Fig. 2. Oncogenic KRAS inactivation differentially impacts the key nodes of the proteostasis regulatory network.

(A) Immunoblot with indicated antibodies in whole-cell lysates of iKras^P at different time points after Kras^G12D inactivation by Dox-withdrawal (Off Dox) until the cells acquired resistance to Kras^G12D inactivation (iKras^R cell). (B and C) Immunoblot with indicated antibodies in whole-cell lysates of parental or sotorasib-resistant MIA-PaCa-2 (B) or H358 (C) cells treated with DMSO or 30 nM sotorasib. (D) Immunohistochemical (IHC) staining with indicated antibodies in iKras GEMM tumors treated with doxycycline (On Dox), Dox withdrawal for 3 days (Off Dox), or relapsed after 30 weeks of Dox-withdrawal (Off Dox, relapsed). (E) IHC staining with indicated antibodies in MIA-PaCa-2 xenograft tumors treated with vehicle, sotorasib (30mg/kg for 1 day), or relapsed after 9 weeks of sotorasib treatment (30mg/kg). (F) Relative HSE luciferase activity in iKras^P or iKras^R cells cultured in the presence or absence of Dox for 2 days. Data are shown as mean ± SD, n=3. (G and H) Representative images (G) and quantification (H) of PROTEOSTAT (magenta) and DAPI (blue) staining in iKras^P or iKras^R cells infected with lentiviruses encoding scramble shRNA (shScr), Xbp1 shRNA (shXbp1) or Ire1 α shRNA (shIre1α). Cells were treated with 2.5mM TUDCA dissolved in water for 2 days as indicated. Data represent the average fluorescence intensity of PROTEOSTAT/cell from each image acquired and presented as mean ± SD from n=10 (On Dox), n=17 (Off Dox), or n=17 (Off Dox + TUDCA) images. (I) CCK-8 assay was used to quantify cell viability of iKras cells treated as in G and H. Data are presented as mean ± SD relative to shScr, n=3. Ordinary one-way ANOVA with Dunnett’s multiple comparisons test (H and I) or ordinary one-way ANOVA with Tukey’s multiple comparisons test (F) was used to calculate P values. n.s., not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar: 40μm (D and E) or 20μm (G).