Journal: Cell reports

Article Title: Modeling Progressive Fibrosis with Pluripotent Stem Cells Identifies an Anti-fibrotic Small Molecule

doi: 10.1016/j.celrep.2019.11.019

Figure Lengend Snippet:

Article Snippet: Rabbit p-SMAD2/3 , R and D systems , Cat# MAB8935; RRID:AB_2313773.

Techniques: Virus, Recombinant, Luciferase, Imaging, Enzyme-linked Immunosorbent Assay, Hydroxyproline Assay, Software

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Functional Overlap Between Chondroitin and Heparan Sulfate Proteoglycans During VEGF-Induced Sprouting Angiogenesis

doi: 10.1161/atvbaha.111.240622

Figure Lengend Snippet: Figure 1. Vigorous angiogenic sprouting is accompanied by defective pericyte attachment in the absence of heparan sulfate (HS). Wild type (wt), exostosin 1 (Ext1)/, and N-deacetylase/N-sulfotransferases (Ndst)1/2/ embryoid bodies (EBs) were grown in collagen I for 12 days (A–G) or 17 days (H) in the presence of vascular endothelial growth factor A (VEGFA; 30 ng/mL). Endothelial cells (ECs) were visualized by staining for CD31 (green) (A, D), and binary images were used for quantification (B, E). ECs and pericytes (PCs) in wt, Ext1/, and Ndst1/2/ EBs were visualized by staining for CD31 (green) and NG2 (red), respectively (C, F, G, and H). Note the absence of pericytes in Ndst1/2/ EBs, and the presence of detached (star), partially detached (arrow heads) and attached (arrows) pericytes in the Ext1/ EBs. The sprout ing area fractions of individual EBs were quantified (I; n12) as well as the number of NG21 cells and their localization in relation to ECs (J, K; n 11). The ability of TGF1 (5 ng/mL) to induce Smad2 phosphorylation (P-Smad2) (L, M; n3) and platelet-derived growth factor B (PDGFB) (100 ng/mL) to induce platelet-derived growth factor -receptor beta (PDGFR) phosphorylation (N) was studied in wt and Ext1/ EBs. PDGFR protein was immunoprecipitated (IP) followed by immunodetection of phosphorylated tyrosine residues (P-Tyr) and PDGFR. (O) Quantification of PDGFR phoshorylation relative to total PDGFR protein after PDGFB stimulation and IP (n2).

Article Snippet: The membranes were incubated with the following primary antibodies: rabbit anti-phospho Smad2 (antibody kindly provided by Dr Aris Moustakas, the Ludwig Institute for Cancer Research, Uppsala, Sweden), mouse anti-Smad1/2/3 (sc- 7960; Santa Cruz), mouse anti-phosphotyrosine (4G10; Upstate, Lake Placid, NY), rabbit polyclonal anti-PDGFRß antibody (sc-432; Santa Cruz), goat anti-human VEGFR2 (AF357; R&D Systems;), goat anti-mouse VEGFR2 (AF644; R&D Systems), rabbit anti-phospho Akt, rabbit anti-phospho-ERK1/2, rabbit anti-total Akt or rabbit anti-total ERK1/2 (all from Cell Signaling Technology).

Techniques: Histone Deacetylase Assay, Staining, Phospho-proteomics, Derivative Assay, Immunoprecipitation, Immunodetection

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Functional Overlap Between Chondroitin and Heparan Sulfate Proteoglycans During VEGF-Induced Sprouting Angiogenesis

doi: 10.1161/atvbaha.111.240622

Figure Lengend Snippet: Figure 4. Chondroitin sulfate (CS) binds angiogenic factors and supports TGF- and platelet-derived growth factor B (PDGFB)-signaling. A, Vascular endothelial growth factor A (VEGFA)165, PDGFB, and TGF1 bind to 35S-labeled chondroitin sulfate (CS) isolated from wild type (wt) and Exostosin 1 (Ext1)/ EBs. The graphs illustrate the mean of two measuring points. B, Phospho-AKT (P-AKT) and phospho- ERK1/2 (P-ERK1/2) activation induced by PDGFB in human aortic smooth muscle cells (hAoSMCs) after treatment with heparitinase III (HEPase) or chondroitinase (CSase). C, D, Quantification of the results shown in (B). E, Smad2 phosphorylation induced by TGF1 in hAoSMCs after treatment with HEPase or CSase. F, Quantification of P-Smad2 after TGF1 stimulation. G, Activation of platelet-derived growth factor-receptor (PDGFR) by PDGFB in N-deacetylase/N-sulfotransferases (Ndst1)/ mouse embryonic fibroblasts. Treatment with CSase leads to reduced activation of the receptor. n3 for all signaling experiments.

Article Snippet: The membranes were incubated with the following primary antibodies: rabbit anti-phospho Smad2 (antibody kindly provided by Dr Aris Moustakas, the Ludwig Institute for Cancer Research, Uppsala, Sweden), mouse anti-Smad1/2/3 (sc- 7960; Santa Cruz), mouse anti-phosphotyrosine (4G10; Upstate, Lake Placid, NY), rabbit polyclonal anti-PDGFRß antibody (sc-432; Santa Cruz), goat anti-human VEGFR2 (AF357; R&D Systems;), goat anti-mouse VEGFR2 (AF644; R&D Systems), rabbit anti-phospho Akt, rabbit anti-phospho-ERK1/2, rabbit anti-total Akt or rabbit anti-total ERK1/2 (all from Cell Signaling Technology).

Techniques: Derivative Assay, Labeling, Isolation, Activation Assay, Phospho-proteomics, Histone Deacetylase Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Spatial transcriptomics reveals Inhba/Smad2/E2f4 axis in Lrp2 high thecal cell proliferation in androgen-induced PCOS mice

doi: 10.3389/fcell.2025.1633254

Figure Lengend Snippet: Proliferative and androgenic features of Lrp2 high TC in PCOS ovaries. (A) Expression analysis of androgen synthesis genes Cyp11a1, Cyp17a1, and Hsd3b1 across clusters. (B) KEGG pathway enrichment analysis for upregulated genes in Lrp2 high TC, emphasizing cell cycle regulation. (C,D) Intersection and STRING network analysis predict an Inhba/Smad2/E2f4 signaling axis involved in cell cycle regulation within Lrp2 high TC. (E) AUC analysis reveals Lrp2 high TC as having the highest co-expression of Inhba, Smad2, and E2f4. (F) Quantification of co-localized regions for Inhba, Smad2, E2f4 and Lrp2, showing a significant increase in PCOS ovaries. (G) Spatial co-localization of Inhba, Smad2, E2f4 and Lrp2 in control and PCOS ovaries. Co-expressed spots are marked as “TRUE.” Data are represented as mean ± SD, *P < 0.05 by t-test.

Article Snippet: For functional assays, thecal cells were transfected with small interfering RNAs (siRNAs) targeting Inhba (Santa Cruz, Cat. No. sc-39783), Smad2 (Santa Cruz, Cat. No. sc-44338), or E2f4 (Santa Cruz, Catalog No. sc-35248) using Lipofectamine 3,000 (Invitrogen, Cat. No. L3000015) according to the manufacturer’s instructions.

Techniques: Expressing, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Spatial transcriptomics reveals Inhba/Smad2/E2f4 axis in Lrp2 high thecal cell proliferation in androgen-induced PCOS mice

doi: 10.3389/fcell.2025.1633254

Figure Lengend Snippet: Inhba/Smad2/E2f4 Signaling Promotes Thecal Cell Proliferation. (A) Immunofluorescence staining showing the subcellular localization of Inhba (cytoplasmic), Smad2, and E2f4 (nuclear) in primary thecal cells under control and DHEA-treated conditions. DAPI (blue) marks nuclei. Bar = 20 µm. (B) Representative EdU staining images of proliferating thecal cells following DHEA treatment and siRNA-mediated knockdown of Inhba, Smad2, or E2f4. Bar = 200 µm. (C) Quantification of EdU-positive nuclei across treatment groups. Data are presented as mean ± SD. Different lowercase letters denote statistically significant differences (one-way ANOVA followed by Tukey’s post hoc test, P < 0.05).

Article Snippet: For functional assays, thecal cells were transfected with small interfering RNAs (siRNAs) targeting Inhba (Santa Cruz, Cat. No. sc-39783), Smad2 (Santa Cruz, Cat. No. sc-44338), or E2f4 (Santa Cruz, Catalog No. sc-35248) using Lipofectamine 3,000 (Invitrogen, Cat. No. L3000015) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Control, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: Spatial transcriptomics reveals Inhba/Smad2/E2f4 axis in Lrp2 high thecal cell proliferation in androgen-induced PCOS mice

doi: 10.3389/fcell.2025.1633254

Figure Lengend Snippet: Inhba/Smad2/E2f4 Axis Promotes Cell Cycle Progression in TCs. (A) Flow cytometry analysis of cell cycle phases. (B) Distribution of cells in G1, S, and G2/M phases (%). (C–E) qRT-PCR analysis of Inhba, Smad2, and E2f4 mRNA levels. (F) Western blot analysis of protein expression. (G–I) Quantification of protein levels for Inhba, Smad2, and E2f4. Data are represented as mean ± SD. Different lowercase letters at the top of each bar denote significant differences among groups (one-way ANOVA followed by Tukey’s post hoc test, P < 0.05).

Article Snippet: For functional assays, thecal cells were transfected with small interfering RNAs (siRNAs) targeting Inhba (Santa Cruz, Cat. No. sc-39783), Smad2 (Santa Cruz, Cat. No. sc-44338), or E2f4 (Santa Cruz, Catalog No. sc-35248) using Lipofectamine 3,000 (Invitrogen, Cat. No. L3000015) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Quantitative RT-PCR, Western Blot, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Spatial transcriptomics reveals Inhba/Smad2/E2f4 axis in Lrp2 high thecal cell proliferation in androgen-induced PCOS mice

doi: 10.3389/fcell.2025.1633254

Figure Lengend Snippet: Schematic overview of the study design and proposed mechanism. Spatial transcriptomic profiling of ovaries from DHEA-induced PCOS mice revealed expansion of Lrp2 high TC with elevated proliferative and steroidogenic activity. Mechanistically, DHEA stimulates Inhba expression, activating Smad2 and E2f4 signaling to drive thecal cell proliferation, contributing to androgen excess and ovarian dysfunction in PCOS.

Article Snippet: For functional assays, thecal cells were transfected with small interfering RNAs (siRNAs) targeting Inhba (Santa Cruz, Cat. No. sc-39783), Smad2 (Santa Cruz, Cat. No. sc-44338), or E2f4 (Santa Cruz, Catalog No. sc-35248) using Lipofectamine 3,000 (Invitrogen, Cat. No. L3000015) according to the manufacturer’s instructions.

Techniques: Activity Assay, Expressing