Fig 2: ARG2, RGS4, and RGS5 expression in BHP18-21v cells analyzed using real-time PCR and western blotting.(A) MOI-dependent changes in gene expressions quantified by real-time PCR 72 h after adenoviral vector infection (n = 6). Ratios of gene expressions in infected cells to those in non-infected cells 72 h after infection are shown. (B) Time-dependent changes in gene expressions quantified by real-time PCR after 300 MOI adenoviral vector infection (n = 6). Ratios of gene expressions in infected cells to those in pre-infected cells (0 h) are shown. Error bars indicate SEM. *p<0.05 and **p<0.01, significant differences between AdNKX2-1 and AdLacZ. (C) Western blot showing ARG2 and RGS4 expression 72 h after 300 MOI AdLacZ or AdNKX2-1 infection (upper panel). Loading control, GAPDH expression (bottom panel).

Fig 3: Deconvolution of the Mouse Hepatic Mesenchyme Identifies Three Distinct Subpopulations in Liver Homeostasis(A) Overview: representative immunofluorescence image depicts GFP reporting in the liver of healthy Pdgfrb-BAC-eGFP reporter mice. Scale bar, 100 µm; portal vein (*) as indicated. CV, central vein; PV, portal vein. GFP+ cells were processed for droplet- and plate-based scRNA-seq.(B) t-Distributed stochastic neighbor embedding (t-SNE) visualization: 12,533 mesenchymal cells (median nGene = 2,268, nUMI = 5,725) cluster into three subpopulations. Selected marker genes are listed alongside each cluster.(C) Representative immunofluorescence images of healthy murine livers: CD34/Reelin/Calponin 1 (red), PDGFRß (green), PanCK (white). Scale bar, 100 µm; portal vein (*) and central vein (#) as indicated. Yellow arrow indicates CD34+ fibroblasts.(D) Schematic representation of the topography of the three identified mesenchymal subpopulations in the liver. CV, central vein; PV, portal vein; HA, hepatic artery; BD, bile duct.(E) Representative immunofluorescence images of healthy human livers: MFAP4/RGS5/MYH11 (red), PDGFRß (green), DAPI (blue). Scale bar, 100 µm; portal vein (*) as indicated.(F) GO enrichment terms associated with signatures A–C corresponding to the three identified mesenchymal subpopulations.See also Figures S1, S2, and S3.

Fig 4: RGS5 inhibits the expression of the CDC25A protein induced by the MAPK/ERK signaling pathway. (A) The expression of the CDK2, CDC25A and cyclin E proteins was higher compared with the LV-H negative control group under hypoxic conditions, and the expression of the p53 and p21 proteins exhibited no change; the expression of phosphorylated ERK protein in the LV-siRGS5 group was markedly decreased and phosphorylation of p38 was not affected. (B) When the activity of ERK1/2 was inhibited with PD98059, the expression of the CDC25A, CDK2 and cyclin E proteins was decreased compared with the control group, and the effect of PD98059 on downregulating the expression of these proteins was in coordination with RGS5. (C) Treating ODMECs with PD98059 and LV-siRGS5 concurrently significantly reduced the cell proliferation rate (n=3; *P<0.05). RGS5, regulator of G-protein signaling 5; ODMECs, ovarian carcinoma-derived endothelial cells; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; LV, lentiviral vector.

Fig 5: LV-siRGS5 effectively inhibits the expression of RGS5 at the mRNA and protein level in ODMECs. (A) At the mRNA level, the blank group, the negative control group LV-H-siRNA and the LV-siRGS5 three groups of quantitative analysis were 1.07±0.05, 1.32±0.09 and 0.52±0.06, 0.60±0.04, 0.54±0.02, respectively; the expression of negative control LV-H did not affect the RGS5 mRNA (n=3, P<0.05). (B) At the protein level, the blank group, the negative control group LV-H-siRNA and the LV-siRGS5 three groups of quantitative analysis was 0.92±0.08, 0.95±0.21 and 0.17±0.04, 0.22±0.08, 0.19±0.12, respectively; the negative control LV-H-siRNA did not affect the expression of RGS5 (n=3, P<0.05). (C) LV-siRGS5 did not interfere with the expression of RGS2, RGS4 and RGS16 in the B/R4 subfamily. RGS5, regulator of G-protein signaling 5; ODMECs, ovarian carcinoma-derived endothelial cells; LV, lentiviral vector.