Figure 1
From: Red-COLA1: a human fibroblast reporter cell line for type I collagen transcription
Design and generation of Red-COLA1 reporter cell line. (a) TALENs binding sites in COL1A1 genomic locus. TALENs recognize bases + 11 (in red) and + 43 (in blue) base pairs from the start codon (underlined) in endogenous COL1A1 genomic locus. Donor DNA cassette comprises of a nuclear localizing mCherry-reporter (NLS-mCherry) fused to a destabilising proline-glutamate-serine-threonine-rich (PEST) sequence signal, followed by polyA tail (pA) flanked by two homologous recombination arms (5′L and 3′R). (b) Schematic diagram of knock-in allele. Primer binding sites (F1, R1, F2, R2) and their expected band sizes for junctional PCR analysis are denoted with arrows (bottom). (c) Junctional PCR. Amplification of the integrated cassette was detected with 2 independent pair of primers that specifically target endogenous COL1A1 and m-Cherry sequences. Full length gels are presented in Supplementary Fig. 1e. The primer positions are indicated on the schematic of the targeted COL1A1 locus in (a,b). (d) Expression of both mCherry and endogenous collagen (FITC stained) in Red-COLA1 reporter cells at 72 h following 10 ng/mL of TGF-β1 stimulation. Red-COLA1 cells immunostained with rabbit antibodies for type I collagen 1 (COL1) and labelled with anti-rabbit conjugated with FITC (COL1-FITC). Image acquisition (10 ×) was performed 3 days post-induction. (e) Representative live images of Red-COLA1 treated with TGF-β1 for 24, 48 and 72 h. (f) Cells were fixed at the end of the experiment and stained with Hoechst 33342 to reveal the nuclei. 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 72 h). Error bars represent SD from two independent experiments. (g). Red-COLA1 derived from post FACS-enrichment were cultured for 20 passages and imaged for m-Cherry expression. Images were acquired from fixed cells at 72 h following 10 ng/mL of TGF-β1 stimulation. (h) Quantification of TGF-β1-responsive cells at different cell passages. Percentage of m-Cherry positive cells were calculated by applying a nuclei mask and quantifying the number of m-Cherry positive signal that overlaps with nuclei mask over the total nuclei detected.
