Figure 7Ratio of CD86 expression or IFN - I production in 5 . 2 + vs 5 . 1 + pDC isolated from MCMV - infected MBMC of each indicated type . Black , CTR MBMC ; pale pink , Itgal - TST MBMC . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with 3 mice per group . Ratio of CD86 expression or IFN - I production in 5 . 2 + vs 5 . 1 + pDC isolated from CpG - stimulated ( B ) MBMC of each indicated type . Black , CTR MBMC ; pale pink , Itgal - TST MBMC . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with 3 mice per group . ( C ) EYFP expression in splenocytes isolated from uninfected ( NI ) vs MCMV - infected ( MCMV ) IFNbtm1 ( EYFP ) reporter mice . The expression of indicated markers was analyzed on YFP + cells . Plots are representative of one out of 6 mice from 2 independent experiments . ( D - F ) Immunohistological analysis of splenic sections stained with mAb against MOMA1 / CD169 ( blue ) , GFP / EYFP ( green ) , whose expression correlates with that of IFN - β , and MCMV IE - 1 ( red ) . Scale bars , 100µm . The image shown is one representative of 15 obtained from 5 different mice . Optical magnification was 20x , and images were scanned on the microscope at zoom x2 for ( E , F ) . White dotted lines delimit marginal zone . Yellow squares in ( D ) defines zoomed region in ( E , F ) .

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Figure 7Ratio of CD86 expression or IFN-I production in 5.2+ vs 5.1+ pDC isolated from MCMV-infected MBMC of each indicated type. Black, CTR MBMC; pale pink, Itgal-TST MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with 3 mice per group.Ratio of CD86 expression or IFN-I production in 5.2+ vs 5.1+ pDC isolated from CpG-stimulated (B) MBMC of each indicated type. Black, CTR MBMC; pale pink, Itgal-TST MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with 3 mice per group.(C) EYFP expression in splenocytes isolated from uninfected (NI) vs MCMV-infected (MCMV) IFNbtm1(EYFP) reporter mice. The expression of indicated markers was analyzed on YFP+ cells. Plots are representative of one out of 6 mice from 2 independent experiments.(D-F) Immunohistological analysis of splenic sections stained with mAb against MOMA1/CD169 (blue), GFP/EYFP (green), whose expression correlates with that of IFN-β, and MCMV IE-1 (red). Scale bars, 100µm. The image shown is one representative of 15 obtained from 5 different mice. Optical magnification was 20x, and images were scanned on the microscope at zoom x2 for (E, F). White dotted lines delimit marginal zone. Yellow squares in (D) defines zoomed region in (E, F).


Figure 1A . Significantly recurrently mutated genes identified by MutSigCV analysis based on whole - exome sequencing data of 25 STAD patients ' samples . B . Distribution of PWWP2B mutations identified in 25 STAD patients . Distribution of PWWP2B mutations identified in the TCGA PanCancer Atlas stomach adenocarcinoma dataset ( Network , 2014 ) . Distribution of PWWP2B mutations identified in a large - scale colorectal adenocarcinoma genomic study . E . Overall survival curve of 25 gastric cancer patients with and without PWWP2B mutations .

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Figure 1A. Significantly recurrently mutated genes identified by MutSigCV analysis based on whole-exome sequencing data of 25 STAD patients' samples.B. Distribution of PWWP2B mutations identified in 25 STAD patients. Distribution of PWWP2B mutations identified in the TCGA PanCancer Atlas stomach adenocarcinoma dataset (Network, 2014). Distribution of PWWP2B mutations identified in a large-scale colorectal adenocarcinoma genomic study.E. Overall survival curve of 25 gastric cancer patients with and without PWWP2B mutations.


Figure 2B . The interaction between exogenous PWWP2B and UHRF1 . The indicated plasmids were transfected into 293T cells . After 24 hours , the transfected cell lysates were immunoprecipitated ( IP ) using an anti - Myc antibody and subjected to Western blotting analysis using the indicated antibodies . The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies . C . The interaction between endogenous PWWP2B and UHRF1 . Immunoprecipitation reactions were performed using rabbit IgG or anti - PWWP2B antibodies and subjected to Western blotting analysis using the indicated antibodies . The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies . E . Myc - UHRF1 and either GFP - PWWP2B WT or its serial deletion mutants were co - transfected into 293T cells . The cell lysates were immunoprecipitated with the anti - Myc antibody and then immunoblotted with the indicated antibodies . G . SFB - PWWP2B WT and either Myc - UHRF1 WT or its serial deletion mutants were immunoprecipitated with anti - FLAG and then immunoblotted with the indicated antibodies . I Cell lysates of 293T cells transfected with Myc - UHRF1 expression vector were incubated with 2 of purified GST , GST - PWWP2B - N , or GST - PWWP2B - C recombinant proteins for 1 h at 4 ° C . After pull - down with GST - beads , the bound complexes were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( SDS - PAGE ) and subjected to Western blot analysis using the indicated antibodies ( upper panel ) . The Coomassie blue stain shows the purified GST fusion proteins . Cell lysates of 293T expressing Myc - UHRF1 WT or Myc - UHRF1 - D4 mutants were incubated with 2 ug of purified GST or GST - PWWP2B - N for 1 h at 4 ° C . After pull - down with GST - beads , the bound complex was analyzed by Western blots .

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Figure 2B. The interaction between exogenous PWWP2B and UHRF1. The indicated plasmids were transfected into 293T cells. After 24 hours, the transfected cell lysates were immunoprecipitated (IP) using an anti-Myc antibody and subjected to Western blotting analysis using the indicated antibodies. The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies.C. The interaction between endogenous PWWP2B and UHRF1. Immunoprecipitation reactions were performed using rabbit IgG or anti-PWWP2B antibodies and subjected to Western blotting analysis using the indicated antibodies. The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies.E. Myc-UHRF1 and either GFP-PWWP2B WT or its serial deletion mutants were co-transfected into 293T cells. The cell lysates were immunoprecipitated with the anti-Myc antibody and then immunoblotted with the indicated antibodies.G. SFB-PWWP2B WT and either Myc-UHRF1 WT or its serial deletion mutants were immunoprecipitated with anti-FLAG and then immunoblotted with the indicated antibodies.I Cell lysates of 293T cells transfected with Myc-UHRF1 expression vector were incubated with 2 of purified GST, GST-PWWP2B-N, or GST-PWWP2B-C recombinant proteins for 1 h at 4 °C. After pull-down with GST-beads, the bound complexes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to Western blot analysis using the indicated antibodies (upper panel). The Coomassie blue stain shows the purified GST fusion proteins.Cell lysates of 293T expressing Myc-UHRF1 WT or Myc-UHRF1-D4 mutants were incubated with 2 ug of purified GST or GST-PWWP2B-N for 1 h at 4 °C. After pull-down with GST-beads, the bound complex was analyzed by Western blots.


Figure 3E . Comparison of the translocation kinetics for GFP - UHRF1 and mCherry - PWWP2B to the sites of DNA damage . HeLa cells expressing both GFP - UHRF1 and mCherry - PWWP2B were subjected to laser microirradiation . The laser stripes were examined at the indicated time points . F . HeLa cells were transfected with GFP - PWWP2B WT or deletion mutant expression plasmids , and 24 h later , the cells were treated with laser microirradiation ( top panel ) . The cells with positive GFP expression on the laser stripes are presented in the bar graph ( bottom panel ) . The results represent the average of three independent experiments . The error bars indicate the standard deviation . H . GFP - PWWP2B - A2A3 deletion mutant translocates to the DNA damage sites . HeLa cells were transfected with GFP - tagged deletion mutant expression plasmids , and 24 h later , the cells were treated with laser microirradiation ( left panel ) . The cells with positive GFP expression on the laser stripes after 10 minutes were counted and presented in the bar graph ( right panel ) . The results represent the average of three independent experiments . The error bars indicate the standard deviation for the cells transfected with each expression plasmid . I - J . HeLa cells depleted with either control or UHRF1 siRNAs were transfected with the GFP - PWWP2B - A2A3 ( I ) and GFP - PWWP2B WT ( J ) expression plasmid . After 24 h , the cells were subjected to laser microirradiation . The laser stripes were examined at the indicated time points . The intensity of each laser stripes in each time point was measured by averaging values from indicated number of cells and graphed in the right panel . The intensity of each laser stripes in each time point was measured by averaging values from indicated number of cells and graphed in the right panel . K . HeLa cells depleted with either control or PWWP2B siRNA were transfected with GFP - UHRF1 expression plasmids . After 24 h , the cells were subjected to laser microirradiation . The laser stripes were examined at the indicated time points .

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Figure 3E. Comparison of the translocation kinetics for GFP-UHRF1 and mCherry-PWWP2B to the sites of DNA damage. HeLa cells expressing both GFP-UHRF1 and mCherry-PWWP2B were subjected to laser microirradiation. The laser stripes were examined at the indicated time points.F. HeLa cells were transfected with GFP-PWWP2B WT or deletion mutant expression plasmids, and 24 h later, the cells were treated with laser microirradiation (top panel). The cells with positive GFP expression on the laser stripes are presented in the bar graph (bottom panel). The results represent the average of three independent experiments. The error bars indicate the standard deviation.H. GFP-PWWP2B-A2A3 deletion mutant translocates to the DNA damage sites. HeLa cells were transfected with GFP-tagged deletion mutant expression plasmids, and 24 h later, the cells were treated with laser microirradiation (left panel). The cells with positive GFP expression on the laser stripes after 10 minutes were counted and presented in the bar graph (right panel). The results represent the average of three independent experiments. The error bars indicate the standard deviation for the cells transfected with each expression plasmid.I-J. HeLa cells depleted with either control or UHRF1 siRNAs were transfected with the GFP-PWWP2B-A2A3 (I) and GFP-PWWP2B WT (J) expression plasmid. After 24 h, the cells were subjected to laser microirradiation. The laser stripes were examined at the indicated time points. The intensity of each laser stripes in each time point was measured by averaging values from indicated number of cells and graphed in the right panel.The intensity of each laser stripes in each time point was measured by averaging values from indicated number of cells and graphed in the right panel. K. HeLa cells depleted with either control or PWWP2B siRNA were transfected with GFP-UHRF1 expression plasmids. After 24 h, the cells were subjected to laser microirradiation. The laser stripes were examined at the indicated time points.


Figure 4D - G . Quantification of radiation - induced RAD51 , BRCA1 , and 53BP1 foci formation in HeLa cells treated with the indicated siRNAs . All data were obtained from over cells ( open circles ) , and the mean values are shown as red bars ( upper panel ) . Immunofluorescence analysis of irradiation - induced RAD51 , BRCA1 , and 53BP1 foci formation . HeLa cells transfected with either control or PWWP2B siRNAs were synchronized in S phase by a double thymidine block and exposed to 0 or 2 Gy of IR . The cells were incubated for 2 h , fixed , and subjected to staining with RAD51 ( D and E ) , BRCA1 ( F ) , or 53BP1 ( G ) antibodies . The cells in S phase were identified by Cyclin A staining . ( E ) The siPWWP2B - resistant PWWP2B WT expression vector was transfected into HeLa cells after siRNA - mediated PWWP2B depletion . IR - induced RAD51 foci formation was examined by immunofluorescence .

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Figure 4D-G. Quantification of radiation-induced RAD51, BRCA1, and 53BP1 foci formation in HeLa cells treated with the indicated siRNAs. All data were obtained from over cells (open circles), and the mean values are shown as red bars (upper panel). Immunofluorescence analysis of irradiation-induced RAD51, BRCA1, and 53BP1 foci formation. HeLa cells transfected with either control or PWWP2B siRNAs were synchronized in S phase by a double thymidine block and exposed to 0 or 2 Gy of IR. The cells were incubated for 2 h, fixed, and subjected to staining with RAD51 (D and E), BRCA1 (F), or 53BP1 (G) antibodies. The cells in S phase were identified by Cyclin A staining. (E) The siPWWP2B-resistant PWWP2B WT expression vector was transfected into HeLa cells after siRNA-mediated PWWP2B depletion. IR-induced RAD51 foci formation was examined by immunofluorescence.


Figure 5A . Immunoblot analysis of RAD51 , BRCA1 , or 53BP1 protein levels in PWWP2B - depleted HeLa cells . B . RPA phosphorylation at the indicated time points in response to IR exposure . HeLa cells treated with the indicated siRNAs were irradiated and the lysates prepared at the indicated time points were subjected to immunoblot analysis for the presence of S4 / S8 phosphorylation and total RPA . C - D . Quantification of radiation - induced RPA2 or S4 / S8 phosphorylated RPA foci formation in PWWP2B - depleted HeLa cells . All data were obtained from over 100 cells ( open circles ) , and the mean values are shown as red bars ( upper panel ) . Immunofluorescence analysis of irradiation - induced RPA2 or S4 / S8 phosphorylated RPA2 foci formation . HeLa cells treated with either control or PWWP2B siRNAs were exposed to 0 or 2 Gy of IR . The cells were incubated for 2 h , fixed , and subjected to staining with RPA2 ( C ) , or S4 / S8 phosphorylation - specific RPA2 ( D ) antibodies ( bottom panel ) .

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Figure 5A. Immunoblot analysis of RAD51, BRCA1, or 53BP1 protein levels in PWWP2B-depleted HeLa cells.B. RPA phosphorylation at the indicated time points in response to IR exposure. HeLa cells treated with the indicated siRNAs were irradiated and the lysates prepared at the indicated time points were subjected to immunoblot analysis for the presence of S4/S8 phosphorylation and total RPA.C-D. Quantification of radiation-induced RPA2 or S4/S8 phosphorylated RPA foci formation in PWWP2B-depleted HeLa cells. All data were obtained from over 100 cells (open circles), and the mean values are shown as red bars (upper panel).Immunofluorescence analysis of irradiation-induced RPA2 or S4/S8 phosphorylated RPA2 foci formation. HeLa cells treated with either control or PWWP2B siRNAs were exposed to 0 or 2 Gy of IR. The cells were incubated for 2 h, fixed, and subjected to staining with RPA2 (C), or S4/S8 phosphorylation-specific RPA2 (D) antibodies (bottom panel).


Figure 6A . Immunofluorescence analysis for irradiation - induced MRE11 foci formation ( left panel ) . HeLa cells transfected with either control siRNA , siPWWP2B or siUHRF1 were irradiated , fixed and subjected to staining with MRE11 and γH2AX antibodies . More than 100 nuclei per condition were counted . The percentage of MRE11 - positive cells is presented as a bar graph ( right panel ) . The results represent the average of three independent experiments . The error bars indicate standard deviation . B . HeLa cells expressing GFP - MRE11 were treated with control siRNA or siRNAs against PWWP2B . After 48 h , the cells were subjected to laser microirradiation . Stripes on the laser were examined at the indicated time points ( upper panel ) . The intensity of the laser stripes at the time points was determined . The average intensity values from 10 cells were graphed in the bottom panel . C . The interaction between PWWP2B and MRE11 . HA - PWWP2B and Myc - MRE11 - expressing vectors were transfected into 293T cells . After 24 h , the cell lysates were immunoprecipitated with anti - HA antibody , followed by immunoblot analysis with the indicated antibodies . D . The interaction between endogenous PWWP2B and MRE11 . Immunoprecipitation reactions were performed using rabbit IgG or anti - MRE11 antibodies , followed by immunoblot analysis with the indicated antibodies . The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies . E . HA - MRE11 and either GFP - PWWP2B WT or its serial deletion mutants were co - transfected into 293T cells . The cell lysates were immunoprecipitated with anti - HA antibody and then immunoblotted with the indicated antibodies .

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Figure 6A. Immunofluorescence analysis for irradiation-induced MRE11 foci formation (left panel). HeLa cells transfected with either control siRNA, siPWWP2B or siUHRF1 were irradiated, fixed and subjected to staining with MRE11 and γH2AX antibodies. More than 100 nuclei per condition were counted.The percentage of MRE11-positive cells is presented as a bar graph (right panel). The results represent the average of three independent experiments. The error bars indicate standard deviation.B. HeLa cells expressing GFP-MRE11 were treated with control siRNA or siRNAs against PWWP2B. After 48 h, the cells were subjected to laser microirradiation. Stripes on the laser were examined at the indicated time points (upper panel).The intensity of the laser stripes at the time points was determined. The average intensity values from 10 cells were graphed in the bottom panel.C. The interaction between PWWP2B and MRE11. HA-PWWP2B and Myc-MRE11-expressing vectors were transfected into 293T cells. After 24 h, the cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblot analysis with the indicated antibodies.D. The interaction between endogenous PWWP2B and MRE11. Immunoprecipitation reactions were performed using rabbit IgG or anti-MRE11 antibodies, followed by immunoblot analysis with the indicated antibodies. The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies.E. HA-MRE11 and either GFP-PWWP2B WT or its serial deletion mutants were co-transfected into 293T cells. The cell lysates were immunoprecipitated with anti-HA antibody and then immunoblotted with the indicated antibodies.


Figure 7B - C . Comparison of IR - induced foci formation for either RAD51 or BRCA1 in HeLa cells transfected with the indicated siRNAs . Quantification of radiation - induced RAD51 or BRCA1 foci formation in PWWP2B alone , UHRF1 alone or PWWP2B and UHRF1 - depleted HeLa cells . All data were obtained from over 100 cells ( open circles ) , and the mean values are shown as red bars ( upper panel ) . Immunofluorescence analysis of irradiation - induced RAD51 or BRCA1 foci formation . HeLa cells treated with the indicated siRNAs were exposed to 0 or 2 Gy of IR . The cells were incubated for 2 h , fixed , and subjected to staining with RAD51 ( B ) , or BRCA1 ( C ) antibodies ( bottom panel ) .

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Figure 7B-C. Comparison of IR-induced foci formation for either RAD51 or BRCA1 in HeLa cells transfected with the indicated siRNAs. Quantification of radiation-induced RAD51 or BRCA1 foci formation in PWWP2B alone, UHRF1 alone or PWWP2B and UHRF1-depleted HeLa cells. All data were obtained from over 100 cells (open circles), and the mean values are shown as red bars (upper panel).Immunofluorescence analysis of irradiation-induced RAD51 or BRCA1 foci formation. HeLa cells treated with the indicated siRNAs were exposed to 0 or 2 Gy of IR. The cells were incubated for 2 h, fixed, and subjected to staining with RAD51 (B), or BRCA1 (C) antibodies (bottom panel).


Figure 1A . CAF and WI - 38 cells pretreated with EVs from MDA - MB - 231 or MCF10A cells , or PBS for 48 h were unstarved ( U ) or starved ( S ) for 2 h in EBSS prior to addition of a mixture of leucine , arginine , and lysine ( LRK ) or a cocktail of essential AAs ( EAA ) . Cells were analyzed by Western blot analysis in a time course as indicated . B . Western blot analysis of puromycin ( Puro ) - labeled fibroblasts ( CAF , normal human mammary fibroblasts , WI - 38 , and NIH3T3 ) after 2 - h starvation and EAA add - back in the presence or absence of cycloheximide ( CHX ) . C . Western blot analysis of Puro - labeled fibroblasts cultured in growth medium .

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Figure 1A. CAF and WI-38 cells pretreated with EVs from MDA-MB-231 or MCF10A cells, or PBS for 48 h were unstarved (U) or starved (S) for 2 h in EBSS prior to addition of a mixture of leucine, arginine, and lysine (LRK) or a cocktail of essential AAs (EAA). Cells were analyzed by Western blot analysis in a time course as indicated.B. Western blot analysis of puromycin (Puro)-labeled fibroblasts (CAF, normal human mammary fibroblasts, WI-38, and NIH3T3) after 2-h starvation and EAA add-back in the presence or absence of cycloheximide (CHX). C. Western blot analysis of Puro-labeled fibroblasts cultured in growth medium.


Figure 2B . TORCAR FRET reporter assay in WI - 38 or NIH3T3 cells after 2 - h starvation and EAA add - back at time 0 ( n = 3 biological replicates for each group ) . Data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( one - way ANOVA ) .

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Figure 2B. TORCAR FRET reporter assay in WI-38 or NIH3T3 cells after 2-h starvation and EAA add-back at time 0 (n=3 biological replicates for each group). Data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA).


Figure 3A , B . Decreases of leucine , arginine , and lysine ( A ) as well as production of ammonium ( B ) in the conditioned medium ( CM ) of NIH3T3 cells at 30 min after the LRK mixture was added to starved cells that were pretreated with EVs or PBS ( n = 3 biological replicates for each group ) . C . Intracellular levels of leucine , arginine , and lysine in NIH3T3 cells collected 30 min after the LRK mixture was added to starved cells ( n = 3 biological replicates for each group ) .

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Figure 3A,B. Decreases of leucine, arginine, and lysine (A) as well as production of ammonium (B) in the conditioned medium (CM) of NIH3T3 cells at 30 min after the LRK mixture was added to starved cells that were pretreated with EVs or PBS (n=3 biological replicates for each group).C. Intracellular levels of leucine, arginine, and lysine in NIH3T3 cells collected 30 min after the LRK mixture was added to starved cells (n=3 biological replicates for each group).


Figure 4A . Western blot analysis of WI - 38 and NIH3T3 cells treated with EVs or PBS for 48 h . B . Western blot analysis of NIH3T3 cells transfected with indicated siRNA for 48 h . C . Western blot analysis of NIH3T3 cells that had been transfected with indicated siRNA and labeled with Puro in the presence or absence of CHX . D . Western blot analysis of NIH3T3 cells that had been transfected with a pRK5 - RAGC - HA - GST expression plasmid or control vector and treated with EVs or PBS for 48 h , and then labeled with Puro . E . Western blot analysis of CAF that had been treated with PBS or EVs from indicated cells for 48 h .

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Figure 4A. Western blot analysis of WI-38 and NIH3T3 cells treated with EVs or PBS for 48 h.B. Western blot analysis of NIH3T3 cells transfected with indicated siRNA for 48 h.C. Western blot analysis of NIH3T3 cells that had been transfected with indicated siRNA and labeled with Puro in the presence or absence of CHX.D. Western blot analysis of NIH3T3 cells that had been transfected with a pRK5-RAGC-HA-GST expression plasmid or control vector and treated with EVs or PBS for 48 h, and then labeled with Puro.E. Western blot analysis of CAF that had been treated with PBS or EVs from indicated cells for 48 h.


Figure 5A . Western blot analysis of CAF and NIH3T3 cells transfected with indicated anti - miRNAs or control for 24 h and then treated with EVs or PBS for 48 h . C . Dual luciferase assay of the psiCHECK reporters containing the 3 ' UTR of RRAGC that was wild - type , with all three miR - 105 sites mutated , or with the miR - 204 site mutated . NIH3T3 cells were co - transfected with the reporter and indicated miRNA mimic ( n = 3 biological replicates for each group ) . D . Dual luciferase assay of the indicated psiCHECK reporters in NIH3T3 cells treated with EVs or PBS for 48 h ( n = 3 biological replicates for each group ) .

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Figure 5A. Western blot analysis of CAF and NIH3T3 cells transfected with indicated anti-miRNAs or control for 24 h and then treated with EVs or PBS for 48 h.C. Dual luciferase assay of the psiCHECK reporters containing the 3'UTR of RRAGC that was wild-type, with all three miR-105 sites mutated, or with the miR-204 site mutated. NIH3T3 cells were co-transfected with the reporter and indicated miRNA mimic (n=3 biological replicates for each group).D. Dual luciferase assay of the indicated psiCHECK reporters in NIH3T3 cells treated with EVs or PBS for 48 h (n=3 biological replicates for each group).


Figure 6D . A heatmap showing the comparison of log2 fold change ( FC ) of the abundance of each identified 13C6 - labeled protein and its corresponding mRNA level determined by RNA - seq .

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Figure 6D. A heatmap showing the comparison of log2 fold change (FC) of the abundance of each identified 13C6-labeled protein and its corresponding mRNA level determined by RNA-seq.


Figure 7A , B . A breast cancer tissue array was analyzed for correlations among RagC , miR - 105 , and miR - 204 in tumor stroma by IHC / ISH - determined scores . Representative IHC and ISH images ( A ) and correlations determined by Kendall ' s tau correlation tests ( N = 24 independent tumors ; Kendall ' s tau and two - sided P value as indicated ) ( B ) are shown . Bar = 200 μm . C , D . Xenograft mammary tumors formed from MCFDCIS cells overexpressing miR - 105 or empty vector were collected from NSG mice for RagC IHC ( n = 5 tumors for each group ) . The relative intensity of RagC stain in tumor stroma was quantified by ImageJ . Bar = 50 μm . In ( D ) , data are presented as mean ± SD . * P < 0 . 05 ( Student ' s t - test ) .

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

Figure 7A,B. A breast cancer tissue array was analyzed for correlations among RagC, miR-105, and miR-204 in tumor stroma by IHC/ISH-determined scores. Representative IHC and ISH images (A) and correlations determined by Kendall's tau correlation tests (N=24 independent tumors; Kendall's tau and two-sided P value as indicated) (B) are shown. Bar=200 μm.C,D. Xenograft mammary tumors formed from MCFDCIS cells overexpressing miR-105 or empty vector were collected from NSG mice for RagC IHC (n=5 tumors for each group). The relative intensity of RagC stain in tumor stroma was quantified by ImageJ. Bar=50 μm. In (D), data are presented as mean ± SD. *P<0.05 (Student's t-test).


Figure 1Wild - type ( WT ) RAD51 SPR curves for ssDNA dN - 50 and dsDNA dN - 50p . RAD51 was injected at 150 nM in the presence of 2 . 5 mM ATP ( pH 7 . 5 ) and 10 mM CaCl2 . The normalised mean number of WT RAD51 bound to respective DNA oligonucleotides ( N ) are plotted versus time , as measured by SPR , following the equation N = S / ( L * ( MRAD51 / MDNA ) ) , where S is the signal in RU units ( 1 RU ~ 50 pg / mm2 ) , L is the amount of DNA ligand immobilised onto the experimental flow cell ( RU ) , MRAD51 is the molecular weight ( kDa ) of RAD51 ( ~ 37 kDa ) and MDNA is the molecular weight ( kDa ) of the immobilised DNA molecule .

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Figure 1Wild-type (WT) RAD51 SPR curves for ssDNA dN-50 and dsDNA dN-50p. RAD51 was injected at 150 nM in the presence of 2.5 mM ATP (pH 7.5) and 10 mM CaCl2. The normalised mean number of WT RAD51 bound to respective DNA oligonucleotides (N) are plotted versus time, as measured by SPR, following the equation N = S / (L * (MRAD51 / MDNA)), where S is the signal in RU units (1 RU ~ 50 pg/mm2), L is the amount of DNA ligand immobilised onto the experimental flow cell (RU), MRAD51 is the molecular weight (kDa) of RAD51 (~ 37 kDa) and MDNA is the molecular weight (kDa) of the immobilised DNA molecule.


Figure 2Biotinylated ssDNA oligonucleotides of indicated lengths were separately immobilised onto SPR CM5 chips via biotin - streptavidin interaction . Wild - type ( WT ) RAD51 was injected at the indicated concentrations to measure association and dissociation kinetics . The dotted and solid curves show the normalised mean number of RAD51 bound to the ssDNA oligonucleotides , as measured by SPR ( see Fig . 1 for equation ) and the ODE model fits ( see Fig . 3A for model description ) , respectively . As in A , except biotinylated dsDNA molecules of indicated lengths were used to measure association and dissociation kinetics . As in B , the dotted and solid curves show the mean number of RAD51 bound to the dsDNA oligonucleotides , as measured by SPR and the ODE model

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

Figure 2Biotinylated ssDNA oligonucleotides of indicated lengths were separately immobilised onto SPR CM5 chips via biotin-streptavidin interaction. Wild-type (WT) RAD51 was injected at the indicated concentrations to measure association and dissociation kinetics.The dotted and solid curves show the normalised mean number of RAD51 bound to the ssDNA oligonucleotides, as measured by SPR (see Fig. 1 for equation) and the ODE model fits (see Fig. 3A for model description), respectively.As in A, except biotinylated dsDNA molecules of indicated lengths were used to measure association and dissociation kinetics.As in B, the dotted and solid curves show the mean number of RAD51 bound to the dsDNA oligonucleotides, as measured by SPR and the ODE model fits


Figure 3Predicted concentrations of WT RAD51 polymers in solution at equilibrium as a function of WT RAD51 monomer concentration ( 150 nM , 3 µM , 30 µM ) , prior to RAD51 injection onto DNA - coated SPR CM5 chips . Predicted % WT RAD51 within each polymer state in solution at equilibrium as a function of WT RAD51 monomer concentration ( 150 nM , 3 µM , 30 µM ) , prior to RAD51 injection onto DNA - coated SPR CM5 chips . In panels C and D , the KD was fixed to the mean value identified in Figs . 3 A and B ( i . e . KD = 1 . 14 nM ) , and the concentration of WT RAD51 monomer concentration was varied accordingly . In panel D , the % WT RAD51 values were calculated by multiplying the polymer concentrations by their respective polymer length , and dividing each value by the total RAD51 monomer concentration ( i . e . % WT RAD51 = [ n - mer ] * n / [ WT RAD51monomer ] ) . [ WT RAD51monomer ] was calculated via coomassie staining image quantification using a BSA standard curve . It is important to note that the assumed maximum polymer length in solution ( 16 - mer ) leads to an overestimation of the concentration of each n - mer in solution , given that RAD51 is likely to form polymers of length greater than 16 at 150 nM , 3 µM and 30 µM WT RAD51 monomer concentration . However , this overestimation is likely to not alter the conclusion that WT RAD51 forms long polymers in solution at 150 nM - 30 µM concentration .

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Figure 3Predicted concentrations of WT RAD51 polymers in solution at equilibrium as a function of WT RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. Predicted % WT RAD51 within each polymer state in solution at equilibrium as a function of WT RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. In panels C and D, the KD was fixed to the mean value identified in Figs. 3 A and B (i.e. KD = 1.14 nM), and the concentration of WT RAD51 monomer concentration was varied accordingly. In panel D, the % WT RAD51 values were calculated by multiplying the polymer concentrations by their respective polymer length, and dividing each value by the total RAD51 monomer concentration (i.e. % WT RAD51 = [n-mer] * n / [WT RAD51monomer]). [WT RAD51monomer] was calculated via coomassie staining image quantification using a BSA standard curve. It is important to note that the assumed maximum polymer length in solution (16-mer) leads to an overestimation of the concentration of each n-mer in solution, given that RAD51 is likely to form polymers of length greater than 16 at 150 nM, 3 µM and 30 µM WT RAD51 monomer concentration. However, this overestimation is likely to not alter the conclusion that WT RAD51 forms long polymers in solution at 150 nM - 30 µM concentration.


Figure 4Biotinylated DNA molecules of varying flexibility were separately immobilised onto SPR CM5 chips via biotin - streptavidin interaction , and WT RAD51 was injected at the indicated concentrations to measure association and dissociation kinetics . The flexibility of respective DNA , the expected entropic penalties upon RAD51 binding to corresponding DNA and protomer - protomer binding energy contribution of WT RAD51 are indicated in gray , green and blue boxes . H and L in each box denote high and low , respectively . The mean number of WT RAD51 bound to respective DNA oligonucleotides , as measured by SPR ( dotted lines ) and ODE model fits ( solid lines ) . Bar plots of fitted kp values ( D ) and ks values ( E ) for each SPR curves . In panel D , all parameters except kp were fixed to the mean values as identified in Fig . 3 A and B , and kp was fitted using lsqcurvefit ( MATLAB ) . In panel E , all parameters except ks and kp were fixed to the mean values as identified in Fig . 3 A and B , and ks and kp were fitted using lsqcurvefit ( MATLAB ) . Only the ks values are reported here . Mean ± 1 SD of 3 ODE model fits ( n = 3 ) . D : un - paired , one - tailed Mann - Whitney - Wilcoxon tests . E : un - paired , two - tailed Mann - Whitney - Wilcoxon tests . * p ≤ 0 . 05 ; ns = non - significant .

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Figure 4Biotinylated DNA molecules of varying flexibility were separately immobilised onto SPR CM5 chips via biotin-streptavidin interaction, and WT RAD51 was injected at the indicated concentrations to measure association and dissociation kinetics. The flexibility of respective DNA, the expected entropic penalties upon RAD51 binding to corresponding DNA and protomer-protomer binding energy contribution of WT RAD51 are indicated in gray, green and blue boxes. H and L in each box denote high and low, respectively.The mean number of WT RAD51 bound to respective DNA oligonucleotides, as measured by SPR (dotted lines) and ODE model fits (solid lines).Bar plots of fitted kp values (D) and ks values (E) for each SPR curves. In panel D, all parameters except kp were fixed to the mean values as identified in Fig. 3 A and B, and kp was fitted using lsqcurvefit (MATLAB). In panel E, all parameters except ks and kp were fixed to the mean values as identified in Fig. 3 A and B, and ks and kp were fitted using lsqcurvefit (MATLAB). Only the ks values are reported here. Mean ± 1 SD of 3 ODE model fits (n=3). D: un-paired, one-tailed Mann-Whitney-Wilcoxon tests. E: un-paired, two-tailed Mann-Whitney-Wilcoxon tests. * p ≤ 0.05; ns = non-significant.


Figure 5As in Fig . 4B , but F86E RAD51 was injected at the indicated concentration to measure association and dissociation kinetics . The flexibility of respective DNA , the expected relative entropic penalties upon F86E RAD51 binding to each DNA molecule and protomer - protomer binding enegy contribution of F86 RAD51 are indicated in gray , green and blue boxes . H and L shown in each box indicate high and low , respectively . The mean number of F86E RAD51 bound to respective DNA oligonucleotides , as measured by SPR ( dotted lines ) and corresponding model fits ( solid lines ) using the model shown in panel D . Using ABC - SMC , individual forward rates ( kp ) and reverse rates ( kpr ) were fitted to each experimental curve , while a single KD was fitted to all experimental curves . Bar plots of fitted kp values for the SPR curves . Mean values of mode particles ± 1 SD derived from the ABC - SMC fits ( n = 3 ) . Un - paired , one - tailed Mann - Whitney - Wilcoxon tests . * p ≤ 0 . 05 ; ns = non - significant . Predicted concentrations of F86E RAD51 polymers in solution at equilibrium as a function of F86E RAD51 monomer concentration ( 150 nM , 3 µM , 30 µM ) , prior to RAD51 injection onto DNA - coated SPR CM5 chips . Predicted % F86E RAD51 within each polymer state in solution at equilibrium as a function of F86E RAD51 monomer concentration ( 150 nM , 3 µM , 30 µM ) , prior to RAD51 injection onto DNA - coated SPR CM5 chips . In panels F and G , the KD was fixed to the mean value identified in Fig . 5D ( i . e . KD = 10 . 2 µM ) , and the concentration of F86E RAD51 monomer concentration was varied accordingly . In panel G , the % F86E RAD51 values were calculated by multiplying the polymer concentrations by their respective polymer length , and dividing each value by the total RAD51 monomer concentration ( i . e . % F86E RAD51 = [ n - mer ] * n / [ F86E RAD51monomer ] ) . [ F86E RAD51monomer ] was calculated via coomassie staining image quantification using a BSA standard curve . Bar plots of fitted kpr values for the SPR curves . Mean values of mode particles ± 1 SD derived from the ABC - SMC fits ( n = 3 ) . Un - paired , one - tailed Mann - Whitney - Wilcoxon tests . * p ≤ 0 . 05 ; ns = 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 O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5As in Fig. 4B, but F86E RAD51 was injected at the indicated concentration to measure association and dissociation kinetics. The flexibility of respective DNA, the expected relative entropic penalties upon F86E RAD51 binding to each DNA molecule and protomer-protomer binding enegy contribution of F86 RAD51 are indicated in gray, green and blue boxes. H and L shown in each box indicate high and low, respectively.The mean number of F86E RAD51 bound to respective DNA oligonucleotides, as measured by SPR (dotted lines) and corresponding model fits (solid lines) using the model shown in panel D. Using ABC-SMC, individual forward rates (kp) and reverse rates (kpr) were fitted to each experimental curve, while a single KD was fitted to all experimental curves.Bar plots of fitted kp values for the SPR curves. Mean values of mode particles ± 1 SD derived from the ABC-SMC fits (n=3). Un-paired, one-tailed Mann-Whitney-Wilcoxon tests. * p ≤ 0.05; ns = non-significant.Predicted concentrations of F86E RAD51 polymers in solution at equilibrium as a function of F86E RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. Predicted % F86E RAD51 within each polymer state in solution at equilibrium as a function of F86E RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. In panels F and G, the KD was fixed to the mean value identified in Fig. 5D (i.e. KD = 10.2 µM), and the concentration of F86E RAD51 monomer concentration was varied accordingly. In panel G, the % F86E RAD51 values were calculated by multiplying the polymer concentrations by their respective polymer length, and dividing each value by the total RAD51 monomer concentration (i.e. % F86E RAD51 = [n-mer] * n / [F86E RAD51monomer]). [F86E RAD51monomer] was calculated via coomassie staining image quantification using a BSA standard curve. Bar plots of fitted kpr values for the SPR curves. Mean values of mode particles ± 1 SD derived from the ABC-SMC fits (n=3). Un-paired, one-tailed Mann-Whitney-Wilcoxon tests. * p ≤ 0.05; ns = non-significant.


Figure 1 ( B ) Southern Blot analysis of targeted ES cells . Positions of wild type ( WT - 14 . 7kb ) and targeted ( loxP - 12 . 8kb ) alleles are indicated . ( C ) Genotyping analysis of wild type ( Clpp + / + ) , heterozygous ( Clpp + / - ) and homozygous ( Clpp - / - ) Clpp knockout animals . DNA fragments corresponding to the wild type ( WT - 199 bp ) , and knockout ( KO - 273 bp ) Clpploci are indicated . ( D ) Analysis of Clpp transcript levels in heart ( n = 4 ) . Bars represent mean ± standard deviation ( SD ) . Student ' s t - test was used to determine the level of statistical difference . ( E ) Western blot analysis of CLPPprotein levels in heart ( He ) , quadriceps ( Quad ) , liver ( Li ) , kidney ( Ki ) , lung ( Lu ) , brain ( Br ) , spleen ( Sp ) and pancreas ( Pa ) . TUBULIN and ACTIN are used as a loading control . The arrowhead indicates positions of CLPP , and the star indicates position of cross - reacting bands .

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Figure 1(B) Southern Blot analysis of targeted ES cells. Positions of wild type (WT - 14.7kb) and targeted (loxP - 12.8kb) alleles are indicated.(C) Genotyping analysis of wild type (Clpp+/+), heterozygous (Clpp+/-) and homozygous (Clpp-/-) Clpp knockout animals. DNA fragments corresponding to the wild type (WT-199 bp), and knockout (KO - 273 bp) Clpploci are indicated.(D) Analysis of Clpp transcript levels in heart (n=4). Bars represent mean ± standard deviation (SD). Student's t-test was used to determine the level of statistical difference.(E) Western blot analysis of CLPPprotein levels in heart (He), quadriceps (Quad), liver (Li), kidney (Ki), lung (Lu), brain (Br), spleen (Sp) and pancreas (Pa). TUBULIN and ACTIN are used as a loading control. The arrowhead indicates positions of CLPP, and the star indicates position of cross-reacting bands.


Figure 2 ( C ) Respiratory chain enzyme activities . Relative enzyme activities of respiratory chain enzyme C I ( NADH coenzyme Q reductase ) , C I / III ( NADH cytochrome c reductase ) , C II ( succinate dehydrogenase ) , C II / III ( succinate cytochrome c reductase ) and C IV ( cytochrome c oxidase ) in mitochondrial extracts from wild type and Clpp knockout hearts . ( n = 5 ) . Bars present mean levels ± SD . Statistical difference was calculated by Student ' s t - test and changes are presented in the figure ( n = 5 ) . ( D ) Substrate - dependent oxygen consumption rates in isolated heartmitochondria . Complex I substrates ( pyruvate , malate , glutamate ) ; complex II substrate ( succinate ) . State 3 - ADP stimulated oxygen consumption rate ; State 4 - respiration rate after addition of oligomycin ; Uncoupled - oxygen consumption rate in the presence of FCCP . Bars present mean levels ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 7 ) . ( E ) Decrease of steady - state levels of respiratory chain subunits in Clpp - knockout hearts relative to wild type ( set to 1 ) . VDAC or TOMM20 were used as loading controls . Bars present mean levels ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 4 ) . ( F ) Blue Native Page Analysis ( BN - PAGE ) of the respiratory chain supercomplexes followed by Western blot analysis using antibodies against C I ( anti - NDUFA9 ) ; C III ( anti - UQCRC1 ) and C IV ( anti - COX4A ) .

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Figure 2(C) Respiratory chain enzyme activities. Relative enzyme activities of respiratory chain enzyme C I (NADH coenzyme Q reductase), C I/III (NADH cytochrome c reductase), C II (succinate dehydrogenase), C II/III (succinate cytochrome c reductase) and C IV (cytochrome c oxidase) in mitochondrial extracts from wild type and Clpp knockout hearts. (n=5). Bars present mean levels ± SD. Statistical difference was calculated by Student's t-test and changes are presented in the figure (n=5).(D) Substrate-dependent oxygen consumption rates in isolated heartmitochondria. Complex I substrates (pyruvate, malate, glutamate); complex II substrate (succinate). State 3 - ADP stimulated oxygen consumption rate; State 4 - respiration rate after addition of oligomycin; Uncoupled - oxygen consumption rate in the presence of FCCP. Bars present mean levels ± SD. Student's t-test was used to determine the level of statistical difference (n=7).(E) Decrease of steady-state levels of respiratory chain subunits in Clpp-knockout hearts relative to wild type (set to 1). VDAC or TOMM20 were used as loading controls. Bars present mean levels ± SD. Student's t-test was used to determine the level of statistical difference (n=4).(F) Blue Native Page Analysis (BN-PAGE) of the respiratory chain supercomplexes followed by Western blot analysis using antibodies against C I (anti-NDUFA9); C III (anti-UQCRC1) and C IV (anti-COX4A).


Figure 3 ( A ) Quantification of mtDNA levels after Southern blot analysis . Bars present mean levels ± SD ( n = 4 ) . ( B - C ) Northern blot analysis and corresponding quantification of mitochondrial ( B ) rRNAs and mRNAs or ( C ) tRNAs . 18S rRNA is used as loading control . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( 12S n = 5 and the rest of samples n = 4 ) . ( D ) Western blot analysis of LRPPRC levels . VDAC was used as a loading control . ( E ) In organello transcription analysis upon 60 minutes labelling with - 32P - UTP .

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Figure 3(A) Quantification of mtDNA levels after Southern blot analysis. Bars present mean levels ± SD (n=4).(B-C) Northern blot analysis and corresponding quantification of mitochondrial (B) rRNAs and mRNAs or (C) tRNAs. 18S rRNA is used as loading control. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (12S n=5 and the rest of samples n=4).(D) Western blot analysis of LRPPRC levels. VDAC was used as a loading control.(E) In organello transcription analysis upon 60 minutes labelling with -32P-UTP.


Figure 4 ( A ) De novo synthesized mitochondrial proteins were isolated after labelling with 35S - methionine for 1 hour . Positions of individual mitochondrial encoded proteins are indicated . ( B ) Western blot analysis of RC subunits in MEFs upon 48 hours treatment with actinonin , doxycycline and chloramphenicol ( + antibiotic ) , followed by a 48 hours recovery period ( - antibiotic ) . During both periods , proteins are isolated at 12h , 24h and 48h . Antibodies against subunits of C I ( NDUFB6 ) , C III ( UQCRC1 ) , C IV ( COXI ) , MRPS35 and MRPL37 were used as indicated . ( C ) Quantitative analysis of the COXI recovery upon treatment with antibiotics ( Actinonin , DOXY - doxycycline and CAP - chloramphenicol ) as in B . The values represent ratios of recovered ( 48h , - antibiotics ) versus untreated ( 0h ) COXI levels . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( 12S n = 5 and the rest of samples n = 4 ) . ( Actinonin and CAP n = 3 , DOXY n = 4 ) . ( D ) Sucrose density gradient sedimentation profile of ribosomal proteins and mtRNAs from heartmitochondria . The migration of ribosomal subunits ( 28S and 39S ) and mitoribosome ( 55S ) was detected by immunobloting with antibodies against MRPS35 and MRPL12 . The profile of a given mt - RNA is calculated after qRT - PCR analysis of individual fractions and corrected for its abundance in the cell according to results of Northern blot analysis . Results of individual experiments are provided for 12S and 16S rRNA , and for mt - mRNAs , results of distribution from two independent experiments using probes against ATP6 , COXIII and ND5 were combined . Error bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 3 ) . ( E ) Relative association of 12S rRNA , 16S rRNA and mt - mRNAs with mitoribosomes ( 55S ) in Clpp knockout mitochondria comparing to wild type . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( 12S and 16S n = 3 ; mRNA n = 6 ) .

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

Figure 4(A) De novo synthesized mitochondrial proteins were isolated after labelling with 35S-methionine for 1 hour. Positions of individual mitochondrial encoded proteins are indicated.(B) Western blot analysis of RC subunits in MEFs upon 48 hours treatment with actinonin, doxycycline and chloramphenicol (+ antibiotic), followed by a 48 hours recovery period (- antibiotic). During both periods, proteins are isolated at 12h, 24h and 48h. Antibodies against subunits of C I (NDUFB6), C III (UQCRC1), C IV (COXI), MRPS35 and MRPL37 were used as indicated.(C) Quantitative analysis of the COXI recovery upon treatment with antibiotics (Actinonin, DOXY - doxycycline and CAP - chloramphenicol) as in B. The values represent ratios of recovered (48h, - antibiotics) versus untreated (0h) COXI levels. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (12S n=5 and the rest of samples n=4). (Actinonin and CAP n=3, DOXY n=4).(D) Sucrose density gradient sedimentation profile of ribosomal proteins and mtRNAs from heartmitochondria. The migration of ribosomal subunits (28S and 39S) and mitoribosome (55S) was detected by immunobloting with antibodies against MRPS35 and MRPL12. The profile of a given mt-RNA is calculated after qRT-PCR analysis of individual fractions and corrected for its abundance in the cell according to results of Northern blot analysis. Results of individual experiments are provided for 12S and 16S rRNA, and for mt-mRNAs, results of distribution from two independent experiments using probes against ATP6, COXIII and ND5 were combined. Error bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=3).(E) Relative association of 12S rRNA, 16S rRNA and mt-mRNAs with mitoribosomes (55S) in Clpp knockout mitochondria comparing to wild type. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (12S and 16S n=3; mRNA n=6).


Figure 5 ( B ) Western blot analysis and subsequent quantification of ERAL1 and EFG1 levels from Clpp knockout ( Clpp - / - ) heartmitochondria relative to wild type ( Clpp + / + ) . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 4 ) . The arrowhead indicates positions of EFG1 . ( C ) Expression levels of potential CLPP substrates from Clpp knockout ( Clpp - / - ) heartmitochondria relative to wild type ( set to 1 ) . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 4 ) . ( D ) Cycloheximide ( CHX ) chase assay to determine the stability of ERAL1 and EFG1 . Proteins are visualized by Western blot with respective antibodies . Proteins are isolated after 2 , 4 , 6 , 8 , 10 and 12 hours after beginning of chase ( 0 ) . The arrowhead indicates positions of ERAL1 . ( E ) Western blot analysis and subsequent quantification of protein levels in wild type ( C1 , C2 ) and Clpp knockout ( KO1 , KO2 ) MEFs upon Clpx siRNA ( Clpx ) relative to untreated ( Ctrl ) wild type . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 6 ) . The arrowhead indicates positions of either CLPX or ERAL1 , and stars indicate position of cross - reacting bands .

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Figure 5(B) Western blot analysis and subsequent quantification of ERAL1 and EFG1 levels from Clpp knockout (Clpp-/-) heartmitochondria relative to wild type (Clpp+/+). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=4). The arrowhead indicates positions of EFG1.(C) Expression levels of potential CLPP substrates from Clpp knockout (Clpp-/-) heartmitochondria relative to wild type (set to 1). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=4).(D) Cycloheximide (CHX) chase assay to determine the stability of ERAL1 and EFG1. Proteins are visualized by Western blot with respective antibodies. Proteins are isolated after 2, 4, 6, 8,10 and 12 hours after beginning of chase (0). The arrowhead indicates positions of ERAL1.(E) Western blot analysis and subsequent quantification of protein levels in wild type (C1, C2) and Clpp knockout (KO1, KO2) MEFs upon Clpx siRNA (Clpx) relative to untreated (Ctrl) wild type. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=6). The arrowhead indicates positions of either CLPX or ERAL1, and stars indicate position of cross-reacting bands.


Figure 6 ( A ) Sucrose density gradient sedimentation profile ( " short gradients " ) of ribosomal proteins and transcripts from control ( Clpp + / + ) and Clpp knockout ( Clpp - / - ) heartmitochondria . The migration of ribosomal subunits ( 28S and 39S ) and mitoribosome ( 55S ) was detected by immunoblotting with antibodies against MRPS35 and MRPL12 . 12S rRNA was analysed by Northern blot . ( B ) Sedimentation profiles of ERAL1 , MRPS28 , EFG1 , CLPP , CLPX , EFTU and MTIF2 in heartmitochondria as in A . ( C ) Relative abundance of proteins bound to the 28S ribosomal subunit ( fractions 3 and up ) . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( ERAL1 n = 4 ; EFG1 , CLPX , MTIF2 n = 3 ; EFTU n = 2 ) . ( D ) Sucrose density gradient sedimentation profile ( " long gradients " as in figure 4D ) of ERAL1 , MRPS35 , MRPL37 , CLPX and EFG1 from control ( Clpp + / + ) and Clpp knockout ( Clpp - / - ) heartmitochondria . The same amount of protein lysates were separated by the gradient and the amount of each individual protein is estimated after Western blot analysis ( Input ) . Only fractions 4 - 11 containing the 28S , 39S and 55S ribosomal fractions were blotted to avoid high signals arising in free fractions ( 1 - 3 ) .

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 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 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 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

Figure 6(A) Sucrose density gradient sedimentation profile ("short gradients") of ribosomal proteins and transcripts from control (Clpp+/+) and Clpp knockout (Clpp-/-) heartmitochondria. The migration of ribosomal subunits (28S and 39S) and mitoribosome (55S) was detected by immunoblotting with antibodies against MRPS35 and MRPL12. 12S rRNA was analysed by Northern blot.(B) Sedimentation profiles of ERAL1, MRPS28, EFG1, CLPP, CLPX, EFTU and MTIF2 in heartmitochondria as in A.(C) Relative abundance of proteins bound to the 28S ribosomal subunit (fractions 3 and up). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (ERAL1 n=4; EFG1, CLPX, MTIF2 n=3; EFTU n=2).(D) Sucrose density gradient sedimentation profile ("long gradients" as in figure 4D) of ERAL1, MRPS35, MRPL37, CLPX and EFG1 from control (Clpp+/+) and Clpp knockout (Clpp-/-) heartmitochondria. The same amount of protein lysates were separated by the gradient and the amount of each individual protein is estimated after Western blot analysis (Input). Only fractions 4 - 11 containing the 28S, 39S and 55S ribosomal fractions were blotted to avoid high signals arising in free fractions (1-3).


Figure 7 ( A ) Coimmunoprecipitation analysis using antibodies raised against ERAL1 and CLPX proteins . Western blot analysis of input ( I ) , control ( C ) and eluate ( E ) is shown . Asterix indicates the cross - reacting band . ( B ) Sucrose density gradient migration profile - association of ERAL1 with the 28S ribosomal subunit ( MRPS35 ) upon Eral1 siRNA . TOMM20 was used as a loading control . ( C ) De novo synthesis of mitochondrial proteins in wild type ( Clpp + / + ) and Clpp knockout ( Clpp - / - ) MEFs upon Eral1 siRNA . Proteins were isolated after labeling with 35S - methionine for 1 hour from cells on control siRNA ( Ctrl ) or cells on Eral1 siRNA ( Eral1 ) . Positions of individual mitochondrial encoded proteins are indicated . Coomassie blue staining was used to ensure equal loading . ( D ) Quantification of Western blot analysis of respiratory chain subunits upon knockdown of Eral1 . Levels relative to wild type cells treated with control siRNA ( set to 1 ) are presented . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( COXI n = 4 ; ATP5A1 n = 2 ; the rest n = 6 ) . Combined results from 2 - 3 blots were used for quantification as only 2 samples of each conditions are used in a single experiment . Numbers within bars represent statistical difference to control wild type cells and numbers above bars represent difference in Clpp - knockout cells upon Eral1 siRNA . ( E ) Western blot analysis of RC subunits upon Eral1 knockdown in MEFs . Cells ( 0 ) were treated with doxycycline for 48 hours ( T ) and upon doxycycline removal allowed to recover for 24 hours ( R ) . ( F ) Quantification of the relative recovery of COXI and NDUFB6 upon Eral1 siRNA . The values are calculated as ratio between recovered ( R ) to doxycycline untreated ( 0 ) protein levels in Eral1 - knockdown cells ( Eral1 ) and compared to the same results obtained in control cells ( Ctrl ) . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( COXI n = 4 ; NDUFB6 n = 3 ) . Numbers within bars represent statistical difference between control / Eral1 siRNA treated cells and numbers above bars represent difference between + / + ; Eral1 siRNA and - / - ; Eral1 siRNA .

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

Figure 7(A) Coimmunoprecipitation analysis using antibodies raised against ERAL1 and CLPX proteins. Western blot analysis of input (I), control (C) and eluate (E) is shown. Asterix indicates the cross-reacting band.(B) Sucrose density gradient migration profile - association of ERAL1 with the 28S ribosomal subunit (MRPS35) upon Eral1 siRNA. TOMM20 was used as a loading control.(C) De novo synthesis of mitochondrial proteins in wild type (Clpp+/+) and Clpp knockout (Clpp-/-) MEFs upon Eral1 siRNA. Proteins were isolated after labeling with 35S-methionine for 1 hour from cells on control siRNA (Ctrl) or cells on Eral1 siRNA (Eral1). Positions of individual mitochondrial encoded proteins are indicated. Coomassie blue staining was used to ensure equal loading.(D) Quantification of Western blot analysis of respiratory chain subunits upon knockdown of Eral1. Levels relative to wild type cells treated with control siRNA (set to 1) are presented. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (COXI n=4; ATP5A1 n=2; the rest n=6). Combined results from 2-3 blots were used for quantification as only 2 samples of each conditions are used in a single experiment. Numbers within bars represent statistical difference to control wild type cells and numbers above bars represent difference in Clpp-knockout cells upon Eral1 siRNA.(E) Western blot analysis of RC subunits upon Eral1 knockdown in MEFs. Cells (0) were treated with doxycycline for 48 hours (T) and upon doxycycline removal allowed to recover for 24 hours (R).(F) Quantification of the relative recovery of COXI and NDUFB6 upon Eral1 siRNA. The values are calculated as ratio between recovered (R) to doxycycline untreated (0) protein levels in Eral1-knockdown cells (Eral1) and compared to the same results obtained in control cells (Ctrl). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (COXI n=4; NDUFB6 n=3). Numbers within bars represent statistical difference between control/Eral1 siRNA treated cells and numbers above bars represent difference between +/+; Eral1 siRNA and -/-; Eral1 siRNA.


Figure 1B . ERK71 mutants have elevated triacylglycerol ( TAG ) levels ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) . C . Fat body specific depletion of ERK7 by RNAi ( BDSC 56939 ) leads to increased TAG levels ( N = 4 replicates of 10 larvae / replicate for each genotype ) . D . Representative immunofluorescent images of lipid droplet ( LipidTOX ) and nuclear ( DAPI ) staining in control and ERK71 mutant fat bodies of third instar larvae . Scale bar : 50 µm . E . ERK71 mutant fat body cells contain more lipid droplets than control cells ( N = 30 cells for each genotype ) . F . After fed [ 13C ] glucose , the ERK71 mutant fat bodies display elevated [ 13C ] TAG / PE and unlabeled TAG / PE molar ratios ( measured by mass spectrometry - based lipidomics , N = 3 replicates of 15 fat bodies / replicate , TAG = triacylglycerol , PE = phosphatidylethanolamine ) G . ERK7 overexpression in the fat body results in reduced TAG levels , while kinase - dead ( K54R ) and activation loop phosphorylation - deficient ( T190A / Y192F ) mutants of ERK7 do not influence the TAG storage ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) . H - ERK7 expressing , GFP - marked , fat body clone ( H , marked by yellow dotted line ; scale bar : 50 µm ) visualized by LipidTOX staining ( N = 11 for control and 4 for ERK7 overexpression ) . ERK7 expressing , fat body clone contains less ( I ) and smaller ( J ) lipid droplets than control cells ( N = 11 for control and 4 for ERK7 overexpression ) . K . qRT - PCR based expression analysis of ERK7 from fat bodies of w1118 larvae upon 6 h starvation . Expression of RP49 was used for normalization ( N = 3 replicates of 10 fat bodies / replicate for each genotype ) .

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

Figure 1B. ERK71 mutants have elevated triacylglycerol (TAG) levels (N=4 replicates of ≥10 larvae/ replicate for each genotype). C. Fat body specific depletion of ERK7 by RNAi (BDSC 56939) leads to increased TAG levels (N=4 replicates of 10 larvae/replicate for each genotype). D. Representative immunofluorescent images of lipid droplet (LipidTOX) and nuclear (DAPI) staining in control and ERK71 mutant fat bodies of third instar larvae. Scale bar: 50 µm. E. ERK71 mutant fat body cells contain more lipid droplets than control cells (N=30 cells for each genotype). F. After fed [13C]glucose, the ERK71 mutant fat bodies display elevated [13C]TAG/PE and unlabeled TAG/PE molar ratios (measured by mass spectrometry-based lipidomics, N=3 replicates of 15 fat bodies/replicate, TAG=triacylglycerol, PE=phosphatidylethanolamine)G. ERK7 overexpression in the fat body results in reduced TAG levels, while kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence the TAG storage (N=4 replicates of ≥10 larvae/replicate for each genotype).H- ERK7 expressing, GFP-marked, fat body clone (H, marked by yellow dotted line; scale bar: 50 µm) visualized by LipidTOX staining (N=11 for control and 4 for ERK7 overexpression).ERK7 expressing, fat body clone contains less (I) and smaller (J) lipid droplets than control cells (N=11 for control and 4 for ERK7 overexpression).K. qRT-PCR based expression analysis of ERK7 from fat bodies of w1118 larvae upon 6 h starvation. Expression of RP49 was used for normalization (N=3 replicates of 10 fat bodies/replicate for each genotype).


Figure 2A . Fatty acid composition of total lipids in CG > control and CG > ERK7 larvae , measured by gas chromatography ( N = 3 replicates of 15 larvae / replicate for each genotype ) . C . Amount of glucose - derived [ 13C ] - labeled carbons incorporated into the TAG pool of control and CG > ERK7 fat bodies ( N = 3 replicates of 15 fat bodies / replicate for each genotype and diet ) . D . Ectopic ERK7 expression in the fat body results in elevated hemolymph glucose levels ( N = 3 replicates of 15 larvae / replicate for each genotype ) . E . Circulating glucose levels are decreased in ERK71 mutants ( N = 4 replicates of 15 larvae / replicate for each genotype ) .

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Figure 2A. Fatty acid composition of total lipids in CG>control and CG>ERK7 larvae, measured by gas chromatography (N=3 replicates of 15 larvae/replicate for each genotype).C. Amount of glucose-derived [13C]-labeled carbons incorporated into the TAG pool of control and CG>ERK7 fat bodies (N=3 replicates of 15 fat bodies/replicate for each genotype and diet).D. Ectopic ERK7 expression in the fat body results in elevated hemolymph glucose levels (N=3 replicates of 15 larvae/replicate for each genotype). E. Circulating glucose levels are decreased in ERK71 mutants (N=4 replicates of 15 larvae/replicate for each genotype).


Figure 3A . Four - way plot ( t - statistics ) presenting the ERK7 - dependent fasting - responsive genes ( adj . p . val < 0 . 05 ) . Dark blue indicates significant in both comparisons ; dark turquoise , significant in one comparison ; light turquoise , not significant . D . Heatmaps displaying expression patterns of selected genes involved in lipid anabolism and lipid catabolism . Color key displays scaled log2 gene expression . E , F . Expression analysis of Seipin , Agpat3 , ATPCL , fabp , Lip4 and CG34448 by qRT - PCR . RP49 was used for normalization ( N = 3 replicates of ≥ 10 larvae / replicate for each genotype ) .

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Figure 3A. Four-way plot (t-statistics) presenting the ERK7-dependent fasting-responsive genes (adj.p.val < 0.05). Dark blue indicates significant in both comparisons; dark turquoise, significant in one comparison; light turquoise, not significant.D. Heatmaps displaying expression patterns of selected genes involved in lipid anabolism and lipid catabolism. Color key displays scaled log2 gene expression.E, F. Expression analysis of Seipin, Agpat3, ATPCL, fabp, Lip4 and CG34448 by qRT-PCR. RP49 was used for normalization (N=3 replicates of ≥10 larvae/replicate for each genotype).


Figure 4A . Weight of control and ERK71 mutant larvae at 72h after egg deposition ( AED ) ( N = 4 replicates of 12 larvae / replicate for each genotype ) . B . Pupariation kinetics of control and ERK71 mutants ( N = 4 replicates of 30 larvae / replicate for each genotype ) . p < 0 . 0001 , analyzed by Log - rank test . C . ERK71 mutants fail to suppress growth upon 24 h of starvation ( N ≥ 4 replicates of 15 larvae / replicate for each genotype ) . D . ERK71 mutant larvae ( N = 4 replicates of 24 larvae / replicate for each genotype , p < 0 . 01 analyzed by Log - rank test ) are sensitive to nutrient deprivation . Fat body specific knockdown of ERK7 by RNAi ( BDSC 56939 ) leads to a moderately accelerated pupariation rate ( E , p < 0 . 001 analyzed by Log - rank test , N = 8 replicates of 30 larvae / replicate for each genotype ) F . Fat body specific knockdown of ERK7 by RNAi ( BDSC 56939 ) leads to an increase in larval weight at 72h AED ( F , N = 5 replicates of 15 larvae / replicate for each genotype ) . G . A representative image showing that fat body - specific overexpression of ERK7 by the CG - GAL4 driver ( CG > ERK7 ) impairs larval growth . Scale bar : 0 . 5 mm . H . Ectopic expression of ERK7 leads to delayed pupariation ( p < 0 . 0001 , analyzed by Log - rank test ) , while kinase - dead ( K54R ) and activation loop phosphorylation - deficient ( T190A / Y192F ) mutants of ERK7 do not influence pupariation rate ( N = 4 replicates of 30 larvae / replicate for each genotype ) . I . In contrast to ERK7 wt , kinase - dead ( K54R ) and activation loop phosphorylation - deficient ( T190A / Y192F ) mutants of ERK7 do not influence the pupal volume ( N = 4 replicates of 10 pupae / replicate for each genotype ) .

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Figure 4A. Weight of control and ERK71 mutant larvae at 72h after egg deposition (AED) (N=4 replicates of 12 larvae/replicate for each genotype).B. Pupariation kinetics of control and ERK71 mutants (N=4 replicates of 30 larvae/replicate for each genotype). p<0.0001, analyzed by Log-rank test.C. ERK71 mutants fail to suppress growth upon 24 h of starvation (N≥4 replicates of 15 larvae/replicate for each genotype).D. ERK71 mutant larvae (N=4 replicates of 24 larvae/replicate for each genotype, p<0.01 analyzed by Log-rank test) are sensitive to nutrient deprivation.Fat body specific knockdown of ERK7 by RNAi (BDSC 56939) leads to a moderately accelerated pupariation rate (E, p<0.001 analyzed by Log-rank test, N=8 replicates of 30 larvae/replicate for each genotype)F. Fat body specific knockdown of ERK7 by RNAi (BDSC 56939) leads to an increase in larval weight at 72h AED (F, N=5 replicates of 15 larvae/replicate for each genotype).G. A representative image showing that fat body-specific overexpression of ERK7 by the CG-GAL4 driver (CG>ERK7) impairs larval growth. Scale bar: 0.5 mm.H. Ectopic expression of ERK7 leads to delayed pupariation (p<0.0001, analyzed by Log-rank test), while kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence pupariation rate (N=4 replicates of 30 larvae/replicate for each genotype).I. In contrast to ERK7 wt, kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence the pupal volume (N=4 replicates of 10 pupae/replicate for each genotype).


Figure 5A . Representative immunofluorescent images of control and ERK71 mutant fat bodies with DAPI staining to visualize nucleus . Scale bar : 30 µm . B , C . ERK71 mutant fat bodies display increased nuclear area ( B , N > 160 cells , obtained from 5 independent fat bodies ) and increased nucleolar / nuclear area ratio ( C , N > 80 cells for nucleolus / nucleus ratio , obtained from 5 independent fat bodies ) , when compared to control fat bodies . D - F . ERK7 expressing fat body clones ( D , marked by GFP , scale bar : 20 µm ) display reduced nuclear area ( E ) and reduced nucleolar / nuclear area ratio ( F ) , when compared to control cells . Nuclei were visualized by DAPI staining , nucleoli were visualized by anti - Fibrillarin antibodies ( N = 10 ) . G , H . Expression analysis of Pol I ( G ) and Pol III ( H ) target RNAs by qRT - PCR . Expression of CDK7 was used for normalization ( N = 3 replicates of 10 fat bodies / replicate for each genotype ) .

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Figure 5A. Representative immunofluorescent images of control and ERK71 mutant fat bodies with DAPI staining to visualize nucleus. Scale bar: 30 µm. B, C. ERK71 mutant fat bodies display increased nuclear area (B, N>160 cells, obtained from 5 independent fat bodies) and increased nucleolar/nuclear area ratio (C, N>80 cells for nucleolus/nucleus ratio, obtained from 5 independent fat bodies), when compared to control fat bodies.D-F. ERK7 expressing fat body clones (D, marked by GFP, scale bar: 20 µm) display reduced nuclear area (E) and reduced nucleolar/nuclear area ratio (F), when compared to control cells. Nuclei were visualized by DAPI staining, nucleoli were visualized by anti-Fibrillarin antibodies (N=10).G, H. Expression analysis of Pol I (G) and Pol III (H) target RNAs by qRT-PCR. Expression of CDK7 was used for normalization (N=3 replicates of 10 fat bodies/replicate for each genotype).


Figure 6A . ERK7 overexpression leads to phosphorylation of PWP1 , as shown by appearance of an extra band on Phos - tag Western blot ( marked with red arrow ) . Insulin - inducible bands ( dark blue ) remain unresponsive to ERK7 . B . Representative immunofluorescent images of PWP1 localization in fat bodies of ERK71 mutant and control third instar larvae . Scale bar : 20 µm . C . PWP1 localization displays increased nucleolar / nuclear ratio in ERK71 mutant fat bodies compared to control ( N = 20 cells from 5 independent fat bodies per genotype ) . D . Representative immunofluorescent images of PWP1 localization in fat bodies of third instar larvae . ERK7 overexpression ( GFP marked clones ) alters the subcellular localization of PWP1 . Scale bar : 30 µm . E . PWP1 localization displays reduced nuclear / cytoplasmic ratio in ERK7 overexpressing clones , compared to control cells ( N = 10 cells from at least 8 different fat bodies per genotype ) . F . Fat body specific depletion of PWP1 by RNAi leads to decreased TAG levels ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) . G . Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK7 and PWP1 fat body ( CG - GAL4 ) knockdown with LipidTOX staining and DAPI to visualize lipid droplets and nucleus , respectively . Scale bar : 50 µm . H , I . Fat body specific knockdown of PWP1 suppresses increased lipid droplet number ( H ; N > 30 cells per genotype ) and increased nuclear area ( I ; N > 45 cells per genotype ) caused by ERK7 knockdown ( BDSC 56939 ) .

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Figure 6A. ERK7 overexpression leads to phosphorylation of PWP1, as shown by appearance of an extra band on Phos-tag Western blot (marked with red arrow). Insulin-inducible bands (dark blue) remain unresponsive to ERK7.B. Representative immunofluorescent images of PWP1 localization in fat bodies of ERK71 mutant and control third instar larvae. Scale bar: 20 µm. C. PWP1 localization displays increased nucleolar/nuclear ratio in ERK71 mutant fat bodies compared to control (N=20 cells from 5 independent fat bodies per genotype). D. Representative immunofluorescent images of PWP1 localization in fat bodies of third instar larvae. ERK7 overexpression (GFP marked clones) alters the subcellular localization of PWP1. Scale bar: 30 µm. E. PWP1 localization displays reduced nuclear/cytoplasmic ratio in ERK7 overexpressing clones, compared to control cells (N=10 cells from at least 8 different fat bodies per genotype). F. Fat body specific depletion of PWP1 by RNAi leads to decreased TAG levels (N=4 replicates of ≥10 larvae/replicate for each genotype).G. Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK7 and PWP1 fat body (CG-GAL4) knockdown with LipidTOX staining and DAPI to visualize lipid droplets and nucleus, respectively. Scale bar: 50 µm. H, I. Fat body specific knockdown of PWP1 suppresses increased lipid droplet number (H; N>30 cells per genotype) and increased nuclear area (I; N>45 cells per genotype) caused by ERK7 knockdown (BDSC 56939).


Figure 7A , B . Expression of sugarbabe is downregulated in fat bodies upon knockdown of PWP1 ( A ) and ectopic expression of ERK7 ( B ) ( N = 3 replicates of 10 fat bodies / replicate for each genotype ) . C . ERK71 mutants fail to maximally downregulate sugarbabe expression upon fasting ( N = 4 replicates of 10 larvae / replicate for each genotype ) . D . Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK71 and sug mutants ( sug17Δ / Df ( 2R ) Exel7123 ) with LipidTOX and DAPI staining to visualize lipid droplets and nucleus , respectively . Scale bar : 50 µm . E . sug mutant ( sug17Δ / Df ( 2R ) Exel7123 ) suppresses increased lipid droplet number observed in ERK71 mutants ( N > 20 ) . F . Ectopic sugarbabe expression in the fat body partially rescues TAG levels in the CG > ERK7 larvae ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) . G . sug mutant ( sug17Δ / Df ( 2R ) Exel7123 ) suppresses increased nuclear size phenotype of ERK71 mutant fat body cells ( N ≥ 25 cells per genotype ) . Ectopic Sugarbabe expression in the fat body partially restores the pupariation rate ( H ) ( p < 0 . 001 for the rescue , analyzed by Log - rank test , N = 4 replicates of 30 larvae / replicate for each genotype ) of CG > ERK7 larvaeI . Ectopic Sugarbabe expression in the fat body partially restores the pupal volume of CG > ERK7 larvae ( I ) ( N = 4 replicates of 10 pupae / replicate for each genotype ) . J . Addition of 20 % sucrose improves the growth rate and survival of CG > ERK7 larvae . Pupariation kinetics of CG > ERK7 larvae on 20 % yeast or 20 % yeast + 20 % sucrose diet ( p < 0 . 0001 for the rescue , analyzed by Log - rank test , N = 4 replicates of 30 larvae / replicate for each genotype and diet ) .

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Figure 7A, B. Expression of sugarbabe is downregulated in fat bodies upon knockdown of PWP1 (A) and ectopic expression of ERK7 (B) (N=3 replicates of 10 fat bodies/replicate for each genotype).C. ERK71 mutants fail to maximally downregulate sugarbabe expression upon fasting (N=4 replicates of 10 larvae/replicate for each genotype).D. Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK71 and sug mutants (sug17Δ/Df(2R)Exel7123) with LipidTOX and DAPI staining to visualize lipid droplets and nucleus, respectively. Scale bar: 50 µm. E. sug mutant (sug17Δ/Df(2R)Exel7123) suppresses increased lipid droplet number observed in ERK71 mutants (N>20). F. Ectopic sugarbabe expression in the fat body partially rescues TAG levels in the CG>ERK7 larvae (N=4 replicates of ≥10 larvae/replicate for each genotype).G. sug mutant (sug17Δ/Df(2R)Exel7123) suppresses increased nuclear size phenotype of ERK71 mutant fat body cells (N ≥25 cells per genotype).Ectopic Sugarbabe expression in the fat body partially restores the pupariation rate (H) (p<0.001 for the rescue, analyzed by Log-rank test, N=4 replicates of 30 larvae/replicate for each genotype) of CG>ERK7 larvaeI. Ectopic Sugarbabe expression in the fat body partially restores the pupal volume of CG>ERK7 larvae (I) (N=4 replicates of 10 pupae/replicate for each genotype).J. Addition of 20% sucrose improves the growth rate and survival of CG>ERK7 larvae. Pupariation kinetics of CG>ERK7 larvae on 20% yeast or 20% yeast + 20% sucrose diet (p<0.0001 for the rescue, analyzed by Log-rank test, N=4 replicates of 30 larvae/replicate for each genotype and diet).


Figure 1F . Microscopic images of the Q97 - GFP fluorescence . Bars , 5 µm . G . The percentage of cells containing or lacking detectable aggregates was quantified . Cell boundaries are indicated by dashed lines .

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Figure 1F. Microscopic images of the Q97-GFP fluorescence. Bars, 5 µm. G. The percentage of cells containing or lacking detectable aggregates was quantified. Cell boundaries are indicated by dashed lines.


Figure 2E . Q97 - GFP was expressed in mia40 - 3 and corresponding wild type cells for the times indicated . Cells were harvested , washed and survivors were counted after plating on glucose plates . Mean values and standard deviations of three replicates are shown .

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Figure 2E. Q97-GFP was expressed in mia40-3 and corresponding wild type cells for the times indicated. Cells were harvested, washed and survivors were counted after plating on glucose plates. Mean values and standard deviations of three replicates are shown.


Figure 3B . Cell extracts were analyzed by Western blotting after shifting cultures to galactose for 16 h . 25 , Q25 - GFP ; 97 , Q97 - GFP ; ev , empty vector . C . Microscopy images of the indicated strains 12 h after shifting them to galactose . Note that in GAL - Mia40 cells the form and number of aggregates is very different to WT cells . Bar , 5 µm . E . PolyQ proteins were expressed for 24 h before survivors were counted . Mean values and standard deviations from three independent experiments are shown .

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Figure 3B. Cell extracts were analyzed by Western blotting after shifting cultures to galactose for 16 h. 25, Q25-GFP; 97, Q97-GFP; ev, empty vector.C. Microscopy images of the indicated strains 12 h after shifting them to galactose. Note that in GAL-Mia40 cells the form and number of aggregates is very different to WT cells. Bar, 5 µm.E. PolyQ proteins were expressed for 24 h before survivors were counted. Mean values and standard deviations from three independent experiments are shown.


Figure 4B . Still frames after different times of growth indicate a uniform fluorescence in Q25 - GFP - expressing cells . Q97 - GFP expression in wild type leads to many small and scattered aggregates per cell . In GAL - Mia40 cells , Q97 - GFP accumulates to much larger intensities but then suddenly collapses into one single aggregate per cell . The arrow depicts aggregate formation . Bars , 4 µm . C . Quantified signal intensities in cells of the indicated mutants . In top and middle graphs , each curve corresponds to a single cell . Averaged signals ( mean + SEM ) in bottom graph ( N = 20 for WT , N = 17 cells for GAL - Mia40 ) .

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Figure 4B. Still frames after different times of growth indicate a uniform fluorescence in Q25-GFP-expressing cells. Q97-GFP expression in wild type leads to many small and scattered aggregates per cell. In GAL-Mia40 cells, Q97-GFP accumulates to much larger intensities but then suddenly collapses into one single aggregate per cell. The arrow depicts aggregate formation. Bars, 4 µm. C. Quantified signal intensities in cells of the indicated mutants. In top and middle graphs, each curve corresponds to a single cell. Averaged signals (mean + SEM) in bottom graph (N=20 for WT, N=17 cells for GAL-Mia40).