calculated in each setting (data are mean ± s.e.m., n = 200; 5 independent experiments, **P 0.01; *P 0.05).


( b ) UVRAG enhances the acquisition of CD63 by autophagosomes . The GFP - LC3 - transfected HeLa . Vec and HeLa . UVRAG cells were treated as described in a and stained for CD63 and the percentage of CD63 - positive autophagosomes was calculated ( data are mean ± s . e . m . , n = 200 , * * P 0 . 01 ; * P 0 . 05 ) .

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(b) UVRAG enhances the acquisition of CD63 by autophagosomes. The GFP-LC3-transfected HeLa.Vec and HeLa.UVRAG cells were treated as described in a and stained for CD63 and the percentage of CD63-positive autophagosomes was calculated (data are mean ± s.e.m., n = 200, **P 0.01; *P 0.05).


( c ) UVRAG promotes autophagic degradation of long - lived proteins . HeLa . Vec and HeLa . UVRAG cells were incubated for 16 h with L - 3H - Leu ( 1 μCi ml - 1 ) . The degradation of long - lived proteins was measured at the indicated time points in complete medium ( NC ) , EBSS alone ( Starv . ) , or EBSS + 0 . 1 μM bafilomycin A1 ( Starv . + BalfA1 ) . Results ( data are mean ± s . e . m . of triplicates ) are representative of 2 independent experiments . Scale bars , 5 μm .

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(c) UVRAG promotes autophagic degradation of long-lived proteins. HeLa.Vec and HeLa.UVRAG cells were incubated for 16 h with L-3H-Leu (1 μCi ml-1). The degradation of long-lived proteins was measured at the indicated time points in complete medium (NC), EBSS alone (Starv.), or EBSS + 0.1 μM bafilomycin A1 (Starv.+BalfA1). Results (data are mean ± s.e.m. of triplicates) are representative of 2 independent experiments. Scale bars, 5 μm.


( a ) Colocalization of GFP - LC3 and LAMP1 . HeLa cells expressing wild - type or mutant UVRAG were transfected with GFP - LC3 and incubated under normal conditions or treated with rapamycin ( 2 μM ) . Autophagosome fusion with LAMP + structures was analysed by confocal microscopy ( left panel ; rapamycin - treated ) and quantified ( right upper panel ; data are mean ± s . e . m . , n = 100 ) . Arrows and insets show the LAMP1 + autophagosomes . The number of GFP - LC3 - positive dots per cell was counted using a fluorescence microscope ( right bottom panel ; data are mean ± s . e . m . , n = 60 , * P 0 . 05 ; * * P 0 . 0001 ) .

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(a) Colocalization of GFP-LC3 and LAMP1. HeLa cells expressing wild-type or mutant UVRAG were transfected with GFP-LC3 and incubated under normal conditions or treated with rapamycin (2 μM). Autophagosome fusion with LAMP+ structures was analysed by confocal microscopy (left panel; rapamycin-treated) and quantified (right upper panel; data are mean ± s.e.m., n = 100). Arrows and insets show the LAMP1+ autophagosomes. The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope (right bottom panel; data are mean ± s.e.m., n = 60, *P 0.05; **P 0.0001).


( b ) Vps16 or Vps18 knockdown inhibits UVRAG - mediated autophagosome maturation . HeLa . Vec ( left ) and HeLa . UVRAG ( right ) cells were transfected with GFP - LC3 together with control siRNA , Vps16 siRNA , or Vps18 siRNA , followed by confocal microscopy . Arrows in the top panel denote the autophagosomes with LAMP1 staining . Immunoblotting of Vps16 , or Vps18 and tubulin are shown in the middle panel . The percentage of LAMP1 + autophagosomes was calculated ( data are mean ± s . e . m . , n = 50 , * P 0 . 05 ) . Scale bars , 5 μm . The raw data of the immunoblots are shown in Supplementary Information , Fig . S6 .

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(b) Vps16 or Vps18 knockdown inhibits UVRAG-mediated autophagosome maturation. HeLa.Vec (left) and HeLa.UVRAG (right) cells were transfected with GFP-LC3 together with control siRNA, Vps16 siRNA, or Vps18 siRNA, followed by confocal microscopy. Arrows in the top panel denote the autophagosomes with LAMP1 staining. Immunoblotting of Vps16, or Vps18 and tubulin are shown in the middle panel. The percentage of LAMP1+ autophagosomes was calculated (data are mean ± s.e.m., n = 50, *P 0.05). Scale bars, 5 μm. The raw data of the immunoblots are shown in Supplementary Information, Fig. S6.


( a ) UVRAG expression promotes the recruitment of Rab7 GTPase to autophagosomes . HeLa . Vec and HeLa . UVRAG cells transfected with GFP - Rab7 and RFP - LC3 were maintained either under normal conditions ( NC ) or treated with rapamycin , followed by confocal microscopy . Insets highlight the Rab7 acquisition of autophagosomes . The percentage of Rab7 + - autophagosomes was quantified ( right panel ; data are mean ± s . e . m . , n = 60 , * * * P 0 . 001 ; * * P 0 . 01 ) . Scale bars , 5 μm .

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(a) UVRAG expression promotes the recruitment of Rab7 GTPase to autophagosomes. HeLa.Vec and HeLa.UVRAG cells transfected with GFP-Rab7 and RFP-LC3 were maintained either under normal conditions (NC) or treated with rapamycin, followed by confocal microscopy. Insets highlight the Rab7 acquisition of autophagosomes. The percentage of Rab7+-autophagosomes was quantified (right panel; data are mean ± s.e.m., n = 60, ***P 0.001; **P 0.01). Scale bars, 5 μm.


( b ) Rab7 GTPase activity is increased with UVRAG expression . At 48 h post - transfection with GFP - Rab7 together with vector , UVRAG or UVRAGΔN ( 1 - 147 ) vector , 293T WCLs were used for immunoprecipitation with anti - GFP , followed by the Rab7 GTPase activity assay . Left panel : Autoradiographs of the GTP hydrolysis products analysed by TLC . Right panel : Quantification of the percentage of GTP hydrolyzed by Rab7 ( data are mean n = 2 independent experiments ) .

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(b) Rab7 GTPase activity is increased with UVRAG expression. At 48 h post-transfection with GFP-Rab7 together with vector, UVRAG or UVRAGΔN(1-147) vector, 293T WCLs were used for immunoprecipitation with anti-GFP, followed by the Rab7 GTPase activity assay. Left panel: Autoradiographs of the GTP hydrolysis products analysed by TLC. Right panel: Quantification of the percentage of GTP hydrolyzed by Rab7 (data are mean n = 2 independent experiments).


( c ) Increased Rab7 - RILP interaction UVRAG is expressed . HCT116 . vector , HCT116 . UVRAG and HCT116 . UVRAGΔN ( 1 - 147 ) cells were transfected with GFP - Rab7 together with the GST - tagged Rab7 binding domain of RILP ( GST - RILPRBD , residues 243 - 318 ) . WCLs were used for GST pulldown , followed by immunoblotting with an anti - GFP antibody . The raw data of the immunoblots are shown in Supplementary Information , Fig . S6 .

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(c) Increased Rab7-RILP interaction UVRAG is expressed. HCT116.vector, HCT116.UVRAG and HCT116.UVRAGΔN(1-147) cells were transfected with GFP-Rab7 together with the GST-tagged Rab7 binding domain of RILP (GST-RILPRBD, residues 243-318). WCLs were used for GST pulldown, followed by immunoblotting with an anti-GFP antibody. The raw data of the immunoblots are shown in Supplementary Information, Fig. S6.


( a ) EGF - stimulated endocytosis and post - endocytic trafficking . Uptake of Alexa Fluor 488 - EGF ( green ) by HCT116 . Vec ( left panel ) and HCT116 . UVRAG ( right panel ) cells was followed by confocal microscopy . At the indicated times , cells were fixed and stained with antibodies to LAMP1 ( red ) , and Topro - 3 ( blue , to stain the nucleus ) . Magnified images of the insets ( fourth row ) highlight trafficking of EGF towards LAMP1 - positive structures .

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(a) EGF-stimulated endocytosis and post-endocytic trafficking. Uptake of Alexa Fluor 488-EGF (green) by HCT116.Vec (left panel) and HCT116.UVRAG (right panel) cells was followed by confocal microscopy. At the indicated times, cells were fixed and stained with antibodies to LAMP1 (red), and Topro-3 (blue, to stain the nucleus). Magnified images of the insets (fourth row) highlight trafficking of EGF towards LAMP1-positive structures.


( b ) EGFR degradation . HCT116 . Vec , HCT116 . UVRAG and HCT116 . UVRAGΔN ( 1 - 147 ) cells were treated with EGF ( 200 ng ml − 1 ) at 37 ° C for the indicated times . WCLs were subjected to immunoblotting with an anti - EGFR antibody . EGFR at each time point was quantified and indicated as a percentage relative to that at time zero ( right panel ; data are mean ± s . e . m . , n = 3 ) .

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(b) EGFR degradation. HCT116.Vec, HCT116.UVRAG and HCT116.UVRAGΔN(1-147) cells were treated with EGF (200 ng ml−1) at 37 °C for the indicated times. WCLs were subjected to immunoblotting with an anti-EGFR antibody. EGFR at each time point was quantified and indicated as a percentage relative to that at time zero (right panel; data are mean ± s.e.m., n = 3).


( c ) DQ - green BSA dequenching assay . Representative confocal microscopy images of DQ - BSA proteolysis ( left panel ) and flow cytometry analysis ( right panel ) of RAW264 . 7 cells expressing an empty vector ( RAW264 . 7 . Vec ; dotted black line ) , wild - type UVRAG ( red line ) or UVRAGΔN ( 1 - 147 ) mutant ( green line ) loaded with 10 μg ml − 1 DQ green - BSA for the indicated times . Background ( grey line ) represents samples without the BSA load .

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(c) DQ-green BSA dequenching assay. Representative confocal microscopy images of DQ-BSA proteolysis (left panel) and flow cytometry analysis (right panel) of RAW264.7 cells expressing an empty vector (RAW264.7.Vec; dotted black line), wild-type UVRAG (red line) or UVRAGΔN(1-147) mutant (green line) loaded with 10 μg ml−1 DQ green-BSA for the indicated times. Background (grey line) represents samples without the BSA load.


( d ) Effect of UVRAG on endosome fusion . RAW264 . 7 . Vec and RAW264 . 7 . UVRAG cells were pulse - labelled with Oregon green 514 - avidin and dextran - 568 for 10 min and chased for 30 min . Cells were then labelled with biotin - BSA for 10 min , and processed for confocal microscopy ( left panel ) . In the absence of biotin - BSA , Oregon green514 - avidin showed very low fluorescence ( first row of confocal image ) . The percentage of dextran - 568 - labelled endosomes with visible Oregon green staining ( denoted by arrows in the left panel ) was calculated from approximately 20 different cells in 2 independent experiments ( mean , right panel ) . Scale bars , 5 μm . The raw data of the immunoblots in b are shown in the Supplementary Information , Fig . S6 .

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(d) Effect of UVRAG on endosome fusion. RAW264.7.Vec and RAW264.7.UVRAG cells were pulse-labelled with Oregon green 514-avidin and dextran-568 for 10 min and chased for 30 min. Cells were then labelled with biotin-BSA for 10 min, and processed for confocal microscopy (left panel). In the absence of biotin-BSA, Oregon green514-avidin showed very low fluorescence (first row of confocal image). The percentage of dextran-568-labelled endosomes with visible Oregon green staining (denoted by arrows in the left panel) was calculated from approximately 20 different cells in 2 independent experiments (mean, right panel). Scale bars, 5 μm. The raw data of the immunoblots in b are shown in the Supplementary Information, Fig. S6.


( a ) Haematoxylin and eosin ( H & E ) staining of kidneys from control ( Flcnflox / flox ) , Flcn KO , Flcn / Tfeb DKO , Flcn / Tfe3 DKO , Flcn / Tfe3 DKO ; Tfeb - HET and Flcn KO ; Tfeb - HET mice at p18 ( replicated three times ) . Scale bars , 3 mm ( upper panels ) . Boxed areas are magnified in the bottom panels . Arrowhead indicates tubular papillary atypical hyperplasia . Scale bars , 100 μm ( lower panels ) .

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(a) Haematoxylin and eosin (H & E) staining of kidneys from control (Flcnflox/flox), Flcn KO, Flcn/Tfeb DKO, Flcn/Tfe3 DKO, Flcn/Tfe3 DKO; Tfeb-HET and Flcn KO; Tfeb-HET mice at p18 (replicated three times). Scale bars, 3 mm (upper panels). Boxed areas are magnified in the bottom panels. Arrowhead indicates tubular papillary atypical hyperplasia. Scale bars, 100 μm (lower panels).


( b ) Blood urea nitrogen ( BUN ) levels in mice of the indicated genotypes at p18 ( mean ± SD , n = 3 ) . One Way ANOVA and the Tukey ' s HSD posthoc test ( corrected for multiple comparisons ) was applied . Significance for each comparison is provided in Methods .

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(b) Blood urea nitrogen (BUN) levels in mice of the indicated genotypes at p18 (mean± SD, n=3). One Way ANOVA and the Tukey's HSD posthoc test (corrected for multiple comparisons) was applied. Significance for each comparison is provided in Methods.


( c ) Kaplan - Meyer survival analysis of the indicated genotypes ( n = 20 for each genotype ) ; log - rank test , p - value < 0 . 0001 .

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(c) Kaplan-Meyer survival analysis of the indicated genotypes (n=20 for each genotype); log-rank test, p-value < 0.0001.


( d ) Representative immunohistochemical ( IHC ) analysis of Tfeb or Tfe3 and Cadherin - 16 ( Cdh16 ) on adjacent kidney sections from mice of the indicated genotypes at p2 . Tfeb and Tfe3 were stained in DAB , Cdh16 was stained in teal . Nuclei were stained with haematoxylin II ( blue ) . Magnification : 10x , scale bar : 100 µm . Magnification : 20x ( in inset image ) , scale bar : 25 µm . ( e ) Quantification of TFEB or TFE3 levels relative to cadherin 16 ( indicated as ratio ) ; values represent mean ± S . E . M ( n = 4 biological replicates for each genotype ) . A Welch ' s One Way ANOVA test with the Dunnett ' s T3 multiple comparisons test was applied . Significance for each comparison is provided in Methods . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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(d) Representative immunohistochemical (IHC) analysis of Tfeb or Tfe3 and Cadherin-16 (Cdh16) on adjacent kidney sections from mice of the indicated genotypes at p2. Tfeb and Tfe3 were stained in DAB, Cdh16 was stained in teal. Nuclei were stained with haematoxylin II (blue). Magnification: 10x, scale bar:100 µm. Magnification: 20x (in inset image), scale bar:25 µm. (e) Quantification of TFEB or TFE3 levels relative to cadherin 16 (indicated as ratio); values represent mean ± S.E.M (n=4 biological replicates for each genotype). A Welch's One Way ANOVA test with the Dunnett's T3 multiple comparisons test was applied. Significance for each comparison is provided in Methods. *P < 0.05; **P < 0.01; ***P < 0.001.


( a ) Abdominal imaging , H & E staining and immunohistochemical staining for TFE3 , TFEB , GPNMB and NPC1 in Hybrid RCC tumors from BHD patients 1 and 2 , BHD Chromophobe RCC tumor ( patient 3 ) , BHD papillary / clear RCC ( patients 4 , 5 and 6 ) , a BHD patient with clear / eosinophilic / papillary RCC ( patient 7 , origin of the BHD RCC cell line UOK257 ) and an area of normal kidney from the BHD patient 3 . In the row 1 - 7 , the arrows point to the tumor in the right or left kidney of BHD patients 1 - 7 ; in the bottom row , the arrow points to uninvolved , normal kidney in BHD patient 3 .

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(a) Abdominal imaging, H&E staining and immunohistochemical staining for TFE3, TFEB, GPNMB and NPC1 in Hybrid RCC tumors from BHD patients 1 and 2, BHD Chromophobe RCC tumor (patient 3), BHD papillary/clear RCC (patients 4, 5 and 6), a BHD patient with clear/eosinophilic/papillary RCC (patient 7, origin of the BHD RCC cell line UOK257) and an area of normal kidney from the BHD patient 3. In the row 1-7, the arrows point to the tumor in the right or left kidney of BHD patients 1-7; in the bottom row, the arrow points to uninvolved, normal kidney in BHD patient 3.


( d ) Correlation between transcriptomic and proteomic analysis of BHD - associated kidney tumor samples ( n = 7 biological replicates ) relative to control kidney tissues ( n = 5 biological replicates ) .

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(d) Correlation between transcriptomic and proteomic analysis of BHD-associated kidney tumor samples (n=7 biological replicates) relative to control kidney tissues (n=5 biological replicates).


Plots show tumor volumes in mice injected with UOK257 / shLuc ( n = 8 ) or UOK257 / shTFEB ( n = 10 ) ( b ) p value = 0 . 036 for plot in ( b )

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Plots show tumor volumes in mice injected with UOK257/shLuc (n=8) or UOK257/shTFEB (n=10) (b) p value = 0.036 for plot in (b)