Fig 2: The effects EGFR/Akt inhibition on cell proliferation and signaling protein expression. HCT116 cells were treated with 20 μM gefitinib, 20 μM LY294002, and 100 μg/mL SAE for 24 h. (A) Cell viability was measured by WST-1 assays. Statistical analyses were performed using t-tests; *p < 0.05, **p < 0.01, ***p < 0.001. (each experiment, n = 3). N., Negative control; S., SAE; G., Gefitinib; L., LY294002. (B) The cells were treated with 20 μM gefitinib, 20 μM LY294002, and 100 μg/mL SAE for 24 h. The expressions of p-EGFR, p-caveolin, P-Src, p-Akt, p53, Bcl-2, Bak, PARP, and cleaved PARP. Pro-caspase 3, cleaved-caspase 3, and β-actin were analyzed by Western blotting (each experiment, n=3). All western blotting images were quantitatively represented using ImageJ (National Institutes of Health). Statistical analyses were performed using t-tests; *P < 0.05, **P < 0.01, ***p < 0.001 compared to N groups (each experiment, n= 3). The error bars represent the standard error. N., Negative control; S., SAE; G., Gefitinib; L., LY294002.

Fig 3: Bakuchiol induced apoptosis in MCF-7 cells. MCF-7 cells were treated with 0–10 µg/ml of bakuchiol for 24 h. (A) Morphology observation of bakuchiol treated MCF-7 under bright field. Small apoptotic bodies are labeled with red arrows. The white bar indicates 100 µm. (B) Representative flow cytometry results of annexin V/PI analysis. (C) JC-1 staining for analysis of mitochondrial membrane potential. Western blot analysis of expression levels of (D) Caspase family protein, including Cleaved Caspase 7, Cleaved Caspase 9, and Cleaved PARP, and (E) Bcl-2 family protein, including Bak, Bax, and Bim. ß-Actin was used as loading control.

Fig 4: Bakuchiol induced alteration in cell cycle and cell cycle regulator expression in breast cancer cells. (A,B) Cells were treated with varying concentrations of bakuchiol as labeled in the figure for 24 h. (A) Cell cycle analysis with PI staining. (B) Expression levels of P-Cdc2 (Tyr15), Cyclin B1, P-Wee1 (Ser 642), Myt1, p21 (Waf1/Cip1) and β-Actin analyzed with Western blot (C) p21, p53, ATM and ATR mRNA expressions in cells treated with ethanol or 7 μg/ml of bakuchiol for 24 h. The data represent the mean ± SD of the values from three separate RNA samples (∗p < 0.05, student’s t-test). Ctrl, control; BAK, bakuchol.

Fig 5: Mcl-1 is downregulated in IDH1-mutated gliomas. a Protein was extracted from human tumor samples of anaplastic astrocytoma expressing IDH1-WT or IDH1-R132H. Western blot analysis was performed for Mcl-1, Bcl-xL, Bcl-2, Noxa, and Bim. Actin served as loading control. b Scatter plot demonstrating Mcl-1 protein expression among IDH1-WT (n = 5) and IDH1-R132H (n = 6) expressing anaplastic astrocytoma samples. Densitometric analysis was performed using imageJ 1.47 v (http://imagej.nih.gov/ij). Pixel density was normalized to the respective Actin control. c Whole-cell extracts of U87MG IDH1-WT/IDH1-R132H cells were collected to determine base line protein expression of Mcl-1, Noxa, Bcl-2, Bcl-xL, and BIM by western blot analysis. Actin was used to ensure equal loading. d, e U87MG glioblastoma cells (d) and GBM12 patient-derived glioblastoma xenograft cells (e) were treated for 3 h (U87MG) or 24 h (GBM12) with increasing concentrations of 2-HG. Whole-cell extracts were collected prior to performing western blot analysis for Mcl-1, Bcl-2, and Bcl-xL. Western blot for Actin or 14-3-3 was performed to confirm equal loading. f, g U87MG (f) and LN229 (g) cells were treated with non-targeting (n.t.)-siRNA or 4 different Mcl-1-siRNAs and subjected to treatment with solvent or ABT263, for 24 h. Staining for propidium iodide was performed prior to flowcytometry. The fraction of subG1 cells was determined. Column: mean. Error bar: SD, n = 3. Western blot analysis was performed to confirm Mcl-1 knockdown. h, i Quantitative representation of LN229 cells treated with non-targeting (n.t.)-siRNA, Noxa-siRNA (h) or Bak-siRNA (i) prior to treatment with ABT263/2-HG or solvent. Staining for propidium iodide and flowcytometry were performed to determine the fraction of subG1 cells. Column: mean. Error bar: SD, n = 3. Whole-cell extracts were collected and western blot analysis was performed for Noxa and Bak to confirm appropriate knockdown