Journal: Cell Communication and Signaling : CCS

Article Title: The natural L370F ERα variant confers endocrine resistance and sensitivity to ATRA in metastatic breast cancer cells

doi: 10.1186/s12964-025-02639-5

Figure Lengend Snippet: The effect of ATRA in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells. Growth curve analyses in parental ( A ), CRISPR/CAS9 engineered L370F ( B ) and Y537S ( C ) MCF-7 cells plated in growing conditions (i.e., 10% FBS) and treated with the indicated doses of all-trans retinoic acid (ATRA) for the indicated time. Experiments were performed using the xCelligence RTCA system. Curves were calculated at the maximum proliferation the untreated sample (control, CTR) in each cell line. D Dose response curves of ATRA effect were calculated at the maximum proliferation of the control sample (CTR). The effect of ATRA is the difference between the normalized cell index (NCI) value in the untreated samples and the NCI value in the treated samples at the time point when cells reach the maximal growth (i.e., confluency). Growth curve analyses in parental ( E ), CRISPR/CAS9 engineered L370F ( F ) and Y537S ( G ) MCF-7 cells plated in growing conditions (i.e., 10% FBS) and treated with the indicated doses of all-trans retinoic acid (ATRA) for the indicated time. Arrows indicate the time point where the medium was changed and ATRA administration was repeated. Experiments were performed using the xCelligence RTCA system. Curves were calculated at the maximum proliferation of the untreated sample (control – CTR) in each cell line. H Western blot and ( H’ and H’’ ) relative densitometric analyses of PARP and phosphorylated γH2AX levels in parental, and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells treated for 3 days with the indicated doses of ATRA. Each dot indicates an experimental replicate. Significant differences in the effect of ATRA were calculated using the Student’s t-test. ** ( p < 0.01) and *** ( p < 0.001) indicates differences with respect the untreated (0) sample in each cell line while °° ( p < 0.01) indicates differences between the corresponding sample in parental MCF-7 cells. I Measurement of β-galactosidase activity (βGal) in parental and in CRISPR/CAS9 engineered Y537S MCF-7 cells treated with the indicated doses of ATRA for 12 days. As positive control parental MCF-7 cells were treated with etoposide (Eto 12.5 µM) and allowed to recover for 4 days. Each dot indicates an experimental replicate. Significant differences in the effect of ATRA were calculated using the Student’s t-test. **** ( p < 0.0001) indicates differences with respect to the untreated (0) sample in each cell line. ( J ) β-Galactosidase staining at pH 6.0 on parental and on CRISPR/CAS9 engineered Y537S MCF-7 cells treated with ATRA (10 − 7 M for MCF-7 cells and 10 − 6 M for Y537S MCF-7 cells) for 12 days. Scale bar = 100 μm

Article Snippet: The following compounds were used in selected experiments: all-trans retinoic acid (ATRA), digoxin (Digo) and bafilomycin A1 from Sigma-Aldrich; fulvestrant (Ful), 4OH-tamoxifen (Tam), disulfiram, necrostatin, and ferrostatin from Selleck Chemicals (USA).

Techniques: CRISPR, Control, Western Blot, Activity Assay, Positive Control, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: The natural L370F ERα variant confers endocrine resistance and sensitivity to ATRA in metastatic breast cancer cells

doi: 10.1186/s12964-025-02639-5

Figure Lengend Snippet: ATRA-dependent regulation of RARα and ERα expression and transcriptional activity in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells. A Western blot and ( A’ ) relative densitometric analyses of ERα levels in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells treated for 72 h with the indicated doses of all-trans retinoic acid (ATRA). Each dot indicates an experimental replicate. Significant differences in the effect of ATRA were calculated using the Student’s t-test. *** and **** indicates p < 0.001 and p < 0.0001 with respect to control (-) samples. ° and °°°° indicates p < 0.05 and p < 0.0001 with respect to the corresponding samples in parental MCF-7 cells. B Western blot and ( B’ and B’’ ) relative densitometric analyses of RARα levels in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells treated for 72 h with the indicated doses of ATRA. Each dot indicates an experimental replicate. Significant differences in the effect of ATRA were calculated using the Student’s t-test. **** indicates p < 0.0001 with respect to control (-) samples. ° and °°°° indicates p < 0.05 and p < 0.0001 with respect to the corresponding samples in parental MCF-7 cells. Dose-response of RARα transcriptional activity ( C ) and ERα transcriptional activity ( D ) in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells stably expressing the RARE-NLuc reporter construct (NLuc) or the ERE-NLuc reporter construct (NLuc), respectively, treated with the indicated doses of ATRA. Experiments were performed three times, and each condition was tested in triplicate

Article Snippet: The following compounds were used in selected experiments: all-trans retinoic acid (ATRA), digoxin (Digo) and bafilomycin A1 from Sigma-Aldrich; fulvestrant (Ful), 4OH-tamoxifen (Tam), disulfiram, necrostatin, and ferrostatin from Selleck Chemicals (USA).

Techniques: Expressing, Activity Assay, CRISPR, Western Blot, Control, Stable Transfection, Construct

Journal: Cell Communication and Signaling : CCS

Article Title: The natural L370F ERα variant confers endocrine resistance and sensitivity to ATRA in metastatic breast cancer cells

doi: 10.1186/s12964-025-02639-5

Figure Lengend Snippet: The senolytic activity of digoxin in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells. Growth curve analyses in parental ( A ), and in CRISPR/CAS9 engineered Y537S ( B ) MCF-7 cells plated in growing conditions (i.e., 10% FBS) and treated with the indicated doses of all-trans retinoic acid (ATRA) or digoxin (Digo) for the indicated time. Black arrows indicate the time point in which media was changed and ATRA was re-administered together with the indicated doses of Digo and the proliferation profile was followed up to 15 days. Experiments were performed twice in duplicate using the xCelligence RTCA system. Curves were calculated at the maximum proliferation the untreated sample in each cell line. A Time dependent inhibitory concentration 50 (IC 50 ) -Log 2 transformed to indicate the sensitivity of parental ( A’ ) and CRISPR/CAS9 engineered Y537S ( B’ ) MCF-7 cells to Digo both in the absence (CTR) and in the presence of the indicated dose of ATRA. C Synergy map of 12-day-treated parental MCF-7 cells with different doses of 4OH-Tamoxifen (Tam) and ATRA. D Synergy map of 10-day-treated MCF-7 cells with different doses of fulvestrant (Ful) and ATRA. E Synergy map of 5-day-treated L370F MCF-7 cells with different doses of fulvestrant (Ful) and ATRA. F Synergy map of 4-day-treated Y537S MCF-7 cells with different doses of fulvestrant (Ful) and ATRA

Article Snippet: The following compounds were used in selected experiments: all-trans retinoic acid (ATRA), digoxin (Digo) and bafilomycin A1 from Sigma-Aldrich; fulvestrant (Ful), 4OH-tamoxifen (Tam), disulfiram, necrostatin, and ferrostatin from Selleck Chemicals (USA).

Techniques: Activity Assay, CRISPR, Concentration Assay, Transformation Assay