Figure 2: PERK ablation sensitizes melanoma cells to ER stress-induced paraptosis.

(A) Annexin V⁺ mean percentage ± SEM in Scramble and PERK^KO B16 (left) or SM1 (right) tumors cultured for 24 hours with or without Thaps. n=3-7 of 3 independent repeats.

(B) Representative histograms from 3 distinct repeats showing ER Tracker Green in Scramble and PERK^KO B16 cells treated as in (A).

(C) Mean fluorescence intensity (MFI) via flow cytometry ± SEM of NIAD4-bound protein aggregates in Scramble and PERK^KO B16 cells treated as in (A). n=4-7/group of 3 independent repeats.

(D) Representative histograms from 3 repeats for fluorescent click chemistry-based detection of O-propargyl-puromycin (OPP) incorporation in Scramble and PERK^KO B16 cells treated as in (A).

(E) Light microscopy morphology (100μm and 20X digital magnification) of Scramble and PERK^KO B16 cells cultured in the presence of vehicle, Thaps, or Tunicamycin; in the absence of Glucose or Serum; or with 25% TES.

(F) SEC61β (MFI ± SEM) in eGFP⁺ Scramble and PERK^KO B16 tumors from mice. n=6/group.

(G) Annexin V⁺ percentage ± SEM in Scramble and PERK^KO B16 cells transfected with untargeted control or Sec61b siRNA and treated as in (A). n=3/group.

(H) Light microscopy morphology in PERK^KO B16 cells transfected with mock or Sec61b siRNA prior to Thaps exposure.

(I) Percentage ± SEM of DiOC₂ low cells in B16 cells transfected with untargeted or Atf4 siRNA and treated as in (A). n=4-5/group.

(J) ATP in supernatants from Scramble or PERK^KO B16 cells incubated or not with Thaps. Data are the normalized relative light units (nRLU) ± SEM of n=12-15/group.

(K) ExoCRT fold change (MFI ± SEM) in Scramble or PERK^KO B16 cells cultured as in (A). n=4-7/group.

(L-M) Extracellular ATP (L) and ExoCRT (M) ± SEM in Scramble or PERK^KO B16 cells carrying mock or Sec61b siRNA and cultured with or without Thaps. n=6-10/group (L); n=3-4/group (M).

Statistics applied using one-way ANOVA (A, C, F, G, I – M) or Student’s t-test (F), *, p<0.05; **, p<0.01; ****, p < 0.0001. Please also see Figure S2.