Journal: Endocrinology

Article Title: ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

doi: 10.1210/en.2017-00652

Figure Lengend Snippet: Figure 3. D1-9-G129R-hPRL PRLR antagonist abrogates the PRL-mediated increase in SKBR3 cell viability. (A) Western blot of MCF7 and SKBR3 whole cell extracts probed with the anti-PRLR extracellular domain antibody, 1A2B1. (B) Western blot of nuclear extracts (10 mg/well) of SKBR3 cells starved in serum-free media for 16 hours then treated with 5 mg/mL oPRL or 5 mg/mL D1-9-G129R-hPRL PRLR antagonist (Antag) for 1 hour or left untreated, probed for p-STAT5, STAT5 (rabbit-anti-STAT5), and histone-H3 (HH3) (loading control). (C) A general schematic for treatments used in WST-1 assays including a 24-hour pretreatment, 2 hours of TOPOII poison, followed by a 48-hour recovery period. (D, E, and F) WST-1 assays using SKBR3 cells pretreated or not with 5 mg/mL oPRL 6 D1-9-G129R-hPRL for 24 hours followed by 2 hours of doxorubicin (DOX) treatment, with or without PRL or D1-9-G129R-hPRL, then a 48-hour recovery with or without PRL or D1-9-G129R-hPRL as indicated. (D) The effect of oPRL or PRLR antagonist alone. (E) The effect of DOX 6 PRL and (F) the effect of DOX + Antag 6 PRL. Graphs represents pooled experiments, n = 12. Data were analyzed with a one-way ANOVA followed by Bonferroni posttests. *P , 0.05; **P , 0.01; ***P , 0.001.

Article Snippet: Cell culture and cell lines PRL-responsive SKBR3 (HER2 overexpressing, p53mutant) and MCF7 (estrogen receptor+, p53 wild type) human breast cancer cell lines, obtained and authenticated from American Type Culture Collection, were used within 6 months when revived from frozen storage.

Techniques: Western Blot, Control

Journal: Endocrinology

Article Title: ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

doi: 10.1210/en.2017-00652

Figure Lengend Snippet: Figure 4. HSP90 inhibition abrogates PRL-mediated resistance. (A, B, and C) SKBR3 or (D, E, and F) MCF7 cells were pretreated or not with 5 mg/mL oPRL, 15 nM (SKBR3) or 100 nM (MCF7) HSP90 inhibitor 17-AAG, or a combination of the two for 24 hours followed by 2 hours of doxorubicin (DOX) treatment, 6PRL or 17-AAG, and a 48-hour recovery 6 PRL. (A–C) WST-1 assays of SKBR3 (A) with PRL or 17-AAG alone, (B) with DOX 6 PRL, or (C) with DOX + 17-AAG 6 PRL. (D–F) WST-1 assays of MCF7 (D) with PRL or 17-AAG alone, (E) with DOX 6 PRL, or (F) with DOX + 17-AAG 6 PRL. Graphs represent pooled experiments for SKBR3 n = 12, and for MCF7 n = 18. (G–I) WST-1 assays of MCF7 (G) with oPRL or 800 nM BIIB021 alone, (H) with DOX 6 PRL, or (I) with DOX + BIIB021 6 PRL. n = 18. Data were analyzed with a one-way ANOVA followed by Bonferroni posttests. *P , 0.05; **P , 0.01; ***P , 0.001.

Article Snippet: Cell culture and cell lines PRL-responsive SKBR3 (HER2 overexpressing, p53mutant) and MCF7 (estrogen receptor+, p53 wild type) human breast cancer cell lines, obtained and authenticated from American Type Culture Collection, were used within 6 months when revived from frozen storage.

Techniques: Inhibition

Journal: Endocrinology

Article Title: ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

doi: 10.1210/en.2017-00652

Figure Lengend Snippet: Figure 6. JAK2 inhibition abrogates the PRL increased cell viability. (A) Western blot of JAK2 inhibitor dose response. MCF7 cells were treated with twofold increasing concentrations of G6 for 12 hours. p-STAT5 levels were determined (mouse anti-p-STAT5) with total STAT5 and histone H3 (HH3) loading controls. Results are representative of two independent experimental replicates. (B) Dose-response curves of JAK2 inhibitor G6. MCF7 cells were treated with G6 at indicated time points and concentrations. The cell viability was determined with WST-1 cell viability assay. WST-1 assays of (C–E) MCF7 or (F–H) SKBR3 cells pretreated with 25 ng/mL hPRL for 24 hours or 25 mM JAK2 inhibitor G6 for 12 hours, followed by

Article Snippet: Cell culture and cell lines PRL-responsive SKBR3 (HER2 overexpressing, p53mutant) and MCF7 (estrogen receptor+, p53 wild type) human breast cancer cell lines, obtained and authenticated from American Type Culture Collection, were used within 6 months when revived from frozen storage.

Techniques: Inhibition, Western Blot, Viability Assay

Journal: Endocrinology

Article Title: ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

doi: 10.1210/en.2017-00652

Figure Lengend Snippet: Figure 7. HSP90 inhibition decreases ATM and p-ATM protein but not its mRNA. (A) MCF7 cells were treated or not with threefold increasing concentrations of 17-AAG for 24 hours followed by 2 hours of doxorubicin (DOX) treatment and recovery. Proteins (30 mg per well) were resolved on SDS-PAGE gel, and blot was probed for p-ATM, ATM, HSP90a, and GRB2. Gel is representative of two experiments. Fold difference of p-ATM with DOX treatment was calculated, in ImageJ, compared with untreated cells (set to 1). Fold difference of (B) total ATM and (C) p- ATM levels with 17-AAG and DOX treatment were calculated compared with DOX alone (set to 1). Representative blot of two experiments. (D) MCF7 cells were pretreated with oPRL (5 mg/mL) or 17-AAG for 24 hours, followed by 2 hours DOX treatment (0.2 mM). ATM expression was detected by qPCR, and the results were normalized to YWHAZ control [mean 6 standard error of the mean (SEM); n = 5]. The DDCt method was used to analyze the relative changes in gene. The letter “a” above SEM bars denotes that there were no statistically significant differences (one-way ANOVA followed by Bonferroni test, P , 0.05). (E) SKBR3 cells as above, with (F) ATM and (G) p-ATM band intensities quantified by ImageJ. Representative blot of two experiments.

Article Snippet: Cell culture and cell lines PRL-responsive SKBR3 (HER2 overexpressing, p53mutant) and MCF7 (estrogen receptor+, p53 wild type) human breast cancer cell lines, obtained and authenticated from American Type Culture Collection, were used within 6 months when revived from frozen storage.

Techniques: Inhibition, SDS Page, Expressing, Control

Journal: Endocrinology

Article Title: ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

doi: 10.1210/en.2017-00652

Figure Lengend Snippet: Figure 11. ATM is needed for PRL-induced increase in cell viability after DNA damage in 3D culture. Cells were pretreated with 25 ng/mL hPRL or vehicle for 24 hours followed by 2 hours of 1 mM doxorubicin (DOX) treatment (or vehicle) with or without PRL. Cells were transferred to collagen 3D culture and allowed to recover for 48 hours. Cell viability was determined with WST-1 assay, and the treatments were normalized to vehicle controls: (A) SKBR3 or (B) MCF7. For ATM knockdown experiments, (C and E) SKBR3 and (D and F) MCF7 cells were transfected with (C and D) siNT or (E and F) siATM, followed by 24 hours of 25 ng/mL hPRL and 2 hours of DOX treatment (3 mM for SKBR3, 2 mM for MCF7 cells). Cells were transferred to collagen gels to allow recovery for 96 hours (SKBR3) or 48 hours (MCF7). Bars in the graphs represent six independent experiments. Statistically significant analysis by ANOVA: *P , 0.05; **P , 0.01; ***P , 0.001.

Article Snippet: Cell culture and cell lines PRL-responsive SKBR3 (HER2 overexpressing, p53mutant) and MCF7 (estrogen receptor+, p53 wild type) human breast cancer cell lines, obtained and authenticated from American Type Culture Collection, were used within 6 months when revived from frozen storage.

Techniques: WST-1 Assay, Knockdown, Transfection