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. Author manuscript; available in PMC: 2023 Oct 23.
Published in final edited form as: Cell Rep. 2023 Aug 30;42(9):113042. doi: 10.1016/j.celrep.2023.113042

Figure 2. DOCK8 is ectopically expressed in a subset of pancreatic cancer tumors and is required for cancer cell invasion in vivo and in vitro.

Figure 2.

(A–D) Relative DOCK8 mRNA expression in (A) normal, healthy pancreas (GTEx) and pancreatic cancer tumors (TCGA), patient-derived organoids (PDOs) generated at (B) the Mayo Clinic or (C) Crown Bioscience, and (D) pancreatic cancer cell lines (Cancer Cell Line Encyclopedia).

(E) Immunoblotting of DOCK8 in pancreatic cancer cell lines. RAW 264.7 macrophages: positive control; non-cancerous HPDE (human pancreatic ductal epithelial) cells: negative control.

(F) Immunoblotting of DOCK8 in three independent control or DOCK8 CRISPR knockout mKPC cell clones. GAPDH: loading control.

(G) Control and DOCK8 knockout average tumor weight (mg) from syngeneic orthotopic tumors in male (blue squares) and female (red triangles) mice.

(H) Representative images of control or DOCK8 knockout metastases and H&E staining in male mice. Scale bar, 50 μm.

(I and J) Mean number of metastatic tumors at indicated sites in (I) male and (J) female mice.

(K) Total number of metastases per mouse, male (blue squares) and female (red triangles).

(G, I, J, and K) Graphs: mean ± SEM of n = 3 male and 3 female mice for control clone 1 and DOCK8 knockout (KO) clone 2, n = 5 each control clone 3, and n = 2 for DOCK8 KO clone 1.

(L) Lysosome activity is required for tumor cell invasion. Quantitation of Transwell invasion assay of 6741-P and mKPC cells treated with DMSO or bafilomycin A1 (1 μM).

(M–R) Mean ± SEM of three biological replicates. Immunoblotting of DOCK8 and quantitation of Transwell invasion following DOCK8 knockdown in (M) iKRAS, (N) Panc04.03, (O) mKPC, (P) 6741-P, (Q) Capan-1, and (R) mKPC control and DOCK8 KO clones 1–3.

(S) Immunoblotting of Panc1 cells stably expressing FLAG, FLAG-DOCK8, or FLAG-DOCK8-V1985A, and quantitation of Transwell cell invasion.

(L–S) NTsi, Nontargeting siRNA. Mean ± SEM from at least three independent biological replicates. p values were calculated using a Student’s t test or one-way ANOVA with the Tukey’s post hoc test (G, K, and R). *p < 0.05; **p < 0.01; ***p < 0.001; ****p ≤ 0.0001.

See also Figures S2–S4.