Fig 2: PTH activates the PERK-CHOP ER stress pathway as detected by Western blot analysis. (A) HASMCs were treated with 1 × 10-8–1 × 10-6 mol/L PTH for three days. (B) Data are the mean ± SD of three independent experiments. (C) HASMCs were treated with 1 × 10-6 mol/L PTH for 0–14 days. (D) Data are the mean ± SD of three independent experiments. HASMCs cultured in normal medium for the same days as control. *p < .05 versus control.

Fig 3: A PERK inhibitor could inhibit the cell survival of BRAFi–resistant melanoma with impaired PTEN.WM88 (a) and A375sm (b) cells with wild-type PTEN or WLH6215 (c) and Sk-mel28 (d) cells with impaired PTEN and their PLX4032-resistant counterparts were treated with different concentrations of PLX4032 BRAF inhibitor (BRAFi) and GSK2606414 PERK inhibitor (PERKi). The cell viability curves of parental and resistant human melanoma cells were determined using CCK8 assays. Error bars denote s.d. for biological three repeats.

Fig 4: PERK was upregulated in BRAFi-resistant human melanoma cells with impaired PTEN.a The western blotting showed the protein level of PTEN, pAKT, and AKT in BRAFV600E human melanoma cell lines with various PTEN genetic backgrounds. The protein level of ß-actin is the loading control. b The values of 50%-inhibitory concentration (IC50) of 4 pairs of parental and resistant human melanoma cells were determined using CCK8 assays. Error bars denote s.d. for biological three repeats. Results are statistically significant between parental and resistant groups by Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001). c The cell viability curves of 4 pairs of parental (WM88, A375sm, Sk-mel28, and WLH6215) and resistant (WM88R, A375smR, Sk-mel28R, and WLH6215R) human melanoma cells were determined using CCK8 assays. Error bars denote s.d. for biological three repeats. d The western blotting showed the indicated protein level in BRAF inhibitor-resistant melanoma cell lines with various PTEN genetic backgrounds, the protein level of tubulin as the loading control.

Fig 5: Inhibition of PERK by a pharmacological inhibitor GSK2606414 blocked tumor growth in vivo.a The growth curves of the resistant Sk-mel28R tumor cells treated with mock (Sk-mel28R Mock, n = 8), BRAF inhibitor PLX4032 (Sk-mel28R BRAFi, n = 8), PERK inhibitor GSK2606414 (Sk-mel28R PERKi, n = 8), or a combination of BRAF inhibitor PLX4032 with PERK inhibitor GSK2606414 (Sk-mel28R BRAFi + PREKi, n = 8). b The growth curves of WLH6215R tumor cells treated with mock (WLH6215R Mock, n = 8), BRAF inhibitor PLX4032 (WLH6215R BRAFi, n = 8), PERK inhibitor GSK2606414 (WLH6215R PERKi, n = 9), or combination of BRAF inhibitor PLX4032 with PERK inhibitor GSK2606414 (WLH6215R BRAFi + PREKi, n = 8). Each point represents mean tumor volume ± s.e.m. ND, no statistical difference; *p < 0.05; **p < 0.01; ***p < 0.001. c, d The tumor weight (g) of resistant Sk-mel28R (c) and WLH6215R (d) tumor cells treated with mock (Mock), BRAF inhibitor (PLX 4032), PERK inhibitor (GSK2606414), or a combination of PERK inhibitor GSK2606414 with BRAF inhibitor PLX4032 (PLX4032 + GSK2606414). ND, no statistical difference; *p < 0.05; **p < 0.01; ***p < 0.001.