Extended Data Fig. 7
From: Hepatocellular senescence induces multi-organ senescence and dysfunction via TGFβ
Liver-derived SASP factors affect the TGFΒ and LIF/JAK-STAT signalling pathways in the kidney. (a) GSEA plot for a SASP gene set on the significant differentially expressed genes from the bulk liver RNA-seq data in the ΔMdm2Hep model versus control at day 4. (b) UMAP plots showing the distribution of the cells that have a positive score for the TGFβ gene signature in the control (left) and the ΔMdm2Hep model (right) renal cells. Inset images are representative kidney sections stained for Smad7 by ISH (RNAscope) n=3/3 for control/ ΔMdm2Hep respectively, dashed lines highlight renal tubules. (c) Representative images of dual p21 IHC/Smad7 ISH in the kidney; pink arrows highlight examples of Smad7 detection in red, whilst p21 is detected by brown 3,3′-Diaminobenzidine (DAB) staining; n=3/3 for control/ ΔMdm2Hep respectively. (d) UMAP plots showing the distribution of the cells that have a positive score for the JAK-STAT gene signature in the control (left) and the ΔMdm2Hep model (right) renal cells. Inset images are representative kidney sections stained for pSTAT3 by IHC; n=3/3 for control/ ΔMdm2Hep respectively. Scale bars are 50μm and 5μm in inset magnification. (e) Automated quantification of pSTAT3+ in renal cortical cells; data are presented as percentage of total cortical cells, n=6 and 7 in control and ΔMdm2Hep mice respectively; two-tailed Mann-Whitney test. Bars are mean ± S.E.M. and the numbers on the graphs are p values. (f) GSEA plot for a TGFβ signalling pathway gene set on the significant differentially expressed genes from the bulk liver RNA-seq in the ΔMdm2Hep model versus control at day 4. (g) Western blots for pSMAD2 and pSMAD3 and their respective total protein on whole kidney lysates from ΔMdm2Hep model and control mice at day 4. n=5 mice in each group; see experimental schematic in Fig. 1a. 1 gel was run for pSMAD2/SMAD2 and another one for pSMAD3/SMAD3. β-actin was used as a loading control on both gels. (h) Representative images of ISH (RNAScope) for Tgfb1 and Tgfb2 on liver sections of ΔMdm2Hep and control mice detected by DAB (brown); n=3/3 for control/ ΔMdm2Hep respectively for each ISH. (i) Representative images of dual p21 IHC/Tgfb1 ISH (RNAScope) in the liver; Tgfb1 ligand detection in red, whilst p21 is detected by brown DAB staining; n=3/3 for control/ ΔMdm2Hep respectively. Normalised Tgfbr1, Tgfbr2 and Tgfbr3 transcript counts (FPKMs) from bulk liver (j) and kidney (k) at day 4 in the ΔMdm2Hep model versus control, n=4 except n=3 in liver control; both unpaired two-tailed t-test. All bars are mean ± S.E.M and the numbers on the graphs are p values. (l) Representative images of ISH (RNAScope) for Tgfbr1 on kidney sections of ΔMdm2Hep model and control mice at day 4 detected by brown DAB staining; n=3/3 for control/ ΔMdm2Hep respectively. Dashed lines highlight renal tubules. Scale bars are 50μm. Source numerical data and unprocessed blots are available in source data.
