Fig 2: PRLR level in PDAC tissue correlates with clinicopathological findings. (A-D) Relative PRLR mRNA level of paired NP and PDAC tissues in GSE15471 (A), GSE16515 (B), GSE28735 (C) and GSE102238 (D). P values were determined by paired two-tailed t-test. (E) Kaplan-Meier survival analysis for all PDAC patients divided into low and high PRLR mRNA level group based on TCGA data. P value was calculated with log-rank (Mantel-Cox) test. (F, G) Kaplan-Meier survival analysis for male PDAC patients (F) or female PDAC patients (G) divided into low and high PRLR mRNA level group based on TCGA data. P value was calculated with log-rank (Mantel-Cox) test. (H) Representative immunohistochemical staining images for showing lower (left) and higher (right) PRLR expression levels using patient-derived PDAC tissues. Scale bar: 100µm. (I) Correlation analyses for PRLR expression with TNM stage of patients with PDAC from Ren Ji cohort. P value was determined by ?2 test. (J) Relative PRLR mRNA level for T1+T2 stages and T3+T4 stages of patients based on TCGA data. P values were determined by unpaired two-tailed t-test. (K) Ratio of Prlr-SF: Prlr-LF in pancreas of non-pregnant mice (Ctrl), pregnant mice (pregnancy), and lactating mice (lactation), based on 3 samples from each group and 3 replicates of each sample. P values were determined using the unpaired 2-tailed test. (L, M) Ratios of PRLR-SF: PRLR-LF in subcutaneous xenografts grown from Panc02 cells in non-pregnant (Ctrl) and pregnant mice (L) and in non-lactating (Ctrl) vs lactating mice (M). Five samples from each group and 3 replicates were analyzed for each sample. P values were determined by unpaired 2-tailed test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: PRLR activates Hippo pathway via interaction with NEK9. (A) Linear regression analysis for PRLR and NEK9 mRNA level of PDAC patients based on TCGA database. n = 182. Pearson correlation coefficients (R) are shown. (B, C) Relative NEK9 mRNA level of paired non-PDAC ('NP') and PDAC tissues in GSE15471 (B) and GSE16515 (C). P values were determined by paired t test. (D) Kaplan-Meier survival analysis for PDAC patients divided into low or high NEK9 mRNA level group based on TCGA. P value was calculated with log-rank (Mantel-Cox) test. (E) Immunoblot analyses for co-immunoprecipitation of NEK9 and PRLR using AsPC-1 cells co-transfected with HA-tagged NEK9 and Flag-tagged PRLR. (F) Immunofluorescence for co-localization of PRLR (green) and NEK9 (red) in AsPC-1 cells (top) and BxPC-3 (bottom) cells. (G) Immunofluorescence for the interactions (red dots) of PRLR and NEK9 detected by in situ proximity ligation assays using AsPC-1 cells (top) and BxPC-3 cells (bottom). (H) Immunoblot analyses for the effects of interfering NEK9 on Hippo signaling pathway using AsPC-1 (left) and BxPC-3 (right) cells stimulated with PRL (0.5 µg/ml). Scale bar: 10 µm. *P < 0.05, ***P < 0.001.

Fig 4: hPRLr heterodimer versus homodimer differential stability.Chinese hamster ovary (CHO) cells (PRLr-null) were transiently-transfected with either hPRLrL, hPRLrI, or both isoforms concurrently (at a 1:1 ratio). a Transfectants were simultaneously stimulated with cycloheximide (CHX; 100 µg/mL) and prolactin (PRL; 250 ng/mL), and lysates were harvested once per hour for 5 consecutive hours. Protein degradation was quantified, via densitometry of band intensity, in b and t1/2 was extrapolated from each respective slope. c Phosphorylation status of the hPRLr phosphodegron was assessed using a pS349-specific antibody (gift of Dr. Serge Fuchs), and d band intensity was quantified via densitometry. *p < 0.05, **p < 0.01, ***p < 0.005, n = 3. Data is shown as mean ± SEM.

Fig 5: Hippo signaling pathway is activated by PRLR-SF in PDAC cells. (A, B) Relative levels of CYR61, CTGF, and ANKRD1 mRNAs in control (Ctrl) AsPC-1 cells (A) and BxPC-3 cells (B) vs those that overexpress PRLR-SF (n = 3). (C, D) Immunoblots showing levels and phosphorylation (p) of AKT, ERK1/2, and proteins in the Hippo signaling pathway in AsPC-1 cells and BxPC-3 cells incubated with 500 ng/mL prolactin (PRL) for 0-120 mins in serum-free media. (E, F) Representative immunofluorescence images of AsPC-1 cells (E) or BxPC-3 cells (F) infected with Lenti-control (Ctrl) or Lenti-PRLR-SF, and transfected transiently with empty vectors (vector) or vectors that overexpress Flag-YAPS127A. Flag labeled in green, nuclei labeled in blue, YAP labeled in red, and combined images below. Scale bar = 25 µm. Quantitative histograms show percentages of nuclear YAP in each cell type (n = 3). (G, H) Histograms of relative incorporation of 13C-labeled R-5-P, AXP, CXP, GXP, and UXP (X = M, D and T) (G) or ratio of NADP+ to NADPH (H) in AsPC-1 cells infected with Lenti- Ctrl or Lenti-PRLR-SF, transfected with empty vector vs vector expressing YAPS127A. (I, J) Histograms of relative incorporation of 13C-labeled R-5-P, AXP, CXP, GXP, and UXP (I) or ratio of NADP+ to NADPH (J) in AsPC-1 cells infected with Lenti-sh-Ctrl or Lenti-sh-PRLR, transfected with empty vector vs vector expressing YAP?PDZ. P values were determined by unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001.