Fig 2: Loss of GR up regulates Wnt/β-catenin pathway by enhancing autophagy flux. a Quantitative results indicate that the number of autophagolysosomes increased with GR knockdown, implying loss of GR enhanced autophagy flux (t-test). Rapamycin could increase the number of both autophagolysosomes and autophagosomes; however, after treatment with chloroquine, the number and the ratio of autophagolysosomes was smaller than that from cells treated with rapamycin, indicating that autophagy flux was blocked. (One-way ANOVA) (n = 5/group). b Representative western blots from MLECs treated with either HBSS (4 h), rapamycin (4 h), chloroquine (16 h) or Wnt3a (4 h). Rap: rapamycin; CQ: chloroquine. c Western blot densitometry of HBSS/CQ and Rap/CQ treatment conditions (n = 3/group). Both HBSS starvation and rapamycin treatment could upregulate Wnt/β-catenin pathway, while chloroquine suppressed the expression of Wnt-related components (One-way ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns not significant. siCT MLECs transfected with control siRNA, GRsi MLECs transfected with control siRNA

Fig 3: PVT1 promotes the Wnt/ß-catenin signaling pathway through enhancing Pygo2 expression. a and b The protein and mRNA expression levels of Pygo2 in PANC-1 and ASPC-1 cells transfected with PVT1 overexpression plasmid or PVT1 siRNA were analyzed by western blotting and real-time qRT-PCR, respectively. c The mRNA expression level of Pygo2 in PANC-1, SW1990 and the gemcitabine resistant PANC-1/Gem, SW1990/Gem cell lines was examined by real-time qPCR. d The mRNA and protein expression levels of Pygo2 in PANC-1 cells transfected with PVT1 siRNA with or without gemcitabine treatment were analyzed. e The expression levels of C-myc, CyclinD1 and Axin2 in PANC-1 cells transfected with PVT1 siRNA or HA-Pygo2 plasmid treated with or without gemcitabine were analyzed by real-time qRT-PCR. f The whole cell lysates of PANC-1 and ASPC-1 cells transfected with PVT1 overexpression plasmid or PVT1 siRNA combined with Pygo2 overexpression plasmid or Pygo2 siRNA transfection were analyzed by western blotting using the indicated antibodies. g and h The cytoplasmic and nuclear distributions of ß-catenin after PVT1 or Pygo2 siRNA transfection were assessed by immunofluorescence. The ratio of nuclear fluorescence intensity/total fluorescence intensity was quantified using GraphPad Prism 6.0 software. Scale bars: 50 µm. i and j TCF transcriptional activity was compared between PVT1 knockdown and Pygo2 overexpressing cells with or without Wnt3a (100 ng/ml). Cells were transiently transfected with TOP/FOP Flash reporter, and the luciferase activity was measured. k-n The protein and mRNA levels of MDR1 (P-gp) were measured in PANC-1 cells with PVT1 overexpression or knockdown with or without Pygo2/ß-catenin knockdown treated with gemcitabine (1 µM) or XAV-939 (10 µM). Data were represented as mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001

Fig 4: Membrane order preference of the canonical Wnt ligand and its receptors. (A) Western blot of detergent extracts of the plasma membrane for Fz8 (detected by its Flag tag), Lrp6, Lypd6 (detected by its GFP tag), Wnt3 (detected by its GFP tag), Wnt3a [from conditioned media (CM)], recombinant Wnt3a (Wnt3a rec), Wnt8a, and the controls caveolin1 (Cav1 with known detergent‐resistant fraction preference) and transferrin‐receptor (TfR2 with known detergent soluble fraction preference) with DRM and soluble positions marked. (B) A schematic of SPIM‐FCS measurements. A focal volume is formed with a single plane illumination. Signal is recorded from the apical membrane of the cells. Later, the signal is correlated with different bin sizes to form varying sizes of observation areas. With larger area, the diffusion time is higher while with a smaller area, the diffusion time is lower. The extent of this change depends on the diffusion mode of the molecule, whether it is free or hindered. Dependence of diffusion time to observation area is shown for different diffusion modes; domain, free, and meshwork diffusion. (C) Wnt3 undergoes domain‐like diffusion unlike DiI‐C18 (used as a control for free diffusion). Statistical significance between the two treatments is determined by Kolmogorov–Smirnov (KS) test statistics. Error bars represent the standard error of the mean at each binned area (number of data points is 36 for 1 × 1 binning, 25 for 2 × 2 binning, and 16 for 3 × 3 binning).

Fig 5: Plasma membrane order is altered upon canonical Wnt stimulation. (A) Representative generalized polarization (GP) maps in the plasma membrane of HEK293T cells treated with either control‐ (top) or Wnt3a‐conditioned media (CM) (bottom) using the polarity sensitive dye Di‐4‐AN(F)EPPTEA and confocal microscopy in combination with spectral detection (large GP values indicate high membrane ordering; red in the color scale). (B) Average and standard deviation (error bars) of GP values determined from 10 images (each image contained multiple cells) for both Wnt3a CM and Wnt3a rec. (C) Average and standard deviation (error bars, three independent experiments) of fluorescence emission spectra of SL2 in HEK293T cells treated with either control‐conditioned (black) or Wnt3a‐conditioned (red) media (for panel C, P value was determined using the intensity at λ = 475 nm). Statistical significance was determined using unpaired t‐test. ***P < 0.001, **P < 0.01, *P < 0.05.