EMBO/SourceData huggingface dataset:
Figure 1c, Knockdown of NetB in the eye disc of GMR-RasV12 flies enhances survival. d, NetB heterozygous mutant flies survive better over Rasv12-induced oncogenic stress. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparisons test (c) and two-tailed unpaired t-test d, *P < 0.05, **P <0.01, ****P <0.0001.f, qRT-PCR with mRNA from the eye disc confirms higher expression of NetB in GMR-RasV12 flies Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by two-tailed unpaired t-test *P < 0.05, **P <0.01, ****P <0.0001.g-h, Oncogenic Ras expression in the eye disc induces NetB protein, which was detected by GFP signal using CPTI-000748, a protein trap of NetB. Single confocal z-section images are shown. i, Quantification of GFP signals in g and h. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by two-tailed unpaired t-test *P < 0.05, **P <0.01, ****P <0.0001. Scale bar, 50 μm.
Figure 2a-b, The amount of NetB protein increases in the fat body GMR-RasV12 (a) compared to control flies (b). CPTI-000748, a protein trap of NetB, labels endogenous NetB. Data information: Scale bar, 20 μm (a-be-h, Ectopic expression of GFP-tagged NetB in the eye disc induces high levels GFP-NetB protein in the fat body. Single confocal z-section are shown images (e and g). Data information: Scale bar, 20 μm f-h 50 μmj-k, Ectopic expression of GFP-tagged NetB in the eye disc leads to detection of GFP signals in the hemolymph. Data information: Scale bar, 20 μm j-k)
Figure 3a, Knockdown of InR (InR) or expressing a dominant-negative form of InR (InR-DN) in the fat body using CG-Gal4 increases survival over oncogenic Ras expression in the imaginal disc. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparisons test (a NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.c, qRT-PCR demonstrates that TMLHE expression in the fat body is reduced by Ras expression in the eye disc and reversed by insulin inhibition in the fat body. d, qRT-PCR demonstrates that NetB knockdown in the eye disc increases TMLHE expression in the fat body. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test (c and d) NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.e, Knockdown of unc-5, but not other NetB receptors (Fra or Dscam1), in the fat body increases TMLHE expression. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparisons test e). NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.
Figure 4b, Local expression of oncogenic Ras in the eye disc decreases the amount of carnitine in the fat body. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by two-tailed unpaired t-test NS, not significant, **P <0.01, ***P <0.001, ****P <0.0001.c, TMLHE knockdown in the fat body aggravates organismal survival over the oncogenic stress. d, In the absence of GMR-RasV12, inhibition of TMLHE in the fat body is not sufficient to induce organismal death. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by two-tailed unpaired t-test NS, not significant, **P <0.01, ***P <0.001, ****P <0.0001.g, Feeding of acetyl-CoA precursor (acetate) makes GMR-RasV12 flies survive even with TMLHE knockdown in the fat body. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by two-tailed unpaired t-test NS, not significant, **P <0.01, ***P <0.001, ****P <0.0001.
Figure 5e, qRT-PCR with mRNA from the adult gut confirms higher expression of NetB in esg ts>RasV12 flies after oncogenic Ras induction for 10 days. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and n represents the number of flies that were analyzed. The statistical significance was determined by two-tailed unpaired t-test NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.f-g, Oncogenic Ras expression in the adult gut induces NetB protein, which was detected by GFP signal using CPTI-000748, a protein trap of NetB. Ras was induced for 10 days. Single confocal z-section images are shown. h, Quantification of GFP signals in f and g. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and n represents the number of flies that were analyzed. The statistical significance was determined by two-tailed unpaired t-test NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001. Scale bar, 50 μm.i, Knockdown of NetB in the adult gut of esg ts>RasV12 flies enhances survival. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and n represents the number of flies that were analyzed. The experiments were repeated independently at least twice with similar results The statistical significance was determined by log-rank (Mantel-Cox) test NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.l, Knockdown of TMLHE in the fat body of adult flies shortened lifespan of esgts>RasV12 flies but not of control flies. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and n represents the number of flies that were analyzed. The experiments were repeated independently at least twice with similar results The statistical significance was determined by log-rank (Mantel-Cox) test l). NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.
Figure 1B Light transmittance of tissue increased more rapidly with FEC than with CLARITY. Each datapoint plotted is the mean from three neighboring transmittance readings (hence resulting in 10 datapoints from the 30 measurements/brain, technical replicates) (One-way ANOVA *** p<0.001, **** p<0.0001) (n = 3 per group, biological replicates).C Comparison of mouse brains cleared by FEC (6 hrs) and passive (3 days). Passive clearing was incomplete even after 3 days.D After whole-brain electrophoretic staining with polythiophenes, sagittal 500 µm slices were cut and plaques were counted. By passively re-staining the same slice, no additional plaques were detected ("FEC + passive"). Focal shifts after slice reprocessing account for the slight differences between the images. One biological replicate. Scale bars: 100 µm
Figure 4A, B The distinct significant effect of each of the strongest treatments on plaque size cluster, NB360 5-month and LIN5044 14-month, as well as their overlap, using either (A) neuroanatomical boundaries, or (B) SAVs.
Figure 1(B) MPL-MIL HCM assay in HeLaWT and HeLaHexaKO expressing HT-LC3B, starved in EBSS for 90min ± 100 nM BafA1. Cells were sequentially incubated with HT ligands MIL and MPL, and HCM quantification carried out. Plasma membrane permeabilization was carried out with 4 nM Agilent XF Plasma Membrane Permeabilizer/PMP. PMP was used during the MIL staining and prior to fixation in 4% paraformaldehyde. MPL staining followed the fixation. (B i) MPL-accessible membrane-bound HT-LC3B (remaining available to MPL after MIL saturation). (B ii) MIL-accessible membrane-bound HT-LC3B; (B iii) MIL/MPL puncta ratio. Circles, control samples (full medium); rectangles (EBSS); triangles (EBSS+BafA1); green, MIL; red, MPL; gray, MIL/MPL ratios. Images and statistics, HCM images (masks: white, primary objects/cells; red, MPL profiles; green, MIL profiles), one of 60 fields/well, 1,000 primary objects (cells) per well; 6 wells per plate (sampling error), triplicate plates (independent biological replicates). Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. All values are mean ± SD, n = 3 biologically independent replicates.(C-E) Selected electron microscopy (EM) images of HeLaWT and HeLaHexaKO EBSS-induced for autophagy for 90 min and their quantification (graph in F). AP, autophagosomes (phagophores or double membrane autophagosomes with content of similar electron-density to surrounding cytosol); Amph, amphisomes; AL, autolysosomes; MVB, multivesicular bodies; LELy, late endosomes or lysosomes. Statistics, unpaired t-test, two groups: HeLaWT and HeLaHexaKO (93 images each; sample mean, SE). Blue single asterisk, cytoplasmic cargo inside amphisome or autolysosome; double asterisk, phagophore structures; arrows, phagophores or autophagosomes. (F) Super-resolution (dSTORM) of HeLaHexaKO HT-LC3B stained with MIL (note staining of the interiors of the globular structures).
Figure 2(B) Representative images of SolVit assay with HeLaWT and HeLaHexaKO as donors, and HeLaHexaKO HT-LC3B as acceptor. SolVit HCM images (example). MPL+ (red), sealed LC3B+ membranes MIL+ (green), unsealed LCB+ membranes. (C) Quantification of MPL+ and MIL+ profiles (60 fields/well acquired): (C i) MPL+ (red) puncta per field. (C ii) MIL+ (green) puncta per field. (C iii) MIL+/MPL+ profile ratios (gray). Data information: Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. All values are mean ± SD, n = 3 biologically independent replicates, each HCM experiment: 1,000 valid primary objects/cells per well (max fields per well, 60), 5 wells/sample.
Figure 3(B) Cells were starved in EBSS for 90 min ±100 nM BafA1, sequentially incubated with MIL and MPL. MPL (B i; red), marker of sealed membranes; MIL (B ii; green), marker of unsealed membranes; profiles quantified by HCM. (B iii) MIL+/MPL+ puncta ratio (gray),(C) HelaWT, HeLaHexaKO, HeLaLC3TKO, HeLaGABATKO stably expressing HT-LC3B were starved with EBSS for 90min or 6h. Cell were incubated with ± HaloTag ligand fluorescently labeled with TMR. In-gel fluoresce (TMR) and Western blots (anti-HaloTag antibody) of freed HaloTagTMR were imaged and band intensity quantified. (D) Quantification of in-gel fluorescence band intensity and Western blot with anti-Halo antibody.
Figure 4(C) SolVit complementation/rescue analysis (quantification) of acceptor HeLaLC3TKO-HT-LC3B PNS with donor PNS from HeLaWT, HeLaLC3TKO or HelaGABATKO cells ±ATP, 1 h, 37˚C. (D) SolVit complementation/rescue analysis (quantification) of acceptor HeLaGABATKO-HT-LC3B PNS with donor PNS from HeLaWT, HeLaLC3TKO and HelaGABATKO cells ±ATP, 1 h, 37˚C.
Figure 5(A) GST pulldown analysis of in vitro translated and radiolabeled [35S] Myc-VPS37A with GST or GST tagged LC3A, LC3B, LC3C, GABARAP, GABARAP-L1 and GABARAP-L2. 2% of input was loaded.(B) Co-IP analysis of interactions between FLAG-VPS37A, GFP-LC3A and GFP-GABARAP(GABA) in HEK293T cells. 5% of input was loaded.(F) Co-IP between GFP-LC3A with FLAG-VPS37AFL, UEVVPS37A and VPS37AdeltaUEV.(G) Co-IP analyses in HEK293T cells expressing FLAG-VPS37AFL, FLAG-VPS37AdeltaN (1-90), VPS37AdeltaN(1-20) and FLAG-Nter-EGFP and co-transfected with GFP-GABA. 5% of input was loaded. (H) Co-IP analyses in HEK293T cells expressing FLAG-VPS37AFL, FLAG-VPS37AdeltaN (1-90), VPS37AdeltaN(1-20) and FLAG-Nter-EGFP and co-transfected with GFP-LC3A. 5% of input was loaded.(I) Complementation of VPS37A KO with VPS37A constructs that have or lack mATG8-binding region. Quantification of MIL+ puncta in Huh7WT HT-LC3B (circles) and Huh7VPS37AKO HT-LC3B cells (squares), transfected with empty FLAG, full length VPS37A (FLAG-VPS37AFL) or mutant VPS37A (FLAG-VPS37AdeltaN(1-20)). Data information: Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. All values are means ± SD, n = 3 biologically independent replicates, each HCM experiment: 1,000 valid primary objects/well, 6 wells/sample. (J) HCM images, examples corresponding to Fig 5I, scale bar, 10 μm.
Figure 6(B) MPL-MIL HCM assay in Huh7WT and Huh7VPS37AKO stably expressing HT-LC3B. Quantification: (B i) membrane-permeant HT ligand (MPL) staining of LC3B-II sequestered within sealed membranes. (B ii) MIL staining of membrane-bound HT-LC3B-II accessible to the cytosol and (B iii) MIL/MPL puncta ratio.(F-H) MPL-MIL HCM assay in Huh7WT (control or CHMP2A siRNA-treated cells; immunoblot in F) stably expressing HT-LC3B during CCCP-induced mitophagy in ± BafA1 (100 nM). (G) Quantification: (G i) MPL+ sealed membranes; (G ii) MIL+ unsealed membranes; (G iii) MIL/MPL puncta ratio. (H) HCM images corresponding to panel G.
Figure 7(B) Quantification of puncta: (B-i) MPL+ puncta (red), sealed LCB+ membranes; (B-ii) MIL+ (green) puncta, unsealed/ligand-accessible LCB+ membranes; (B-iii) MIL/MPL ratios (gray).(C) Quantification of MIL+ puncta (unsealed/ligand-accessible LC3B+ membranes) in Huh7WT (circles) or Huh7VPS37AKO (squares) cells stably expressing HT-LC3B. Cells were transfected with empty FLAG vector, FLAG-VPS37A full length (VPS37AFL), FLAG-VPS37AdeltaN(1-90), or FLAG-VPS37AdeltaN(1-20) constructs. CCCP, 6 h treatment. (D) HCM images of Huh7VPS37AKO HT-LC3B with VPS37A constructs (VPS37AFull, FLAG-VPS37AdeltaN(1-90), FLAG-VPS37AdeltaN(1-20) and FLAG-VPS37ANter-GFP). FLAG-Tag VPS37A constructs, expression detected as red; and MIL+ puncta, pseudo color green. Data information: Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. All values are mean ± SD, n = 3 biologically independent experiments, each HCM experiment: 1,000 valid primary objects/cells per well, 6 wells/sample, Scale bar, 10μm.
Figure 116 days after tumor inoculation, B16F10 tumors from WT and Ythdf2 cKO mice were digested for flow cytometry analysis. D, Percentage (left) and absolute numbers (right) of CD8+ T cells in tumors (n=4). Each data point represents one mouse. Data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.16 days after tumor inoculation, B16F10 tumors from WT and Ythdf2 cKO mice were digested for flow cytometry analysis. E, Representative plots (left) and the percentage (right) of IFNγ producing CD8+ T cells (n=5). Each data point represents one mouse. Data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.16 days after tumor inoculation, B16F10 tumors from WT and Ythdf2 cKO mice were digested for flow cytometry analysis. G, Percentage (left) and absolute numbers (right) of Treg cells (n=4). Each data point represents one mouse. Data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.16 days after tumor inoculation, B16F10 tumors from WT and Ythdf2 cKO mice were digested for flow cytometry analysis. I, The proportion of Treg cells expressing the indicated surface marker (n=5). Each data point represents one mouse. Data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.
Figure 2splenocytes from 1-year-old WT and Ythdf2 cKO mice were analyzed by flow cytometry (n=4). C, Representative plots (left) and the percentage (right) of naïve (CD62LhiCD44lo), central memory (cMem, CD62LhiCD44hi), and effector memory (eMem, CD62LloCD44hi) T cells. Data are presented as means ± s.d. and were analyzed by two-tailed unpaired Student's t-test.splenocytes from 1-year-old WT and Ythdf2 cKO mice were analyzed by flow cytometry (n=4). D, Representative plot (left) of FOXP3 expression level and the FOXP3 expression MFI (right) in splenic Treg cells. Data are presented as means ± s.d. and were analyzed by two-tailed unpaired Student's t-test.splenocytes from 1-year-old WT and Ythdf2 cKO mice were analyzed by flow cytometry (n=4). E, Representative plots (left) and percentage (right) of FOXP3 expressing Treg cells in CD4 T cells. Data are presented as means ± s.d. and were analyzed by two-tailed unpaired Student's t-test.splenocytes from 1-year-old WT and Ythdf2 cKO mice were analyzed by flow cytometry (n=4). F, Representative plot (left) of FOXP3 Treg cells PD-1 expression and the MFI value (right) in WT and Ythdf2 cKO Treg cells. Data are presented as means ± s.d. and were analyzed by two-tailed unpaired Student's t-test.
Figure 3B, Functional assay with WT (n=5) or Ythdf2 cKO Treg (n=5) cells isolated from B16F10 tumors on day 16. B, Percentage of IFNγ producing CD8+ T cells after coculture. Each data point represents one mouse. Data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.E, F, Ratio of Annexin V+ Treg cells in the B16F10 tumor (E, n=4) or spleen (F, n=3). Each data point represents one mouse. Data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.G, Representative plots (left) and ratio (right) of GFP+/GFP- Treg cells in B16F10 tumors from Foxp3CreGFP/+Ythdf2fl/+ or Foxp3CreGFP/+Ythdf2fl/fl mice.
Figure 4E, IGV visualization showing m6A and YTHDF2 binding at Nlrc3, Nfkbie, and Traf3 loci in CD4+ T cells (GSE188853).
Figure 5B16F10 tumors were developed in mice receiving anti-TNFα or control IgG treatments and harvested on day 15 after tumor injection (n=5, WT + anti-TNFα n=4). C, Ratio of tumor-infiltrating Treg cells in CD4+ T cells.B16F10 tumors were developed in mice receiving anti-TNFα or control IgG treatments and harvested on day 15 after tumor injection (n=5, WT + anti-TNFα n=4). D, Percentage of apoptotic Treg cells in the tumor. In C and D, data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.E, Relative mRNA levels of indicated genes in WT or Ythdf2 cKO Treg cells before or after 24 hours TNFα stimulation. Each data point represents one mouse. Data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.F, WT or Ythdf2 cKO Treg cells from the spleen were stimulated with 10 ng/mL TNFα in vitro. 24 hours post TNFα stimulation, cells were treated with Actinomycin D. Decay of Nlrc3, Nfkbie, and Traf3 was monitored by RT-qPCR (n=3, biological replicates). Data are presented as means ± s.d. and analyzed two-way ANOVA.H, Functional assay with WT (n=4) or Ythdf2 cKO Treg (n=5) cells isolated from B16F10 tumors on day 16. H, Percentage of IFNγ producing CD8 T cells after coculture. Each data point represents one mouse. Data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.
Figure 1B Representative images of Drosophila adult wings that resulted from larvae wing imaginal discs expressing a control RNAi (mCherry RNAi), a lds RNAi, or co-expressing san RNAi with control RNAi (san RNAi control RNAi) or with lds RNAi (san RNAi lds RNAi).
Figure 2A Representative images of cells in the wing disc pouch upon nub-Gal4-mediated RNAi for lds (bottom) compared to a nub-Gal4 control (top). Cells also express HisH2AvD-mRFP1 (magenta) and centromeric marker cid-EGFP (green); Times (min:sec) are relative to NEBD; scale bars 5 µm and applies to all images. B Mitotic timing defined from NEBD to anaphase onset; each dot represents a different cell (n=77 cells from at least 6 independent movies per condition); black lines represent mean; statistical analysis was performed using the non-parametric Mann-Whitney test. Data information: Scale bars are 10 µm and apply to all images.E Localisation of Lds-EGFP (green) throughout mitosis in Drosophila syncytial embryos (E). Times are relative to NEBD, flies also express HisH2AvDmRFP1 (magenta). Data information: Scale bars are 10 µm and apply to all images.
Figure 3A Analysis of ongoing transcription monitored by MCP-EGFP labelled nascent transcripts on a reporter containing MS2 loops (green), in the referred experimental conditions. Flies also express H2AvD-mRFP1 (magenta). Scale bar is 10 µm and applies to all images.
Figure 4B Segregation defects upon inhibition of transcription in luciferase and lds RNAi. Graph depicts mean ± s.e.m. of the frequencies of defects scored in 11 (luciferase RNAi, H2O and lds RNAi, α-amanitin) or 8 (lds RNAi, H2O and Luc RNAi, α-amanitin) independent embryos; a total of ~800 nuclei were scored per condition.
Figure 5A Wing morphology phenotype obtained upon depletion of Lds combined with control co-depletion (luciferase RNAi) or expression/depletion of chromosome architecture modulators. Wing morphology defects were classified according to their severity (1= normal wings and 5= no wing); depicted numbers indicate the average morphology grade and the number of flies counted, from at least 2 independent experiments.B Live cell imaging analysis of the kinetics of Rad21-EGFP (green) removal from mitotic chromosomes in luciferase- and lds-depleted embryos. DNA is marked with H2AvD-mRFP1 (magenta); times are relative to NEBD; scale bar is 10 µm and applies to all images.
Figure 6(A, Chromatin localisation of chromosome assembly factors (Top2 (A) in green) upon RNAi for luciferase (left) or lds (right) in early embryos. DNA is marked with H2AvD-mRFP1 (magenta); scale bars are 10 µm and apply to all images.C) Chromatin localisation of Condensin I subunit Barren (C), in green) upon RNAi for luciferase (left) or lds (right) in early embryos. DNA is marked with H2AvD-mRFP1 (magenta); scale bars are 10 µm and apply to all images.G Telophase images of embryos depleted for luciferase (left), Cap-D2 (middle) and wapl (right) RNAi, and expressing Lds-EGFP (green) and HisH2AvDmRFP1 (magenta). Bottom panels depict Lds-EGFP alone. Scale bar is 10 µm and applies to all images.
Figure 7B, C Loading of EGFP-Top (B) and Lds-EGFP (C) (labelled in green) on mitotic chromatin of metaphase arrested embryos (UbcH10C114S injection), upon subsequent injection of DMSO/ICRF-193; DNA is marked with H2AvD-mRFP1 (magenta); times (min:sec) are relative to time of injection. Graphs depict integrated intensities normalized to the first time point, represented as mean (dots) ± SD (grey area); n=54 (Lds+ ICRF-193, Lds+DMSO, Top2 + ICRF-193) or 47 (Top2 + DMSO), derived from 8-9 independent embryos per experimental condition. Data information: Scale bar is 10 µm and applies to all images.E-F FRAP analysis with and without ICRF-193 treatment for EGFP-Top2 (E) and Lds-EGFP (F). Metaphase-arrested embryos were microinjected with DMSO/ICRF-193 and an entire metaphase plate was bleached 10 min after. Graphs depict recovery of fluorescence signal over time. Dots represent average and grey areas SD; Top2: n= 14 (DMSO) and 18 (ICRF-193) metaphases; Lds: n= 11 (DMSO) and 9 (ICRF-193) metaphases, derived from 3-7 independent embryos Data information: Scale bar is 10 µm and applies to all images.
Figure 8A, Representative images of mitotic nuclei in prophase (1 min before NEBD) and metaphase (1 min before anaphase onset), upon RNAi for luciferase/lds, microinjected with water (A) Scale bars are 5 µm and refer to all images.B Representative images of mitotic nuclei in prophase (1 min before NEBD) and metaphase (1 min before anaphase onset), upon RNAi for luciferase/lds ICRF-193 (B). Scale bars are 5 µm and refer to all images.
Figure 1A Representative G9A immunostaining (left) and semi-quantitative results (right) of human kidney samples. AKI (acute kidney injury), n = 3; Con (control), n = 6.C Representative renal H&E images (left) with quantitative pathological scores (right) of WT (n = 8) and Ehmt2Ksp mice (n = 7) at day three after renal I/R injury (I/R 3D).G, H Representative immunoblots (G) and quantitative results (H) of p-Rip3 and p-Mlkl of WT (n = 3) and Ehmt2Ksp mice (n = 3) under I/R 3D injury.I Representative Sirius Red staining (left) with quantitative results (right) on the kidney sections of WT (n = 7) and Ehmt2Ksp (n = 4) mice at day seven after renal I/R injury (I/R 7D).K Representative TUNEL staining (left) with quantitative results (right) of WT (n = 8) and Ehmt2Ksp mice (n = 4) under I/R 7D injury.