Reporters to mark and eliminate basal or luminal epithelial cells in culture and in vivo
Fig 3
Generation and characterization of 4T1 cell lines expressing K14 and K8 reporters.
(A) Cartoon of the K14 promoter–driving expression of tRFP-P2A-TK (K14.tRPT) reporter. (B) Cartoon of the K8 promoter–driving expression of tGFP-P2A-DTR (K8.tGPD) reporter construct. (C) Phase contrast images of K14+, K14−, and K8+ cells grown in monolayer (upper panels, scale bar 100 μm) and 3D (lower panels, scale bar 200 μm) on M/Col-I culture for 4 days. (D) Quantification of invasive structures in C, represented as means ± SEM from 3 independent experiments; *p < 0.001 by unpaired t test. (E) K14+ and K8+ cells were treated with DT (2.5 ng/ml), GCV (1 μg/ml), or media. Column bars indicate the percentage of reporter-positive and negative cells after treatments for the indicated cell lines, as determined by flow cytometry. (F) Immunoblots of lysates from K14+ and K14− reporter cell lines were analyzed for changes in expression of E-cadherin, β-catenin, fibronectin, vimentin, and α-SMA. Blots were also probed with antibodies for GAPDH and α-tubulin as loading controls. Right panel shows quantification of vimentin for 3 independent western blot p = 0.0317 by paired t test; mean ± SD is shown. (G) Immunofluorescence of K14+ (upper panel) and K14− (lower panel) monolayers for vimentin, β-catenin, or detection of endogenous tRFP signal. Scale bar 25 μm. α-smooth muscle actin; DT, diphtheria toxin; DTR, diphtheria toxin receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GCV, ganciclovir; K, cytokeratin; K8.tGPD, keratin-8 promoter followed by turbo green fluorescent protein and diphtheria toxin receptor; K14.tRPT, keratin-14 promoter followed by a turbo red fluorescent protein and herpes simplex virus thymidine kinase; M/Col-I; 1:1 mixture of Matrigel/Collagen-I; pA, polyadenylation signal sequence; TK, thymidine kinase; tGFP, turbo green fluorescent protein; tRFP, turbo red fluorescent protein