Fig. 2: Cell death following proteasome inhibition occurs in the absence of RIPK1 and NFκB-dependent cytokine production.

A, B WT, Casp8-KO, and MLKL-KO LLC-OVA cells were treated for 8 h with DOX (1 μg/mL) followed by treatment with MG132 (4 μM). Three hours after treatment with MG132, RNA was prepped using tumor cell lysates. Tumor cytokine expression was measured via Nanostring using the mouse tumor 360 signaling panel. A Cytokine production in tumor cells with DOX + MG132. B Tumor cytokine production in WT cells with DMSO, MG132, DOX + DMSO, or DOX + MG132. For A, B, data is from a single experiment. The red solid line indicates the mean read count for the negative controls and the dashed black line indicates the mean read count for the lowest positive control. C, D WT cells were treated in a similar fashion to (A, B). After 4.5 h of treatment with MG132, RNA was prepared from tumor cell lysates for qPCR. C Gene expression for Ccl2 and Cxcl1 following treatments as indicated. Each point represents an average of technical replicates from an individual experiment. Treatment groups were compared using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. D WT cells were treated with DOX (1 μg/mL) 8 h prior to treatment with MG132 (4 μM). Cell lysates were collected immediately, 30 mins, 1 hour, 2 hours, and 6 h after treatment with MG132 for western blot. Data is representative of two independent experiments. E, F RIPK1 knockout in WT cells was performed using the CRISPR/Cas9 system. E RIPK1-KO and WT cells were treated with DOX (1 μg/mL) for 7 h, cells were pre-treated with zVAD-fmk (20 μM) 30 min prior to GSK’843 (20 μM) then MG132 (4 μM) after another 30 min. Cell death was measured using Incucyte. Data is representative of two independent experiments. F RIPK1-KO and WT cells were treated with MG132 (4 μM) following an 8-h induction with DOX (1 μg/mL). Lysates were collected 4 h following treatment with MG132 for western blot. Data is a from a single experiment.