Journal: Gene therapy

Article Title: Targeted inhibition of platelet-derived growth factor receptor-beta subunit in hepatic stellate cells ameliorates hepatic fibrosis in rats.

doi: 10.1038/gt.2008.93

Figure Lengend Snippet: Figure 3 Localization of LacZ driven by CMV promoter and GFAP promoter in normal rats, acute liver injury rats and chronic liver injury rats. (a) Representative graphs of b-galactosidase (b-gal) immunofluorescence showed the distribution of b-gal-positive cells in normal rats, acute liver injury rats and chronic liver injury rats treated with pCMV-shRNA-LacZ or pGfa-shRNA-LacZ. Magnification of 20. The scale bar represents 80 mm. (b) Representative graphs of a-SMA and GFAP immunofluorescence showed that in the chronic injured liver, a-SMA and GFAP proteins distributed mainly around the portal area and the hyperplastic bile duct, whereas in the acute injured liver, both the proteins revealed a much more diffuse distribution in the hepatic lobule. In addition, the GFAP-positive cells were more than the a-SMA- positive cells both in the acute injured liver and in the chronic injured liver. Magnification of 20. The scale bar represents 80 mm. (c) Representative graphs of b-gal and a-SMA double-staining revealed that b-gal protein in pCMV-shRNA-LacZ-treated livers could be expressed in hepatocytes (blue arrow) and activated HSCs stained by a-SMA (white arrow), magnification of 63. The scale bar represents 40 mm. (d) Representative graphs of b-gal and GFAP double-staining showed that b-gal-positive cells were all GFAP-positive HSCs (pink arrow) in pGfa-shRNA-LacZ-treated livers, magnification of 63. The scale bar represents 40 mm. (e) Representative graphs of b-gal and a-SMA double-staining showed that some b-gal protein was expressed in activated HSCs stained by a-SMA (white arrow) in pGfa-shRNA- LacZ-treated livers, magnification of 63. The scale bar represents 40 mm. CMV, cytomegalovirus; GFAP, glial fibrillary acidic protein; HSCs, hepatic stellate cells; PDGFR-b, platelet-derived growth factor receptor-b subunit; a-SMA, a-smooth muscle actin; shRNA, short hairpin RNA; RT-PCR, reverse transcriptional-PCR.

Article Snippet: Frozen liver sections (8 mm in thickness) were incubated with mouse anti-b-galactosidase monoclonal antibody (Promega, Madison, WI, USA), rabbit anti-GFAP polyclonal antibody (Boster, Wuhan, China) or rabbit anti-a-SMA polyclonal antibody (Bios, Beijing, China) for 20 h at 4 1C, then washed in phosphate-buffered saline and incubated for 30 min with Cy5-labeled anti-rabbit IgG antiserum and FITC-labeled anti-mouse IgG antiserum (Jackson, West Grove, PA, USA) at 1:100 dilution in phosphate-buffered saline.

Techniques: shRNA, Double Staining, Staining, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Cell reports

Article Title: Mitf is a Schwann cell sensor of axonal integrity that drives nerve repair.

doi: 10.1016/j.celrep.2023.113282

Figure Lengend Snippet: Figure 1. Network analysis links Mit/Tfe to SC response in CMT4J (A) Diagram of SC development and injury response. Mitf has a biphasic expression pattern. Egr2/Krox20:CRE is expressed in immature SCs, and Dhh:CRE is expressed by myelinating and non-myelinating SCs. Recombination from SC CRE expression will indelibly label subsequent cellular lineages (e.g., repair cells). (B) Workflow for the identification of differentially expressed genes (DEGs) and TF motif enrichment (GSEA) in sciatic nerves from Fig4plt mice. (C) Volcano plot showing log fold change versus adjusted p value of sciatic nerves of Fig4plt at P4. Each dot represents a gene. Blue and purple dots are differentially expressed. n = 3, each sample is pooled from both sciatic nerves of 3 animals. (D) Heatmap of expression levels (Z score) of DEGs from (C). Each row corresponds to the indicated gene. (E) Biological process (BP) GO terms from DEGs. (F and G) Gene set enrichment analysis (GSEA) of predicted transcription factor (TF) binding motifs in genes (F) down in Fig4plt and (G) up in Fig4plt. (H) Schematic of sciatic nerve protein lysate of postnatal WT mice. (I) Protein lysates of sciatic nerves from WT animals analyzed at P0, P7, P14, P21, P35, and P56 by western blot. (J) Quantification of (I) showing Mitf protein expression normalized against GAPDH and relative to expression at P0 (n = 4). Ordinary one-way ANOVA with Dunnett’s multiple-comparisons test. The data are represented as the mean ± SEM. (K) A schematic of sciatic nerve immunofluorescence. (L) Mitf KI construct and genetic strategy to convert the Mitf KI allele (MitfKI) to the Mitf floxed allele (MitfFL). (M–N000) Longitudinal sections of adult sciatic nerves (3 months) were co-immunostained for Mitf (magenta) and GFAP (green) and stained with Hoechst (blue). Yellow arrowheads show Mitf colocalization with GFAP. White arrowheads show Mitf staining without GFAP. (M–M000) are WT. (N–N000) are MitfKrox20 mutants, in which Mitf is absent from both GFAP and GFAP+ SCs. Scale bar, 50 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Chicken Polyclonal Anti-Glial Fibrillary Acidic Protein (GFAP) Aviva Systems Biology Cat# OAPC00115; RRID: N/A Donkey anti-Goat IgG (H + L) CrossAdsorbed Secondary Antibody; Alexa FluorTM 555 Thermo Fisher Scientific A-21432; RRID:AB_2535853 Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody; Alexa FluorTM 555 Thermo Fisher Scientific Cat# A-31570; RRID:AB_2536180 Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody; Alexa FluorTM 647 Thermo Fisher Scientific Cat# A-31573; RRID:AB_2536183 Goat anti-Chicken IgY (H + L) Secondary Antibody; Alexa FluorTM 555 Thermo Fisher Scientific A-21437; RRID:AB_2535858 Goat Anti-Human Sox10 Polyclonal antibody R and D Systems Cat# AF2864; RRID:AB_442208 Goat anti-Mouse IgG (H + L) Highly CrossAdsorbed Secondary Antibody; Alexa FluorTM Plus 680 Thermo Fisher Scientific Cat# A32729; RRID:AB_2633278 Goat anti-Mouse IgG (H + L) Secondary Antibody; DyLightTM 800 4X PEG Thermo Fisher Scientific Cat# SA5-35521; RRID:AB_2556774 Goat anti-Rabbit IgG (H + L) Highly CrossAdsorbed Secondary Antibody; Alexa FluorTM 680 Thermo Fisher Scientific Cat# A-21109; RRID:AB_2535758 Goat anti-Rabbit IgG (H + L) Secondary Antibody; DyLightTM 800 4X PEG Thermo Fisher Scientific Cat# SA5-35571; RRID:AB_2556775 Goat Polyclonal Anti-GFAP Antibody Aviva Systems Biology Cat# OAEB01041; RRID:AB_10885969 Mouse Anti-Paxillin Monoclonal Antibody; Unconjugated; Clone 349 BD Biosciences Cat# 610051; RRID:AB_397463 Mouse Anti-Rabbit Glyceraldehyde-3phosphate dehydrogenase (GAPDH) Monoclonal Antibody; Clone 6C5 Fitzgerald Industries International Cat# 10R-G109a; RRID:AB_1285808 Rabbit Polyclonal Anti-MiTF antibody Abcam Cat# ab122982; RRID:AB_10902226 Rabbit Polyclonal anti-MITF C terminus (CGTMPESSPAYSIPRKMGSNLEDILMD) This paper # 7416; RRID: N/A Rabbit Polyclonal anti-MITF N terminus (DLVNRIIKQEPVLENCSQE) This paper # 7414; RRID: N/A Rabbit Polyclonal anti-S100 Abcam Cat# ab868; RRID:AB_306716 Biological samples Mouse: Germplasm; Mtmr2tm1a(KOMP)Mbp KOMP/IKMC Project # 30275 Chemicals, peptides, and recombinant proteins Alexa FluorTM 488 Phalloidin Thermo Fisher Scientific Cat# A12379 BIO Sigma-Aldrich Cat# B1686; CAS 667463-62-9 Canertinib (CI-1033) Selleck Chemicals LLC Cat# S1019; CAS 267243-28-7 Enzastaurin (LY317615) Selleck Chemicals LLC Cat# S1055; CAS 170364-57-5 (Continued on next page) Cell Reports 42, 113282, November 28, 2023 19

Techniques: Expressing, Binding Assay, Western Blot, Construct, Staining