human skin fibroblasts hs 27 cell line  (ATCC)

Journal: Frontiers in Oncology

Article Title: Stromal Fibroblasts Counteract the Caveolin-1-Dependent Radiation Response of LNCaP Prostate Carcinoma Cells

doi: 10.3389/fonc.2022.802482

Figure Lengend Snippet: Stromal fibroblasts limit the radiosensitizing effect of SRC inhibition by dasatinib in LNCaP spheroid co-cultures. CAV1 expressing stromal fibroblasts (HS5) were co-cultured with LNCaP control (Ctrl) cells expressing low endogenous CAV1 levels and the reporter green fluorescent protein (GFP) in hanging drops for 24 h. After formation of spheroids, cells were plated in growth factor-reduced Matrigel mixed with normal growth medium (1/2, v/v) supplemented with dasatinib or vehicle control (VC) and irradiated with 0 Gy or 10 Gy. Pictures were taken at the time of irradiation (0 h) and 48 h later. (A) Spheroid growth was measured and the respective volumes were calculated for 0 and 48 h after irradiation. Data were derived from 3 to 5 individual experiments, where at least 10 spheroids per condition and per experiment each were measured. (B) Cell death was analyzed afterwards by fluorescence microscopy using propidium iodide. Hoechst 33342 was used for nuclei staining. Representative fluorescent images from the individual experiments are shown (48 h time point). Scale bars represent 100 µm.

Article Snippet: The human PCa cell line LNCaP and the human skin fibroblast cell line HS5 were from ATCC (Manassas, VA) and cultured in RPMI Medium (Gibco, ThermoFisher, Waltham, MA) supplemented with 10% fetal bovine serum and 100 U/ml Penicillin/Streptomycin (Sigma-Aldrich, St. Louis, MO) under standard cell culture conditions (37°C, 5% CO2, 95% humidity).

Techniques: Inhibition, Expressing, Cell Culture, Control, Irradiation, Derivative Assay, Fluorescence, Microscopy, Staining

Journal: Frontiers in Oncology

Article Title: Stromal Fibroblasts Counteract the Caveolin-1-Dependent Radiation Response of LNCaP Prostate Carcinoma Cells

doi: 10.3389/fonc.2022.802482

Figure Lengend Snippet: Dasatinib treatment does not impact on survival or growth of stromal fibroblasts, nor in combination with RT. CAV1 expressing stromal fibroblasts (HS5) were cultured as monolayers (A, B) or spheroids (C, D) with or without radiation treatment (0 and 10 Gy) in the presence of dasatinib or vehicle control (VC), (A) Apoptotic cells (subG1) were analyzed 48 h post treatment by flow cytometry. Graphs consist of data from 3 individual experiments (with SEM). P-values indicate ***p ≤0.005 as estimated by one-way ANOVA with Tukey’s multiple comparison test. (B) Clonogenic survival was evaluated 10 days post treatment by counting respective colonies. Data show the surviving fractions from 3 independent experiments (means ± SD) plated in triplicates each. (C) Spheroid growth was measured and the respective volumes were calculated for 0 and 48 h after irradiation from 3 to 4 individual experiments where at least 10 spheroids per condition and per experiment each were measured. (D) Cell death was analyzed afterwards by fluorescence microscopy using propidium iodide. Hoechst 33342 was used for nuclei staining. Representative fluorescent images from individual experiments are shown (48 h time point). Scale bar represents 100 µm.

Article Snippet: The human PCa cell line LNCaP and the human skin fibroblast cell line HS5 were from ATCC (Manassas, VA) and cultured in RPMI Medium (Gibco, ThermoFisher, Waltham, MA) supplemented with 10% fetal bovine serum and 100 U/ml Penicillin/Streptomycin (Sigma-Aldrich, St. Louis, MO) under standard cell culture conditions (37°C, 5% CO2, 95% humidity).

Techniques: Expressing, Cell Culture, Control, Flow Cytometry, Comparison, Irradiation, Fluorescence, Microscopy, Staining

Journal: Frontiers in Oncology

Article Title: Stromal Fibroblasts Counteract the Caveolin-1-Dependent Radiation Response of LNCaP Prostate Carcinoma Cells

doi: 10.3389/fonc.2022.802482

Figure Lengend Snippet: RT treatment causes an increased metabolic potential of fibroblasts, an effect that is not affected upon SRC inhibition; LNCaP PCa cells are not affected. Extracellular flux analyses of LNCaP and fibroblast cell cultures were performed following RT treatment (0 and 10 Gy) in the presence of dasatinib or vehicle control (VC) 24 h post treatments. OCR (A) and ECAR (B) levels of LNCaP PCa cells are shown. Data were summarized as mean values ± SD (measured in 4–7 replicates each). (C) OCR levels of stromal HS5 fibroblasts are shown. (D) Basal respiration, ATP production and proton leak were depicted in separate bar diagrams. (E) Respective ECAR measurement over time are shown. (F) Basal and stressed (following oligomycin treatment) measurements of ECAR were depicted in separate bar diagrams. Data were summarized as mean values ± SD (measured in 4–7 replicates each). P-values indicate: *p ≤ 0.05, by one-way ANOVA with Tukey’s multiple comparison test and additionally by unpaired (two-tailed) t-tests depicted as # p ≤0.05.

Article Snippet: The human PCa cell line LNCaP and the human skin fibroblast cell line HS5 were from ATCC (Manassas, VA) and cultured in RPMI Medium (Gibco, ThermoFisher, Waltham, MA) supplemented with 10% fetal bovine serum and 100 U/ml Penicillin/Streptomycin (Sigma-Aldrich, St. Louis, MO) under standard cell culture conditions (37°C, 5% CO2, 95% humidity).

Techniques: Inhibition, Control, Comparison, Two Tailed Test