17 dmag

AR113Q knock-in mice were generated using exon 1-specific gene targeting^{50 (link),59 (link)}, backcrossed to C56BL/6 (≥10 generations), and housed in a specific pathogen-free facility on a 12-h light/dark cycle with chow and water ad libitum. For analysis, AR113Q males were used at 3 months of age and randomly assigned to treatment groups. JG-294 was dissolved in 70% propylene glycol (W294004, Sigma-Aldrich) and 30% PBS (pH 7.4, Life Technologies), heated to 95 °C to dissolve compound, aliquoted and frozen. Mice received intraperitoneal injections (3 mg kg⁻¹) every other day for 2 weeks. 17-DMAG (S1142, Selleckchem) was dissolved in 1% DMSO (D2650, Sigma-Aldrich), 30% polyethylene glycol (202371; Sigma-Aldrich) and 1% Tween80 (P4780; Sigma-Aldrich), and administered by i.p. injection (10 mg kg⁻¹) every other day for 2 weeks. At the end of treatment, mice were euthanized and quadriceps were harvested and flash frozen in liquid nitrogen or mounted on OCT (Tissue-Plus, Fisher Health Care) for cryosectioning. For western blot, lysates were obtained by homogenizing tissue in RIPA containing phosphatase and protease inhibitors. All procedures involving mice were approved by the University of Michigan Committee on Use and Care of Animals (PRO00008133) and conducted in accordance with institutional and federal ethical guidelines for animal testing and research.

The antibodies (FITC-CD3, PE-CD56, APC-NKG2D, BV421-CD8, and APC-MICA/B) and their respective isotype antibodies were purchased from BioLegend (San Diego, CA, USA). The Annexin V-FITC -7AAD (7-Amino-Actinomycin D) kit was also purchased from BioLegend (San Diego, CA, USA). The HSP90 inhibitors 17-DMAG and ganetespib (STA-9090) were purchased from Selleckchem (Boston, MA, USA). These HSP90 inhibitors were dissolved in DMSO and stored at −80 °C at a concentration of 50 mM (please note that the control DMSO concentration of 17-DMAG was 0.1‰, and that the control DMSO concentration of ganetespib was 0.02‰). The FxCycle™ Violet stain and CellTrace™ CFSE Cell Proliferation Kit (Invitrogen, Thermo Fisher Scientific, Eugene, OR, USA) were used to distinguish tumor cells from CIK cells using flow cytometry. Hoechst 34580 (Merck, Sigma, Darmstadt, Germany) was added prior to flow cytometry to stain the dead cells. The CellEvent Tm Caspase-3/7 Green flow cytometry assay kit was purchased from Invitrogen (Thermo Fisher Scientific Inc., Waltham, MA, USA).

When JIMT-1 tumor volume reached about 100~200 mm³, mice (n = 5/group) were intraperitoneally injected with heat shock protein 90 inhibitor (17-DMAG, Selleck Chemicals, Houston, TX, USA). Mice were administered a total of 150 mg/kg of 17-DMAG dissolved in 10% DMSO and 10% ethanol over 24 h in three doses of 50 mg/kg each. The control (vehicle, n = 5/group) mice were injected with an equal amount of saline in 10% DMSO and 10% ethanol. Tumor volume was calculated by long diameter × (short diameter)²/2, and body weight was measured thrice a week.

Senolytic drugs used are s follows: JQ1, ARV825, and ABT263 were obtained from Medchem Express (cat#: HY-13030, HY-16954, HY-10087, respectively). OTX015 and 17-DMAG were purchased from Selleck (cat#: S7360 and S1142, respectively)). Quercetin and Dasatinib were purchased from Cayman (cat#: 10005169) and LC Laboratories (cat#: D-3307), respectively.

Imatinib mesylate and ponatinib hydrochloride were obtained from NCE Biomedical Co., Ltd. ApoE-mimetic peptide COG112 (acetyl-LRVRLASHLRKLRKRLL-amide) was synthesized by ChinaPeptides Co., Ltd and purified to > 95% purity. Antibodies to BCR-ABL (b2a2 Junction Specific) (#3908), p-Abl (Tyr245) (#2861) and PP2Ac (#2038) were from CST; antibodies to p-PP2Ac (Y307) (#sc-12615-R), EphB2 (#sc-1763), PDGFRα (#sc-338), β-actin (#sc-47778) and β-tubulin (#sc-9104) were from Santa Cruz; antibodies to SET (#ab1183) was from Abcam. 17-DMAG was acquired from Selleck Chemicals. Okadaic acid (OA) and other reagents and solvents were analytical grade and from Sigma-Aldrich unless noted otherwise.

To evaluate the HER2 expression level using ⁸⁹Zr radiolabeled pertuzumab in breast cancer cell lines, in vitro cell binding assay was done. ⁸⁹Zr radiolabeled pertuzumab (100 ng) was added to 1 × 10⁶ of breast cancer cells at 4 °C for 1 h. To determine whether pertuzumab binding to HER2 was inhibited by pretreatment of trastuzumab and herzuma, trastuzumab biosimilar, or not, trastuzumab and herzuma (10 μg) pretreated in JIMT-1 cells for 1 h at 4 °C and ⁸⁹Zr radiolabeled pertuzumab (100 ng) was added at 4 °C for 1 h. Nonspecific binding was determined in the presence of 100-fold excess of pertuzumab. After incubation, the samples were washed twice in cold PBS containing 1% BSA. Each tube was counted in a gamma counter (WIZARD 1480, Perkin–Elmer, Waltham, MA, USA). Cell-bound radioactivity (%) was calculated by (cell-bound radioactivity—nonspecific binding radioactivity)/total radioactivity × 100.
To evaluate the correlation of HER2 expression by various concentrations of 17-DMAG (Selleck Chemicals, Houston, TX, USA) treatments, correlation analysis between flow cytometry and a cell-binding assay was performed by Prism^® Ver. 5.0 software (GraphPad Software, San Diego, CA, USA).