Figure 2.

BHLHE40 promotes PCa proliferation and liver metastases in vitro and in vivo. A) Western blot of BHLHE40 knockdown efficiency in PANC‐1 cell lines. B,C) Effects of BHLHE40 knockdown on the proliferation and migration ability of the PANC‐1 cell lines were measured using CCK‐8 and wound‐healing assays. D) PCa, PANC‐1, and BxPC‐3 (gross anatomy also shown in Figure S3, Supporting Information) cells stably transfected with BHLHE40 sh ctrl or sh #1 were injected subcutaneously into 4 week old BALB/c male nude mice (n = 5 for each group). Representative images are of dissected xenogeneic tumors from nude mice in each group. E) Tumor volumes were measured and calculated after subcutaneous injection. F) Tumor tissues were subjected to Ki‐67 staining. Scale bar, 100 µm. G) Representative micro‐PET/CT image of the metastatic xenograft mouse model. H) BHLHE40 knockdown decreased the mean SUV value of metastatic liver tumors, reinforcing the role of BHLHE40 in promoting metastasis in vivo. I) Western blot of BHLHE40 overexpression efficiency in MIA PaCa‐2 cell lines. J,K) Effects of BHLHE40 overexpression on the proliferation ability of the MIA PaCa‐2 cell line was measured using the CCK‐8 and colony formation assays. L–N) Representative pictures of two PCa organoids (PTO1 and PTO2) transfected with BHLHE40 knockdown vectors or control lentivirus for 10 d (scale bar, 200 µm, left panel) and quantified via organoid diameters (right panel). PET, positron emission tomography; mean SUV, mean standardized uptake value; "B sh" or “BHLHE40 sh,” short for BHLHE40 short hairpin RNA. Data presentation: B,E,J,M,N) data were the mean ± s.d. of n = 6 independent experiments; E) data were the mean ± s.d. of n = 5 independent experiments; H) data were the mean ± s.d. of n = 4 independent experiments; C,K) Data were the mean ± s.d. of n = 3 independent experiments. Statistical analysis: two‐way ANOVA for (B,E,J); unpaired two‐sided t‐test for (C,H,K,M,N). * P < 0.05, ** P < 0.01, *** P < 0.001.