Journal: Scientific Reports

Article Title: Impact of Escherichia coli K12 and O18:K1 on human platelets: Differential effects on platelet activation, RNAs and proteins

doi: 10.1038/s41598-018-34473-w

Figure Lengend Snippet: Western blot and ELISA analysis of HMBS and ATP2C1. ( a ) Cell lysates were applied to SDS-PAGE gels under reducing conditions. HMBS and ATP2C1 protein were detected in platelets using a polyclonal antibody. Incubation of platelets with E. coli K12 and E. coli O18:K1 in 1:5 or 1:10 platelet-bacteria ratios converted HMBS 47 kDa form to a 40 kDa protein. ATP2C1 was not affected. GAPDH was used as a loading control. ( b ) The releasates of the platelet-bacteria mix were collected after centrifugation (500 g, 10 minutes without break). HMBS, ATP2C1 and LRCH4 levels were measured by ELISA. Data represents the mean of three independent experiments (n = 3). ATP2C1 was detectable in platelet supernatants, while HMBS and LRCH4 proteins were either not released from platelets or in concentrations below the detection level of the ELISA (data not shown). BDL, below detection limit. Western blot results are representative image of three replications. The same exposure was applied equally across the entire image. The original pictures of the full-length western blots can be found in Supplementary Fig. .

Article Snippet: The blots were incubated overnight with primary anti-human antibodies (rabbit polyclonal ATP2C1 (1:750), LRCH4 (1:500), HMBS (1:1000) or GAPDH (1:600) from Atlas Antibodies, Bromma, Sweden).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, SDS Page, Incubation, Bacteria, Control, Centrifugation

Journal: Heliyon

Article Title: Cell autonomous TLR4 signaling modulates TGF-β induced activation of human cardiac fibroblasts

doi: 10.1016/j.heliyon.2025.e42452

Figure Lengend Snippet: Fig. 1. The expression of TLRs in adult cardiac fibroblasts: (A) FACS analysis for TLR4 in human cardiac fibroblasts with unstained control and cardiac fibroblasts stained with anti-TLR4 APC antibody. (B) Western blot and quantitative analysis showing the expression of TLR4 in CF in growth medium with 10 % serum, 2 % serum and with LPS stimulation.(C) Dot plot representing the expression of different TLRs in human cardiac fibroblast clusters. (D) Dot plot representing the average expression and percentage cells expressing different Tlr genes in healthy mouse adult Col1- GFP labelled cardiac fibroblasts by single-cell RNA seq data. (E) Temporal changes in gene expression of Tlr4 following cardiac injury: UMAP demonstrating the expression of Tlr4 in Col1-GFP single-cell RNA seq data of healthy and at different time points at 7 days, 14 days, and 30 days following injury. (F) Dot plot demonstrating the expression of Tlr4, Myd88, Tril, Traf6, Tab2, Mapk14, Nfkb1, and Jun in healthy control and injured Col1-GFP labelled cardiac fibroblasts at 7 days, 14 days and 30 days post-injury by single-cell sequencing (n = 3).

Article Snippet: The membranes were blocked with 5 % skimmed milk in Tris Buffered Saline with Tween-20 (TBS-T) and incubated overnight with primary antibodies, anti-TLR4 monoclonal antibody (Santa Cruz Biotechnology), and anti-GAPDH antibody (Origin Biosciences).

Techniques: Expressing, Control, Staining, Western Blot, RNA Sequencing, Gene Expression, Sequencing

Journal: Heliyon

Article Title: Cell autonomous TLR4 signaling modulates TGF-β induced activation of human cardiac fibroblasts

doi: 10.1016/j.heliyon.2025.e42452

Figure Lengend Snippet: Fig. 3. Inhibiting TLR4 signaling reduces differentiation and migration of myofibroblasts: (A) qPCR showing the expression of fibrotic genes, Collagen1A1 and α-Sma in cardiac fibroblasts treated with TGF-β, n = 8. (B) qPCR showing the expression of fibrotic markers Collagen1A1 and α-Sma in TGF-β plus TAK-242 treated cells compared to TGF-β with vehicle alone control,n = 5.(C) Scratch wound assay to determine the migration of cardiac fibroblasts in the presence of TGF−β plus TAK-242, TGF-β alone and controls cells in medium with 10 % FBS and 2 % FBS. (D) Percentage area of cardiac fibroblast migration in TGF-β condition versus TGF-β plus TAK-242 treatment and controls,n = 4. (E) Cardiac fibroblasts seeded on contractable collagen gel matrices were assayed for gel contraction after treatment with TGF-β and TGF-β plus TAK-242 for 24 h, n = 4. Data are represented as the mean ± SE, *p < 0.05, **p < 0.01, ***p < 0.0001, ns = p > 0.05.

Article Snippet: The membranes were blocked with 5 % skimmed milk in Tris Buffered Saline with Tween-20 (TBS-T) and incubated overnight with primary antibodies, anti-TLR4 monoclonal antibody (Santa Cruz Biotechnology), and anti-GAPDH antibody (Origin Biosciences).

Techniques: Migration, Expressing, Control, Scratch Wound Assay Assay

Journal: Heliyon

Article Title: Cell autonomous TLR4 signaling modulates TGF-β induced activation of human cardiac fibroblasts

doi: 10.1016/j.heliyon.2025.e42452

Figure Lengend Snippet: Fig. 4. Inhibiting TLR4 signaling reduces the expression of extracellular matrix genes: (A) GO enrichment analysis to determine the biological processess that are significantly altered with TGF-β treatment (for 48 h) in cardiac fibroblasts compared to control cells without TGF-β. (B) GO enrichment analysis to determine the biological processess significantly inhibited by TAK-242 in TGF-β treated cardiac fibroblasts. (C) Heatmap showing the scaled expression of extracellular matrix genes with TAK-242 treatment. (D) Expression of genes encoding profibrotic factors and extracellular matrix by qPCR in TGF-β+TAK-242 compared to TGF-β only, n = 4. Data are represented as the mean ± SE, *p < 0.05, **p < 0.01, ***p < 0.0001.

Article Snippet: The membranes were blocked with 5 % skimmed milk in Tris Buffered Saline with Tween-20 (TBS-T) and incubated overnight with primary antibodies, anti-TLR4 monoclonal antibody (Santa Cruz Biotechnology), and anti-GAPDH antibody (Origin Biosciences).

Techniques: Expressing, Control