Cryoprep instrument

For cell lines, quantitative reverse transcriptase PCR analysis was performed using the Cells to C_T kit (Life Technologies), and the lysates were processed according to the manufacturer’s instructions. qRT-PCR was performed using the one-step master mix and TaqMan™ probes (Applied Biosystems). For in vivo pharmacodynamic studies, end of study flash-frozen tumors were pulverized using the cryoPREP instrument (Covaris). From the pulverized tissue, total RNA was extracted using the RNeasy Mini Kit (Qiagen); qRT-PCR was performed using the TaqMan Fast Virus One-Step Master Mix and TaqMan™ probes (Applied Biosystems). The C_T values were analyzed to assess the relative changes in the expression of the TFF1 (trefoil factor 1/breast cancer estrogen-inducible protein; Hs00907239_m1), GREB1 (gene regulated by estrogen in breast cancer 1; Hs00536409_m1), and PGR (progesterone receptor; Hs01556702_m1) genes, with GAPDH (4310884E) as an internal control, using the 2^−ΔΔC_T method [47 (link)].

Tumors were excised, snap frozen and stored at − 80 °C. Tumors were subsequently pulverized with the automated CryoPrep instrument (Covaris) and 10 mg was transferred to a CK mix bead beating tube (Bertin, France) with 350 µL RLT buffer (Qiagen) supplemented with 1% β-mercaptoethanol (Sigma). Tissue was homogenized in the Precellys 24 instrument (Bertin) 6500 rpm for 20 s. Samples were centrifuged for 3 min at 16,000g and the supernatant was collected. RNA was extracted using the RNeasy Mini kit (Qiagen) following the manufacturer’s guidelines. RNA concentration was measured with the NanoDrop One (Thermo Scientific) and RNA integrity confirmed with the 2100 Bioanalyzer RNA Nano Chip (Agilent).

Cells were harvested and lysed in CelLytic^MT lysis buffer (Sigma-Aldrich) after either 24 or 48 h of treatment, and total protein was separated by SDS-PAGE and transferred to the membranes and immunostained using antibodies specific to the indicated proteins. For in vivo pharmacodynamic studies, end of study flash-frozen tumors (4 h post-last dose) were fractured using a cryoPREP instrument (Covaris), and pulverized tissue was lysed in CelLytic^MT lysis buffer (Sigma-Aldrich). Total protein was analyzed by Western blot analysis as described above. Protein expression was analyzed using standard practice and antibodies as follows: ERα, PR, E2F1, CCNE1, CCNE2, CCND1, total Rb, phospho-Rb S807/811, CDK2, CDK4, CDK6, Actin (Cell Signaling Technologies, Catalog #13258, #3153, #3742, #20808, #4132, #2978, #9309, #8516, #2546, #12790, #13331, #4970, respectively), phospho-p107, p107, phospho-p130, p130 (Abcam: ab111348, ab76255, ab168458, ab6545, respectively), GREB1 (Millipore, MAB562), and Vinculin (Sigma-Aldrich, #v9131). Protein expression was quantified using the AzureSpot software and normalized to the expression of the vinculin protein.