Figure 1E Western blot analysis showing protein levels of p53 and genes related to mitochondrial homeostasis and function in GA of mice receiving PBS or indicated EVs . Data information : Western blot signals are quantified and normalized to Gapdh . F RT - qPCR showing relative levels of miR - 122 and indicated mRNAs in GA of indicated groups of mice . Data were normalized to U6 ( for miR - 122 ) or Ppib ( for all mRNAs ) and compared to control group receiving PBS ( one - way ANOVA , n = 4 or 5 mice per group ) . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant . H ROS levels in GA from indicated groups of mice detected by DHE staining ( red ) . DAPI ( blue ) shows the nuclei . Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis ( one - way ANOVA , n = 4 mice per group ) . Scale bar : 50 µm . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant . I ROS levels in GA from mice that had received indicated EV treatment for 5 weeks . Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis ( one - way ANOVA , n = 4 mice per group ) . Scale bar : 50 µm . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant . J RT - qPCR showing relative levels of indicated mRNAs in GA of indicated groups of mice ( one - way ANOVA , n = 5 mice per group ) . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant . K Relative mtDNA / nDNA ratio in GA determined by PCR ( one - way ANOVA , n = 5 mice per group ) . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant .

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Figure 1E Western blot analysis showing protein levels of p53 and genes related to mitochondrial homeostasis and function in GA of mice receiving PBS or indicated EVs. Data information: Western blot signals are quantified and normalized to Gapdh.F RT-qPCR showing relative levels of miR-122 and indicated mRNAs in GA of indicated groups of mice. Data were normalized to U6 (for miR-122) or Ppib (for all mRNAs) and compared to control group receiving PBS (one-way ANOVA, n=4 or 5 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.H ROS levels in GA from indicated groups of mice detected by DHE staining (red). DAPI (blue) shows the nuclei. Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis (one-way ANOVA, n=4 mice per group). Scale bar: 50 µm. Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.I ROS levels in GA from mice that had received indicated EV treatment for 5 weeks. Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis (one-way ANOVA, n=4 mice per group). Scale bar: 50 µm. Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.J RT-qPCR showing relative levels of indicated mRNAs in GA of indicated groups of mice (one-way ANOVA, n=5 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.K Relative mtDNA/nDNA ratio in GA determined by PCR (one-way ANOVA, n=5 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.


Figure 2C Responsiveness of the reporters to miR - 122 mimic in transfected C2C12 ( t - test , n = 3 biological replicates ) . Data information : In bar graph , values are shown as mean ± SD . * * P < 0 . 01 , ns : not significant . D CFSE signals in C2C12 myotubes treated with indicated CFSE - labeled Evs for 24 h , indicating EV uptake . Scale bar : 100 µm . Data information : In bar graph , values are shown as mean ± SD . * * P < 0 . 01 , ns : not significant .

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Figure 2C Responsiveness of the reporters to miR-122 mimic in transfected C2C12 (t-test, n=3 biological replicates). Data information: In bar graph, values are shown as mean ± SD. **P<0.01, ns: not significant.D CFSE signals in C2C12 myotubes treated with indicated CFSE-labeled Evs for 24 h, indicating EV uptake. Scale bar: 100 µm. Data information: In bar graph, values are shown as mean ± SD. **P<0.01, ns: not significant.


Figure 3A Western blot showing levels of indicated proteins in C2C12 treated with indicated EVs , or transfected as indicated with miR - 122 mimic or TP53 expression plasmid carrying silent mutation of the miR - 122 binding site . Data information : Western blot signals are quantified and normalized to Gapdh . C Relative ATP levels in C2C12 myotubes treated as indicated ( one - way ANOVA , n = 3 biological replicates ) . Data information : In bar graphs , values are shown as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant . D Relative ROS levels in C2C12 myotubes treated as indicated ( one - way ANOVA , n = 3 - 5 biological replicates ) . Data information : In bar graphs , values are shown as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant .

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Figure 3A Western blot showing levels of indicated proteins in C2C12 treated with indicated EVs, or transfected as indicated with miR-122 mimic or TP53 expression plasmid carrying silent mutation of the miR-122 binding site. Data information: Western blot signals are quantified and normalized to Gapdh.C Relative ATP levels in C2C12 myotubes treated as indicated (one-way ANOVA, n=3 biological replicates). Data information: In bar graphs, values are shown as mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.D Relative ROS levels in C2C12 myotubes treated as indicated (one-way ANOVA, n=3-5 biological replicates). Data information: In bar graphs, values are shown as mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.


Figure 4B Indicated MDA - MB - 231 - derived BC cells were injected into the mammary fat pad of female NSG mice to form tumors . Tumor - bearing and age - matched tumor - free mice were sacrificed for muscle collection at week 6 . Western blots show protein levels in GA . Western blot signals are quantified and normalized to Gapdh . D RT - qPCR indicated mRNAs ( D ) in GA of tumor - free and tumor - bearing mice . Data were normalized to Ppib ( for all mRNAs ) and compared to the tumor - free group ( one - way ANOVA , n = 4 or 5 mice per group ) . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant . F Representative TEM images showing SS and IMF mitochondria and their quantification in GA ( one - way ANOVA , n = 3 mice per group for the left subpanel and n = 154 - 164 mitochondria analyzed per group for the right subpanel ) . Scale bar : 1 µm . Yellow bars : depths of SS mitochondrial areas ; arrows : IMF mitochondrial area . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant . H Western blots of GA from female BALB / c mice bearing 4T1 - derived tumors with tumor - free mice as controls . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant . Western blot signals are quantified and normalized to Gapdh . I RT - qPCR showing levels of indicated genes in of GA from indicated BALB / c mice ( one - way ANOVA , n = 4 or 5 mice ) . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant .

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

Figure 4B Indicated MDA-MB-231-derived BC cells were injected into the mammary fat pad of female NSG mice to form tumors. Tumor-bearing and age-matched tumor-free mice were sacrificed for muscle collection at week 6. Western blots show protein levels in GA. Western blot signals are quantified and normalized to Gapdh.D RT-qPCR indicated mRNAs (D) in GA of tumor-free and tumor-bearing mice. Data were normalized to Ppib (for all mRNAs) and compared to the tumor-free group (one-way ANOVA, n=4 or 5 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.F Representative TEM images showing SS and IMF mitochondria and their quantification in GA (one-way ANOVA, n=3 mice per group for the left subpanel and n=154-164 mitochondria analyzed per group for the right subpanel). Scale bar: 1 µm. Yellow bars: depths of SS mitochondrial areas; arrows: IMF mitochondrial area. Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.H Western blots of GA from female BALB/c mice bearing 4T1-derived tumors with tumor-free mice as controls. Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant. Western blot signals are quantified and normalized to Gapdh.I RT-qPCR showing levels of indicated genes in of GA from indicated BALB/c mice (one-way ANOVA, n=4 or 5 mice). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.


Figure 5A GA were collected from female NSG mice that had received indicated EVs or PBS for 5 weeks . Western blot analysis showing protein levels of p53 and genes related to mitochondrial homeostasis and function . Data information : Western blot signals are quantified and normalized to Gapdh . B Relative mtDNA / nDNA ratio in GA determined by PCR ( one - way ANOVA , n = 4 mice per group ) . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , ns : not significant . C RT - qPCR showing indicated mRNAs in GA of indicated groups of mice . Data were normalized to Ppib and compared to control group receiving PBS ( one - way ANOVA , n = 4 mice per group ) . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , ns : not significant .

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Figure 5A GA were collected from female NSG mice that had received indicated EVs or PBS for 5 weeks. Western blot analysis showing protein levels of p53 and genes related to mitochondrial homeostasis and function. Data information: Western blot signals are quantified and normalized to Gapdh.B Relative mtDNA/nDNA ratio in GA determined by PCR (one-way ANOVA, n=4 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ns: not significant.C RT-qPCR showing indicated mRNAs in GA of indicated groups of mice. Data were normalized to Ppib and compared to control group receiving PBS (one-way ANOVA, n=4 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ns: not significant.


Figure 6AAV8 - TP53 virus or AAV8 - eGFP control virus were injected into GA of both sides twice during the entire experiments , first immediately following implantation of MDA - MB - 231 cells into the mammary fat pad , and then one month later . Muscles were collected at 6 weeks after tumor implantation . A Western blot showing indicated protein levels in the GA . Western blot signals are quantified and normalized to Gapdh . AAV8 - TP53 virus or AAV8 - eGFP control virus were injected into GA of both sides twice during the entire experiments , first immediately following implantation of MDA - MB - 231 cells into the mammary fat pad , and then one month later . Muscles were collected at 6 weeks after tumor implantation . C Relative mtDNA / nDNA ratio in GA determined by PCR ( two - tailed Student ' s t - test , n = 8 mice per group ) . Data information : In all bar and line graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant . AAV8 - TP53 virus or AAV8 - eGFP control virus were injected into GA of both sides twice during the entire experiments , first immediately following implantation of MDA - MB - 231 cells into the mammary fat pad , and then one month later . Muscles were collected at 6 weeks after tumor implantation . D Representative TEM images showing SS and IMF mitochondria and their quantification in GA ( two - tailed Student ' s t - test , n = 3 mice per group for the left subpanel and n = 166 or 181 mitochondria analyzed per group for the right subpanel ) . Scale bar : 1 µm . Yellow bars : depths of SS mitochondrial areas ; arrows : IMF mitochondrial area . Data information : In all bar and line graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant . AAV8 - TP53 virus or AAV8 - eGFP control virus were injected into GA of both sides twice during the entire experiments , first immediately following implantation of MDA - MB - 231 cells into the mammary fat pad , and then one month later . Muscles were collected at 6 weeks after tumor implantation . E ROS levels in GA from indicated groups of mice . Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis ( two - tailed Student ' s t - test , n = 4 mice per group ) . Scale bar : 50 µm . Data information : In all bar and line graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant .

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Figure 6AAV8-TP53 virus or AAV8-eGFP control virus were injected into GA of both sides twice during the entire experiments, first immediately following implantation of MDA-MB-231 cells into the mammary fat pad, and then one month later. Muscles were collected at 6 weeks after tumor implantation. A Western blot showing indicated protein levels in the GA. Western blot signals are quantified and normalized to Gapdh.AAV8-TP53 virus or AAV8-eGFP control virus were injected into GA of both sides twice during the entire experiments, first immediately following implantation of MDA-MB-231 cells into the mammary fat pad, and then one month later. Muscles were collected at 6 weeks after tumor implantation. C Relative mtDNA/nDNA ratio in GA determined by PCR (two-tailed Student's t-test, n=8 mice per group). Data information: In all bar and line graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.AAV8-TP53 virus or AAV8-eGFP control virus were injected into GA of both sides twice during the entire experiments, first immediately following implantation of MDA-MB-231 cells into the mammary fat pad, and then one month later. Muscles were collected at 6 weeks after tumor implantation. D Representative TEM images showing SS and IMF mitochondria and their quantification in GA (two-tailed Student's t-test, n=3 mice per group for the left subpanel and n=166 or 181 mitochondria analyzed per group for the right subpanel). Scale bar: 1 µm. Yellow bars: depths of SS mitochondrial areas; arrows: IMF mitochondrial area. Data information: In all bar and line graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.AAV8-TP53 virus or AAV8-eGFP control virus were injected into GA of both sides twice during the entire experiments, first immediately following implantation of MDA-MB-231 cells into the mammary fat pad, and then one month later. Muscles were collected at 6 weeks after tumor implantation. E ROS levels in GA from indicated groups of mice. Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis (two-tailed Student's t-test, n=4 mice per group). Scale bar: 50 µm. Data information: In all bar and line graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.


Figure 1Fluorescent microscopy of cultured Gfp - Lc3 GMPs ( a ) and HSCs ( b ) Electron microscopy images of WT HSCs . Arrowheads indicate autophagic vesicles . d , GFP - LC3 loss in cultured Gfp - Lc3 HSCs ( n = 3 ) . Results are expressed as percentage of GFP - LC3 MFI in - BafA compared to + BafA conditions . GFP - LC3 levels in HSCs of fed and starved Gfp - Lc3 mice ( n = 3 ) . Results are expressed as percent of GFP - LC3 MFI in fed mice . Data are means ± s . d . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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Figure 1Fluorescent microscopy of cultured Gfp-Lc3 GMPs (a) and HSCs (b)Electron microscopy images of WT HSCs. Arrowheads indicate autophagic vesicles.d, GFP-LC3 loss in cultured Gfp-Lc3 HSCs (n = 3). Results are expressed as percentage of GFP-LC3 MFI in -BafA compared to +BafA conditions.GFP-LC3 levels in HSCs of fed and starved Gfp-Lc3 mice (n = 3). Results are expressed as percent of GFP-LC3 MFI in fed mice. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


Figure 2Apoptosis levels in cultured WT HSCs ( n = 3 - 5 ) . Results are expressed as percent caspase activation compared to + cytokines conditions . Apoptosis levels in cultured control ( Cnt ) and Atg12cKO HSCs and GMPs ( n = 3 ) . Results are expressed as percent caspase activation compared to + cytokines Cnt HSCs ( 100 % ) . Absolute number of HSCs in fed and starved Cnt and Atg12cKO mice ( n = 4 - 8 mice per group ) . Results are expressed as percent of HSCs in fed mice . Data are means ± s . d . Hatching indicates starved conditions . * P ≤ 0 . 05 , * * P ≤ 0 . 01 .

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Figure 2Apoptosis levels in cultured WT HSCs (n = 3-5). Results are expressed as percent caspase activation compared to +cytokines conditions.Apoptosis levels in cultured control (Cnt) and Atg12cKO HSCs and GMPs (n = 3). Results are expressed as percent caspase activation compared to +cytokines Cnt HSCs (100%).Absolute number of HSCs in fed and starved Cnt and Atg12cKO mice (n = 4-8 mice per group). Results are expressed as percent of HSCs in fed mice. Data are means ± s.d. Hatching indicates starved conditions. *P ≤ 0.05, **P ≤ 0.01.


Figure 3Status of the autophagy machinery in WT HSCs ( n = 3 ) . Results are expressed as log2 fold expression compared to WT GMPs ( set to 0 ) . b , c , qRT - PCR analyses of pro - autophagic genes in WT ( b ) and FoxO3a − / − ( c ) HSCs ( n = 3 ) . Results are expressed as log2 fold expression compared to WT GMPs ( b ) or FoxO3a + / + HSCs ( c ) ( set to 0 ) . Autophagy flux in cultured FoxO3a − / − : : Gfp - Lc3 HSCs ( n = 3 ) . Results are expressed as percent GFP - LC3 MFI in - BafA vs . + BafA conditions , and normalized to FoxO3a + / + − : : Gfp - Lc3 HSCs . Data are means ± s . d . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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Figure 3Status of the autophagy machinery in WT HSCs (n = 3). Results are expressed as log2 fold expression compared to WT GMPs (set to 0).b, c, qRT-PCR analyses of pro-autophagic genes in WT (b) and FoxO3a−/− (c) HSCs (n = 3). Results are expressed as log2 fold expression compared to WT GMPs (b) or FoxO3a+/+ HSCs (c) (set to 0).Autophagy flux in cultured FoxO3a−/−::Gfp-Lc3 HSCs (n = 3). Results are expressed as percent GFP-LC3 MFI in -BafA vs. +BafA conditions, and normalized to FoxO3a+/+−::Gfp-Lc3 HSCs. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


Figure 4Ongoing autophagy in old HSCsElectron microscopy images of young and old HSCs . Arrowheads indicate autophagic vesicles .

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Figure 4Ongoing autophagy in old HSCsElectron microscopy images of young and old HSCs. Arrowheads indicate autophagic vesicles.


Figure 5a , qRT - PCR analyses of pro - autophagic genes in old HSCs ( n = 3 - 5 ) . Results are expressed as log2 fold expression compared to young HSCs ( set to 0 ) . Apoptosis levels in cultured young and old HSCs ( n = 3 ) . Results are expressed as percent caspase activation compared to + cytokines young HSCs . Hatching indicates - cytokines conditions . 2 - NBD glucose uptake in cultured young and old HSCs ( n = 3 ) . Percent colony formation in young and old WT HSCs cultured for 32h ± BafA and methylpyruvate ( M - Pyr ) ( n = 3 ) . Colonies were counted at day 10 and normalized to - BafA conditions . Data are means ± s . d . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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Figure 5a, qRT-PCR analyses of pro-autophagic genes in old HSCs (n = 3-5). Results are expressed as log2 fold expression compared to young HSCs (set to 0).Apoptosis levels in cultured young and old HSCs (n = 3). Results are expressed as percent caspase activation compared to +cytokines young HSCs. Hatching indicates -cytokines conditions.2-NBD glucose uptake in cultured young and old HSCs (n = 3).Percent colony formation in young and old WT HSCs cultured for 32h ± BafA and methylpyruvate (M-Pyr) (n = 3). Colonies were counted at day 10 and normalized to -BafA conditions. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


Figure 1 . Recovery of infectious recombinant MHV - GFP . Cell culture supernatants containing viruses produced after virus rescue from two MHV - GFP YAC clones ( Clone1 , Clone 2 ) were used to infect 17Cl - 1 cells . At 48 hours post - infection , infected cells were visualised for GFP expression ( left panels ) and by bright field microscopy ( right panels ) . Mock represents 17Cl - 1 cells inoculated with supernatant from BHK - MHV - N cells electroporated without viral RNAs . Replication kinetics of parental MHV - GFP and recombinant MHV - GFP Clone 1 and Clone 2 . L929 cells were infected at an MOI of 0 . 1 . Cell culture supernatants were harvested at indicated time points post - infection and titrated by plaque assay . Data are representative of three independent experiments with two replicates per virus in each experiment . Error bars show standard deviation ( SD ) from the mean . pfu / ml , plaque forming units per millilitre .

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Figure 1.Recovery of infectious recombinant MHV-GFP. Cell culture supernatants containing viruses produced after virus rescue from two MHV-GFP YAC clones (Clone1, Clone 2) were used to infect 17Cl-1 cells. At 48 hours post-infection, infected cells were visualised for GFP expression (left panels) and by bright field microscopy (right panels). Mock represents 17Cl-1 cells inoculated with supernatant from BHK-MHV-N cells electroporated without viral RNAs.Replication kinetics of parental MHV-GFP and recombinant MHV-GFP Clone 1 and Clone 2. L929 cells were infected at an MOI of 0.1. Cell culture supernatants were harvested at indicated time points post-infection and titrated by plaque assay. Data are representative of three independent experiments with two replicates per virus in each experiment. Error bars show standard deviation (SD) from the mean. pfu/ml, plaque forming units per millilitre.


Figure 2 . Rescue of recombinant MERS - CoV and MERS - CoV - GFP . Following delivery of viral RNAs into BHK - 21 cells via electroporation the cells were co - cultivated with VeroB4 cells and supernatants containing recombinant viruses produced were used to infect new VeroB4 cells . Infected cells were visualised by bright field microscopy for recombinant MERS - CoV ( upper panel ; 5 days post infection ) , and for GFP expression for recombinant MERS - CoV - GFP ( lower panel , 3 days post infection ) . Mock represents VeroB4 cells inoculated with supernatant from BHK - 21 cells electroporated without viral RNAs . Replication kinetics of the MERS - CoV - EMC ( laboratory adapted isolate ) , rMERS - CoV and rMERS - CoV - GFP . VeroB4 cells were infected at an MOI of 0 . 01 . Cell culture supernatants were harvested at indicated time points post infection and titrated by TCID50 assay . Data are representative of three independent experiments with two replicates per virus in each experiment . Error bars show standard deviation ( SD ) from the mean . TCID50 / ml , 50 % tissue culture infectious dose per millilitre .

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Figure 2.Rescue of recombinant MERS-CoV and MERS-CoV-GFP. Following delivery of viral RNAs into BHK-21 cells via electroporation the cells were co-cultivated with VeroB4 cells and supernatants containing recombinant viruses produced were used to infect new VeroB4 cells. Infected cells were visualised by bright field microscopy for recombinant MERS-CoV (upper panel; 5 days post infection), and for GFP expression for recombinant MERS-CoV-GFP (lower panel, 3 days post infection). Mock represents VeroB4 cells inoculated with supernatant from BHK-21 cells electroporated without viral RNAs.Replication kinetics of the MERS-CoV-EMC (laboratory adapted isolate), rMERS-CoV and rMERS-CoV-GFP. VeroB4 cells were infected at an MOI of 0.01. Cell culture supernatants were harvested at indicated time points post infection and titrated by TCID50 assay. Data are representative of three independent experiments with two replicates per virus in each experiment. Error bars show standard deviation (SD) from the mean. TCID50/ml, 50% tissue culture infectious dose per millilitre.


Figure 3 . Rescue of recombinant rSARS - CoV - 2 and rSARS - CoV - 2 - GFP . The experimental setting is illustrated in the upper panel . Two clones of each construct 1 - 6 were used to prepare YAC DNAs and in - vitro transcribed viral genome RNAs were electroporated into BHK - 21 cells and / or BHK - SARS - N together with an mRNA encoding the SARS - CoV - 2 N protein . Electroporated cells were co - cultivated with VeroE6 cells in a T25 flask and cell culture supernatants were transferred to 12 - well plates to perform plaque assay for rSARS - CoV - 2 constructs 1 - 3 and fluorescence microscopy for rSARS - CoV - 2 - GFP constructs 4 - 6 . Plaque assays are shown in the lower panel for SARS - CoV - 2 clones 1 . 1 , 2 . 2 , and 3 . 1 . VeroE6 cells were infected with 10 − 1 , 10 − 2 , and 10 − 3 dilutions of 1 ml supernatant from the individual rescue experiments and are compared to non - infected cells ( Mock ) . The expression of GFP in SARS - CoV - 2 - GFP infected VeroE6 cells is shown for clones 4 . 1 , 5 . 2 , and 6 . 1 . Mock represents non - infected VeroE6 cells .

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Figure 3.Rescue of recombinant rSARS-CoV-2 and rSARS-CoV-2-GFP. The experimental setting is illustrated in the upper panel. Two clones of each construct 1-6 were used to prepare YAC DNAs and in-vitro transcribed viral genome RNAs were electroporated into BHK-21 cells and/or BHK-SARS-N together with an mRNA encoding the SARS-CoV-2 N protein. Electroporated cells were co-cultivated with VeroE6 cells in a T25 flask and cell culture supernatants were transferred to 12-well plates to perform plaque assay for rSARS-CoV-2 constructs 1-3 and fluorescence microscopy for rSARS-CoV-2-GFP constructs 4-6. Plaque assays are shown in the lower panel for SARS-CoV-2 clones 1.1, 2.2, and 3.1. VeroE6 cells were infected with 10−1, 10−2, and 10−3 dilutions of 1 ml supernatant from the individual rescue experiments and are compared to non-infected cells (Mock). The expression of GFP in SARS-CoV-2-GFP infected VeroE6 cells is shown for clones 4.1, 5.2, and 6.1. Mock represents non-infected VeroE6 cells.


Figure 1 ( D ) Kaplan - Meier survival curves . Statistics : log rank Gehan Breslow Wilcoxon test , p < 0 . 0001 , n = 20 per group .

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Figure 1(D) Kaplan-Meier survival curves. Statistics: log rank Gehan Breslow Wilcoxon test, p < 0.0001, n=20 per group.


Figure 2 ( C ) Kaplan - Meier survival curves of the 4 groups of mice shown in panel B . Statistics : Gehan - Breslow - Wilcoxon test .

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Figure 2(C) Kaplan-Meier survival curves of the 4 groups of mice shown in panel B. Statistics: Gehan-Breslow-Wilcoxon test.


Figure 3 ( B ) Representative immunohistochemical staining for calreticulin in tumor masses of control ( PBS ) and BoxA - treated mice . Scale bar 50 μm . The distribution of calreticulin positive pixels describes the location of calreticulin ( intracellular vs ecto - calreticulin ) . Statistics : Mann - Whitney test , n = 4 . ( C ) HMGB1 levels in the medium of AB1 cells treated with BoxA or 1 μM MTX for 24 hours . Statistics : One - way Anova with Dunnett ' s post - test ; n = 3 . Data information : columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( D ) Ratio of phosphorylated eIF2α ( p - eIF2α ) over eIF2α in MM cells treated with increasing concentrations of BoxA or 1 μM MTX for 24 hours ; β - actin is shown as loading control . Statistics : One - way Anova with Dunnett ' s post - test ; n = 2 . Data information : columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( G ) Confocal immunofluorescence microscopy of cleaved caspase 3 ( red ) in MM cells treated for 24 hours with MTX ( 1 μM ) or increasing concentrations of BoxA . Nuclei were stained with Hoechst ( blue ) and cytosol with phalloidin ( green ) . Scale bar 50 μm . ( I ) Kaplan - Meier survival curve of mice injected with MM cells pretreated with PBS or BoxA shown in panel H ( Log rank Gehan Breslow Wilcoxon test , p = 0 . 59 ) .

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Figure 3(B) Representative immunohistochemical staining for calreticulin in tumor masses of control (PBS) and BoxA-treated mice. Scale bar 50 μm. The distribution of calreticulin positive pixels describes the location of calreticulin (intracellular vs ecto-calreticulin). Statistics: Mann-Whitney test, n=4.(C) HMGB1 levels in the medium of AB1 cells treated with BoxA or 1 μM MTX for 24 hours. Statistics: One-way Anova with Dunnett's post-test; n=3. Data information: columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.(D) Ratio of phosphorylated eIF2α (p-eIF2α) over eIF2α in MM cells treated with increasing concentrations of BoxA or 1 μM MTX for 24 hours; β-actin is shown as loading control. Statistics: One-way Anova with Dunnett's post-test; n=2. Data information: columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.(G) Confocal immunofluorescence microscopy of cleaved caspase 3 (red) in MM cells treated for 24 hours with MTX (1 μM) or increasing concentrations of BoxA. Nuclei were stained with Hoechst (blue) and cytosol with phalloidin (green). Scale bar 50 μm.(I) Kaplan-Meier survival curve of mice injected with MM cells pretreated with PBS or BoxA shown in panel H (Log rank Gehan Breslow Wilcoxon test, p=0.59).


Figure 4 ( E ) Representative immunofluorescence staining of CD47 ( red ) in unpermeabilized and permeabilized MM cells treated or not with 800 nM BoxA for 24 hours . Nuclei are counterstained with DAPI . Scale bar 50 μm .

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Figure 4(E) Representative immunofluorescence staining of CD47 (red) in unpermeabilized and permeabilized MM cells treated or not with 800 nM BoxA for 24 hours. Nuclei are counterstained with DAPI. Scale bar 50 μm.


Figure 5 ( A ) Ecto - calreticulin exposure ( expressed as MFI ) in CT26 cells treated with 800 nM BoxA or 1 μM MTX for 2 and 4 hours . Error bars indicate standard deviation . Data were pooled from two different experiments . Data information : bars and error bars represent mean ± SD ; statistics : One - way Anova with Dunnett ' s post - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( I ) Kaplan - Meier survival curves of mice shown in panel H . Statistics : Gehan - Breslow - Wilcoxon test .

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Figure 5(A) Ecto-calreticulin exposure (expressed as MFI) in CT26 cells treated with 800 nM BoxA or 1 μM MTX for 2 and 4 hours. Error bars indicate standard deviation. Data were pooled from two different experiments. Data information: bars and error bars represent mean ± SD; statistics: One-way Anova with Dunnett's post-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.(I) Kaplan-Meier survival curves of mice shown in panel H. Statistics: Gehan-Breslow-Wilcoxon test.


Figure 6 ( A ) Confocal images of Proximity Ligation Assays performed on CD47 and CXCR4 , CD47 and RAGE , CD47 and TLR4 . Representative images from one experiment out of 3 performed are shown . MM cells were incubated overnight with 400 nM BoxA or PBS . Nuclei are in blue ( DAPI ) , phalloidin is in green . Red dots represent physical contact of CD47 with CXCR4 or RAGE or TLR4 . Scale bar , 20 μm . The intensity of red signal in PLA was quantified as described in Materials and Methods in individual cells ( n as indicated ) . Insets are enlargements of the y axis . Mean and SD are indicated . Statistics : Kolmogorov - Smirnov test .

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Figure 6(A) Confocal images of Proximity Ligation Assays performed on CD47 and CXCR4, CD47 and RAGE, CD47 and TLR4. Representative images from one experiment out of 3 performed are shown. MM cells were incubated overnight with 400 nM BoxA or PBS. Nuclei are in blue (DAPI), phalloidin is in green. Red dots represent physical contact of CD47 with CXCR4 or RAGE or TLR4. Scale bar, 20 μm. The intensity of red signal in PLA was quantified as described in Materials and Methods in individual cells (n as indicated). Insets are enlargements of the y axis. Mean and SD are indicated. Statistics: Kolmogorov-Smirnov test.


Figure 7 ( C ) Surface CD47 on MM cells after incubation for 24 hours with the indicated concentrations of CXCL12 . The experiment shown is representative of 2 performed , in biological triplicate . Data information : bars and error bars represent mean ± SD ; statistics : one - way Anova plus Dunnett ' s post - test . In all panels , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 .

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Figure 7(C) Surface CD47 on MM cells after incubation for 24 hours with the indicated concentrations of CXCL12. The experiment shown is representative of 2 performed, in biological triplicate. Data information: bars and error bars represent mean ± SD; statistics: one-way Anova plus Dunnett's post-test. In all panels, *p<0.05, **p<0.01, ****p<0.0001.


Figure 1A - B : Percentage of pDCs ( A ) and mDCs ( B ) from total PBMCs collected at D1 was quantified by flow cytometry and grouped to self - reported duration of symptoms prior to D1 ( A - B ; 0 - 4 n = 22 ; 5 - 8 n = 39 ; 9 - 12 n = 39 ; ≥ 13 n = 16 ) . Data information : Each dot represents a patient , lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Kruskal - Wallis test * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant . PBMCs and plasma samples were collected from SARS - CoV - 2 infected individuals at time of hospitalization ( D1 ) and after 5 days ( D5 ) . C - D : Percentage of pDCs ( C ) and mDCs ( D ) at D1 and D5 matched samples from all patients independent of symptom duration ( n = 93 ) . Data information : Each dot represents a patient , lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Wilcoxon matched pairs signed rank test * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant . PBMCs and plasma samples were collected from SARS - CoV - 2 infected individuals at time of hospitalization ( D1 ) and after 5 days ( D5 ) . E - J : Matched D1 and D5 plasma samples were available from 55 donors and protein levels were determined for IFNα2a ( E , n = 30 ) , IL - 3 ( F , n = 38 ) , IL - 8 ( G , n = 44 ) , IL - 6 ( H , n = 51 ) , TNFα ( I , n = 42 ) and CXCL10 ( J , n = 46 ) , note that some samples could not be quantified and were excluded from the analysis . Data information : Each dot represents a patient , lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Wilcoxon matched pairs signed rank test * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant . K - L : Percentage of pDCs grouped by COVID - 19 severity , as determined by oxygen supplementation ( no oxygen n = 43 , nasal oxygen n = 65 and high flow oxygen n = 5 ) . Percentage of pDCs ( K ) was correlated to COVID - 19 disease severity ( L ) using simple linear regression . Data information : Each dot represents a patient , lines with error bars show the median values with interquartile ranges mean values with the standard error of the mean Statistical significance was determined using the Kruskal - Wallis test and simple linear regression * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant .

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Figure 1A-B: Percentage of pDCs (A) and mDCs (B) from total PBMCs collected at D1 was quantified by flow cytometry and grouped to self-reported duration of symptoms prior to D1 (A-B; 0-4 n= 22; 5-8 n=39; 9-12 n= 39; ≥13 n=16). Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Kruskal-Wallis test *<p0.05, **<p0.01 ***<p0.001, ns= not significant.PBMCs and plasma samples were collected from SARS-CoV-2 infected individuals at time of hospitalization (D1) and after 5 days (D5). C-D: Percentage of pDCs (C) and mDCs (D) at D1 and D5 matched samples from all patients independent of symptom duration (n=93). Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Wilcoxon matched pairs signed rank test *<p0.05, **<p0.01 ***<p0.001, ns= not significant.PBMCs and plasma samples were collected from SARS-CoV-2 infected individuals at time of hospitalization (D1) and after 5 days (D5). E-J: Matched D1 and D5 plasma samples were available from 55 donors and protein levels were determined for IFNα2a (E, n=30), IL-3 (F, n=38), IL-8 (G, n=44), IL-6 (H, n=51), TNFα (I, n=42) and CXCL10 (J, n=46), note that some samples could not be quantified and were excluded from the analysis. Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Wilcoxon matched pairs signed rank test *<p0.05, **<p0.01 ***<p0.001, ns= not significant.K-L: Percentage of pDCs grouped by COVID-19 severity, as determined by oxygen supplementation (no oxygen n=43, nasal oxygen n=65 and high flow oxygen n=5). Percentage of pDCs (K) was correlated to COVID-19 disease severity (L) using simple linear regression. Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges mean values with the standard error of the mean Statistical significance was determined using the Kruskal-Wallis test and simple linear regression *<p0.05, **<p0.01 ***<p0.001, ns= not significant.


Figure 2C - J : The FR2020 strain was used in subsequent experiments where pDC were either mock treated ( mock , grey ) , exposed to SARS - CoV - 2 at 1 MOI ( SARS - 2 , purple ) , TLR7 ( 2 . 5 μg / mL R837 , blue ) or TLR3 agonist ( 800 ng / mL poly ( I : C ) , pink ) . Supernatants were collected after 24 hrs and analyzed for type I IFNα ( C ) , IFNβ ( D ) , type II IFNγ ( E ) , type III IFNλ1 ( F ) , IL - 6 ( G ) , IL - 8 ( H ) , CXCL10 ( I ) and TNFα ( J ) expression by ELISA . Data information : Bars represent mean values with standard error of the mean and symbols represent individual pDC donors ( n = 3 - 4 ) . Equal symbols represent equal donors Statistical significance was determined using the ratio paired student T test and compared the treated condition with the time point - matched mock condition * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 .

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Figure 2C- J: The FR2020 strain was used in subsequent experiments where pDC were either mock treated (mock, grey), exposed to SARS-CoV-2 at 1 MOI (SARS-2, purple), TLR7 (2.5 μg/mL R837, blue) or TLR3 agonist (800 ng/mL poly(I:C), pink). Supernatants were collected after 24 hrs and analyzed for type I IFNα (C), IFNβ (D), type II IFNγ (E), type III IFNλ1 (F), IL-6 (G), IL-8 (H), CXCL10 (I) and TNFα (J) expression by ELISA. Data information: Bars represent mean values with standard error of the mean and symbols represent individual pDC donors (n=3-4). Equal symbols represent equal donors Statistical significance was determined using the ratio paired student T test and compared the treated condition with the time point-matched mock condition *<p0.05, **<p0.01 ***<p0.001.


Figure 4Using CRISPR / Cas9 , MyD88 knock - out ( KO ) and AAVS1KO ( control ) pDCs were generated . MyD88 protein levels in KO and control pDCs were analyzed by western blottingC - D : MyD88KO and control pDCs were either mock treated ( mock ) or exposed to SARS - CoV - 2 ( SARS - 2 , 1 MOI ) , supernatants were collected at indicated time points and analyzed for type I IFNα ( C ) and CXCL10 ( D ) . Done with n = 4 Data information : Bars represent mean values and equal symbols represent equal donors . Statistical significance was determined using Mann - Whitney one - tailed T test in ( C ) as MyD88 - KO data had no variance and values of 0 . For ( D ) a Mann - Whitney two - tailed T test was used for comparison between conditions of mock and MyD88 KO donors . E - F : Type I IFNα production was then determined in cell culture supernatant from SARS - CoV - 2 exposed TRIFKO or TRIG + MyD88KO ( E ) and RIG - IKO or RIG - I + MyD88KO ( F ) pDCs . Done with n =

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Figure 4Using CRISPR/Cas9, MyD88 knock-out (KO) and AAVS1KO (control) pDCs were generated. MyD88 protein levels in KO and control pDCs were analyzed by western blottingC-D: MyD88KO and control pDCs were either mock treated (mock) or exposed to SARS-CoV-2 (SARS-2, 1 MOI), supernatants were collected at indicated time points and analyzed for type I IFNα (C) and CXCL10 (D). Done with n=4 Data information: Bars represent mean values and equal symbols represent equal donors. Statistical significance was determined using Mann-Whitney one-tailed T test in (C) as MyD88-KO data had no variance and values of 0. For (D) a Mann-Whitney two-tailed T test was used for comparison between conditions of mock and MyD88 KO donors.E-F: Type I IFNα production was then determined in cell culture supernatant from SARS-CoV-2 exposed TRIFKO or TRIG+MyD88KO (E) and RIG-IKO or RIG-I+MyD88KO (F) pDCs. Done with n=2


Figure 1 ( A ) Representative micro - CT images and H & E staining of trabecular bone from the femoral metaphysis of 6 - week - old male Sp7 - Cre ; Cdc20f / f and littermate control mice . Scale bar , 500 μm . ( B ) Histomorphometric analyses of 6 - week - old male femurs . ( n = 6 ) ( C ) Representative micro - CT images and H & E staining of trabecular bone from the femoral metaphysis of 6 - week - old female Sp7 - Cre ; Cdc20f / f and littermate control mice . Scale bar , 500 μm . ( D ) Histomorphometric analyses of 6 - week - old female femurs . ( n = 6 ) ( E ) Representative micro - CT images of tibial cortical bone defects in Sp7 - Cre ; Cdc20f / f and littermate control mice . The green lines show the position of the original defect position . The green circle represents the regenerated bone . Scale bar , 500 μm . ( F ) Histomorphometric analysis of the regenerated bone in tibial cortical gaps . ( n = 5 )

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Figure 1(A) Representative micro-CT images and H&E staining of trabecular bone from the femoral metaphysis of 6-week-old male Sp7-Cre;Cdc20f/f and littermate control mice. Scale bar, 500 μm. (B) Histomorphometric analyses of 6-week-old male femurs. (n=6)(C) Representative micro-CT images and H&E staining of trabecular bone from the femoral metaphysis of 6-week-old female Sp7-Cre;Cdc20f/f and littermate control mice. Scale bar, 500 μm. (D) Histomorphometric analyses of 6-week-old female femurs. (n=6) (E) Representative micro-CT images of tibial cortical bone defects in Sp7-Cre;Cdc20f/f and littermate control mice. The green lines show the position of the original defect position. The green circle represents the regenerated bone. Scale bar, 500 μm. (F) Histomorphometric analysis of the regenerated bone in tibial cortical gaps. (n=5)


Figure 2 ( B , C ) The ALP staining ( B ) and ALP quantification ( C ) of Cdc20f / f and Sp7 - Cre ; Cdc20f / f BMSCs after 7 days osteogenic differentiation . ( n = 6 ) ( E , F ) The ALP staining ( E ) and ALP quantification ( F ) of NC and CDC20sh hBMSCs after 7 days osteogenic differentiation . ( n = 5 ) ( H , I ) The ALP staining ( H ) and ALP quantification ( I ) of Vector and truncated fragments overexpression of CDC20 in CDC20sh hBMSCs after 7 days osteogenic differentiation . ( n = 5 )

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Figure 2(B, C) The ALP staining (B) and ALP quantification (C) of Cdc20f/f and Sp7-Cre;Cdc20f/f BMSCs after 7 days osteogenic differentiation. (n=6)(E, F) The ALP staining (E) and ALP quantification (F) of NC and CDC20sh hBMSCs after 7 days osteogenic differentiation. (n=5)(H, I) The ALP staining (H) and ALP quantification (I) of Vector and truncated fragments overexpression of CDC20 in CDC20sh hBMSCs after 7 days osteogenic differentiation. (n=5)


Figure 3 ( E ) Western blot analyses of the degradation of nuclear p65 protein after the overexpression of CDC20 . The cytoplasmic and nuclear protein of overexpressed Myc - CDC20 and Vector HEK293T cells were extracted after TNF - α treatment for 30 minutes . ( G ) Western blot analyses of the degradation of p65 protein after the overexpression of Myc - CDC20 . hBMSCs were transfected with Vector and Myc - CDC20 plasmids for 36 h , cells were treated with or without 10 μM MG132 for 6 h before collected .

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Figure 3(E) Western blot analyses of the degradation of nuclear p65 protein after the overexpression of CDC20. The cytoplasmic and nuclear protein of overexpressed Myc-CDC20 and Vector HEK293T cells were extracted after TNF-α treatment for 30 minutes.(G) Western blot analyses of the degradation of p65 protein after the overexpression of Myc-CDC20. hBMSCs were transfected with Vector and Myc-CDC20 plasmids for 36 h, cells were treated with or without 10 μM MG132 for 6 h before collected.


Figure 4 ( E , F ) Co - immunoprecipitation of endogenous CDC20 with endogenous p65 in HEK293T cells . ( G ) Co - immunoprecipitation of purified GST - CDC20 protein with endogenous p65 in HEK293T cells and the construct of GST - CDC20 . Whole cell lysates ( WCL ) were subjected to perform GST pull - down analyses with purified GST - CDC20 and GST as a negative control .

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Figure 4(E, F) Co-immunoprecipitation of endogenous CDC20 with endogenous p65 in HEK293T cells.(G) Co-immunoprecipitation of purified GST-CDC20 protein with endogenous p65 in HEK293T cells and the construct of GST-CDC20. Whole cell lysates (WCL) were subjected to perform GST pull-down analyses with purified GST-CDC20 and GST as a negative control.


Figure 5 ( G , H ) Co - immunoprecipitation of ectopically expressed Myc - CDC20 171 - 499 with ectopically expressed Flag - p65 1 - 310 fragment in HEK293T cells .

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Figure 5(G, H) Co-immunoprecipitation of ectopically expressed Myc-CDC20 171-499 with ectopically expressed Flag-p65 1-310 fragment in HEK293T cells.


Figure 6 ( C ) Western blot analyses of the degradation of p65 protein in NC and APC11si HEK293T cells . ( F ) Western blot analyses of p65 expression in NC or CDC20sh HEK293T cells after the overexpression of Vector or HA - APC11 plasmids . ( H , I ) The ALP staining ( H ) and ALP quantification ( I ) of NC and APC11si hBMSCs after 7 days osteogenic differentiation . ( n = 6 )

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Figure 6(C) Western blot analyses of the degradation of p65 protein in NC and APC11si HEK293T cells.(F) Western blot analyses of p65 expression in NC or CDC20sh HEK293T cells after the overexpression of Vector or HA-APC11 plasmids.(H, I) The ALP staining (H) and ALP quantification (I) of NC and APC11si hBMSCs after 7 days osteogenic differentiation. (n=6)


Figure 7 ( A ) Immunoblot of Flag - p65 - linked ubiquitination promoted by Myc - CDC20 . HEK293T cells were co - transfected with Flag - p65 , HA - Ubiquitin , with or without Myc - CDC20 or Vector plasmids for 36 h . Transfected cells were then treated with 10 μM MG132 for 6 h before collection . ( B ) Immunoblot of Flag - p65 - linked ubiquitination promoted by HA - APC11 . HEK293T cells were transfected with Flag - p65 , His - Ubiquitin , with or without HA - APC11 or Vector plasmids for 36 h . Transfected cells were then treated with 10 μM MG132 for 6 h before collection . ( F ) Immunoblot of Flag - p65 - linked ubiquitination promoted by HA - APC11 in NC and CDC20sh cells . NC or CDC20sh HEK293T cells were co - transfected with Flag - p65 , His - Ubiquitin , with or without HA - APC11 and Vector plasmids . Transfected cells were treated with 10 μM MG132 for 6 h before collection .

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Figure 7(A) Immunoblot of Flag-p65-linked ubiquitination promoted by Myc-CDC20. HEK293T cells were co-transfected with Flag-p65, HA-Ubiquitin, with or without Myc-CDC20 or Vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection. (B) Immunoblot of Flag-p65-linked ubiquitination promoted by HA-APC11. HEK293T cells were transfected with Flag-p65, His-Ubiquitin, with or without HA-APC11 or Vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection. (F) Immunoblot of Flag-p65-linked ubiquitination promoted by HA-APC11 in NC and CDC20sh cells. NC or CDC20sh HEK293T cells were co-transfected with Flag-p65, His-Ubiquitin, with or without HA-APC11 and Vector plasmids. Transfected cells were treated with 10 μM MG132 for 6 h before collection.


Figure 8 ( A ) Representative micro - CT images of trabecular bone from the femoral metaphysis of Cdc20f / f with negative control lentivirus , Sp7 - Cre ; Cdc20f / f with negative control lentivirus , and Sp7 - Cre ; Cdc20f / f with p65sh lentivirus . Scale bar , 500 μm . ( B ) Histomorphometric analyses of Cdc20f / f with negative control lentivirus , Sp7 - Cre ; Cdc20f / f with negative control lentivirus , and Sp7 - Cre ; Cdc20f / f with p65sh lentivirus . ( n = 6 ) ( C , D ) The ALP staining ( C ) and ALP quantification ( D ) of BMSCs from Cdc20f / f mice and Sp7 - Cre ; Cdc20f / f mice after 7 days osteogenic differentiation treated with negative control or p65si . ( n = 5 ) ( G ) The expression of RUNX2 in BMSCs from Cdc20f / f mice and Sp7 - Cre ; Cdc20f / f mice after 7 days osteogenic differentiation treated with negative control or p65si by western blot analyses .

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Figure 8(A) Representative micro-CT images of trabecular bone from the femoral metaphysis of Cdc20f/f with negative control lentivirus, Sp7-Cre;Cdc20f/f with negative control lentivirus, and Sp7-Cre;Cdc20f/f with p65sh lentivirus. Scale bar, 500 μm. (B) Histomorphometric analyses of Cdc20f/f with negative control lentivirus, Sp7-Cre;Cdc20f/f with negative control lentivirus, and Sp7-Cre;Cdc20f/f with p65sh lentivirus. (n=6) (C, D) The ALP staining (C) and ALP quantification (D) of BMSCs from Cdc20f/f mice and Sp7-Cre;Cdc20f/f mice after 7 days osteogenic differentiation treated with negative control or p65si. (n=5)(G) The expression of RUNX2 in BMSCs from Cdc20f/f mice and Sp7-Cre;Cdc20f/f mice after 7 days osteogenic differentiation treated with negative control or p65si by western blot analyses.


Figure 2 ( C ) ChIP - qPCRs at ARAP1 , RFX5 - GBAT2 , WDR74 and HAUS5 promoters for IgG and DVL3 in MDA - MB - 468 ( NTC , shA DVL3 , and shB DVL3 ) . ( D ) ChIP - qPCRs at RFX5 - GBAT2 , COX14 , HLA - E and PSMB8 promoters for IgG and DVL3 in MCF7 ( NTC , shA DVL3 , and shB DVL3 ) . ( E ) RT - qPCR - based analysis of expression changes of RFX5 , GBAT2 , WDR74 , HCG15 , POU2F3 , SNORD3B , APOC1P1 , HLA - A , TAP1 , PSMD8 , PDG1 and JUNB genes in MDA - MB - 468 ( NTC , shA DVL3 , and shB DVL3 ) cells . ( F ) RT - qPCR - based analysis of expression changes of RFX5 , GBAT2 , PCAT6 , ZNF672 , SSC5D , APOC1P1 , HLA - A , HLA - F and TAP1 genes in MCF7 ( NTC , shA DVL3 , and shB DVL3 ) cells . D

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Figure 2(C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, WDR74 and HAUS5 promoters for IgG and DVL3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).(D) ChIP-qPCRs at RFX5-GBAT2, COX14, HLA-E and PSMB8 promoters for IgG and DVL3 in MCF7 (NTC, shA DVL3, and shB DVL3).(E) RT-qPCR-based analysis of expression changes of RFX5, GBAT2, WDR74, HCG15, POU2F3, SNORD3B, APOC1P1, HLA-A, TAP1, PSMD8, PDG1 and JUNB genes in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (F) RT-qPCR-based analysis of expression changes of RFX5, GBAT2, PCAT6, ZNF672, SSC5D, APOC1P1, HLA-A, HLA-F and TAP1 genes in MCF7 (NTC, shA DVL3, and shB DVL3) cells. D


Figure 4 ( C ) ChIP - qPCRs at ARAP1 , RFX5 - GBAT2 , KDM5B , APOC1P1 , and PSMB8 promoters for IgG and H3K4me3 in MDA - MB - 468 ( NTC , shA DVL3 , and shB DVL3 ) . ( D ) ChIP - qPCRs at RFX5 - GBAT2 , PCAT6 , APOC1P1 and HAUS5 promoters for IgG and H3K4me3 in MCF7 ( NTC , shA DVL3 , and shB DVL3 ) .

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

Figure 4(C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, KDM5B, APOC1P1, and PSMB8 promoters for IgG and H3K4me3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).(D) ChIP-qPCRs at RFX5-GBAT2, PCAT6, APOC1P1 and HAUS5 promoters for IgG and H3K4me3 in MCF7 (NTC, shA DVL3, and shB DVL3).


Figure 5 ( C ) ChIP - qPCRs at ARAP1 , RFX5 - GBAT2 , and PSMB8 promoters for IgG and KMT2D in MDA - MB - 468 ( NTC , shA DVL3 , and shB DVL3 ) . ( D ) An assembly of IgG ( first row ) , DVL3 ( second row ) , H3K27ac ( third row ) , and H4K8ac peak ( fourth row ) ChIP - Seq data in MDA - MB - 468 for the PRDM10 , TRIM26 , BTN2A1 , PNRC2 , and MR1 genes , visualized by IGV . The green rectangular bars are the locations of the statistically - qualified peak of H3K27ac or H4K8ac in MDA - MB - 468 . ( E ) An assembly of IgG ( first row ) , DVL3 ( second row ) , H3K27ac ( third row ) , and H4K8ac peak ( fourth row ) ChIP - Seq data in MCF7 for the WDR74 , YWHAQ , CDNF / HSPA14 , BTN2A1 , and MR1 genes , visualized by IGV . The turquoise and purple rectangular bars are the locations of the statistically - qualified peak of H3K27ac or H4K8ac in MCF7 , respectively .

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

Figure 5(C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, and PSMB8 promoters for IgG and KMT2D in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).(D) An assembly of IgG (first row), DVL3 (second row), H3K27ac (third row), and H4K8ac peak (fourth row) ChIP-Seq data in MDA-MB-468 for the PRDM10, TRIM26, BTN2A1, PNRC2, and MR1 genes, visualized by IGV. The green rectangular bars are the locations of the statistically-qualified peak of H3K27ac or H4K8ac in MDA-MB-468.(E) An assembly of IgG (first row), DVL3 (second row), H3K27ac (third row), and H4K8ac peak (fourth row) ChIP-Seq data in MCF7 for the WDR74, YWHAQ, CDNF/HSPA14, BTN2A1, and MR1 genes, visualized by IGV. The turquoise and purple rectangular bars are the locations of the statistically-qualified peak of H3K27ac or H4K8ac in MCF7, respectively.


Figure 6 ( D ) RT - qPCR - based analysis of expression changes of DVL3 in MDA - MB - 468 ( NTC , shA DVL3 , and shB DVL3 ) cells . ( E ) RT - qPCR - based analysis of expression changes of KMT2D in MDA - MB - 468 ( NTC , shA DVL3 , and shB DVL3 ) cells . ( F ) RT - qPCR - based analysis of expression changes of DVL3 in MCF7 ( NTC , shA DVL3 , and shB DVL3 ) cells . ( G ) RT - qPCR - based analysis of expression changes of KMT2D in MCF7 ( NTC , shA DVL3 , and shB DVL3 ) cells . ( H ( H ) Immunofluorescence of KMT2D localization in MDA - MB - 468 cells . The cells were probed with KMT2D antibody ( red ) . The nucleus was stained with DAPI ( blue ) and the actin filaments ( green ) were stained with Phalloidin . Merge of KMT2D ( red ) and nuclear staining ( blue ) is shown as KMT2D / DAPI , merge of KMT2D ( red ) and actin ( green ) is shown as KMT2D / ACTIN , and , merge of KMT2D ( red ) , nuclear staining ( blue ) and actin ( green ) is shown as MERGE for NTC , shA DVL3 and shB DVL3 cells . Scale bar :

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

Figure 6(D) RT-qPCR-based analysis of expression changes of DVL3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (E) RT-qPCR-based analysis of expression changes of KMT2D in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (F) RT-qPCR-based analysis of expression changes of DVL3 in MCF7 (NTC, shA DVL3, and shB DVL3) cells. (G) RT-qPCR-based analysis of expression changes of KMT2D in MCF7 (NTC, shA DVL3, and shB DVL3) cells. (H (H) Immunofluorescence of KMT2D localization in MDA-MB-468 cells. The cells were probed with KMT2D antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of KMT2D (red) and nuclear staining (blue) is shown as KMT2D/DAPI, merge of KMT2D (red) and actin (green) is shown as KMT2D/ACTIN, and, merge of KMT2D (red), nuclear staining (blue) and actin (green) is shown as MERGE for NTC, shA DVL3 and shB DVL3 cells. Scale bar: 10µm


Figure 7 ( C ) IVIS images showing representative tumors generated by injecting luciferase - expressing MDA - MB - 468 cells ( NTC , shA DVL3 or shB DVL3 ) in the mammary fat pad of SCID mice . Color scale ( luminescent signal intensity ) : blue , least intense signal ; red , most intense signal . ( D ) Quantification of luminescence changes in tumors generated by injecting luciferase - expressing MDA - MB - 468 cells ( NTC , shA DVL3 or shB DVL3 ) in the mammary fat pad of SCID mice , Average luminescence was used to measure volume of tumor ; n ≥ 9 . ( ( G ) IVIS images showing representative tumors generated by injecting luciferase - expressing MCF7 cells ( NTC , shA DVL3 ) in the mammary fat pad of SCID mice . Color scale ( luminescent signal intensity ) : blue , least intense signal ; red , most intense signal . ( H ) Quantification of luminescence changes in tumors generated by injecting luciferase - expressing MCF7 cells ( NTC , shA DVL3 ) in the mammary fat pad of SCID mice . Average luminescence was used to measure volume of tumor . n = 7 .

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

Figure 7(C) IVIS images showing representative tumors generated by injecting luciferase-expressing MDA-MB-468 cells (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice. Color scale (luminescent signal intensity): blue, least intense signal; red, most intense signal. (D) Quantification of luminescence changes in tumors generated by injecting luciferase-expressing MDA-MB-468 cells (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice, Average luminescence was used to measure volume of tumor; n ≥ 9. ( (G) IVIS images showing representative tumors generated by injecting luciferase-expressing MCF7 cells (NTC, shA DVL3) in the mammary fat pad of SCID mice. Color scale (luminescent signal intensity): blue, least intense signal; red, most intense signal. (H) Quantification of luminescence changes in tumors generated by injecting luciferase-expressing MCF7 cells (NTC, shA DVL3) in the mammary fat pad of SCID mice. Average luminescence was used to measure volume of tumor. n=7.


Figure 8 ( A ) Immunofluorescence of HA - tagged DVL3 localization in MDA - MB - 468 cells . The cells were probed with an HA antibody ( red ) . The nucleus was stained with DAPI ( blue ) and the actin filaments ( green ) were stained with Phalloidin . Merge of actin ( green ) and nuclear staining ( blue ) is shown as ACTIN / DAPI , merge of DVL3 ( red ) and nuclear staining ( blue ) is shown as HA / DAPI , merge of DVL3 ( red ) and actin ( green ) is shown as HA / ACTIN , and , merge of DVL3 ( red ) , nuclear staining ( blue ) and actin ( green ) is shown as MERGE for empty vector ( EV ) , HA - DVL3 - WT and HA - DVL3 - NESm transient transfected cells . Scale bar : 10µm . ( B ) RT - qPCR - based analysis of expression changes of DVL3 in stable MDA - MB - 468 ( EV , HA - DVL3 - NESm ) cells . ( C ) RT - qPCR - based analysis of expression changes of KMT2D in stable MDA - MB - 468 ( EV , HA - DVL3 - NESm ) cells . ( D ) RT - qPCR - based analysis of expression changes of ARAP1 , RFX5 , GBAT2 , WDR74 , SNORD3D , HAUS5 , TAP1 , HLA - A , PDG1 , and , MR1 in stable MDA - MB - 468 ( EV , HA - DVL3 - NESm ) cells . ( E

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

Figure 8(A) Immunofluorescence of HA-tagged DVL3 localization in MDA-MB-468 cells. The cells were probed with an HA antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of actin (green) and nuclear staining (blue) is shown as ACTIN/DAPI, merge of DVL3 (red) and nuclear staining (blue) is shown as HA/DAPI, merge of DVL3 (red) and actin (green) is shown as HA/ACTIN, and, merge of DVL3 (red), nuclear staining (blue) and actin (green) is shown as MERGE for empty vector (EV), HA-DVL3-WT and HA-DVL3-NESm transient transfected cells. Scale bar: 10µm.(B) RT-qPCR-based analysis of expression changes of DVL3 in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (C) RT-qPCR-based analysis of expression changes of KMT2D in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (D) RT-qPCR-based analysis of expression changes of ARAP1 , RFX5, GBAT2, WDR74, SNORD3D, HAUS5, TAP1, HLA-A, PDG1, and, MR1 in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (E


Figure 1A Dose - response viability curves in presence of increasing concentrations of IMT1 for one - week , viable cells were counted and normalized to DMSO - treated controls . Data are expressed as mean ± SD of n = 3 independent experiments with four technical replicates . IC50 values were calculated with non - linear least square fit , RKO : 521 . 8 nM , MiaPaCa - 2 : 291 . 4 nM and HeLa : 29 . 9 nM . B Mitochondrial transcript levels measured by qRT - PCR in RKO cells after 96 hours of 1 μM IMT1 treatment . Data are relative to DMSO - treated controls and are expressed as mean ± SD of n = 4 independent experiments . Ordinary one - way ANOVA was used for comparisons to DMSO - treated controls ( RNR1 , RNR2 , MT - COX2 , MT - ATP6 , MT - ND1 : * * * p < 0 , 0001 ) . D Volcano plot showing proteomic changes of RKO cells treated with 1 μM IMT1 for 96 hours . Data are plotted as average log2 - fold change versus log10 of adjusted p - value of DMSO - treated controls ; mitochondrial protein are highlighted in red . F CRISPR guides enriched ( positive hits ) in IMT1 - treated RKO cells plotted as log2 fold change ( LFC ) versus log10 of adjusted p - value . mTORC1 and VHL pathway - related genes are reported in green and blue , respectively .

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

Figure 1A Dose-response viability curves in presence of increasing concentrations of IMT1 for one-week, viable cells were counted and normalized to DMSO-treated controls. Data are expressed as mean ± SD of n=3 independent experiments with four technical replicates. IC50 values were calculated with non-linear least square fit, RKO: 521.8 nM, MiaPaCa-2: 291.4 nM and HeLa: 29.9 nM.B Mitochondrial transcript levels measured by qRT-PCR in RKO cells after 96 hours of 1 μM IMT1 treatment. Data are relative to DMSO-treated controls and are expressed as mean ± SD of n=4 independent experiments. Ordinary one-way ANOVA was used for comparisons to DMSO-treated controls (RNR1, RNR2, MT-COX2, MT-ATP6, MT-ND1: ***p<0,0001).D Volcano plot showing proteomic changes of RKO cells treated with 1 μM IMT1 for 96 hours. Data are plotted as average log2-fold change versus log10 of adjusted p-value of DMSO-treated controls; mitochondrial protein are highlighted in red.F CRISPR guides enriched (positive hits) in IMT1-treated RKO cells plotted as log2 fold change (LFC) versus log10 of adjusted p-value. mTORC1 and VHL pathway-related genes are reported in green and blue, respectively.


Figure 2B - D Viable cells count in presence of DMSO , IMT1 alone or in combination with rapamycin in RKO ( B ) , MiaPaCa - 2 ( C ) and HeLa ( D ) cells . Data are expressed as mean values ± SD of n = 6 independent experiments each including four technical intra - plate replicates . Statistical significance was calculated with one - way ANOVA test . Paired IMT1 vs . IMT1 + RAPA comparisons : RKO : * * * p < 0 , 0001 , MiaPaCa - 2 : * * p = 0 , 0014 , HeLa : * p = 0 , 0238 . F - H Viability assessment of RKO ( F ) , MiaPaCa - 2 ( G ) and HeLa ( H ) cells treated with DMSO , IMT1 alone and in combination with FG4592 . Data are expressed as mean values ± SD of n = 6 , 5 , 6 independent experiments for RKO , HeLa and MiaPaCa - 2 , respectively . Statistical significance was calculated with one - way ANOVA . Paired IMT1 vs IMT1 + FG4592 comparisons : RKO : * * p < 0 , 0028 , MiaPaCa - 2 : * * p = 0 , 0011 , HeLa : * p = 0 , 0029 .

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

Figure 2B-D Viable cells count in presence of DMSO, IMT1 alone or in combination with rapamycin in RKO (B), MiaPaCa-2 (C) and HeLa (D) cells. Data are expressed as mean values ± SD of n=6 independent experiments each including four technical intra-plate replicates. Statistical significance was calculated with one-way ANOVA test. Paired IMT1 vs. IMT1 + RAPA comparisons: RKO: ***p<0,0001, MiaPaCa-2: **p= 0,0014, HeLa: *p= 0,0238.F-H Viability assessment of RKO (F), MiaPaCa-2 (G) and HeLa (H) cells treated with DMSO, IMT1 alone and in combination with FG4592. Data are expressed as mean values ± SD of n=6, 5, 6 independent experiments for RKO, HeLa and MiaPaCa-2, respectively. Statistical significance was calculated with one-way ANOVA. Paired IMT1 vs IMT1 + FG4592 comparisons: RKO: **p<0,0028, MiaPaCa-2: **p= 0,0011, HeLa: *p= 0,0029.


Figure 3C Western blot analyses of OXPHOS protein steady state levels of RKO cells after treatment with DMSO , IMT1 , rapamycin , rapamycin with IMT1 , FG4592 and FG4592 with IMT1 . ACTINB is shown as loading control . F , G Cellular OCR measured with Seahorse extracellular flux analyzer after sequential addition of different modulators of mitochondrial function . RKO cells were treated with DMSO , rapamycin ( F ) or FG4592 ( G ) in presence or absence of IMT1 . Data are expressed as the mean ± SEM of n = 3 independent experiments with six technical replicates . Statistical significance was calculated with one - way ANOVA test . DMSO - treated controls vs : IMT1 : p = 0 , 0008 ; RAPA : p = 0 , 1578 ; RAPA + IMT1 : p = 0 , 0034 ; FG4592 : p = 0 , 872 ; FG4592 + IMT1 : p = 0 , 0029 . Paired t - test of IMT1 vs RAPA + IMT1 : p = 0 , 034 ; IMT1 vs FG4592 + IMT1 : non - significant .

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

Figure 3C Western blot analyses of OXPHOS protein steady state levels of RKO cells after treatment with DMSO, IMT1, rapamycin, rapamycin with IMT1, FG4592 and FG4592 with IMT1. ACTINB is shown as loading control.F, G Cellular OCR measured with Seahorse extracellular flux analyzer after sequential addition of different modulators of mitochondrial function. RKO cells were treated with DMSO, rapamycin (F) or FG4592 (G) in presence or absence of IMT1. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test. DMSO-treated controls vs: IMT1: p=0,0008; RAPA: p=0,1578; RAPA + IMT1: p= 0,0034; FG4592: p=0,872; FG4592 + IMT1: p=0,0029. Paired t-test of IMT1 vs RAPA + IMT1: p= 0,034; IMT1 vs FG4592 + IMT1: non-significant.


Figure 4A Differences in sensitivity to increasing IMT1 doses between RKO cells and resistant RKO cells . The graph shows mean values ± SEM of n = 3 independent experiments . B Western blot analyses of OXPHOS protein steady state levels at increasing concentrations of IMT1 of RKO ( left ) and IMT1 - resistant RKO cells ( right ) . The samples were collected and analyzed after eight weeks of chronic IMT1 treatment of the resistant line ; GAPDH is shown as loading control . G Relative mtDNA copy number measured in RKO and IMT1 - resistant cells treated with IMT1 for 96 hours . Data are expressed as average from n = 3 experiments ± SEM . Statistical significance was calculated with one - way ANOVA test . RKO + IMT1 vs Resistant + IMT1 : MT - ND1 * * p = 0 , 0035 , MT - ATP6 * * * p = 0 , 0002 , MT - CYTB * * p = 0 , 0028 . H Cellular OCR measured by Seahorse extracellular flux analysis after sequential addition of different modulators of mitochondrial function in RKO and IMT1 - resistant RKO cells treated with DMSO or IMT1 . Data are expressed as the mean ± SEM of n = 3 independent experiments with six technical replicates . Statistical significance was calculated with one - way ANOVA test . RKO DMSO vs RKO + IMT1 : p = 0 , 0008 ; RKO DMSO vs resistant DMSO : p = 0 , 0343 ; RKO DMSO vs resistant + IMT1 : p = 0 , 0092 . Paired t - test of RKO + IMT1 vs resistant + IMT1 : p = 0 , 135 .

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

Figure 4A Differences in sensitivity to increasing IMT1 doses between RKO cells and resistant RKO cells. The graph shows mean values ± SEM of n=3 independent experiments.B Western blot analyses of OXPHOS protein steady state levels at increasing concentrations of IMT1 of RKO (left) and IMT1-resistant RKO cells (right). The samples were collected and analyzed after eight weeks of chronic IMT1 treatment of the resistant line; GAPDH is shown as loading control.G Relative mtDNA copy number measured in RKO and IMT1-resistant cells treated with IMT1 for 96 hours. Data are expressed as average from n=3 experiments ± SEM. Statistical significance was calculated with one-way ANOVA test. RKO + IMT1 vs Resistant + IMT1: MT-ND1 **p=0,0035, MT-ATP6 ***p=0,0002, MT-CYTB **p=0,0028.H Cellular OCR measured by Seahorse extracellular flux analysis after sequential addition of different modulators of mitochondrial function in RKO and IMT1-resistant RKO cells treated with DMSO or IMT1. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test. RKO DMSO vs RKO + IMT1: p=0,0008; RKO DMSO vs resistant DMSO: p=0,0343; RKO DMSO vs resistant + IMT1: p= 0,0092. Paired t-test of RKO + IMT1 vs resistant + IMT1: p= 0,135.


Figure 5A ECAR measured by Seahorse extracellular flux analysis in DMSO and IMT1 - treated RKO and IMT1 - resistant cells . Data are expressed as the mean ± SEM of n = 3 independent experiments with six technical replicates . Statistical significance was calculated with one - way ANOVA test for multiple comparisons . RKO DMSO vs RKO + IMT1 : * * p = 0 , 0019 ; RKO + IMT1 vs resistant + IMT : * p = 0 , 0214 ; RKO DMSO vs resistant DMSO : non - significant . D , E IMT1 extracellular ( D ) and extracellular ( E ) concentrations measured in the medium at 0 , 2 and 24 hours of IMT1 treatment in RKO and IMT1 - resistant RKO cells . Mean values ± SEM of n = 3 experiments .

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Figure 5A ECAR measured by Seahorse extracellular flux analysis in DMSO and IMT1-treated RKO and IMT1-resistant cells. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test for multiple comparisons. RKO DMSO vs RKO + IMT1: **p=0,0019; RKO + IMT1 vs resistant + IMT: *p=0,0214; RKO DMSO vs resistant DMSO: non-significant.D, E IMT1 extracellular (D) and extracellular (E) concentrations measured in the medium at 0, 2 and 24 hours of IMT1 treatment in RKO and IMT1-resistant RKO cells. Mean values ± SEM of n=3 experiments.


Figure 6B Comparison between proteomic analyses of parental RKO and resistant cells treated with 1 μM IMT1 for 96 hours ( r : sample correlation coefficient ) . Data are expressed as average log2 - fold change of controls ( DMSO - treated RKO cells ) . Mitochondrial proteins are reported in red .

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Figure 6B Comparison between proteomic analyses of parental RKO and resistant cells treated with 1 μM IMT1 for 96 hours (r: sample correlation coefficient). Data are expressed as average log2-fold change of controls (DMSO-treated RKO cells). Mitochondrial proteins are reported in red.


Figure 7A Depleted CRISPR guides ( negative hits ) in IMT1 - treated RKO cells plotted as log2 fold change ( LFC ) versus log10 of adjusted p - value . C Representative images and related plot of IMT1 - resistant RKO spheroid areas in presence of the aforementioned compounds for two weeks ( IMT1 : 1 μM , CAP : 1 μg / ml ) ; Scale bar : 1mm . Data show the mean values ± SD of n = 4 experiments . Statistical significance was calculated with paired t - test : IMT1 vs IMT1 + CAP : * p = 0 , 0242 . G Viable cells counts of IMT1 - resistant RKO cells treated for one - week with either DMSO or IMT1 in presence of controls ( control # 1 , # 2 ) or TFAM ( TFAM # 1 , TFAM # 2 ) siRNAs . Data are expressed as mean values ± SD of n = 5 independent experiments each including six technical intra - plate replicates . Statistical significance was calculated with one - way ANOVA test . Controls + IMT1 vs TFAM # 1 + IMT1 : p = 0 , 2148 ; Controls + IMT1 vs TFAM # 2 + IMT1 : * p = 0 , 0415 .

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

Figure 7A Depleted CRISPR guides (negative hits) in IMT1-treated RKO cells plotted as log2 fold change (LFC) versus log10 of adjusted p-value.C Representative images and related plot of IMT1-resistant RKO spheroid areas in presence of the aforementioned compounds for two weeks (IMT1: 1 μM, CAP: 1 μg/ml); Scale bar: 1mm. Data show the mean values ± SD of n=4 experiments. Statistical significance was calculated with paired t-test: IMT1 vs IMT1+ CAP: *p=0,0242.G Viable cells counts of IMT1-resistant RKO cells treated for one-week with either DMSO or IMT1 in presence of controls (control #1, #2) or TFAM (TFAM #1, TFAM #2) siRNAs. Data are expressed as mean values ± SD of n=5 independent experiments each including six technical intra-plate replicates. Statistical significance was calculated with one-way ANOVA test. Controls + IMT1 vs TFAM #1 + IMT1: p= 0,2148; Controls + IMT1 vs TFAM #2 + IMT1: *p=0,0415.