Fig 2: Lin28B enhances TET3 expression in pancreatic cancer cells. (A) Samples from PC patients were prepared for IHC staining with antibodies against Lin28B and TET3. (B) Analysis of the expression of Lin28B correlates with TET3. The results are presented by a heat map. (C) Relative expression protein and mRNA levels of Lin28B and TET3 were assessed in PANC1, SW1990 and PaTu8988 cells. (D) TET3 mRNA and protein levels were detected by real‐time PCR or Western blotting in PANC1 with Lin28B downregulation. (E) Overexpression of Lin28B in PaTu8988 promotes the expression of TET3. All experiments were repeated three times. Each error bar represents the standard error of the mean. Statistical analysis was performed using the unpaired Student’s t‐test.

Fig 3: TET3 can rescue the effect of Lin28B knockdown in pancreatic cancer cells. (A) Expression of the plasmid sh‐Lin28B was analyzed in PANC1 and SW1990 cells, and sh‐EGFP was used to track the expression of sh‐Lin28B. (B–E) PANC1 and SW1990 cells were co‐transfected Flag‐vector and sh‐EGFP, Flag‐TET3 and sh‐EGFP, sh‐Lin28B and Flag‐vector, or Flag‐TET3 and sh‐Lin28B. (B) Cell proliferation, (C) colony formation capacity, (D) invasive ability and (E) the stem cell markers NANOG, OCT4 and SOX2 were analyzed in the two transfected cells by CCK‐8 assay, the colony formation assay, Transwell assay and Western blotting analysis, respectively. All experiments were repeated three times. Each error bar represents the standard error of the mean. Statistical analysis was calculated using Student’s t‐test or one‐way ANOVA test.

Fig 4: Scheme of PKCβ driven nuclear localization of Lin28B connected to a TET3‐mediated positive feedback loop. KRAS promotes nuclear localization of Lin28B by PKCβ, which directly phosphorylates Lin28B. Nuclear Lin28B indirectly elevates TET3 expression by repressing the bioactivity of let‐7i, and increased TET3 can feed back to promote Lin28B expression, which maintains the stemness of PC cells.

Fig 5: Lin28B maintains the stemness of pancreatic cancer cells via regulation of TET3 expression. (A–E) PaTu8988 and SW1990 cells were co‐transfected Flag‐vector and sh‐EGFP, Flag‐Lin28B and sh‐EGFP, sh‐TET3 and Flag‐vector, or Flag‐Lin28B and sh‐TET3. Relative expression of (A) mRNA levels of Lin28B and TET3, (B) cell proliferation, (C) colony formation capacity, (D) invasive ability and (E) the stem cell markers NANOG, OCT4 and SOX2 were analyzed in the two transfected cells by real‐time PCR, the CCK‐8 assay, the colony formation assay, Transwell assay, or Western blotting analysis, respectively. All experiments were repeated three times. Each error bar represents the standard error of the mean. Statistical analysis was calculated using Student’s t‐test or one‐way ANOVA test.