Figure 2
From: Red-COLA1: a human fibroblast reporter cell line for type I collagen transcription
Red-COLA1 cells accurately depict changes in COL1A1 activation. (a) Relative mRNA expression of COL1A1 and mCherry at 24, 48, or 72 h after stimulation with10 ng/mL TGF-β1. Relative expression of the target genes was calculated with reference to the untreated control. β-actin was used as an internal control. Error bars represent SD of the average of two independent experiments performed in triplicates. (b) Total cell lysates (TCL) from cells treated with TGF-β1 were probed for mCherry and pro-collagen (Pro-Col I). β-tubulin was included as a loading control. Full length blots are presented in Supplementary Fig. 3(c). Intensity of bands corresponding to type I collagen or m-Cherry was quantified using ImageJ and normalised with β-tubulin and expressed relative to respective 0 h control. Error bars showed standard deviation of fold-change averaged from 3 independent experiments. (d) Representative images of Red-COLA1 fibroblasts stimulated with 1.25, 2.5, and 5 ng/mL of TGF-β1. Image acquisition (10 ×) was performed 24 or 72 h post-induction with a high-content imager (ImageXpress) in live cells. (e) The intensity levels of both mCherry and nuclei count from 4 sites of 3 independent wells were calculated using the manufacturer’s analysis software and normalised to their respective untreated controls (24 or 72hours). Error bars represent SD from two independent experiments. (f) Representative images from untreated or TGF-β1 stimulated Red-COLA1 fibroblasts co-treated with 0.1 ng/mL of TNF or 1 µM TGF-β1 receptor inhibitor, SB431542. (g) Quantification of immunofluorescence in (f). The intensity levels of both mCherry and type I collagen-FITC per cell, from 4 sites of 3 independent wells were calculated and normalised to untreated controls. Error bars represent SD from three independent experiments. (h) Untreated and TGF-β1-treated (72 h) Red-COL1A1 cells immunostained with rabbit antibodies for type I collagen 1 (COL1) and labelled with anti-rabbit conjugated with FITC (COL1-FITC). (i) The total intensity levels of both mCherry and type I collagen-FITC per nuclei (DAPI), 5 independent wells were calculated using the manufacturer’s analysis software and normalised to untreated controls. Error bars represent SD.
