pc 100 r d systems  (R&D Systems)

Journal: bioRxiv

Article Title: Differential immune- and apoptosis-related gene signatures in pancreatic alpha and beta cells contribute to their fate in type 1 diabetes

doi: 10.1101/2025.07.26.666935

Figure Lengend Snippet: EndoC-βH1 cells were transfected with small interfering RNA (siCTRL or siMEG3). After recovery, cells were left untreated or exposed to either IL-1β (50U/ml) +IFN-γ (1000U/ml) for 48h, to IFN-α(1000U/ml) alone for 24h or to IFN-γ alone for 2h, 4h, 8h or 24h. (A) pSTAT1 and GAPDH protein expression were analyzed by immunoblot after silencing MEG3 and exposing to the cytokines detailed in the figure. (B) pSTAT1 bands were quantified by densitometry and normalized to GAPDH. (C) Representative blots of pSTAT1 and GAPDH protein expression after silencing MEG3 and exposing to the cytokines detailed in the figure. (D-E) pSTAT1, tSTAT1 and GAPDH protein expression were analyzed by immunoblot after silencing MEG3 and exposing to IFN-γ at different time points. P-STAT1 bands were quantified by densitometry and normalized to tSTAT1. (F) The AUC from the figure shown in (E) . (G) CXCL10 mRNA expression assessed by RT-qPCR, normalized to the geometric mean of ACTIN and VAPA and presented as fold change compared with siCTRL cytokine-treated cells. (H) CXCL10 secretion in the supernatant was measured by ELISA. Results are means ± SEM. Each point represents an independent experiment. *p<0.05, ****p<0.0001 vs siCTRL, ###p<0.001, ####p<0.0001 vs untreated one-way ANOVA, Bonferroni correction. P-STAT1: phosphorylated STAT1, tSTAT1: total STAT1, AUC: Area under the curve, SEM: Standard Error of the Mean, Unt.: untreated.

Article Snippet: The nitrocellulose membranes were probed using specific primary antibodies: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167, Stat1 (9H2) Mouse mAb #9176 or Human/Mouse/Rat GAPDH Antibody R&D Systems # 2275-PC-100 diluted 1:1000 in TBST (TBS, 0.1% Tween 20) with 5% BSA (GAPDH was diluted 1:10 000).

Techniques: Transfection, Small Interfering RNA, Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Endothelial-to-osteoblast transition in normal mouse bone development.

doi: 10.1016/j.isci.2023.105994

Figure Lengend Snippet: Figure 5. RNA-sequencing analysis of RFP+/GFP+versus RFP+ cells (A) Pooled bone marrow cells from DRM were sorted by FACS, and RFP+ and RFP+/GFP+ cells collected. (B) GSEA of differentially expressed genes from RNAseq analyses of RFP+/GFP+ (EC-OSB) versus RFP+ cells. NES, normalized enrichment score. (C) The top upregulated genes in RFP+/GFP+ (EC-OSB) cells relative to RFP+ cells. (D) Hallmark gene set analysis identified EMT as among the top biological processes in RFP+/GFP+ cells. (E) Regulatory target gene set analysis showed that GATA genes, including GATA_Q6, GATA_C, and GATA3_01, were among the top 10 transcription factor gene signatures.

Article Snippet: Antibodies GATA3 R&D Systems MAB6330-S, RRID: AB 10640 GAPDH(D16H11) Cell Signaling Technologies #5174, RRID:AB_10622025 Rat anti-mCD31 (MEC 13.3) BD Pharmingen #553370 Chemicals, peptides, and recombinant proteins

Techniques: RNA Sequencing

Journal: iScience

Article Title: Endothelial-to-osteoblast transition in normal mouse bone development.

doi: 10.1016/j.isci.2023.105994

Figure Lengend Snippet: Figure 6. GATA3 transcription factor in endothelial-to-osteoblast transition (A–C) qRT-PCR of MLEC mRNA for the expression of GATA family genes before and after BMP4 treatment for 14 days (B) GATA family gene expression in 2H11 ECs after BMP4 treatment for 2 days (C) Time course of GATA3 induction during BMP4-induced EC-to-OSB transition of 2H11 ECs. (D) Knockdown of GATA3 by shRNA in 2H11 ECs, as shown by mRNA and protein levels. (E) shRNA knockdown of GATA3 significantly reduced BMP4-induced OSX and osteocalcin (Bglp) expression in 2H11 cells. (F and G) Knockdown of GATA3 in 2H11 cells reduced BMP4-induced mineralization. Left panel, Alizarin Red S (ARS) staining. Right panel, enlarge image (X10) of left panel. The staining was quantified using ImageJ software. Data in A-E are represented as mean GSD (G) Graphical summary. Using lineage tracing, we showed that Tie2-RFP+ and Col1a1-GFP+ dual-positive cells are present in cells lining the calvaria bone, femur, and other bones. We also isolated ECs from bone marrow or lung of DRM and showed that they could become OSBs when cultured in osteogenic medium. These observations suggest that EC-to-OSB transition occurs during normal bone development.

Article Snippet: Antibodies GATA3 R&D Systems MAB6330-S, RRID: AB 10640 GAPDH(D16H11) Cell Signaling Technologies #5174, RRID:AB_10622025 Rat anti-mCD31 (MEC 13.3) BD Pharmingen #553370 Chemicals, peptides, and recombinant proteins

Techniques: Quantitative RT-PCR, Expressing, Gene Expression, Knockdown, shRNA, Staining, Software, Isolation, Cell Culture