Perk 5683

Cells were scraped in radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher) containing 2 mmol/L Na₃VO₄ and protease inhibitors. Supernatants were quantified by BCA assay (Pierce), separated by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane (Thermo Fisher), and blocked in Tris-buffered saline (TBS) with 5% non-fat dry milk and 0.1% Tween 20. Membranes were probed with the antibodies: pSer51 eIF2a #3398, PERK #5683, GCN2 #3202, PKR #12297, and GAPDH #2118 from Cell Signaling Technology (MA, USA); reovirus μ1C 10F6 and reovirus σ3 4F2 were from the Developmental Studies Hybridoma Bank (IA, USA); and ATF4 ab184909 and Beta Actin ab8226 were from Abcam (Cambridge, UK).

Each of the primary antibodies were used according to the manufacturer’s protocol: Phospho-JNK #4671, JNK #9252, IRE1α #3294, PERK #5683, phospho-p38 MAPK #9215, p38 MAPK #9212, phospho-p44/42 MAPK #9106, p44/42 MAPK #4695, (Cell Signaling, Danvers, MA). phospho- IRE1α #PA1-16927, phospho-PERK #MA5-15033, and HCV core (Thermo Scientific, Rockford, IL). ATF6 #ab11909 (Abcam, Cambridge, MA).

Spheroids were rinsed in PBS before fixation in 10% neutral-buffered formalin (NBF). Spheroids were embedded in HistoGel before paraffin embedding and sectioning. In vivo tumors were fixed in NBF before paraffin embedding and sectioning. Heat-induced antigen retrieval was performed using pH 6 sodium citrate buffer. Tissue was blocked using 5% BSA before incubation with the following antibodies: PERK #5683, GRP78 #3177, and PDI #3501 were from Cell Signaling Technologies (MA, USA); KDEL 10C3 was from Enzo (Exeter, UK); and reovirus μ1C 10F6 and reovirus σ3 4F2 were from the Developmental Studies Hybridoma Bank (IA, USA). Alexa Fluor 488, 546, and 647 conjugates of anti-rabbit IgG H+L, anti-mouse IgG H+L, anti-mouse IgG2a, and anti-mouse IgG2b were from Thermo Fisher (UK). Spheroid sections were imaged on a Zeiss 710 confocal (Jena, Germany) with quantification using Cell Profiler v.3. Exposure settings were based on the minimum possible, which still allowed robust spheroid identification for segmentation. Controls were compared during exposure setup to ensure similar intensities across experiments. Stained sections from in vivo tumor samples were imaged on a Perkin Elmer Vectra 3.0 with spectral unmixing of images performed using Perkin Elmer Inform software. Image quantification was performed using Cell Profiler v.3 as described in the Results.