Journal: Cancer Research

Article Title: BRAF Inhibitors Reprogram Cancer-Associated Fibroblasts to Drive Matrix Remodeling and Therapeutic Escape in Melanoma

doi: 10.1158/0008-5472.can-21-0614

Figure Lengend Snippet: Figure 1. BRAFi drives increased nuclear b-catenin in CAFs. A–D, Human melanoma sections [collected from eight patients with melanoma before (A) and after (B) BRAFi/ MEKi therapy] were coimmunostained for aSMA (green) and b-catenin expression (red). Nuclei were stained blue with DAPI. C and D are close-up pictures of the area circled by white boxes in A and B, respectively. Yellow triangles in D indicate aSMAþ fibroblasts expressing nuclear b-catenin. Scale bar, 100 mm. E, The scattered plot represents the numbers of aSMAþ cells per mm2 counted in pre- and posttreated melanoma samples. F, Percentage of aSMAþ cells expressing nuclear b-catenin in pre- and posttreated melanoma samples. Each data point represents the quantification result of one picture. At least five pictures were taken per tumor section. n ≥40. G–L, Immunostaining of b-catenin in M50 treated with DMSO or indicated inhibitors for 72 hours. M, Quantification and comparison of relative fluorescence intensities of nuclear b-catenin in M50 treated with DMSO or indicated BRAFi/MEKi for 72 hours. The fluorescence intensity of nuclear b-catenin in DMSO-treated M50 was used as the control and set at one for comparison. Each data point represents the relative fluorescent intensities of nuclear b-catenin in one treated cell. n ¼ 30. N, Nuclear and cytoplasmic fractions of M50 treated with DMSO or different concentrations of PLX4032. Lamin B was used as a nuclear loading control and tubulin as a cytoplasmic loading control. O and P, Quantification of fluorescence intensities of nuclear and cytoplasmic b-catenin Western blot bands. n ¼ 3. For all graphs, data are represented as mean SEM. , P ≤0.05; , P ≤0.001; ns, not significant.

Article Snippet: Human melanoma cell lines, including 1205Lu (1205 Lu-01–0001, RRID:CVCL_5239), WM278 (WM278–01–0001, RRID:CVCL_6473), and WM1366 (WM1366–01–0001, RRID:CVCL_6789), were purchased from Rockland Immunochemicals.

Techniques: Expressing, Staining, Immunostaining, Comparison, Control, Western Blot

Journal: Cancer Research

Article Title: BRAF Inhibitors Reprogram Cancer-Associated Fibroblasts to Drive Matrix Remodeling and Therapeutic Escape in Melanoma

doi: 10.1158/0008-5472.can-21-0614

Figure Lengend Snippet: Figure 5. POSTN is a direct downstream effector of nuclear b-catenin signaling in CAFs. A, Fibroblasts and POSTN expression in human melanomas pre- and post-BRAFi/MEKi treatment were visualized by coimmunostaining using an anti-TE7 antibody (green) and an anti-POSTN antibody (red). Scale bar, 100 mm. B, The scatter plot represents the percentage of TE7þ cells expressing POSTN in pre- and posttreated melanoma samples. Each data point represents the percentage of TE7þ fibroblasts that are POSTNþ in each 40field. n ≥40. C, Western blotting of POSTN expression levels in indicated melanoma cells and CAFs. Chart shows fluorescence intensities of POSTN bands. n ¼ 3. D, Graph shows qPCR data of POSTN expression in A375, GFP/M50, and bcat-GFP/M50 treated with DMSO or PLX4032. n ¼ 4. E, Illustration of the promoter region of POSTN that contains a potential LEF/TCF–binding site. F, Bar graph shows signals obtained from chromatin immunoprecipitation normalized by signals obtained from the input sample. Axin 2 was used as a positive control. Three different primer sets targeting the TCF/LEF–binding sites in the POSTN promoter region (POSTN1, POSTN2, and POSTN3) and three negative control primers targeting the 30UTR, exon 6, and the 50UTR were used. G, Western blotting of POSTN expression in M50 transfected with scramble siRNA or POSTN siRNA. Graph shows fluorescence intensities of POSTN bands. n ¼ 3. H and I, Graph shows A375 numbers in cocultured spheroids of indicated groups under PLX4032 treatment (H) or PLX4032 and GDC0973 treatment (I). n ¼ 3. J and K, Graph shows A375 numbers in cocultured spheroids of indicated groups with or without recombinant POSTN under PLX4032 treatment (J) or PLX4032 and GDC0973 treatment (K). n ¼ 3. For all graphs, the data are shown as mean SEM. , P ≤0.05; , P ≤0.01; , P ≤0.001. ns, not significant.

Article Snippet: Human melanoma cell lines, including 1205Lu (1205 Lu-01–0001, RRID:CVCL_5239), WM278 (WM278–01–0001, RRID:CVCL_6473), and WM1366 (WM1366–01–0001, RRID:CVCL_6789), were purchased from Rockland Immunochemicals.

Techniques: Expressing, Western Blot, Binding Assay, Chromatin Immunoprecipitation, Positive Control, Negative Control, Transfection, Recombinant

Journal: Nature Communications

Article Title: Non-canonical pathway for Rb inactivation and external signaling coordinate cell-cycle entry without CDK4/6 activity

doi: 10.1038/s41467-023-43716-y

Figure Lengend Snippet: a Density scatterplot of Hoechst and EdU staining in MP41 cells treated with palbociclib (1 µM) for the indicated time ( n = 1000 cells/condition). b Immunoblot showing Rb and GAPDH expression in MP41 cells after palbociclib (1 µM) treatment. c Immunoblot showing p-ERK, t-ERK, c-Myc, and GAPDH expression in H1373, WM983B, WM989, and MP41 cells treated with DMSO or the indicated drug for 24 h (sotorasib, 1 µM; vemurafenib, 1 µM; trametinib, 10 nM). d EU levels 24 h after treatment with the indicated drug. Data are shown as mean ± SD ( n = 3 biological replicates). Asterisks indicate significant differences in the two-tailed unpaired t -test (* p ≤ 0.05; ** p ≤ 0.001). e Representative histograms of EdU levels in H1373 cells showing classification of S-phase cells. 24 h after treatment with the indicated drug (palbociclib, 1 µM; sotorasib, 1 µM), cells were incubated with EdU (10 µM) for 48 h prior to fixation ( n > 3000 cells/condition). f Percentage of S-phase cells in H1373, WM989, and MP41 cells. Data are shown as mean ± SD ( n = 3 biological replicates). Asterisks indicate significant differences in the one-way ANOVA test (* p ≤ 0.05; ** p ≤ 0.001; *** p ≤ 0.0001). g Immunoblot showing Rb, c-Myc, and GAPDH expression in H1373, WM989, and MP41 cells 72 h after treatment with the indicated drug.

Article Snippet: WM989 (Rockland Immunochemicals, #WM989-01-0001) and WM983B cells (Rockland Immunochemicals, #WM983B-01-0001) were cultured in a mixture of 80% MCDB153 (Sigma, #M7403) and 20% Leibovitz’s L15 media (Gibco, #21083027), supplemented with 2% FBS and 1.68 mM CaCl 2 .

Techniques: Staining, Western Blot, Expressing, Two Tailed Test, Incubation