Ipa 3

Small molecule inhibitors were purchased from Tocris Biosciences (R&D Systems): PF-4708671, U 73122, GSK2334470, IPA3, SL0101-1 and XMD 8-92 or from Selleck Chemicals LLC (TX, USA): PF-562271, Enzastaurin (LY317615), AUY922 (NVP-AUY922), 17-AAG (Geldanamycin), PF-04929113 (SNX-5422), AZD6244 (Selumetinib), AT7867, CHIR-98014, LY2228820, BIX 02188, AS703026, PH-797804, SP600125, NU7441. All inhibitors were prepaed in DMSO at 100 mM. Cells were treated with inhibitors prepared in culture media where the final concentration of DMSO was 2% v/v. Vehicle control treatments consisted of culture media containing 2% v/v DMSO. Six days after treatment, cell survival was measured in comparison to vehicle controls using the CellTiter 96® Assay as per manufacturer instructions (MTS assay, Promega Corporation, WI, USA). Data were analyzed in GraphPad Prism® version 5.00 for Windows (GraphPad Software, CA, USA) to measure the log₁₀ of IC50 for each drug. For combination assays, drugs were added simultaneously. Heatmaps for sensitivities (-log₁₀ of IC50) were prepared using D-chip Analyzer software [49 (link)].

Anti-PAK1 pSer204 antibody (11748), anti-PAK1 antibody (Ab-212), anti-Aurora A pThr288 antibody (12301), anti-TACC3 pSer558 antibody (13495), and anti-LIMK1 pThr508 (11123) antibody were purchased from Signalway Antibody (College Park, MD, USA). Anti-Aurora A antibody was purchased from Abcam (Ab13824, Cambridge, UK). Anti-TACC3 (SN73-05) and Rabbit polyclonal anti-LIMK1 (HA500189) antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). IPA-3 was purchased from Selleck Chemicals (S7093, Houston, TX, USA). All other chemicals and reagents were purchased from Sigma-Aldrich, unless explicitly specified or indicated otherwise.

Cell proliferation was assessed using Cell Counting Kit‐8 (CCK8; APExBIO, Houston, TX, USA). Briefly, the cells were seeded in 96‐well plates and incubated with CCK8 solution (10 μL per well) for 2 h; the absorbance was then measured at 450 nm. For growth inhibition analysis, cells were simultaneously incubated with ibrutinib (Selleck Chemicals, S2680, Shanghai, China), IPA‐3 (Selleck Chemicals, S7093), gabapentin (Selleck Chemicals, S2133), BCATc inhibitor 2 (APExBIO, C3463) or a combination of these agents. The synergistic effect of the drugs was determined using the combination index (CI) parameter, wherein a CI value of < 1 indicates synergism, CI = 1 indicates additivity and CI > 1 indicates antagonism [17 (link)]. Quantitative analysis of data was conducted using calcusyn version 2.0 software (Biosoft, Cambridge, UK).

For the Cell Counting Kit-8 (CCK-8) assay, the IPA-3 (Selleck, Houston, TX, United States), ABC294640 (Selleck, Houston, TX, United States), and S1P (Avanti Polar Lipids, Alabaster, AL, United States) were dissolved in dimethyl sulfoxide (DMSO) to generate stock solutions at concentrations of 50, 50, and 10 mM, respectively. The final concentration of DMSO in the treatment medium was below 0.1%. TNBC cells in DMEM containing 10% FBS were seeded into 96-well plates at a concentration of 1 × 10⁴ cells per well and incubated for 24 h. The culture medium was replaced with a fresh medium containing vehicle or testing agents at indicated concentrations. After treating cells with different agents or vehicles for 48 h, CCK-8 solution (10 μl/well) was added to the 96-well plates and incubated for 1 h to detect the viability of TNBC cells. The light absorbance values at 450 nm were measured in a microplate reader (Bio-Rad, Hercules, CA, United States), and cell viability was determined. Relative viability was normalized to the vehicle-treated control cells after background subtraction and was expressed as OD_test/OD_control^∗100%. Each treatment was performed in triplicate wells, and three independent experiments were repeated.

Cytarabine (Ara-C), Idarubicin (IDA), and dimethyl sulfoxide (DMSO) were purchased from Actavis (Nerviano, Italy) and Sigma (Merck, Germany), respectively. IPA-3 was obtained from Selleck (Houston, TX, United States). Antibodies of PAK1 and p-PAK1 was purchased from Abcam (MA, Cambridge, United States). GAPDH, ERK1/2, p-ERK1/2, β-actin and PARP, Cleaved-PARP antibodies were obtained from Cell Signaling Technology (Danvers, MA, United States).