Journal: Cancers

Article Title: PGRMC1 Promotes Progestin-Dependent Proliferation of Breast Cancer Cells by Binding Prohibitins Resulting in Activation of ERα Signaling

doi: 10.3390/cancers13225635

Figure Lengend Snippet: PGRMC1 promotes proliferation of breast cancer cells upon progestin treatment. Relative MTT-signal as surrogate for cell number of ( A ) MCF7/PGRMC1 and MCF7/EVC cells, ( B ) T47D/PGRMC1 and T47D/EVC cells, ( C ) MDA-MB-231/PGRMC1 and MDA-MB-231/EVC cells. Cells were treated with different progestins in the concentration of 10 −6 M. Relative MTT-signal of ( D ) MCF7/PGRMC1 and ( E ) T47D/PGRMC1 cells treated with different concentrations of progestins (10 −6 –10 −9 M) for 72 h. Values were normalized to respective DMSO treated cells. Statistical analysis was performed with twoway ANOVA and Bonferroni post−hoc test. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: PGRMC1-deficient MCF7 cells (MCF7/PGRMC1-KO) were generated by transient transfection of MCF7 cells with ‘PGRMC1 CRISPR/Cas9 KO Plasmid (h)’ (Santa Cruz Biotechnology, CA, USA, sc-401945, containing a mixture of three expression plasmids each encoding for the Cas9 enzyme and a PGRMC1-specific gRNA) using Lipofectamine 3000 (Thermo Fisher Scientific, L3000001).

Techniques: Concentration Assay

Journal: Cancers

Article Title: PGRMC1 Promotes Progestin-Dependent Proliferation of Breast Cancer Cells by Binding Prohibitins Resulting in Activation of ERα Signaling

doi: 10.3390/cancers13225635

Figure Lengend Snippet: PGRMC1 interacts with the ERα-modulators PHB1 and PHB2 upon treatment with NET. Analysis of immunopurified (HAbased) samples of MCF7/PGRMC1-GFP cells (PGRMC1-GFP) and MCF7/PGRMC1 cells (PGRMC1-HA) treated with DMSO or NET (10 −6 M) for co-precipitated proteins. ( A ) Volcano plot showing the result of a Welch’s t -test including 253 proteins with an increased abundance as revealed by a two-way ANOVA after HA-based enrichment. Proteins represented by red dots and blue triangles show a significantly altered abundance (FDR 0.01%). Mass spectrometry results for co-precipitated ( B ) PHB1 or ( C ) PHB2, log2 normalized intensity +: significantly different (Welch’s test). ( D , F ) Western blot analysis for co-precipitated ( D ) PHB1 or ( F ) PHB2 (upper panel) and the protein level of PHB1, PHB2 and PGRMC1 in whole cell lysates from the same cells (lower panel). ( E , G ) Densitometric analysis for precipitated ( E ) PHB1 or ( G ) PHB2. Signal intensity was normalized to PGRMC1-HA/DMSO. Difference between DMSO- and NET-treated samples was calculated with unpaired Student’s t -test. **: p < 0.01.

Article Snippet: PGRMC1-deficient MCF7 cells (MCF7/PGRMC1-KO) were generated by transient transfection of MCF7 cells with ‘PGRMC1 CRISPR/Cas9 KO Plasmid (h)’ (Santa Cruz Biotechnology, CA, USA, sc-401945, containing a mixture of three expression plasmids each encoding for the Cas9 enzyme and a PGRMC1-specific gRNA) using Lipofectamine 3000 (Thermo Fisher Scientific, L3000001).

Techniques: Mass Spectrometry, Western Blot

Journal: Cancers

Article Title: PGRMC1 Promotes Progestin-Dependent Proliferation of Breast Cancer Cells by Binding Prohibitins Resulting in Activation of ERα Signaling

doi: 10.3390/cancers13225635

Figure Lengend Snippet: PGRMC1-S181-phosphorylation is essential for increased cell proliferation and PHB binding upon progestin treatment. ( A ) Western blot analysis of PGRMC1-S181-phosphorylation and PGRMC1 protein levels in whole cell lysates of MCF7/PGRMC1 cells after treatment with progestins (10 −6 M) and DMSO. S181-phosphorylation occurs on both the endogenous PGRMC1 (lower band, ≈25 kDa) and exogenous HA-tagged PGRMC1 (upper band, ≈28 kDa). Densitometric analysis of Western blot results for S181-phosphorylation of ( B ) exogenous PGRMC1 and ( C ) endogenous PGRMC1 relatively to total PGRMC1 protein level. ( D – F ) Relative MTT signal as surrogate for cell number of ( D ) MCF7/PGRMC1-S57A, ( E ) MCF7/PGRMC1-S181A, ( F ) MCF7/PGRMC1-S57A/S181A cells treated with different progestins (all 10 −6 M) or DMSO for 72 h. Values were normalized to DMSO treated cells. ( G ) Western blot analysis of immunopurified HA-tagged PGRMC1 and co-precipitated PHB1 from MCF7/PGRMC1 cells treated with different progestins (10 −6 M) and DMSO (upper panel) and PHB1 protein level in whole cell lysates in the same cells (lower panel). ( H ) Densitometric analysis of co-precipitated PHB1 ( I ) Western blot analysis of immunopurified HA-tagged PGRMC1-variants and co-precipitated PHB1 after treatment with DYD (10 −6 M) or DMSO. ( J ) Densitometric analysis of co-precipitated PHB1. ( B , C , H , J ) Signal intensity was normalized to corresponding DMSO-control and signal intensity of total PGRMC1 ( B , C ) or each precipitated PGRMC1-variant ( H , J ). Statistical analysis was performed by one-way ANOVA ( A – H ) or two-way ANOVA ( J ) and Bonferroni post-hoc tests. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: PGRMC1-deficient MCF7 cells (MCF7/PGRMC1-KO) were generated by transient transfection of MCF7 cells with ‘PGRMC1 CRISPR/Cas9 KO Plasmid (h)’ (Santa Cruz Biotechnology, CA, USA, sc-401945, containing a mixture of three expression plasmids each encoding for the Cas9 enzyme and a PGRMC1-specific gRNA) using Lipofectamine 3000 (Thermo Fisher Scientific, L3000001).

Techniques: Phospho-proteomics, Binding Assay, Western Blot, Control, Variant Assay

Journal: Cancers

Article Title: PGRMC1 Promotes Progestin-Dependent Proliferation of Breast Cancer Cells by Binding Prohibitins Resulting in Activation of ERα Signaling

doi: 10.3390/cancers13225635

Figure Lengend Snippet: PGRMC1-PHBs interaction disturbs PHBs’ binding to ERα. ( A ) Proximity ligation assay (PLA) for PGRMC1- (or ERα-) interactions with PHB1 upon treatment with NET, DYD, P4 (10 −6 M) and DMSO in MCF7 cells. Analysis of PLA for PGRMC1- (or ERα-) interactions with PHB1 in ( B ) MCF7 and ( C ) MCF7/PGRMC1-KO cells upon treatment with progestins and DMSO. Dots per cell were counted for 50-60 cells in each sample. Cell number and PLA signals were quantified using imageJ software. ( D ) PLA for PGRMC1- (or ERα-) interactions with PHB1 upon treatment with progestins and DMSO in MCF7/PGRMC1-KO cells. Each red spot represents a single interaction. Nuclear stain: DAPI. Magnification 40×.

Article Snippet: PGRMC1-deficient MCF7 cells (MCF7/PGRMC1-KO) were generated by transient transfection of MCF7 cells with ‘PGRMC1 CRISPR/Cas9 KO Plasmid (h)’ (Santa Cruz Biotechnology, CA, USA, sc-401945, containing a mixture of three expression plasmids each encoding for the Cas9 enzyme and a PGRMC1-specific gRNA) using Lipofectamine 3000 (Thermo Fisher Scientific, L3000001).

Techniques: Binding Assay, Proximity Ligation Assay, Software, Staining

Journal: Cancers

Article Title: PGRMC1 Promotes Progestin-Dependent Proliferation of Breast Cancer Cells by Binding Prohibitins Resulting in Activation of ERα Signaling

doi: 10.3390/cancers13225635

Figure Lengend Snippet: ERα is activated upon progestin-treatment in a PGRMC1-dependent manner. ( A ) qRT-PCR analysis of TFF1 mRNA expression in MCF7/PGRMC1, MCF7/EVC and MCF7/PGRMC1-S181A cells upon treatment with progestins (10 −6 M) or DMSO (0.01%) for 24 h. ( B ) Relative MTT signal as surrogate for cell number of MCF7/PGRMC1 and MCF7/EVC cells treated with fulvestrant (10 −7 M) and NET (10 −6 M) or DMSO (0.01%). Values were normalized to DMSO treated cells. qRT-PCR analysis of TFF1 mRNA expression in MCF7/PGRMC1 and MCF7/EVC cells upon treatment with ( C ) fulvestrant (10 −7 M) and NET (10 −6 M), ( D ) fulvestrant and DYD (10 −6 M), ( E ) AG-205 (25 × 10 −6 M) and NET, ( F ) AG-205 and DYD, or DMSO, respectively. Signal intensity was normalized to respective DMSO control. Statistical analysis was performed by two-way ANOVA and Bonferroni post-hoc test. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: PGRMC1-deficient MCF7 cells (MCF7/PGRMC1-KO) were generated by transient transfection of MCF7 cells with ‘PGRMC1 CRISPR/Cas9 KO Plasmid (h)’ (Santa Cruz Biotechnology, CA, USA, sc-401945, containing a mixture of three expression plasmids each encoding for the Cas9 enzyme and a PGRMC1-specific gRNA) using Lipofectamine 3000 (Thermo Fisher Scientific, L3000001).

Techniques: Quantitative RT-PCR, Expressing, Control