RCC cell lines ACHN and KRCY were obtained from the American Type Culture Collection. Caki-1 was obtained from Japanese Collection of Research Bioresources Cell Bank. KU19-20 was kindly provided by Dr Mototsugu Oya (Department of Urology, School of Medicine, Keio University, Tokyo, Japan). The cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 1% MEM Non-Essential Amino Acids (Gibco; Thermo Fisher Scientific, Inc.), 1% MEM sodium pyruvate solution 100 mM (Gibco; Thermo Fisher Scientific, Inc.) and 90 µg/ml kanamycin in a 37°C incubator containing 5% CO₂. 9-ING-41 was provided by Actuate Therapeutics, Inc. and used at 0.5-50 µM. Sorafenib was obtained from ChemScence, LLC and used at 3-8 µM. Sunitinib was obtained from Sigma-Aldrich; Merck KGaA and used at 1-4 µM. Cabozantinib was obtained from LC Laboratories and used at 0.5-6 µM. Pazopanib, chloroquine and bafilomycin were obtained from Cayman Chemical Company and used at 0.5-1, 8-30 and 1-10 µM, respectively. Control cells were treated with an equal amount of DMSO. Mycoplasma testing was performed for all cell lines; if mycoplasma was positive, the cells were treated with MC-210 (DS Pharma Promo Co., Ltd.) at 0.5 µg/ml for two weeks, and the reagent was washed out for another one week at 37°C in an incubator.

Cell viability was detected with a colorimetric CellTiter 96^® AQueous One Solution Cell Proliferation assay (Promega Corporation), using a tetrazolium compound according to the manufacturer's instructions using ACHN, Caki-1, KRCY and KU19-20 cell lines. The cells were treated with 0.5-5 µM 9-ING-41 during the assay, and cell viability was measured at 0, 24, 48, 72 and 96 h in 9-ING-41 monotherapy. In the combination treatment, 9-ING-41 was used at 0.5-4 µM during the assay, and cell viability was measured at 0 and 72 h. For the estimation of cell proliferation, a 5-bromo-2-deoxyuridine (BrdU) Cell Proliferation Assay kit (EMD Millipore) was used according to the manufacturer's instructions using ACHN, Caki-1, KRCY and KU19-20 cell lines. The cells were treated with 0-25 µM 9-ING-41. Both experiments were performed in three or four replicates using a flat-bottom 96-well plate (Corning, Inc.) and an iMark™ 96-well microplate reader (Bio-Rad Laboratories, Inc.). Absorbance was measured at 490 nm in the cell viability assay and at 450-595 nm in the cell proliferation assay. GI₅₀, a concentration of the drug that inhibits the proliferation of cancer cells by 50%, was calculated using GraphPad Prism 7 (GraphPad Software, Inc.).

ACHN and KRCY cells were fixed in cold 70% ethanol for 30 min. Propidium iodide (PI) staining of fixed cells was performed for cell cycle analysis and quantification of apoptosis (sub-G₁ population) using FxCycle™ PI/RNase Staining Solution (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Stained cells were analyzed using BD Accuri™ C6 software and a BD Accuri™ C6 Flow Cytometer (BD Biosciences).

Subconfluent cell cultures were washed with cold PBS and lysed in lysis buffer (150 mM sodium chloride, 5 mM EDTA, 1% Triton X-100, 100 mM Tris-HCl and a protease inhibitor). Following clarification of the lysates by centrifugation at 15,000 × g for 30 min at 4°C, protein concentration was detected by the Bradford method, and 30 µg of each protein was electrophoretically separated on a 10% SDS-polyacrylamide gel and transblotted to a PVDF membrane. Immunoblots were blocked with 10% skimmed milk in TBS followed by incubation with primary antibodies. Horseradish peroxidase-labeled ECL™ Anti-mouse IgG (1:2,000-1:5,000) and ECL™ Anti-rabbit IgG (1:5,000-1:20,000) from GE Healthcare were used as secondary antibodies and detected using Clarity Max Western ECL Substrate from Bio-Rad Laboratories, Inc. according to the manufacturer's instructions. Expression of β-actin was used as a loading control. The images were analyzed using Ez-Capture MG (Atto Corporation). The following antibodies were used: Anti-cyclin D1 (cat. no. 2922), anti-cyclin B1 (cat. no. 4135S), anti-E2F transcription factor 1 (E2F-1; cat. no. 3742), anti-cyclin-dependent kinase 1 (CDK1; cat. no. 77055S), anti-GS (cat. no. 3893), anti-phospho-GS (Ser641) (cat. no. 3891), anti-poly (ADP-ribose) polymerase (PARP; cat. no. 9542), anti-GSK-3β (cat. no. 12456), anti-β-actin (cat. no. 12262) from Cell Signaling Technology, Inc.; anti-X-linked inhibitor of apoptosis (XIAP; cat. no. 610716) and anti-Bcl-2 (cat. no. 610538) from BD Biosciences. The dilution ratios of the primary antibodies were 1:250-1:1,000.

ACHN, Caki-1, KRCY and KU19-20 cell lines were treated with 0, 25 and 50 µM 9-ING-41. Total cellular RNA was extracted using the SV Total RNA Isolation System (Promega Corporation) and the first-strand DNA was synthesized using a cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. qPCR was performed in a 7300 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) with 40 cycles of denaturation (95°C for 15 sec), annealing and elongation (60°C for 1 min). Predesigned TaqMan^® Gene Expression assays (Applied Biosystems; Thermo Fisher Scientific, Inc.) targeting human Bcl-2 (assay ID, Hs00236808_s1), E2F1 (assay ID, Hs00153451_m1) mRNA were used, and GAPDH (assay ID, Hs02758991_g1) was used as an endogenous control. Each experiment was performed in triplicate wells for each sample in a final reaction volume of 20 µl using a TaqMan^® Universal PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The expression of the target mRNA was quantified relative to that of the GAPDH mRNA using the 2^-ΔΔCq method as previously described (21) and untreated controls were used as a reference.

Human peripheral blood mononuclear cells (PBMC) were separated from human blood obtained from healthy volunteers using Lymphocyte Separation Solution (Nacalai Tesque, Inc.). For activation, PBMCs were suspended at a concentration of 2×10⁶ cells/ml in RPMI-1640 medium containing 10% FBS, and recombinant human interleukin 2 (IL-2; cat. no. 0617AFC12; Peprotech, Inc.) was added at a concentration of 2,000 IU/ml. PBMCs were cultured for 3 days at 37°C in a 5% CO₂ atmosphere. CytoTox96^® Non-radioactive Cytotoxic Assay (Promega Corporation) was used according to the manufacturer's instructions. The CytoTox96^® colorimetric assay measures lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis. Briefly, RCC cells ACHN and Caki-1 and activated PBMCs were added to a round-bottom 96-well plate (Corning, NY) and mixed at the lymphocyte to cancer cell ratios between 1:2.5 and 1:80. Following 4-h incubation at 37°C, 50 µl of the supernatants were transferred to a fresh 96-well flat-bottom plate (Corning, Inc.), and the absorbance signal was measured at 490 nm using an iMark™ Microplate Reader. Experiments were performed in triplicate.

Continuous variables are presented as the mean ± SD. All continuous variables in this study met the criteria for a normal distribution and were assumed to be parametric. Data were analyzed using one-way ANOVA with Dunnett's test for multiple comparisons. Statistical analysis was performed using GraphPad Prism 7 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Immunoblotting was used to determine GSK-3β expression in ACHN, Caki-1, KRCY and KU19-20 RCC cell lines (Fig. 1A). Treatment with 9-ING-41 resulted in reduced GSK-3β activity in all RCC cell lines, indicated by the decreased expression of phospho-GS, a downstream target of GSK-3β, compared with control cells treated with DMSO (Fig. 1A). The results of the MTS assay demonstrated that treatment with 9-ING-41 decreased the proliferation of RCC cells at low micromolar concentrations in a dose-dependent manner with a GI₅₀ range of 0.5-1.7 µM (Fig. 1B). The results of the BrdU incorporation assay confirmed that treatment with 9-ING-41 inhibited the proliferation of RCC cells compared with the respective control groups (Fig. 1C).

PI-fluorescence-activated cell sorting revealed that treatment with 9-ING-41 for 48 h induced cell cycle arrest at the G₂ phase, and treatment for 96 h induced cell cycle arrest with an increased sub-G₁ cell population, which is an indicator of apoptosis, in ACHN and KRCY cells compared with the control groups (Fig. 2A and B). Mechanistically, the expression of Cyclin B1 and CDK1 proteins, which serve an important role in the transition from G₂ to M phase, was decreased following treatment with 9-ING-41 (Fig. 3A). In addition, the expression of E2F-1 and cyclin D1 proteins, which serve a crucial role in the cell cycle, was decreased following treatment with 9-ING-41 (Fig. 3A). Treatment with 9-ING-41 decreased the expression of antiapoptotic proteins, Bcl-2 and XIAP, leading to an increase in apoptosis indicated by PARP cleavage, which is a marker of apoptosis (Fig. 3B). However, the immunoblotting results also indicated that Bcl-2 was scarcely expressed in KU19-20. RT-qPCR results demonstrated decreased Bcl-2 and E2F1 mRNA expression in RCC cells treated with 9-ING-41 compared with the controls, with the exception of Bcl-2 in KU19-20 (Fig. 3C).

Using RCC cell lines, the effects of 9-ING-41 in combination with first- and second-line RCC targeted therapeutics sunitinib, pazopanib, sorafenib and cabozantinib were investigated (Figs. 4A, C and S1). For combination experiments, a minimally effective and clinically relevant concentration of 0.5-2 µM 9-ING-41 was used for ACHN (GI₅₀=0.8 µM), Caki-1 (GI₅₀=1.7 µM), KRCY (GI₅₀=1 µM) and KU19-20 (GI₅₀=0.5 µM) cells. The results of the MTS assay demonstrated that 9-ING-41 potentiated the antitumor effects of sorafenib (P<0.05), cabozantinib (P<0.05), sunitinib (P<0.05) and pazopanib (P<0.05) in RCC cells (Figs. 4A, C and S1).

To test the hypothesis that an autophagy inhibitor may potentiate the antitumor effects of 9-ING-41, RCC cells were treated with a combination of 9-ING-41 (0.5-2 µM) and the autophagy inhibitors chloroquine (8-30 µM) and bafilomycin (1-10 µM). The results of the MTS assay revealed that the antitumor effect of 9-ING-41 was significantly increased when 9-ING-41 was combined with chloroquine (P<0.05) or bafilomycin (P<0.05) compared with single treatments in RCC cells (Figs. 4B, D and S1).

To investigate whether GSK-3 inhibition affects the antitumor effects of human immune cells in RCC cell lines, ACHN and Caki-1 renal cancer cells were treated with 9-ING-41 for 72 h, and then mixed with activated PBMCs at various ratios. LDH activity of the supernatant was measured to evaluate the cytotoxic effects (Fig. 5). The results demonstrated that treatment with 9-ING-41 significantly increased the cytotoxic effects of activated PBMCs in representative RCC cell lines ACHN and Caki-1 compared with control cells treated with DMSO (Fig. 5).