Supplementary Figures 1 - 7 from Carfilzomib and ONX 0912 Inhibit Cell Survival and Tumor Growth of Head and Neck Cancer and Their Activities Are Enhanced by Suppression of Mcl-1 or Autophagy

PDF file, 1411K, Supplemental Figure 1. Determination of IC50 values for carfilzomib and ONX 0912 in HNSCC cell lines. UMSCC-22A, UMSCC-22B, 1483, UMSCC-1, Cal33, PCI-15A, PCI-15B, and OSC-19 cells were treated in triplicate wells for 48 hours with varying concentrations of carfilzomib (A) or ONX 0912 (B). Following treatment, floating and attached cells were collected and combined, and cell viabilities were determined by trypan blue exclusion assays. Each data point represents the average of values obtained in 3 independent experiments. Error bars represent the standard error of the means. IC50 values (nmol/L) were determined using GraphPad PRISM sofeware (GraphPad Software, Inc.) Supplemental Figure 2. Inhibition of caspase-3 partially attenuates carfilzomib- and ONX 0912-induced apoptosis of HNSCC cell lines. UMSCC-22A (A) and 1483 (B) cells were pre-incubated for 2 hours with 0.1% DMSO (vehicle), or 20 �mol/L z-DEVD or z-VAD, followed by treatment for 36 hours with 100 nmol/L carfilzomib or ONX 0912. Following treatment, cells were collected and analyzed by flow cytometry for Annexin V/PI staining. Numbers indicate the percentage of Annexin V-positive cells. Supplemental Figure 3. Carfilzomib- and ONX 0912-induced apoptosis is mediated by Bik upregulation, and antagonized by Mcl-1 upregulation. 1483 (A) and UMSCC-1 (B) cells were transfected with nonspecific siRNA, Bik siRNA, or Mcl-1 siRNA for 6 hours. Following transfection, 1483 cells were treated for 36 hours with 100 nmol/L carfilzomib or ONX 0912 and UMSCC-1 cells were treated for 24 hours with 100 nmol/L carfilzomib or for 36 hours with 100 nmol/L ONX 0912. Flow cytometry was used to assess Annexin V/PI staining. Numbers represent the percentage of Annexin V-positive cells. Supplemental Figure 4. Carfilzomib and ONX 0912 induction of LC3-II is partially dependent on upregulation of ATF4. 1483 (A) and UMSCC-1 (B) cells transfected with nonspecific siRNA or ATF4 siRNA were treated for 24 hours with 0.1% DMSO, or 100 nmol/L carfilzomib or ONX 0912, then subjected to immunoblotting for ATF4, LC3-II, or β-actin. Supplemental Figure 5. Impact of autophagy inhibition on ONX 0912-induced HNSCC cell death. UMSCC-22A, UMSCC-22B, 1483 and UMSCC-1 cells were treated with the indicated concentrations of ONX 0912 in the absence or presence of 10 �mol/L chloroquine. After 48 hours treatment, cell viabilities were determined by trypan blue exclusion assays. Data represent the mean + SEM from 3 independent experiments. Supplemental Figure 6. Suppression of ATF4 expression enhances apoptosis in carfilzomib- and ONX 0912-treated HNSCC cells. UMSCC-22A (A) or 1483 (B) cells transfected with nonspecific siRNA or ATF4 siRNA were treated for 24 hours with 100 nmol/L carfilzomib or ONX 0912. Following treatment, Annexin V/PI staining was assessed by flow cytometry. Numbers represent the percentage of Annexin V-positive cells. Supplemental Figure 7. Carfilzomib inhibits HNSCC xenograft tumor growth in vivo. HNSCC tumor-bearing mice were randomized into 3 groups on day 0. Treatment with vehicle (1% carboxymethyl cellulose) or carfilzomib (3 or 5 mg/kg) was initiated on day 0. Mice were treated once per day for two consecutive days and the treatment repeated weekly for 2 weeks (arrows represent treatment days). Mean tumor volumes + SEM are presented.