Fig 1: IGF2BP1 sustains mesenchymal-like tumor cell properties. (A) Indicated tumor-derived cell lines were cultured (48 h) and harvested at 80% confluence before analyzing the abundance of indicated proteins by western blotting. Epithelial-like cell lines and marker proteins are labeled in green. Mesenchymal-like cell lines and marker proteins are depicted in red. VCL and HSPB1 (HSP27) served as loading controls. IGF2BP1 is almost exclusively expressed in the following tumor-derived mesenchymal-like cells: ES-2 ovarian carcinoma (ATCC#: CRL-1978); SW480 colorectal carcinoma (ATCC#: CCL228); MDA-MB-231 (ATCC#: HTB-26) and HBL-100 (ATCC#: HTB-124) mammary carcinoma; 1F6 (no ATCC# available) and HT-144 (ATCC#: HTB-63) melanoma. In epithelial-like adenocarcinoma-derived cells [OVCAR (ATCC#: HTB-161) ovarian adenocarcinoma; MCF7 (ATCC#: HTB-22) breast adenocarcinoma; HT-29 (ATCC#: HTB-38) colorectal adenocarcinoma], expression of IGF2BP1 was only observed in OVCAR cells. (B) Melanoma-derived HT-144 cells were transiently transfected with indicated siRNAs for 72 h. The abundance of indicated proteins was analyzed by western blotting. IGF2BP1 as well as LEF1 depletion result in reduced FN1 and SNAI2 protein abundance, whereas CDH1 and VIM levels remain essentially unchanged. (C–F) HT-144 cells were stably transduced by lentiviral vectors encoding IGF2BP1 (shI1-1), LEF1 (shL1-1), SNAI2 (shS2-1) directed or control (shC) shRNAs. Three weeks after transduction, cells were cultured for 48 h before analyzing protein abundance by western blotting with indicated antibodies (C and D). Protein abundance on IGF2BP1 knockdown was determined relative to controls (siC) using VCL and HSPB1 for cross-normalization, as indicated above panels (C). Standard deviation of at least three independent analyses is shown. The stable knockdown of SNAI2, LEF1 and IGF2BP1 promotes the expression of the epithelial marker CDH1, whereas all mesenchymal marker proteins were reduced. Cell morphology was monitored by bright field microscopy (E). Cells were cultured on collagen coated coverslips for 48 h before immunostaining of CTNNB1 and CTNND1 (p120 Catenin) to label cell–cell contacts (F). Enlargements of boxed regions (left panels) are shown in right panels (enlargement). All three stable knockdowns promote the formation of cell–cell contacts, suggesting an enhancement of epithelial-like cell morphology. (G and H) MCF7 cells were stably transduced with GFP-ZBP1 (the chicken ortholog of IGF2BP1) or GFP. Three weeks after transduction, cells were cultured for 48 h before determining the abundance of indicated proteins by western blotting (G). Cell morphology was monitored by bright field microscopy (H, left panel) and immunostaining for CDH1 as well as labeling of F-actin by phalloidin (H, right panel). Neither CDH1 expression nor cell-cell contact formation is compromised by GFP-ZBP1, although cell size appeared modestly increased. Bars, 10 µm.
Fig 2: Small-molecule inhibitors bind to PD-L1 with no impact on cell viability. (A) Thermal shifts indicate the stabilization of PD-L1 by compounds 5 (black), 42 (green), 47 (yellow), 69 (yellow), 75 (black) and 84 (black). Curves represent the fraction of unfolded recombinant human PD-L1 protein, where 0 represents the folded PD-L1 and 1 the unfolded, in the presence of 1% DMSO (light gray), indicated compounds (green, yellow and black) and BMS202 (dark gray) at 100 µM. (B) Different cell lines were incubated with increased concentrations of compounds for 48 hours. Cell viability was normalized to untreated cells. All cell lines, MDA-MB-231 (ATCC# HTB-26), A375 (ATCC# CRL. 1619), and HMEC (ATCC# CRL-3243) showed tolerance to the compounds. Three different concentrations 100 µM (blue), 10 µM (green) and 1 µM (gray) were tested. Data are presented as mean±SD, N=3 and N=1, n=9 or n=3 from three or one independent experiment(s) performed in triplicate. (C–D) Compounds inhibit PD-1/PD-L1 interaction on melanoma and breast cancer cell lines. (C) MDA-MB-231 breast cancer cells (gray) or (D) A375 melanoma cells (gray) were treated with 10 µM of compounds (green, yellow and black), BMS202 (dark gray) and anti-PD-L1 (αPD-L1) (red) for 72 hours. A375 cells were stimulated with 200 ng.mL−1 interferon-ɣ (gray) for 18 hours before treatments to enhance PD-L1 levels. The remaining % of accessible PD-L1 was determined in live cells by flow cytometry. Data are presented as mean±SD, N=3, n=9, or N=1, n=3, from three or one independent experiments performed in triplicate. Statistical analysis: one-way analysis of variance and Tukey’s post test. PD-L1, programmed cell death ligand 1. ATCC, American Type Culture Collection.
Fig 3: Dose–response curves for antiproliferative activity of BIM-23052 analogs determined in MCF-10A (ATCC® CRL-10317TM), MCF-7 (ATCC® HTB-22TM) and MDA-MB-231 (ATCC® HTB-26TM) cell lines. Mean values are presented ± SD from three independent experiments, n = 6.
Fig 4: MTT cell viability assay after exposure to graded doses (0–200 μg.mL−1 in DMSO; 48 h) of PMG fruit extracts (red, pink, white). Data are expressed as mean (triplicate) ± SD. * Statistically different (p < 0.05; one-way ANOVA, Tukey’s post hoc test) vs. unstimulated (0 μg.mL−1) cells; human normal retinal (ARPE-19; CRL-2302TM), breast MDA-MB-231; HTB-26TM), alveolar (A549; CCL-185TM) and colorectal Duke’s type II (LS180; CL-187TM) adenocarcinomas, breast ductal carcinoma (T-47D; HTB-133TM) cell lines (ATCC®, Rockville, MD, USA).
Supplier Page from ATCC for MDA-MB-231