Fig 2: Overexpression of HNF1A promotes CSC properties in PDA cells and normal pancreatic cell lines.(A) NY15 and NY53 cells Western blotted for HNF1A and control gene induction following 48 hr ± doxycycline (Dox). Quantitation of proteins is indicated below the respective blots. (B) NY8 cells were treated 48 hr ± Dox to induce ectopic HNF1A. Lysates were western blotted for HNF1A, Actin, and PCSC markers EPCAM and CD44. Quantitation of proteins is indicated below the respective blots. (C) Representative surface expression of CD24 and EPCAM on NY15 cells expressing GFP or HNF1A. (D) Quantitation of CD24 +NY15 GFP and HNF1A cells and NY8 and NY53 LacZ and HNF1A cells by flow cytometry (n = 3). Statistical difference was determined by one-way ANOVA with Tukey’s multiple comparisons test. (E) NY15 GFP and HNF1A, NY8 and NY53 LacZ and HNF1A cells were grown under sphere-forming conditions ± Dox. The number of tumorspheres formed after 7 days were quantitated (n = 4). Statistical difference was determined by one-way ANOVA with Tukey’s multiple comparisons test. Representative images (100X magnification) of spheres are shown in the upper panels. (F, G) HPNE LacZ and HNF1A cells were plated at 200 cells/6 cm dish and treated ±Dox for 2 weeks, fixed, and stained with crystal violet (F). (G) Resultant colonies were quantitated (n = 3). Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. (H, I) HPDE cells expressing inducible LacZ, LacZ with KRASG12D, HNF1A, or HNF1A with KRASG12D were embedded in soft agar + Dox and monitored for signs of anchorage-independent growth for 21 days. (H) Representative images of resultant colonies (100X magnification) and (I) quantitation of colonies after 21 days (n = 3). Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. Related data can be found in Figure 4—figure supplement 1.10.7554/eLife.33947.017Figure 4—source data 1.Quantitation of CD24 expression and tumorsphere formation in PDA cells with HNF1A overexpression, and quantitation of colony formation in HPNE and HPDE cells expressing HNF1A and oncogenic KRAS.10.7554/eLife.33947.018Figure 4—source data 2.Quantitation of CD44+/CD24+ HPDE and HPNE cells overexpressing HNF1A.

Fig 3: Knockdown of HNF1A depletes CSC numbers.(A) Multiple PDA cells were transfected with HNF1A-targeting siRNA or non-targeting control siRNA for 6 days. Surface expression of CD44 and EPCAM was measured by flow cytometry, and the percentage of CD44High/EPCAMHigh (P2) cells are represented (mean ± SEM, n = 3). Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. (B) Quantitation of CD24 +cells in multiple primary PDA cells following HNF1A knockdown for 6 days, n = 4. Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. (C) NY5, NY8, and NY15 cells expressing LacZ2.1 (L) or two distinct HNF1A-targeting shRNAs (H1 and H2) were lysed and western blotted for HNF1A, CDH17, and Actin, showing effective knockdown of HNF1A and downstream signaling (CDH17). Quantitation of proteins is indicated below the respective blots. (D, E) NY5, NY8, and NY15 cells expressing LacZ2.1 or HNF1A-targeting shRNAs were grown in tumorsphere media on non-adherent plates (1500 cells/well). The number of tumorspheres formed after 6 days were counted (n = 3). Representative images of spheres (100X magnification) are shown in (F) and in Figure 3—figure supplement 1, with quantitation in (E). Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. Related data can be found in Figure 3—figure supplement 1.10.7554/eLife.33947.014Figure 3—source data 1.Quantitation of the P2 subpopulation, CD24 expression, and tumorsphere formation following HNF1A knockdown.

Fig 4: PIASγ translocates HNF-1A to the nuclear periphery and leads to reduced expression of known HNF-1A target genes. (a,b) After RIPA lysis, pelleted fractions from HEK293 cells transiently transfected with V5-tagged HNF-1A in the absence/presence of PIASγ were analyzed by SDS-PAGE and immunoblotting using indicated antibodies. The level of HNF-1A was quantified by densiometric analysis using the Image Lab v.6.0.0 software (BIO-RAD) and normalized to α-tubulin. Full-length blots are presented in Supplementary Fig. S11. Quantifications are presented relative to the level of HNF-1A alone. The results are shown as mean of three technical experiments (n = 1) ±SD. P indicates parallel. (c) HEK293 cells were transiently transfected with constructs expressing V5-tagged HNF-1A and/or Flag-PIASγ alone or in combination as indicated in the figure. HNF-1A (green) and PIASγ (magenta) were visualized by immunostaining for V5- and Flag-tag, respectively, and analyzed by confocal microscopy. Cell nuclei were stained with DAPI (blue). Co-localization of transfected HNF-1A and PIASγ is shown in the lower panel. Scalebar, 10 µm. (d) The mRNA expression of three known HNF1A targets genes (Ace2, Slc2a2 and Crp) was investigated in MIN6 cells overexpressed with HNF1A in the absence or presence of PIASy and was determined by qPCR. Target gene expression was normalized to reference gene Gapdh and represented as mean fold change relative to HNF1A alone ±SD of two biological replicates (n = 2).

Fig 5: PIASγ interacts with HNF-1A. (a) Co-immunoprecipitation analysis of HNF-1A and PIASγ from HEK293 cells transiently transfected with V5-tagged HNF-1A or empty vector together with Flag-tagged PIASγ. Cells were lysed and subjected to immunoprecipitation using anti-V5 antibody. The precipitates (left) and 10 µg input (right) were separated by SDS-PAGE and immunoblotting using anti-Flag and anti-V5 antibodies. This experiment was replicated in two independent experimental days (n = 2). (b) Mutation of K205, K273 and K506 in HNF-1A does not affect the binding of PIASγ to HNF-1A. HEK293 cells were transiently transfected with WT HNF1A, K205R, K273R or K506R, together with Flag-tagged PIASγ. Lysates were analyzed similarly as described in (a) using anti-V5 antibody immunoprecipitation, SDS-PAGE analysis and immunoblotting. Full-length blots are presented in Supplementary Fig. S8. This experiment was replicated in two independent experimental days (n = 2).