Fig. 6: aPKCs support tumor growth and are required for macropinocytosis in vivo.

a Growth curves for AsPC-1-derived heterotopic xenograft tumors expressing the indicated Doxycycline (Dox)-inducible shRNAs. Dox was provided in the drinking water and diet. A total of n = 8 tumors were measured per condition. ^***P(_{shCTRL vs shPRKCZ})< 0.0001; ^***P(_{shCTRL vs shPRKCI})< 0.0001. b Representative photos of the xenograft tumors at termination. c Measurement of tumor weights at termination for n = 8 tumors per condition. ^***P(_{shCTRL vs shPRKCZ})<0.0001; ^***P(_{shCTRL vs shPRKCI}) = 0.0003. d Immunoblots assessing PKCζ and PKCι protein levels in the tumors at termination. n = 5 and n = 4 knockdown tumors for PKCζ and PKCι, respectively, were analyzed relative to two different control tumors. e Representative images of p-HistoneH3 staining in two different tumors per condition. Scale bar, 100 μm. f Quantification of p-HistoneH3 staining in tumor sections of shCTRL (n = 44 fields), shPRKCZ (n = 46 fields) and shPRKCI (n = 43 fields). Fields were quantified from n = 3 tumors for each condition. ^***P(_{shCTRL vs shPRKCZ})< 0.0001; ^***P(_{shCTRL vs shPRKCI})< 0.0001. g Representative images of macropinocytosis in tumors expressing the indicated shRNAs, with macropinosomes labeled with TMR-dextran (red) and tumor cells labeled with CK8 (green). Nuclei are stained with DAPI (blue). Scale bar, 20 µm. h Macropinocytosis quantification in n = 6 tumors (shCTRL and shPRKCZ) and n = 5 tumors (shPRKCI). Data are shown relative to shCTRL. ^***P(_{shCTRL vs shPRKCZ})<0.0001; ^***P(_{shCTRL vs shPRKCI})< 0.0001. i Immunofluorescent staining of p-PKCζT560 or total PKCζ (purple) in control tumors, with CK8 (green) delineating peripheral and non-peripheral regions. Pictures are representative of n = 4 tumors from the control group. Scale bar,100 µm. j Quantification of peripheral and non-peripheral staining for p-PKCζT560 (n = 57 fields/periphery, n = 62 fields/non-periphery) and total PKCζ (n = 50 fields/periphery, n = 51 fields/non-periphery) in control tumors. p-PKCζ^T560: ^***P(_{non-periphery vs periphery})<0.0001); PKCζ: ^nsP(_{non-periphery vs periphery}) = 0.1385. k Representative co-staining images of p-PKCζT560 and total PKCζ (purple) with macropinosomes (red) in peripheral and non-peripheral regions. Nuclei labeled with DAPI (blue). Scale bar, 20 µm Statistical significance was calculated using two-way ANOVA with Tukey’s multiple comparison test for (a) at day 47. For (c, f, h, and j), statistical significance was calculated using an unpaired two-tailed Student’s t test. ns, non-significant, *P < 0.05, **P < 0.01, ***P < 0.001. All data are presented as the mean ± SEM. Source data are provided as a Source Data file.