Lipofectamine 3000

Melanoma cells were trypsinized and counted (1 × 10⁶ cells/well) and transfected with Lipofectamine 3000 and a non targeting control siRNA (AllStars negative Control #SI03650318, QIAGEN) or two different siRNAs targeting PGC1α (Hs_PPARGC1A_2 and 6 FlexiTube siRNA; #SI00101031 and #SI02639833; all from QIAGEN). Two rounds of transfection were performed within 48 h prior to cells being used for RNA analysis or drug response experiments.

MUC1shRNA (MISSION shRNA TRCN0000122938), and a control scrambled shRNA (CshRNA)(Millipore Sigma) were inserted into pLKO-tet-puro (Plasmid #21915; Addgene, Cambridge, MA, USA). XISTshRNA was purchased from GenePharma (Shanghai, China). Control, XIST, and TDP-43 targeted siRNAs (ThermoFisher Scientific) were transfected into cells using Lipofectamine 3000 (ThermoFisher Scientific). DOX-inducible lentiviral shRNA targeting XIST (GE Dharmacon, V3SH11258_245457769) was obtained from Horizon Discovery (Cambridge, UK). DOX-inducible DYRK1A/pTRE3G-FL-hXIST vector was obtained from Addgene (Plasmid #149608; RRID:Addgene_149608). The CshRNA, MUC1shRNA, MUC1shRNA#2 (MISSION shRNA TRCN0000430218), and NF-κBshRNA (MISSION shRNA TRCN0000014687) were produced in HEK293T cells as described [28 (link)]. Flag-tagged MUC1-CD [60 ] was inserted into pInducer20 (Plasmid #44012, Addgene) [61 ]. Transduced cells were selected for growth in 1–2 μg/ml puromycin or 100 μg/ml geneticin. For inducible gene silencing, cells were treated with 0.1% DMSO as the vehicle control or 500 ng/ml doxycycline (DOX; Millipore Sigma). Cells were transfected with a MUC1/ASO (LG00788741; Qiagen, Hilden, Germany) or a control C/ASO (LG00000001; Qiagen) in the presence of Lipofectamine 3000 Reagent.

Human breast adenocarcinoma cell lines, MCF7 cells and MDA-MB-231 cells were purchased from American Type Culture Collection (ATCC). Each cell line was maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% penicillin/streptomycin. Transfection reagent, Effectene or lipofectamine 3000 was from Qiagen, Inc. (Valencia, CA) or Invitrogen (Grand Island, NY), respectively. ON-TARGETplus SMARTpool MMP9 siRNA and siGENOME Non-targeting control siRNA was from GE Dharmacon (Lafayette, CO). Lipopolysaccharide and b-actin antibody was purchased from Sigma (St. Louis, MO). Antibodies against p-p38 (Thr180/Tyr182), p-JNK (Thr183/Tyr185), p-ERK1/2 (Thr202/Tyr204) and p-IkBa (Ser-32) were from Cell Signaling Technology, Inc. (Beverly, MA). Antibodies against TOPK, TLR4, MMP9 or p-serine/threonine, and breast cancer tissue array paired with metastatic tumors or MMP9 inhibitor were purchased from Abcam (Cambridge, MA). Luciferase assay system was from Promega (Madison, WI). IkBa antibody or protein A/G plus-agarose bead was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). TOPK inhibitor, HI-TOPK-032 was from R&D system (Minneapolis, MN). SuperScript III reverse transcriptase was from Invitrogen (Grand Island, NY). GST-IkBa protein was from EMD Millipore (Billerica, MA).

Mouse-derived hippocampal HT22 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and 1% antibiotic-antimycotic in a humidified 5% (v/v) CO₂ incubator at 37 °C. Cells were seeded on the plate at a density of 1.5 × 10⁵ cells/ml, and AdipoR1 shRNA (Qiagen) was transfected using Lipofectamine 3000 according to the manufacturer’s instructions. AdipoR1 knockdown was measured after 72 h of transfection by western blotting. After 72 h of transfection, physiological changes such as cell viability and apoptosis were measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assays and Caspase-Glo^TM 3/7 assays (Promega) according to the manufacturer’s instructions.

Human embryonic kidney 293 (HEK 293) cells and human non‐small‐cell lung carcinoma (H460) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HEK293 or H460 cell line was maintained in DMEM or RPMI 1640 supplemented with 10% FBS, 2 mml‐glutamine, and 1% penicillin/streptomycin, respectively. Paclitaxel, bafilomycin A1, nutlin‐3, anti‐LC3 antibody, and anti‐β‐actin antibody were purchased from Sigma (St. Louis, MO, USA). Z‐VAD‐FMK was purchased from Selleckchem (Houston, TX, USA). HI‐PBK 032 was purchased from Tocris Bioscience (Bristol, UK). Anti‐PBK, anti‐p53, and anti‐Mdm2 antibodies were purchased from Abcam (Cambridge, MA, USA). Anti‐p‐PBK (Thr‐9) and anti‐cleaved PARP antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti‐GFP and anti‐HA antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Alexa Fluor^® 488 goat anti‐rabbit IgG, Alexa Fluor^® 594 goat anti‐rabbit IgG, Alexa Fluor^® 488 goat anti‐mouse IgG, and Alexa Fluor^® 594 goat anti‐mouse IgG antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Transfection reagents, Effectene, or Lipofectamine 3000 were purchased from Qiagen, Inc. (Valencia, CA, USA) or Invitrogen (Grand Island, NY, USA), respectively.