( A ) WISH for cmyb expression at 60hpf in wild type slco2b1 - / - . ( B ) of cmyb - expressing . Each n represents number of cmyb - expressing for each ( biological replicates ) . Each experiment has been repeated three independent times . Data information : Centre values denote mean , error values denote s . e . m . statistical was completed using an unpaired two t . * * * P < . 001 Scale bar 500μm ( A )

O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WISH for cmyb expression at 60hpf in wild type and slco2b1-/- embryos. (B) Quantification of cmyb-expressing cells. Each n represents the number of cmyb-expressing cells for each embryo (biological replicates). Each experiment has been repeated three independent times. Data information: Centre values denote the mean, and error values denote s.e.m. The statistical analysis was completed using an unpaired two tailed t test. ***P < .001 Scale bar 500μm (A)


( C ) Schematic indicating imaging in tail at 60hpf , as indicated by black box ; imaging in CHT of kdrl : mCherry ; cmyb : GFP injected with control - slco2b1 - Mos . ( D ) of HSPCs associated to ECs . Each n represents number of yellow spots for each ( biological replicates ) . Each experiment has been repeated three independent times . Data information : Centre values denote mean , error values denote s . e . m . statistical was completed using an unpaired two t . * * * P < . 001 Scale bar 200μm ( C )

O O O O O O O B-TISSUE O O O O O O O O O O O B-TISSUE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Schematic indicating the imaging area in the tail at 60hpf, as indicated by the black box; fluorescence imaging in the CHT of kdrl:mCherry;cmyb:GFP embryos injected with control- and slco2b1-Mos. (D) Quantification of HSPCs associated to ECs. Each n represents the number of yellow spots for each embryo (biological replicates). Each experiment has been repeated three independent times. Data information: Centre values denote the mean, and error values denote s.e.m. The statistical analysis was completed using an unpaired two tailed t test. ***P < .001 Scale bar 200μm (C)


( E ) Experimental outline of time - lapse live imaging in controls slco2b1 - morphants cmyb : GFP + , using Imaris software . Data information : statistical was completed using an unpaired two t . * * * P < .

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O B-GENEPROD 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) Experimental outline and quantification of time-lapse live imaging in controls and slco2b1-morphants cmyb:GFP+ cells, using Imaris software. Data information: The statistical analysis was completed using an unpaired two tailed t test. ***P < .001


( A ) Anti - GFP pH3 immunostainings of either controls ( Ctrl - Mo ) or slco2b1 - morphants ( slco2b1 - MO ) cmyb : GFP . ( B ) of number of pH3 + HSCs in controls or slco2b1 - morphants . Centre values denote mean , error values denote s . e . m , statistical was completed using an unpaired two t . * * * P < . 001 . Data information : Scale bar 50μm ( A )

O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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) Anti-GFP and pH3 immunostainings of either controls (Ctrl-Mo) or slco2b1-morphants (slco2b1-MO) cmyb:GFP embryos. (B) Quantification of the number of the pH3+ HSCs in controls or slco2b1-morphants. Centre values denote the mean, and error values denote s.e.m, statistical analysis was completed using an unpaired two tailed t test. ***P < .001. Data information: Scale bar 50μm (A)


( D ) of PGE2 in control slco2b1 morphants . statistical was completed using an unpaired two t * P < . 01 . Centre values denote mean , error bars denote s . e . m .

O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantification of PGE2 concentration in control and slco2b1 morphants. The statistical analysis was completed using an unpaired two tailed t test *P < .01. Centre values denote the mean, and error bars denote s.e.m.


( E ) imaging in CHT of cmyb : GFP injected with control - slco2b1 - MOs treated with PGE2 . ( F ) of GFP positive . Statistical : one - way ANOVA , multiple comparison , * P < . 01 ; * * * * P < . 0001 . Centre values denote mean , error bars denote s . e . m . Data information : Scale bar 200μm ( E ) .

O O O O O B-TISSUE O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fluorescence imaging in the CHT of cmyb:GFP embryos injected with control- and slco2b1-MOs and treated with PGE2. (F) Quantification of GFP positive cells. Statistical analysis: one-way ANOVA, multiple comparison, *P < .01; ****P < .0001. Centre values denote the mean, and error bars denote s.e.m. Data information: Scale bar 200μm (E).


( A ) imaging of CHT of double transgenic cd45 : CFP - NTR / runx1 : mcherry injected with control - or slco2b1 - morpholinos , after treatment with DMSO . ( B ) imaging of CHT of double transgenic cd45 : CFP - NTR / runx1 : mcherry injected with control - or slco2b1 - morpholinos , after treatment with DMSO + MTZ . ( C ) imaging of CHT of double transgenic cd45 : CFP - NTR / runx1 : mcherry injected with control - or slco2b1 - morpholinos , after treatment with DMSO + MTZ + PGH2 . ( D ) of runx1 : mcherry positive - . Each dot represents number of red for each ( biological replicates ) . Each experiment has been repeated three independent times . ( E ) of cd45 : CFP positive - . Each dot represents number of blue for each ( biological replicates ) . Each experiment has been repeated three independent times . Data information for ( D ) ( E ) : Statistical was completed using one - way ANOVA , multiple comparison . * * * * P < . 0001 . Centre values denote mean , error bars denote s . e . m Scale bar 200μm ( A - C ) .

O O O O O B-TISSUE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O B-TISSUE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-TISSUE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fluorescence imaging of the CHT of double transgenic cd45:CFP-NTR/runx1:mcherry embryos injected with control- or slco2b1-morpholinos, after treatment with DMSO. (B) Fluorescence imaging of the CHT of double transgenic cd45:CFP-NTR/runx1:mcherry embryos injected with control- or slco2b1-morpholinos, after treatment with DMSO+MTZ. (C) Fluorescence imaging of the CHT of double transgenic cd45:CFP-NTR/runx1:mcherry embryos injected with control- or slco2b1-morpholinos, after treatment with DMSO+MTZ+PGH2. (D) Quantification of runx1:mcherry positive-cells. Each dot represents the number of red cells for each embryo (biological replicates). Each experiment has been repeated three independent times. (E) Quantification of cd45:CFP positive-cells. Each dot represents the number of blue cells for each embryo (biological replicates). Each experiment has been repeated three independent times.Data information for (D) and (E): Statistical analysis was completed using one-way ANOVA, multiple comparison test. ****P<.0001. Centre values denote the mean, and error bars denote s.e.m Scale bar 200μm (A-C).


( A ) WISH for cmyb at 60hpf in kdrl : Gal4 + injected with control - slco2b1 - morpholinos / or co - injected with Tol2 - UAS : slco2b1 vector tol2 mRNA . region indicated by dashed bas been magnified shows in ( B ) . ( C ) statistical were completed using one - way ANOVA , multiple comparison . * * * * P < . 0001 . Centre values denote mean , error bars denote s . e . m . Each dot represents number of cmyb - expressing for each ( biological replicates ) . Each experiment has been repeat three independent times .

O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O

(A) WISH for cmyb at 60hpf in kdrl:Gal4+ embryos injected with control- and slco2b1- morpholinos and/or co-injected with the Tol2-UAS:slco2b1 vector and tol2 mRNA. The region indicated by the dashed bas been magnified and shows in (B). (C) Quantification and statistical analysis were completed using one-way ANOVA, multiple comparison test. ****P< .0001. Centre values denote the mean, and error bars denote s.e.m. Each dot represents the number of cmyb-expressing cells for each embryo (biological replicates). Each experiment has been repeat three independent times.


( A ) Immunohistochemistry on paraffin sections for cKit ( HSPCs marker ) CD31 ( endothelial marker ) at E13 E16 stages of mouse liver . ( B ) distribution between cKithigh endothelial ( n = 79 from 3 E13 livers ; n = 92 from 3 E16 livers ) , binned into 20μm intervals . Statistical was completed using t - . * P < . 01 ; * * P < . 001 ; * * * P < . 0001 . Centre values denote mean , error values denote s . e . m nuclei marked with DAPI . Scale bar

O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O B-ORGANISM B-TISSUE O O O O O O B-GENEPROD B-CELL_TYPE O O O O O O O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O B-SMALL_MOLECULE O O O

(A) Immunohistochemistry on paraffin sections for cKit (HSPCs marker) and CD31 (endothelial cell marker) at E13 and E16 stages of mouse fetal liver. (B) Distance distribution between cKithigh cells and endothelial cells (n=79 from 3 E13 fetal livers; n=92 from 3 E16 fetal livers), binned into 20μm intervals. Statistical analysis was completed using t-test. *P<.01; **P<.001; ***P<.0001. Centre values denote the mean, and error values denote s.e.m All nuclei marked with DAPI. Scale bar 20μm


( C ) Immunohistochemistry on paraffin sections for cKit ( HSPCs marker ) F4 / 80 ( macrophage marker ) at E13 E16 stages of mouse liver . ( D ) distribution between cKithigh macrophages ( n = 54 from 3 E13 livers ; n = 64 from 3 E16 livers ) , binned into 5μm intervals . Statistical was completed using t - . nuclei marked with DAPI . Scale bar

O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O B-ORGANISM B-TISSUE O O O O O O B-GENEPROD B-CELL_TYPE O O O O O O O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O

(C) Immunohistochemistry on paraffin sections for cKit (HSPCs marker) and F4/80 (macrophage cell marker) at E13 and E16 stages of mouse fetal liver. (D) Distance distribution between cKithigh cells and macrophages (n=54 from 3 E13 fetal livers; n=64 from 3 E16 fetal livers), binned into 5μm intervals. Statistical analysis was completed using t-test. All nuclei marked with DAPI. Scale bar 20μm


( E ) Immunohistochemistry on paraffin sections for cKit ( HSPCs marker ) MBP ( neutrophil marker ) at E13 E16 stages of mouse liver . ( F ) distribution between cKithigh neutrophils ( n = 45 from 3 E13 livers ; n = 53 from 3 E16 livers ) , binned into 20μm intervals . Statistical was completed using t - * * * P < . 001 . nuclei marked with DAPI . Scale bar

O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O B-ORGANISM B-TISSUE O O O O O O B-GENEPROD B-CELL_TYPE O O O O O O O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O

(E) Immunohistochemistry on paraffin sections for cKit (HSPCs marker) and MBP (neutrophil cell marker) at E13 and E16 stages of mouse fetal liver. (F) Distance distribution between cKithigh cells and neutrophils (n=45 from 3 E13 fetal livers; n=53 from 3 E16 fetal livers), binned into 20μm intervals. Statistical analysis was completed using t-test ***P<.001. All nuclei marked with DAPI. Scale bar 20μm


A . Representative of HeLa treated with 250 μM lysosomotropic drug LLOMe or equal volume of DMSO ( Ctrl ) for 1 h before fixation immunostained with Hoechst ( blue ) , anti - CHMP4B ( green ) , anti - GAL3 ( red ) anti - LAMP1 ( ) are presented . treated with LLOMe show increased recruitment of ESCRT - III to lysosomes when compared to Ctrl . Number of foci per were quantified are indicated as mean ± SD . Data from > 86 per condition from three independent experiments . CHMP4B , GAL3 , LAMP1 foci per ( Ctrl versus LLOMe treatment ) : p = 0 . 0398 , p = 0 . 0033 , p = 0 . 2400 ( Student " s t ‐ ) , respect Data information : Scale bars : 10 μm 1 μm ( inset )

O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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. Representative fluorescence micrographs of HeLa cells treated with 250 μM lysosomotropic drug LLOMe or equal volume of DMSO (Ctrl) for 1 h before fixation and immunostained with Hoechst (blue), anti-CHMP4B (green), anti-GAL3 (red) and anti-LAMP1 (white) are presented. Cells treated with LLOMe show increased recruitment of ESCRT-III protein to lysosomes when compared to Ctrl cells. Number of foci per cell were quantified and are indicated as mean ± SD. Data from >86 cells per condition from three independent experiments. CHMP4B, GAL3, LAMP1 foci per cell (Ctrl versus LLOMe treatment): p=0.0398, p=0.0033, p=0.2400 (Student"s t‐test), respect Data information: Scale bars: 10 μm and 1 μm (inset)


B . HeLa stably expressing CHMP4B - eGFP mCherry - Galectin3 treated as in ( A ) stained with CD63 antibody are shown . Number of foci per were quantified are indicated as mean ± SD . Data is quantified from > 87 per condition from three independent experiments . CHMP4B , GAL3 , CD63 foci per ( Ctrl versus LLOMe treatment ) : p = 0 . 0111 , p = 0 . 0050 , p = 0 . 3793 ( Student " s t ‐ ) , respect Data information : Scale bars : 10 μm 1 μm ( inset )

O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY O 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-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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. HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 treated as in (A) and stained with CD63 antibody are shown. Number of foci per cell were quantified and are indicated as mean ± SD. Data is quantified from >87 cells per condition from three independent experiments. CHMP4B, GAL3, CD63 foci per cell (Ctrl versus LLOMe treatment): p=0.0111, p=0.0050, p=0.3793 (Student"s t‐test), respect Data information: Scale bars: 10 μm and 1 μm (inset)


To screen for ESCRT that are involved in endolysosomal repair process , were incubated with 250 μM LLOMe for 30 min processed for immunofluorescence . TSG101 , a component of ESCRT - I , EAP30 , an ESCRT - II , are clearly recruited to sites of damage compared to DMSO control ( Ctrl ) . As shown , ESCRT - III together with ALIX is recruited to damaged endolysosomes . In contrast , HRS , a component of ESCRT - 0 , HD - PTP show no recruitment to damaged endomembranes . Number of foci per was quantified from > 85 per condition from three independent experiments are presented as mean ± SD . Statistical significance for number of foci per in Ctrl versus LLOMe treatment was determined using Student ' s t ‐ , p - values for which are : HRS p = 0 . 9257 , GAL3 p = 0 . 0050 ; TSG101 p = 0 . 0243 , GAL3 p = 0 . 0489 ; EAP30 p = 0 . 0006 , GAL3 p = 0 . 0307 ; CHMP2A p = 0 . 0284 , GAL3 p = 0 . 0260 ; CHMP4B p = 0 . 0028 , GAL3 p = 0 . 0414 ; VPS4A p = 0 . 0309 , GAL3 p = 0 . 0461 ; ALIX p = 0 . 0249 , GAL3 p = 0 . 0471 ; HD - PTP p = 0 . 4898 , GAL3 p = 0 . 0105 . Data information : Scale bars : 5 μm 1 μm ( inset ) .

O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O B-EXP_ASSAY 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-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

To screen for ESCRT proteins that are involved in the endolysosomal repair process, cells were incubated with 250 μM LLOMe for 30 min and processed for immunofluorescence. TSG101, a component of the ESCRT-I complex, and EAP30, an ESCRT-II protein, are clearly recruited to the sites of damage compared to the DMSO control (Ctrl). As shown, the ESCRT-III complex together with ALIX is recruited to damaged endolysosomes. In contrast, HRS, a component of the ESCRT-0 complex, and HD-PTP show no recruitment to damaged endomembranes. Number of foci per cell was quantified from >85 cells per condition from three independent experiments and are presented as mean ± SD. Statistical significance for number of foci per cell in Ctrl versus LLOMe treatment was determined using Student's t‐test, the p-values for which are: HRS p=0.9257, GAL3 p=0.0050; TSG101 p=0.0243, GAL3 p=0.0489 ; EAP30 p=0.0006, GAL3 p=0.0307; CHMP2A p=0.0284, GAL3 p=0.0260; CHMP4B p=0.0028, GAL3 p=0.0414; VPS4A p=0.0309, GAL3 p=0.0461; ALIX p=0.0249, GAL3 p=0.0471; HD-PTP p=0.4898, GAL3 p=0.0105. Data information: Scale bars: 5 μm and 1 μm (inset).


HeLa stably expressing CHMP4B - eGFP were transfected with siRNAs against : ( A ) HR 48 h post - transfection were used for live - imaging experiments . LLOMe was added using perfusion system . A . Depletion of HRS does not alter dynamics of CHMP4B - eGFP recruitment as compared to siCtrl during treatment with LLOMe Data information : graphs represent average CHMP4B foci per quantified from three independent live - imaging experiments per condition . Error bars correspond to 95 % confidence intervals . Data from > 56 per condition were analyzed in each experiment . Right panels show knockdown efficiency of siRNA oligos as detected by Western blot ( * , nonspecific immunoreactivity )

B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O

HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs against: (A) HR 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system. A. Depletion of HRS does not alter the dynamics of CHMP4B-eGFP recruitment as compared to siCtrl during treatment with LLOMe Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)


HeLa stably expressing CHMP4B - eGFP were transfected with siRNAs agains ( B ) TSG10 48 h post - transfection were used for live - imaging experiments . LLOMe was added using perfusion system B . Downregulation of TSG101 , with two independent siRNAs causes a delay in CHMP4B - eGFP recruitment as compared to siCtrl , indicating an important role of ESCRT - I in recruiting downstream components Data information : graphs represent average CHMP4B foci per quantified from three independent live - imaging experiments per condition . Error bars correspond to 95 % confidence intervals . Data from > 56 per condition were analyzed in each experiment . Right panels show knockdown efficiency of siRNA oligos as detected by Western blot ( * , nonspecific immunoreactivity )

B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O

HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs agains (B) TSG10 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system B. Downregulation of TSG101, with two independent siRNAs causes a delay in CHMP4B-eGFP recruitment as compared to siCtrl, indicating an important role of the ESCRT-I complex in recruiting the downstream components Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)


HeLa stably expressing CHMP4B - eGFP were transfected with siRNAs agains ( C ) CHMP2 48 h post - transfection were used for live - imaging experiments . LLOMe was added using perfusion system C . CHMP2A depletion accumulates stabilizes CHMP4B - eGFP positive foci Data information : graphs represent average CHMP4B foci per quantified from three independent live - imaging experiments per condition . Error bars correspond to 95 % confidence intervals . Data from > 56 per condition were analyzed in each experiment . Right panels show knockdown efficiency of siRNA oligos as detected by Western blot ( * , nonspecific immunoreactivity )

B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O

HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs agains (C) CHMP2 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system C. CHMP2A depletion accumulates and stabilizes CHMP4B-eGFP positive foci Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)


HeLa stably expressing CHMP4B - eGFP were transfected with siRNAs against ( D ) ALI 48 h post - transfection were used for live - imaging experiments . LLOMe was added using perfusion system D . siRNA mediated depletion of ALIX shows no significant change on dynamics of CHMP4B recruitment Data information : graphs represent average CHMP4B foci per quantified from three independent live - imaging experiments per condition . Error bars correspond to 95 % confidence intervals . Data from > 56 per condition were analyzed in each experiment . Right panels show knockdown efficiency of siRNA oligos as detected by Western blot ( * , nonspecific immunoreactivity )

B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O

HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs against (D) ALI 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system D. siRNA mediated depletion of ALIX shows no significant change on dynamics of CHMP4B recruitment Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)


HeLa stably expressing CHMP4B - eGFP were transfected with siRNAs against ( E ) both TSG101 ALIX 48 h post - transfection were used for live - imaging experiments . LLOMe was added using perfusion system E . Simultaneous depletion of TSG101 ALIX caused almost no recruitment of CHMP4B upon induction of endolysosomal damage Data information : graphs represent average CHMP4B foci per quantified from three independent live - imaging experiments per condition . Error bars correspond to 95 % confidence intervals . Data from > 56 per condition were analyzed in each experiment . Right panels show knockdown efficiency of siRNA oligos as detected by Western blot ( * , nonspecific immunoreactivity )

B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O

HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs against (E) both TSG101 and ALIX 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system E. Simultaneous depletion of TSG101 and ALIX caused almost no recruitment of CHMP4B upon induction of endolysosomal damage Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)


A . HeLa stably expressing CHMP4B - eGFP were treated with 250 μM LLOMe 75 nM Lysotracker DND - 99 fixed at different time points as indicated . After 10 min of LLOMe treatment CHMP4B is recruited whereas number of Lysotracker positive foci is reduced . After 30 min , lysosomes gain back functionality ( judging by increased number of Lysotracker foci ) appear recovered after 1 h indicating that ESCRT is able to seal damaged endolysosomal membranes . Representative confocal for each time point are shown . Scale bars : 5

O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O

A. HeLa cells stably expressing CHMP4B-eGFP were treated with 250 μM LLOMe and 75 nM Lysotracker DND-99 and fixed at different time points as indicated. After 10 min of LLOMe treatment CHMP4B is recruited whereas the number of Lysotracker positive foci is reduced. After 30 min, lysosomes gain back functionality (judging by the increased number of Lysotracker foci) and appear recovered after 1 h indicating that the ESCRT complex is able to seal the damaged endolysosomal membranes. Representative confocal images for each time point are shown. Scale bars: 5 μm


B . graph ( > 250 per condition from four independent experiments ) showing CHMP4B Lysotracker positive foci per at different time points . Error bars correspond to 95 % confidence

O O O O O O O O O O O O O O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O O O

B. Quantification graph (>250 cells per condition from four independent experiments) showing CHMP4B and Lysotracker positive foci per cell at different time points. Error bars correspond to 95% confidence intervals


C . graph showing dynamics of Lysotracker recovery in control ( siCtrl ) both TSG101 ALIX depleted . HeLa stably expressing CHMP4B - eGFP were co - transfected with siRNAs against TSG101 ALIX . 48 h post - transfection , were pre - treated for 20 min with 75 nM Lysotracker Deep - Red , which was used as a read - out . While decrease in number of Lysotracker spots is quickly recovered in siCtrl , simultaneous depletion of ALIX TSG101 leads to a severe impairment in lysosomal repair . Data from > 40 per condition from four independent live - imaging experiments are shown . Graph is normalized to occupied by . Error bars correspond to 95 % confidence intervals .

O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD O O B-CELL_LINE O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

C. Quantification graph showing dynamics of Lysotracker recovery in control (siCtrl) and both TSG101 and ALIX depleted cells. HeLa cells stably expressing CHMP4B-eGFP were co-transfected with siRNAs against TSG101 and ALIX. 48 h post-transfection, cells were pre-treated for 20 min with 75 nM Lysotracker Deep-Red, which was used as a read-out. While the decrease in the number of Lysotracker spots is quickly recovered in siCtrl, simultaneous depletion of ALIX and TSG101 leads to a severe impairment in lysosomal repair. Data from >40 cells per condition from four independent live-cell imaging experiments are shown. Graph is normalized to the area occupied by the cells. Error bars correspond to 95% confidence intervals.


Representative movie montage of a live - imaging experiment illustrating dynamics of CHMP4B - eGFP mCherry - Galectin3 recruitment at different time points before after LLOMe treatment . Using perfusion system , 250 μM LLOMe was added to HeLa stably expressing CHMP4B - eGFP mCherry - Galectin3 were acquired every 20 sec for a period of 20 min . As shown in representative movie montage graph , CHMP4B recruitment precedes Galectin3 , which is an established marker for detection of damaged endolysosomal membranes . graph is normalized to occupied by . Data were obtained from > 40 per condition from three independent experiments . Error bars correspond to 95 % confidence intervals . Data information : Scale bars : 5 μm .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O 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 movie montage of a live-cell imaging experiment illustrating the dynamics of CHMP4B-eGFP and mCherry-Galectin3 recruitment at different time points before and after LLOMe treatment. Using the perfusion system, 250 μM LLOMe was added to HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 and images were acquired every 20 sec for a period of 20 min. As shown in the representative movie montage and the quantification graph, CHMP4B recruitment precedes Galectin3, which is an established marker for detection of damaged endolysosomal membranes. The quantification graph is normalized to the area occupied by the cells. Data were obtained from >40 cells per condition from three independent experiments. Error bars correspond to 95% confidence intervals. Data information: Scale bars: 5 μm.


A . HeLa stably expressing CHMP4B - eGFP mCherry - Galectin3 were incubated with 250 μM LLOMe , fixed at different time points as indicated stained for ubiquitin . As presented , CHMP4B is recruited before ubiquitin Galectin3 upon lysosomal damage Data information : graphs are normalized to number where error bars represent 95 % confidence intervals . Data were obtained from > 150 per condition from three independent

O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-SMALL_MOLECULE B-GENEPROD O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

A. HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 were incubated with 250 μM LLOMe, fixed at different time points as indicated and stained for ubiquitin. As presented, CHMP4B is recruited before ubiquitin and Galectin3 upon lysosomal membrane damage Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments


B . HeLa stably expressing CHMP4B - eGFP were treated as in ( A ) stained for p62 . CHMP4B is recruited before p62 on damaged lysosomes . number of LAMP1 - positive foci appears stable independently of LLOMe treatment Data information : graphs are normalized to number where error bars represent 95 % confidence intervals . Data were obtained from > 150 per condition from three independent

O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-SUBCELLULAR O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

B. HeLa cells stably expressing CHMP4B-eGFP were treated as in (A) and stained for p62. CHMP4B is recruited before p62 on the damaged lysosomes. The number of LAMP1-positive foci appears stable independently of LLOMe treatment Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments


C . Experimental setup as in ( A B ) where HeLa stably expressing CHMP4B - eGFP mCherry - Galectin3 were stained for LC3 . As shown , CHMP4B is recruited prior to LC3 Data information : graphs are normalized to number where error bars represent 95 % confidence intervals . Data were obtained from > 150 per condition from three independent

O O O O O O O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

C. Experimental setup as in (A and B) where HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 were stained for LC3. As shown, CHMP4B is recruited prior to LC3 Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments


A . HeLa were transfected with different siRNAs as indicated . 48 h post - transfection were treated with 250 μM LLOMe for 3 , 6 , 10 hours harvested stained for Annexin V . were immediately processed for flow cytometry . Already after 3 h of treatment with LLOMe , co - transfected with TSG101 + ALIX siRNA showed an increase in apoptotic , whereas TSG101 siRNA transfected showed elevated Annexin V after 6 h of treatment Data information : Graphs show average data from a set of three independent experiments where > 10 , 000 per condition were analyzed . Error bars represent . Statistical significance was determined using one - way ANOVA . p - values are presented where significant . * p - value ≤ 0 . 05 , * * p - value ≤ 0 . 01 * * * p - value ≤ 0 .

O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HeLa cells were transfected with different siRNAs as indicated. 48 h post-transfection cells were treated with 250 μM LLOMe for 3, 6, 10 hours harvested and stained for Annexin V. Cells were immediately processed for flow cytometry. Already after 3 h of treatment with LLOMe, cells co-transfected with TSG101+ALIX siRNA showed an increase in apoptotic cell death, whereas TSG101 siRNA transfected cells showed elevated Annexin V staining after 6 h of treatment Data information: Graphs show average data from a set of three independent experiments where >10,000 cells per condition were analyzed. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. p-values are presented where significant. *p-value≤ 0.05, **p-value≤ 0.01 and ***p-value≤ 0.001


B . Experimental set up as in ( A ) with additional propidium iodide ( PI ) of transfected HeLa Data information : Graphs show average data from a set of three independent experiments where > 10 , 000 per condition were analyzed . Error bars represent . Statistical significance was determined using one - way ANOVA . p - values are presented where significant . * p - value ≤ 0 . 05 , * * p - value ≤ 0 . 01 * * * p - value ≤ 0 .

O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Experimental set up as in (A) with additional propidium iodide (PI) staining of transfected HeLa cells Data information: Graphs show average data from a set of three independent experiments where >10,000 cells per condition were analyzed. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. p-values are presented where significant. *p-value≤ 0.05, **p-value≤ 0.01 and ***p-value≤ 0.001


A . HeLa stably expressing CHMP4B - eGFP were transfected with mCherry - Galectin3 plasmid . 24 h later , were infected with WT Coxiella burnetti . Live - imaging started 24 h post - infection several time points are indicated during next 24 h of observatio Arrows indicate Coxiella - containing vacuole ( DRAQ5 labeling ) becoming positive for CHMP4B - eGFP mCherry - Galectin3 . Data information : Scale bars : 50

O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-ORGANISM I-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O

A. HeLa cells stably expressing CHMP4B-eGFP were transfected with the mCherry-Galectin3 plasmid. 24 h later, cells were infected with WT Coxiella burnetti. Live-cell imaging started 24 h post-infection and several time points are indicated during the next 24 h of observatio Arrows indicate Coxiella-containing vacuole (DRAQ5 labeling) becoming positive for CHMP4B-eGFP and mCherry-Galectin3. Data information: Scale bars: 50 μm


B . viability replication upon TSG101 KD . HeLa were treated with either siCtrl or with two different siTSG101 for 48 h . Then were infected with mCherry - Coxiella burnetii for 48 h before lysis serial dilutions were made to infect Vero . 72 h later , infected were fixed , DAPI stained processed for quantitative image . Between 30 40 fields representing more than 9 , 000 per condition of three independent experiments were analyzed . Error bars represent SD . Statistical significance was determined using one - way ANOVA . * * p - value ≤ 0 . 01 * * * p - value ≤ 0 . 001 .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-GENEPROD O B-ORGANISM I-ORGANISM O O O O O O O O O O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Bacterial viability and replication assay upon TSG101 KD. HeLa cells were treated with either siCtrl or with two different siTSG101 for 48 h. Then cells were infected with mCherry-Coxiella burnetii for 48 h before lysis and serial dilutions were made to infect Vero cells. 72 h later, infected cells were fixed, DAPI stained and processed for quantitative image analysis. Between 30 and 40 fields representing more than 9,000 cells per condition of three independent experiments were analyzed. Error bars represent SD. Statistical significance was determined using one-way ANOVA test. **p-value≤ 0.01 and ***p-value≤ 0.001.


b - d ) Comparative characterization of CHO bulk transfection cultures , selected for stable expression of UnaG - or EGFP - encoding reporter constructs . b ) Flow cytometry revealed efficient induction of green , after treatment of both UnaG EGFP - expressing CHO bulk cultures with CoCl2 , while growth under hypoxia ( 1 % oxygen ) for 12 hours , selectively induced green only in dUnaG expressing . Under normoxia ( 21 % oxygen ) only background was observed . c ) Assessment of mean ( MFI ) to determine activity of 5xHRE - CMV promoter after 12 24hrs . increased between 12 24hrs .

O O O O O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O

b - d) Comparative characterization of CHO cell bulk transfection cultures, selected for stable expression of the UnaG- or EGFP-encoding reporter constructs.b) Flow cytometry revealed efficient induction of green fluorescence, after treatment of both UnaG and EGFP-expressing CHO bulk cultures with CoCl2, while growth under hypoxia (1% oxygen) for 12 hours, selectively induced green fluorescence only in dUnaG expressing cells. Under normoxia (21% oxygen) only background fluorescence was observed.c) Assessment of the mean fluorescence intensity (MFI) to determine the activity of the 5xHRE-CMV promoter after 12 and 24hrs. Fluorescence intensity increased between 12 and 24hrs.


d ) Representative of dUnaG - or dEGFP - expressing CHO grown either under normoxia ( 21 % oxygen ) , CoCl2 - treatment or hypoxia ( 1 % oxygen ) for 12 hours . Scale bars =

O O O O O O O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O

d) Representative images of dUnaG- or dEGFP-expressing CHO cells grown either under normoxia (21% oxygen), CoCl2-treatment or hypoxia (1% oxygen) for 12 hours. Scale bars= 50µm


e ) in dUnaG - or dEGFP - expressing CHO bulks grown under increasingly hypoxic conditions of 10 % , 5 % 1 % oxygen for 24 hrs indicated efficient induction of dUnaG sensor at oxygen concentrations below 5 % .

O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O

e) Fluorescence intensity in dUnaG- or dEGFP-expressing CHO cell bulks grown under increasingly hypoxic conditions of 10%, 5% and 1% oxygen for 24 hrs indicated efficient induction of the dUnaG sensor at oxygen concentrations below 5%.


f ) Maximal of UnaG - hypoxia reporter in CHO grown at 1 % oxygen for 24 hrs hours was only observed at serum concentrations of 5 % FCS or higher , reflecting dependence of UnaG on its serum cofactor bilirubin .

O O O O B-GENEPROD O O O O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O

f) Maximal fluorescence of the UnaG-based hypoxia reporter in CHO cells grown at 1% oxygen for 24 hrs hours was only observed at serum concentrations of 5% FCS or higher, reflecting the dependence of UnaG on its serum cofactor bilirubin.


b ) Microscopic assessment of averaged ( AFI ) of CHO bulk cultures stably expressing indicated UnaG variants or destabilized EGFP . Expression under hypoxia was induced by incubation in 1 % oxygen for 16 hrs then culture was continued for 24 hrs under normoxia . hysteresis after switching to normoxia was markedly reduced in singly - destabilized UnaG - variants , while doubly destabilized doUnaG displayed most rapid switching behavior , but least brightness . To reduce phototoxicity , illumination was kept minimal . Data points represent one viewfield of 425 µm2 ± .

O O O O O O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O 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-GENEPROD O O O O O O O O O O O O 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) Microscopic assessment of the averaged fluorescence intensity (AFI) of CHO cell bulk cultures stably expressing the indicated UnaG variants or destabilized EGFP. Expression under hypoxia was induced by incubation in 1% oxygen for 16 hrs and then culture was continued for 24 hrs under normoxia. Fluorescence hysteresis after switching to normoxia was markedly reduced in the singly-destabilized UnaG-variants, while the doubly destabilized doUnaG displayed the most rapid switching behavior, but the least brightness. To reduce phototoxicity, illumination intensity was kept minimal. Data points represent one viewfield of 425 µm2 ± SEM.


c ) Flow cytometry of induction decay kinetics of in CHO stably expressing either dUnaG or oUnaG . Open bars represent control cultures grown under normoxia . Solid bars indicate culture for 4 , 8 , 12 , 16 , 20 or 24 hrs under hypoxia ( 1 % oxygen ) . To assess deactivation , cultures previously kept under hypoxia ( 1 % oxygen ) for 24hrs were shifted to normoxia ( 21 % oxygen ) for indicated times . Error bars represent standard errors .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

c) Flow cytometry analysis of the induction and decay kinetics of the fluorescence intensity in CHO cells stably expressing either dUnaG or oUnaG. Open bars represent control cultures grown under normoxia. Solid bars indicate culture for 4, 8, 12, 16, 20 or 24 hrs under hypoxia (1% oxygen). To assess deactivation, cultures previously kept under hypoxia (1% oxygen) for 24hrs were shifted to normoxia (21% oxygen) for the indicated times. Error bars represent standard errors.


d ) Representative maximum projections ( MIPs ) from life cultures depicted in b . were grown for indicated times at hypoxic ( 1 % oxygen ) conditions subsequently switched for indicated times to normoxia ( 21 % oxygen ) ( red arrows in b ) to illustrate dynamics of different UnaG - hypoxia sensors . Increasing destabilization is associated with reduced brightness but , also reduced background improved switching behavior . Scale bars = 50 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

d) Representative maximum intensity projections (MIPs) from the life cell cultures depicted in b. Cells were grown for the indicated times at hypoxic (1% oxygen) conditions and subsequently switched for the indicated times to normoxia (21% oxygen)(red arrows in b) to illustrate the dynamics of the different UnaG-based hypoxia sensors. Increasing destabilization is associated with reduced brightness but, also reduced background and improved switching behavior. Scale bars = 50 µm.


a ) UnaG - hypoxia sensing is applicable in vivo as shown here using human Gli36 glioblastoma model . 500 Gli36 glioblastoma , constitutively expressing mCherry stably transfected with HRE - dUnaG sensor construct , were stereotactically transplanted into cortex of a SCIDmouse . Shown is a 30 µm cryosection of a growing 10 days after transplantation . are distinguished from surrounding cortex by mCherry expression . Blood vessels were contrasted by immunostaining against PECAM - 1 . Expression of dUnaG was visualized by its green predominates in areas with reduced vascular density ( outlined by in composite panel ( bottom right ) . b ) Representative from shown in a ) , which is located outside viewfield in a ) positioned more closely to border . Also at border UnaG - expressing are preferentially observed at a to PECAM - 1 + vessels .

O O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD O O B-TISSUE I-TISSUE O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O

a) UnaG-based hypoxia sensing is applicable in vivo as shown here using the human Gli36 glioblastoma model. 500 Gli36 glioblastoma cells, constitutively expressing mCherry and stably transfected with the HRE-dUnaG sensor construct, were stereotactically transplanted into the cortex of a SCIDmouse. Shown is a 30 µm cryosection of a growing tumor 10 days after transplantation. Tumor cells are distinguished from the surrounding cortex by mCherry expression. Blood vessels were contrasted by immunostaining against PECAM-1. Expression of dUnaG was visualized by its green fluorescence and predominates in areas with reduced vascular density (outlined by white line in the composite panel (bottom right).b) Representative area from the tumor shown in a), which is located outside the viewfield in a) positioned more closely to the tumor border. Also at the tumor border UnaG-expressing cells are preferentially observed at a distance to PECAM-1+ vessels.


c ) Areas of dUnaG expression correlate well with HIF - 1α stabilization . Immunostaining for HIF - 1α ( cyan ) revealed predominantly localization of stabilized HIF - 1α in that were also dUnaG - positive ( green ) . d ) of dUnaG expressing in c ) for mCherry HIF - 1α stabilization . dUnaG - positive were classified according to their average into background level above background level expression for mCherry HIF - 1α threshold was set in either case to channel 70 of 256 channels . Using this classification > 60 % of UnaG positive displayed only background level mCherry . On other , > 98 % of UnaG expressing also expressed HIF - 1α , which together suggests that here UnaG acts preferentially as a hypoxia sensor in vivo .

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O

c) Areas of dUnaG expression correlate well with HIF-1α stabilization. Immunostaining for HIF-1α (cyan) revealed the predominantly nuclear localization of stabilized HIF-1α in cells that were also dUnaG-positive (green).d) Analysis of dUnaG expressing cells in c) for mCherry fluorescence and HIF-1α stabilization. dUnaG-positive cells were classified according to their average fluorescence intensity into background level and above background level expression for mCherry and HIF-1α The threshold was set in either case to channel 70 of 256 intensity channels. Using this classification >60% of UnaG positive cells displayed only background level mCherry fluorescence. On the other hand, >98% of the UnaG expressing cells also expressed HIF-1α , which together suggests that here UnaG acts preferentially as a hypoxia sensor in vivo.


e ) dUnaG - senor was efficiently applied in intravital microscopy . 500 Gli36 glioblastoma stably expressing mCherry harboring dUnaG - sensor construct were positioned at 200µm depth subcortically in a SCIDmouse observed 4 days later through a cranial window using multiphoton microscopy . We noted two distinct areas of intense UnaG ( red lines ) . Shown is a MIP of a 60 µm z stack . vasculature was contrasted by intravenous injection of non - functionalized Qdots . f ) Depth color coding of MIP shown in e ) revealed spatial relation between vasculature , ( contrasted by Qdots ) , areas of increased dUnaG expression ( bordered by green lines ) expressing residual of mCherry . hypoxic areas were located towards top of imaged tissue cube therefore at different compared to large vessel at lower left corner ( red asterisk ) smaller vessels with a more axial orientation ( arrows ) . Scale bars =

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-TISSUE O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O 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) The dUnaG-based senor was efficiently applied in intravital microscopy. 500 Gli36 glioblastoma cells stably expressing mCherry and harboring the dUnaG-sensor construct were positioned at 200µm depth subcortically in a SCIDmouse and observed 4 days later through a cranial window using multiphoton microscopy. We noted two distinct areas of intense UnaG fluorescence (red lines). Shown is a MIP of a 60 µm z stack. The vasculature was contrasted by intravenous injection of non-functionalized Qdots.f) Depth color coding of the MIP shown in e) revealed the spatial relation between the vasculature, (contrasted by Qdots), areas of increased dUnaG expression (bordered by green lines) and tumor cells expressing residual levels of mCherry. The hypoxic areas were located towards the top of the imaged tissue cube and therefore at different levels compared to the large vessel at the lower left corner (red asterisk) and the smaller vessels with a more axial orientation (white arrows). Scale bars = 100µm


b ) Microscopic assessment of averaged ( AFI ) in CHO stably transfected with dUnOHR sensor . Hypoxia was induced by incubation in 1 % oxygen for 18 hrs , then culture was switched to normoxia for 24 hrs , followed by another 14 hrs hypoxia finally normoxia again . As expected UnaG is efficiently induced under hypoxia , while mOrange only appears after switch to normoxia only . increase in both green orange is limited under normoxia by subsiding HRE - mCMV promoter activity . This behavior is repeated in subsequent hypoxia / normoxia cycles . increase in absolute is due to proliferation during 72hrs culture period . Plotted is average of mean ± . c ) Visualization of of dUnOHR reporter during alternating hypoxia / normoxia cycles as described in b . ) MIPs of life cultures stably transfected with dUnOHR sensor construct illustrate temporally asynchronous of UnaG mOrange . Scale bars = 100 µm .

O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O 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-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD O O O O O O O

b) Microscopic assessment of the averaged fluorescence intensity (AFI) in CHO cells stably transfected with the dUnOHR sensor. Hypoxia was induced by incubation in 1% oxygen for 18 hrs, then the culture was switched to normoxia for 24 hrs, followed by another 14 hrs hypoxia and finally normoxia again. As expected UnaG fluorescence is efficiently induced under hypoxia, while mOrange fluorescence only appears after the switch to normoxia only. The increase in both green and orange fluorescence is limited under normoxia by the subsiding HRE-mCMV promoter activity. This behavior is repeated in subsequent hypoxia / normoxia cycles. The increase in absolute fluorescence intensity is due to proliferation during the 72hrs culture period. Plotted is the average of the mean ± SEM.c) Visualization of the fluorescence of the dUnOHR reporter during alternating hypoxia / normoxia cycles as described in b.) MIPs of life cell cultures stably transfected with the dUnOHR sensor construct illustrate the temporally asynchronous fluorescence of UnaG and mOrange. Scale bars = 100 µm.


a ) Overview of a induced by deep cortical deposition of approximately 500 Gli36 stably transfected with dUnOHR sensor construct . perimeter of developing ( in merged picture , lower right panel ) was delineated by Hoechst 33342 . As observed for dUnaG reoxygenation sensor was expressed in compact areas with low vascular density . Vascular endothelium was identified by PECAM - 1 . 30 µm cryosection . Scale bar = 500µm . b ) Details of shown in a ) . Sparse , distinct are characterized by intense green orange ( shown as orange ) are located preferentially to edge of hypoxic phalanx ( arrows ) . A number of in close proximity of nearest blood vessel display weak green orange ( asterisks ) , indicative of previously hypoxic environment subsequent reoxygenation . Scale bar = 50 µm .

O O O O O O O O B-TISSUE O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Overview of a tumor induced by deep cortical deposition of approximately 500 Gli36 cells stably transfected with the dUnOHR sensor construct. The perimeter of the developing tumor (white line in the merged picture, lower right panel) was delineated by Hoechst 33342 staining. As observed for dUnaG the reoxygenation sensor was expressed in compact areas with low vascular density. Vascular endothelium was identified by PECAM-1 staining. 30 µm cryosection. Scale bar = 500µm.b) Details of the tumor shown in a). Sparse, distinct cells are characterized by intense green and orange fluorescence (shown as orange) and are located preferentially to the edge of the hypoxic phalanx (white arrows). A number of cells in close proximity of the nearest blood vessel display weak green and orange fluorescence (asterisks), indicative of previously hypoxic environment and subsequent reoxygenation. Scale bar = 50 µm.


c ) Recently reoxygenated ( arrows ) cluster at edge of hypoxic areas are often detected in proximity of vessels with a homogeneous caliber ( double arrows ) . Scale bar = 100 µm .

O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O

c) Recently reoxygenated cells (arrows) cluster at the edge of hypoxic areas and are often detected in the proximity of vessels with a homogeneous caliber (double arrows). Scale bar = 100 µm.


d ) Previously hypoxic , frequently undergo apoptosis after reoxygenation . We noted an accumulation of intensely orange fluorescing spots at edges of compact UnaG positive areas . Scale bar = 50µm .

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-GENEPROD O O O O O O O O

d) Previously hypoxic cells, frequently undergo apoptosis after reoxygenation. We noted an accumulation of intensely orange fluorescing spots at the edges of compact UnaG positive areas. Scale bar = 50µm.


e ) TUNEL of sections revealed that tissue areas rich in intense mOrange colocalized with areas of increased apoptosis . Scale bar = 50µm .

O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O O

e) TUNEL staining of tumor sections revealed that the tissue areas rich in intense mOrange aggregates colocalized with areas of increased apoptosis. Scale bar = 50µm.


A - B . Correlation of MCU MCUb expression with breast cancer clinical stages . Median - centered log2 mRNA expression of MCU MCUb were collected from TCGA breast cancer dataset ( http : / / tcga - data . nci . nih . gov / docs / publications / brca _ 2012 / ) . Data were plotted analysed against ( T1 - T4 ) ( A ) regional lymph node infiltration ( N0 - N3 ) ( B ) , according to AJCC Cancer Staging Manual ( 7th edition ) . Linear regression with different stages was implemented . Parameters of linear regression are shown . Number of samples for each stage are shown in parentheses .

O O O O O O B-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-TISSUE O O O O O O O O O O O O O O O O O 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-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O 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. Correlation of MCU and MCUb expression levels with breast cancer clinical stages. Median-centered log2 mRNA expression levels of MCU and MCUb were collected from the TCGA breast cancer dataset (http://tcga-data.nci.nih.gov/docs/publications/brca_2012/). Data were plotted and analysed against tumor size (T1-T4) (A) and regional lymph node infiltration (N0-N3) (B), according to the AJCC Cancer Staging Manual (7th edition). Linear regression analysis with different stages was implemented. Parameters of linear regression are shown. Number of samples for each stage are shown in parentheses.


C - E . MCU silencing reduces Ca2 + mit uptake in TNBC . were transfected with siMCU or siControl . After 48h , [ Ca2 + ] mit uptake upon ATP ( C , E ) or Histamine ( D ) stimulation was measured ( n = 10 ) . P - values : * * * p = 0 . 0008 , * * * p < 0 . 0001 , * * * p = 0 . 0001 , respectively .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O 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. MCU silencing reduces Ca2+mit uptake in TNBC cells. Cells were transfected with siMCU or siControl. After 48h, [Ca2+]mit uptake upon ATP (C, E) or Histamine (D) stimulation was measured (n=10). P-values: ***p= 0.0008, ***p<0.0001, ***p= 0.0001, respectively.


F - H . MCU silencing impairs TNBC migration . were transfected with siMCU or siControl . day after transfection , a linear scratch was obtained on monolayer through a vertically held P200 tip ( time - point 0h ) . migration into scratched was monitored 48 hours later . covered was measured expressed as a relative to 0 - hour time - point ( n = 12 ) . P - value : * * * p < 0 . 0001 .

O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O 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-H. MCU silencing impairs TNBC cell migration. Cells were transfected with siMCU or siControl. The day after transfection, a linear scratch was obtained on the cell monolayer through a vertically held P200 tip (time- point 0h). Cell migration into the scratched area was monitored 48 hours later. The covered area was measured and expressed as a percentage relative to 0-hour time-point (n=12). P-value: ***p<0.0001.


I - K . proliferation is mainly unaffected by MCU depletion . were transfected with siMCU or siControl . number was counted every 24 hours for 3 days ( 72 - hour time point corresponds to 48 - hour time - point of wound healing ) . Results are expressed as R / R0 where R0 is number of at time of transfection ( 0 hour time - point ) ( n = 6 ) . P - value : * p = 0 . 05 .

O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K. Cell proliferation is mainly unaffected by MCU depletion. Cells were transfected with siMCU or siControl. Cell number was counted every 24 hours for 3 days (the 72-hour time point corresponds to the 48-hour time-point of wound healing assay). Results are expressed as ratio R/R0 where R0 is the number of cells at the time of transfection (0 hour time-point) (n=6). P-value: *p=0.05.


A . Re - expression of mMCU rescues motility of MCU - silenced . were transfected with siMCU or siControl . Ad - mMCU was used to re - express MCU ( Ad - GFP was used as control ) . MCU expression was verified by western blot . day after transduction a linear scratch was made ( time - point 0h ) . migration into wounded was monitored at 48 hour time - point covered was measured ( n = 12 ) . P - value : * * * p < 0 . 0001 .

O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O 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. Re-expression of mMCU rescues cell motility of MCU-silenced cells. Cells were transfected with siMCU or siControl. Ad-mMCU was used to re-express MCU (Ad-GFP was used as control). MCU protein expression was verified by western blot. The day after transduction a linear scratch was made (time-point 0h). Cell migration into the wounded area was monitored at 48 hour time-point and the covered area was measured (n=12). P-value: ***p<0.0001.


B . MCU silencing blunts invasiveness . Stable shMCU shControl - expressing spheroids were plated let grow into collagen - I ( 0 hour time - point ) . Spheroid was measured at 0 - hour 48 - hour ( n = 8 ) . Scale bar 300 μm . P - value : * * * p = 0 . 0003 .

O O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O 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. MCU silencing blunts cell invasiveness. Stable shMCU and shControl-expressing spheroids were plated and let grow into collagen-I (0 hour time-point). Spheroid area was measured at 0-hour and 48-hour (n=8). Scale bar 300 μm. P-value: ***p=0.0003.


C . MCU silencing reduces clonogenic potential of MDA - MB - 231 . Stable shMCU shControl - expressing were plated at low confluence ( 2x103 / well of a 6 - well plate ) . After 7 days number of colonies was counted ( minimum 30 / colony , n = 8 ) . P - value : * * p = 0 . 0027 .

O O B-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. MCU silencing reduces the clonogenic potential of MDA-MB-231 cells. Stable shMCU and shControl-expressing cells were plated at low confluence (2x103/well of a 6-well plate). After 7 days the number of colonies was counted (minimum 30 cells/colony, n=8). P-value: **p=0.0027.


D . MCU depletion does not induce . were transfected with siMCU or siControl . 72 hours later , apoptosis necrosis were measured by FITC - Annexin V Propidium Iodide ( PI ) detection ( Q1 : PI positive , Q2 : PI FITC - Annexin V positive , Q3 : PI FITC - Annexin V negative , Q4 : FITC - Annexin V positive . n = 6 ) .

O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

D. MCU depletion does not induce cell death. Cells were transfected with siMCU or siControl. 72 hours later, cell apoptosis and necrosis were measured by FITC-Annexin V and Propidium Iodide (PI) detection (Q1: PI positive, Q2: PI and FITC-Annexin V positive, Q3: PI and FITC-Annexin V negative, Q4: FITC-Annexin V positive. n=6).


E . MCU depletion does not alter cycle . were transfected with siMCU or siControl . 72 hours later , cycle distribution was monitored by Propidium Iodide ( PI ) detection ( n = 6 ) .

O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O

E. MCU depletion does not alter cell cycle. Cells were transfected with siMCU or siControl. 72 hours later, cell cycle distribution was monitored by Propidium Iodide (PI) detection (n=6).


A . mass volume was measured at specific time - points until day of sacrifice ( day 39 post - injection for control , day 46 56 p . i . for MCU - / - cl . 1 cl . 2 , respectively ) . P - values : * * * p = 0 . 0001 , * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O 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. Tumor mass volume was measured at specific time-points until the day of sacrifice (day 39 post-injection for control, day 46 and 56 p.i. for MCU-/- cl.1 and cl.2, respectively). P-values: ***p=0.0001, ***p<0.0001.


B . left : in vivo metastasis at homolateral axillary of three representative mice per group at time of sacrifice . Right : total flux . P - values : * * p = 0 . 01 , * p = 0 . 02 .

O O O O O O O O O O O O O B-ORGANISM O O O O O O O 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. left: in vivo metastasis at the homolateral axillary area of three representative mice per group at the time of sacrifice. Right: total flux analysis. P-values: **p=0.01, *p=0.02.


C . Lymph nodes at time of sacrifice . P - values : * * * p = 0 . 0010 , * * p = 0 . 0014 .

O O B-TISSUE I-TISSUE O O 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. Lymph nodes weight at the time of sacrifice. P-values: ***p=0.0010, **p=0.0014.


D . Human cytokeratin 7 ( CK7 ) of three representative lymph nodes per group . Scale bar = 500 µm .

O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O O O O

D. Human cytokeratin 7 (CK7) IHC staining of three representative lymph nodes per group. Scale bar= 500 µm.


E . Left : of three representative lungs per group collected ex vivo at time of sacrifice . Right : total flux . P - values : * * p = 0 . 0031 , * * * p = 0 . 0004 .

O O O O O O O B-TISSUE O O O O O O O O O O O O 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. Left: images of three representative lungs per group collected ex vivo at the time of sacrifice. Right: total flux analysis. P-values: **p=0.0031, ***p= 0.0004.


A - C . FLIM of NADH / NADPH . lifetimes of NAD ( P ) H autofluorescence in stable shControl shMCU expressing were imaged . Representative of distribution of τbound on an weighted pseudocolored scale ( 2 . 2 - 2 . 5 ns ) are shown . Scale bars = 20 μm ( A ) . Mean + / - SE of τbound ( B ) relative NADH NADPH intensities ( C ) calculated from equation in ( Blacker et al . , 2014 ) are shown ( n = 3 ) . P - values : * * p = 0 . 01 , * * p = 0 . 002 , respectively .

O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O 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-C. FLIM analysis of cellular NADH/NADPH levels. Fluorescence lifetimes of NAD(P)H autofluorescence in stable shControl and shMCU expressing cells were imaged. Representative images of the distribution of τbound on an intensity weighted pseudocolored scale (2.2-2.5 ns) are shown. Scale bars = 20 μm (A). Mean +/- SE of τbound (B) and relative NADH and NADPH intensities (C) calculated from equation in (Blacker et al., 2014) are shown (n=3). P-values: **p=0.01, **p=0.002, respectively.


D - E . Measurement of redox state of NADH / NAD + couple . Representative measurements of NADH at steady state at minimal maximal reduced state ( D ) . of steady state redox state ( E ) ( n = 3 ) .

O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O

D-E. Measurement of the redox state of the NADH/NAD+ couple. Representative measurements of NADH intensity at steady state and at minimal and maximal reduced state (D). Percentage of the steady state redox state (E) (n=3).


F . MCU depletion impairs mitochondrial rate of ATP production . were transfected with siMCU or siControl . 48 hours later , were treated with 5 . 5 mM 2 - deoxy - D - glucose for 1 hour ATP were quantified ( n = 6 ) . P - value : * * * p = 0 . 0009 .

O O B-GENEPROD O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

F. MCU depletion impairs the mitochondrial rate of ATP production. Cells were transfected with siMCU or siControl. 48 hours later, cells were treated with 5.5 mM 2-deoxy-D-glucose for 1 hour and cellular ATP levels were quantified (n=6). P-value: ***p=0.0009.


A . Antioxidant treatments decrease migration . A linear scratch was obtained on monolayer through a vertically held P200 tip ( time - point 0h ) . were treated for 48 hours with N - Acetylcysteine ( NAC ) or Dithioerythritol ( DTE ) . migration into wounded was monitored at 48 - hour time - point covered was measured ( n = 12 ) . P - values : * * p = 0 . 008 , * * * p < 0 . 0001 , * * p = 0 . 005 .

O O O O O B-EXP_ASSAY O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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. Antioxidant treatments decrease cell migration. A linear scratch was obtained on cell monolayer through a vertically held P200 tip (time-point 0h). Cells were treated for 48 hours with N-Acetylcysteine (NAC) or Dithioerythritol (DTE). Cell migration into the wounded area was monitored at 48-hour time-point and the covered area was measured (n=12). P-values: **p=0.008, ***p<0.0001, **p=0.005.


B . Scavenging of mitochondrial ROS decreases migration . A linear scratch was obtained on a monolayer through a vertically held P200 tip ( time - point 0h ) . were treated for 48 hours with 50 µM MitoTEMPO . migration into wounded was monitored at 48 hour time - point covered was measured ( n = 12 ) . P - value : * * * p < 0 . 0001 .

O O O O B-SUBCELLULAR B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O 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-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O 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. Scavenging of mitochondrial ROS decreases cell migration. A linear scratch was obtained on a cell monolayer through a vertically held P200 tip (time-point 0h). Cells were treated for 48 hours with 50 µM MitoTEMPO. Cell migration into the wounded area was monitored at 48 hour time-point and the covered area was measured (n=12). P-value: ***p<0.0001.


C . MCU silencing does not affect matrix pH . were transfected with siMCU or siControl SypHer2 probe . 48 hours later , matrix pH was measured ( n = 22 ) .

O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O O O O O O

C. MCU silencing does not affect matrix pH. Cell were transfected with siMCU or siControl and SypHer2 probe. 48 hours later, matrix pH was measured (n=22).


D - E . Mitochondrial H2O2 are critically blunted after MCU depletion . were transfected with shMCU or shControl , together with ratiometric YFP - biosensor pHyper - dMito ( D ) or mitochondrial H2O2 - sensitive HyPerRed probe ( E ) . 48 hours later , H2O2 production was measured ( n = 35 ) . P - values : * p = 0 . 02 , * p = 0 . 05 , respectively .

O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-E. Mitochondrial H2O2 levels are critically blunted after MCU depletion. Cells were transfected with shMCU or shControl, together with the ratiometric YFP-based biosensor pHyper-dMito (D) or the mitochondrial H2O2-sensitive HyPerRed probe (E). 48 hours later, H2O2 production was measured (n=35). P-values: *p=0.02, *p=0.05, respectively.


F . Mitochondrial superoxide are critically blunted after MCU silencing . were transfected with siMCU or siControl . 48 hours later , were loaded with red dye MitoSOXTM superoxide anion were measured ( n = 25 ) . P - value : * p = 0 . 04 .

O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

F. Mitochondrial superoxide levels are critically blunted after MCU silencing. Cells were transfected with siMCU or siControl. 48 hours later, cells were loaded with the red dye MitoSOXTM and superoxide anion levels were measured (n=25). P-value: *p=0.04.


G . Mitochondrial GSSG / GSH is critically reduced after MCU silencing . were transfected with shMCU or shControl , together with mitochondrial targeted mitGrx1 - roGFP2 probe . 96 hours later , glutathione redox potential ( EGSH ) was measured ( n = 46 ) . P - value : * * * p < 0 . 0001 .

O O B-SUBCELLULAR B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O 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. Mitochondrial GSSG/GSH ratio is critically reduced after MCU silencing. Cells were transfected with shMCU or shControl, together with the mitochondrial targeted mitGrx1-roGFP2 probe. 96 hours later, the glutathione redox potential (EGSH) was measured (n=46). P-value: ***p<0.0001.


A . MCU silencing reduces HIF - 1α . were transfected with siMCU or siControl . HIF - 1α were detected 48 hours later .

O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

A. MCU silencing reduces HIF-1α protein levels. Cells were transfected with siMCU or siControl. HIF-1α protein levels were detected 48 hours later.


B . MCU silencing reduces MG132 - mediated HIF - 1α hydroxylated HIF - 1α accumulation . were transfected with siMCU or siControl . 48 hours later , were treated with 10 μM of proteasome inhibitor MG132 . Left : were revealed by western blot . Right : by densitometry ( n = 5 ) .

O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

B. MCU silencing reduces MG132-mediated HIF-1α and hydroxylated HIF-1α protein accumulation. Cells were transfected with siMCU or siControl. 48 hours later, cells were treated with 10 μM of the proteasome inhibitor MG132. Left: Protein levels were revealed by western blot. Right: quantification by densitometry (n=5).


C . ROS increase HIF - 1α transcription . were treated O / N with 100 µM paraquat to induce ROS production . HIF - 1α mRNA were measured by RealTime - PCR ( n = 3 ) . P - value : * * p = 0 . 002 .

O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

C. ROS increase HIF-1α transcription. Cells were treated O/N with 100 µM paraquat to induce ROS production. HIF-1α mRNA levels were measured by RealTime-PCR (n=3). P-value: **p=0.002.


D - J . MCU silencing reduces mRNA of HIF - 1α , HIF - 2α , HIF - 1α target genes . were transfected with siMCU or siControl . mRNA expression was measured by RealTime - PCR ( n = 3 ) . P - values : ( HIF - 1α ) * * p = 0 . 0031 , * * p = 0 . 009 , ( HIF - 2α ) * * p = 0 . 01 , ( LOX ) * * * p = 0 . 001 , * * * p = 0 . 0005 , ( PDK1 ) * p = 0 . 02 , * * p = 0 . 009 , ( G6PI ) * p = 0 . 02 , * * p = 0 . 005 , ( CA IX ) * * p = 0 . 0026 , * * p = 0 . 0022 , ( HK2 ) * * p = 0 . 0024 , * p = 0 . 03 .

O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

D-J. MCU silencing reduces mRNA levels of HIF-1α, HIF-2α, and HIF-1α target genes. Cells were transfected with siMCU or siControl. mRNA expression was measured by RealTime-PCR (n=3). P-values: (HIF-1α) **p=0.0031, **p=0.009, (HIF-2α)**p=0.01, (LOX)***p=0.001, ***p=0.0005, (PDK1)*p=0.02, **p=0.009, (G6PI)*p=0.02, **p=0.005, (CA IX)**p=0.0026, **p=0.0022, (HK2) **p=0.0024, *p=0.03.


K . HIF - 1α overexpression rescues siMCU - mediated migration impairment . were transfected with siMCU or siControl . Wild type ( wt ) constitutively active ( ca ) HIF - 1α were expressed by retroviral infection ( pBABE was used as control ) . day after transduction , were scratched ( time - point 0h ) . migration into wounded was monitored at 48 hour time - point covered was measured ( n = 12 ) . P - values : * * * p < 0 . 0001 , * p = 0 . 04 .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K. HIF-1α overexpression rescues siMCU-mediated migration impairment. Cells were transfected with siMCU or siControl. Wild type (wt) and constitutively active (ca) HIF-1α were expressed by retroviral infection (pBABE was used as control). The day after transduction, cells were scratched (time-point 0h). Cell migration into the wounded area was monitored at 48 hour time-point and the covered area was measured (n=12). P-values: ***p<0.0001, *p=0.04.


L - M . MCU expression correlate with HIF1α ( L ) HIF1α - regulated genes ( M ) . A linear model ( lm ) to power of MCU expression predicting expression of HIF1α HIF1α - regulated genes was calculated plotted for each of 532 samples of TCGA database ( see Figure 1 ) . Equation R2 values of linear regression significance indicating deviation from 0 are shown . of 95 % prediction limit is shaded below above linear regression . HIF1α - regulated gene set was compiled from Broad Institute GSEA database ( http : / / www . broadinstitute . org / gsea / msigdb / cards / V $ HIF1 _ Q5 . html , merged sets of V $ HIF1 _ Q3 V $ HIF1 _ Q5 ) .

O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L-M. MCU expression levels correlate with HIF1α (L) and HIF1α-regulated genes (M). A linear model (lm) to test the power of MCU expression levels predicting the expression of HIF1α and HIF1α-regulated genes was calculated and plotted for each of the 532 samples of the TCGA database (see Figure 1). Equation and R2 values of the linear regression and significance indicating deviation from 0 are shown. The area of 95% prediction limit is shaded below and above the linear regression line. The HIF1α-regulated gene set was compiled from Broad Institute GSEA database (http://www.broadinstitute.org/gsea/msigdb/cards/V$HIF1_Q5.html, merged sets of V$HIF1_Q3 and V$HIF1_Q5).


( C ) Decreased of free carnitine modified carnitine species in PKM2 KO lines as compared with their parental counterparts ( n = 4 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - Student ' s t - , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 ∗ ∗ ∗ p < 0 . 001 . n . s means no significance .

O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Decreased levels of free carnitine and modified carnitine species in PKM2 KO cell lines as compared with their parental counterparts (n=4). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. n.s means no significance.


( E ) Increased acetyl - CoA in PKM2 KO MCF7 MDA - MB 231cells as compared with their parental counterparts ( n = 4 ) . ( F ) Decreased total cholesterol in PKM2 KO MCF7 MDA - MB 231cells as compared with their parental counterparts ( n = 3 ) . ( Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - Student ' s t - , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 ∗ ∗ ∗ p < 0 . 001 . n . s means no significance .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Increased acetyl-CoA in PKM2 KO MCF7 and MDA-MB 231cells as compared with their parental counterparts (n=4). (F) Decreased total cholesterol in PKM2 KO MCF7 and MDA-MB 231cells as compared with their parental counterparts (n=3). ( Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. n.s means no significance.


( G ) Western blot of TMEM33 , ABCA1 , SREBP1 , HMGCR , SCAP , FASN , LDLR , FDFT1 , I DI1 , RNF5 , INSIG 1 2 , Calnexin in PKM2 KO MCF7 MDA - MB - 231 , their respective parental . Arrow indicates SREBP1 . P : precursor , N : .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O

(G) Western blot analysis of TMEM33, ABCA1, SREBP1, HMGCR, SCAP, FASN, LDLR, FDFT1, I DI1, RNF5, INSIG 1 and 2, and Calnexin in PKM2 KO MCF7 and MDA-MB-231 cells, and their respective parental cells. Arrow indicates nuclear SREBP1. P: precursor, N: nuclear.


( A ) Immunofluorescence of SREBP1 in MCF7 parental PKM2 KO by confocal imaging . Scale bar , 5μm . ( B ) Quantitation of SREBP1 in A . n > = 25 as in ( A ) from three different fields . ( Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - Student ' s t - , ∗ p < 0 . 05 ∗ ∗ ∗ p < 0 . 001 .

O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of SREBP1 in MCF7 parental and PKM2 KO cells by confocal imaging. Scale bar, 5μm. (B) Quantitation of nuclear SREBP1 intensity in A. n>=25 cells as in (A) from three different fields. ( Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05 and ∗∗∗p < 0.001.


( G ) Western blot of TMEM33 , RNF5 , SCAP , SREBP1 , HMGCR , LDLR , FDFT1 , IDI1 , PKM2 in wild type , PKM2 KO , TMEM33 OE ( pool ) , two TMEM33OE clones ( clones # 1 # 8 ) MDA - MB - 231 . Arrow indicates SREBP1 . N : nucleus P : precursor .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O B-SUBCELLULAR O O O O

(G) Western blot analysis of TMEM33, RNF5, SCAP, SREBP1, HMGCR, LDLR, FDFT1, IDI1, and PKM2 in wild type, PKM2 KO, TMEM33 OE (pool), and two TMEM33OE clones (clones #1 and #8) MDA-MB-231 cells. Arrow indicates nuclear SREBP1. N: nucleus and P: precursor.


( B ) Western blot of PKM2 , PKM1 , NRF1 , TMEM33 , RNF5 SCAP in MEF ( PKM2fl / fl , Cre - ERT2 ) treated with 4 - OHT in a time course experiment .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-CELL_TYPE O B-GENEPROD O O O B-GENEPROD O B-SUBCELLULAR O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O

(B) Western blot analysis of PKM2, PKM1, NRF1, TMEM33, RNF5 and SCAP levels in MEF (PKM2fl/fl, Cre-ERT2) cells treated with 4-OHT in a time course experiment.


( C ) Co - immunoprecipitation of endogenous SCAP RNF5 in Flag - tagged TMEM33 stably expressed MCF7 . SEC61β is an ER , which is found to interact with TMEM33 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE O B-GENEPROD O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O B-GENEPROD O

(C) Co-immunoprecipitation of endogenous SCAP and RNF5 in Flag-tagged TMEM33 stably expressed MCF7 cells. SEC61β is an ER protein, which is found to interact with TMEM33.


( H ) RNF5 is involved in TMEM33 mediated SCAP polyubiquitination in 293T . 293T were stably transfected with shCtrl or shRNF5 constructs , then indicated plasmids were co - transfected into shCtrl or shRNF5 expressing 293T for 18 hours followed by MG - 132 treatment for 8 hours . FLAG M2 beads were used for pull - down .

O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(H) RNF5 is involved in TMEM33 mediated SCAP polyubiquitination in 293T cells. 293T cells were stably transfected with shCtrl or shRNF5 constructs, then the indicated plasmids were co-transfected into shCtrl or shRNF5 expressing 293T cells for 18 hours followed by MG-132 treatment for 8 hours. FLAG M2 beads were used for pull-down.


( D ) qRT - PCR analyses of selected SREBPs downstream genes in shCtrl shRNF5 PKM2 KO MDA - MB - 231 ( n = 3 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - Student ' s t - , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 ∗ ∗ ∗ p < 0 . 001 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) qRT-PCR analyses of selected SREBPs downstream genes in shCtrl and shRNF5 PKM2 KO MDA-MB-231 cells (n=3). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.


( G ) proliferation of Ctrl HA - RNF5 OE MCF7 measured by MTT assays ( n = 8 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - Student ' s t - , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 ∗ ∗ ∗ p < 0 . 001 .

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell proliferation of Ctrl and HA-RNF5 OE MCF7 cells measured by MTT assays (n=8). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.


( E ) Western blot analyses of TMEM33 RNF5 in NRF1 overexpressed MCF7 clones . ( F ) Western blot analyses of TMEM33 RNF5 in shCtrl shNRF1 PKM2 KO MCF7 . (

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-CELL_LINE O O

(E) Western blot analyses of TMEM33 and RNF5 in NRF1 overexpressed MCF7 clones. (F) Western blot analyses of TMEM33 and RNF5 in shCtrl and shNRF1 PKM2 KO MCF7 cells. (


( G ) Co - immunoprecipitation of VCP with FLAG - tagged PKM1 / 2 in PKM2 KO 293T . FLAG - tagged PKM1 or PKM2 construct was transiently transfected into PKM2 KO or parental 293T for 24 hours , followed by pull - down using FLAG - M2 beads . PKM1 , PKM2 VCP antibodies were used for immunoblotting .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O

(G) Co-immunoprecipitation of VCP with FLAG-tagged PKM1/2 in PKM2 KO 293T cells. FLAG-tagged PKM1 or PKM2 construct was transiently transfected into PKM2 KO or parental 293T cells for 24 hours, followed by pull-down using FLAG-M2 beads. PKM1, PKM2 and VCP antibodies were used for immunoblotting.


( E ) Representative H & E of TMEM33 SCAP in WT TMEM33 KO mouse livers . Scale bars , 100µm .

O O O O O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-ORGANISM B-TISSUE O O O O O O

(E) Representative H&E and IHC staining images of TMEM33 and SCAP in WT and TMEM33 KO mouse livers. Scale bars, 100µm.


( F , G ) Significant decrease of total plasma cholesterol ( F ) LDL - C ( G ) in TMEM33 - / - + / - mice relative to WT counterparts ( n > = 5 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - Student ' s t - , ∗ p < 0 . 05 ∗ ∗ ∗ p < 0 . 001 . n . s denotes no significance .

O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Significant decrease of total plasma cholesterol (F) and LDL-C (G) levels in TMEM33-/- and +/- mice relative to WT counterparts (n>=5). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05 and ∗∗∗p < 0.001. n.s denotes no significance.


( B ) growth curves of PKM2 WT or KO py8119 allografts in systemic PKM2 KO or WT mice ( n = 5 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using a two - Student ' s t - , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 ∗ ∗ ∗ p < 0 . 001 .

O O O B-EXP_ASSAY O O B-GENEPROD O O O B-CELL_LINE O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Tumor growth curves of PKM2 WT or KO py8119 cell line allografts in systemic PKM2 KO or WT mice (n=5). Data information: Data are displayed as means ± SD. Statistical significance was assessed using a two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.


( E ) growth curves of py8119 allografts treated with vehicle TEPP - 46 ( 50mg / kg ) for 17 days in mice ( n = 7 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using a two - Student ' s t - , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 ∗ ∗ ∗ p < 0 . 001 .

O O O B-EXP_ASSAY O O B-CELL_LINE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The growth curves of py8119 allografts treated with vehicle and TEPP-46 (50mg/kg) for 17 days in mice (n=7). Data information: Data are displayed as means ± SD. Statistical significance was assessed using a two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.


( A ) Electrophoretic ( under non - denaturing conditions ) of association between indicated IκBα species association reconstituted nucleosome particles ( NCP ) .

O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O

(A) Electrophoretic analysis (under non-denaturing conditions) of the association between the indicated IκBα species association and reconstituted nucleosome core particles (NCP).