Fig 2: Schematic depiction of the role of Gli2 in cisplatin (DDP) resistance in ovarian cancer. Under normal circumstances, the Hh signaling pathway is inhibited and therefore, Gli2 cannot bind to the downstream target genes. This maintains DDP sensitivity in ovarian cancer cells and cell proliferation is inhibited. When the Hh signaling pathway is abnormally activated, Gli2 is overexpressed, enters the nucleus in the activated form of Gli2A and directly binds to the downstream target gene MDR1. This promotes DDP resistance in ovarian cancer cells, thereby significantly enhancing cell proliferation.

Fig 3: GLI2 mediates the regulation of melanogenesis by primary cilia.Melan-a cells were transfected with scrambled control (siNC) or GLI2 siRNA. After 24 h, the cells were treated with cytochalasin D (0.5 µM) or serum-free media. After 48 h, the proportion of ciliated cells was counted (A). Cellular melanin content was analyzed (B). GLI2, tyrosinase, and TRP1 were analyzed (C). B16F1 cells were transfected with siNC or GLI2 siRNA. After 1 day, the cells were treated with cytochalasin D (0.5 µM) or serum-free media for 2 days. Ciliated cells and cellular melanin content were analyzed (D, E). GLI2, tyrosinase, and TRP1 were analyzed (F). Data represent the mean ± SE of 3 experiments (* p < 0.05, ** p < 0.01).

Fig 4: Wdr4 promotes the ubiquitination and degradation of Arhgap17 to activate Rac1.A The Volcano plot of proteins identified by LC-MS/MS from GNPs isolated from the P7 Wdr4 A-cKO; Ai14 and control cerebella. Differentially expressed proteins (p < 0.05 and fold change > 1.5) are marked in blue (for up-regulated) and green (for down-regulated). Among the upregulated proteins, those with a known anti-proliferative function are marked in red, orange or yellow with their names. Data were from 3 cerebella in each group and were analyzed using pairwise-ratio-based, Student’s t-test by the Proteome Discoverer 2.3 software. B, C Immunoprecipitation analysis for the interaction between exogenous (B) or endogenous (C) Wdr4 with indicated proteins. D–E Western blot (D) and RT-qPCR (E) analyses for the expression of Arhgap17 protein or Arhgap17 mRNA in N2a cells expressing control or Wdr4 shRNAs. Data in (E) were from four repeats in each group and analyzed using one-way ANOVA post hoc Dunnett’s test, p = 0.3302 in shWdr4#1 v.s. shCtrl, and 0.1855 in shWdr4#2 v.s. shCtrl. Data are represented as individual points and mean. F Western blot analysis for Arhgap17 and Gli2 expression in the purified GNPs from the P7 Wdr4 A-cKO and control cerebella. G, H Western blot analysis for Arhgap17 expression in N2a cells stably expressing Wdr4 (G) or Wdr4 shRNAs (H) and treated with 1 µM MG132 for 16 h. I Western blot analysis for Arhgap17 expression in N2a cells stably expressing Wdr4 and treated with 100 µg/ml CHX for indicated time points. The levels of Arhgap17 were normalized to the 0 h time point in the control or Wdr4-expressing group, respectively, and indicated at the bottom. J, K In vivo ubiquitination assay using N2a cells (J) or N2a cells expressing Wdr4 shRNAs (K) and transfected with indicated constructs. L Rac1 activity assay using N2a cells expressing Wdr4 shRNAs or transfected with Flag-Arhgap17. The western blot results were quantified using ImageJ software. The protein levels were normalized first to the Gapdh protein level in each group and then to the corresponding control groups, and expressed as fold changes at the bottom. All western blot analyses were done at least twice.

Fig 5: OPN loss impairs growth of basal-like PDA.(A) CRISPR-Cas9-mediated knockout of SPP1 in KP4 cells leads to a decrease in mRNA expression and secreted Osteopontin in two independent clones. (B,C) Immunoblot of KRT14 and E-cadherin (B) and GATA6 (C) levels following KO of SPP1 in KP4 cells. (D) Brightfield images of KP4 cell morphology when grown in 2D monolayer following KO of SPP1. Note the switch to a more epithelial phenotype in the KO cells. Scale bars, 100 µm. (E) In vitro growth rate of KP4 control versus SPP1 KO cell lines. Error bars represent s.e.m. (F) Growth rate of control and SPP1 KO KP4 cells following subcutaneous injection into the flank of NOD/SCID mice. Error bars represent s.e.m. (G) Relative size of control and SPP1 KO KP4 xenografts resected at day 25 (left) and comparative tumor weight (graph at right). (H) Representative H and E stained sections of lungs at endpoint following tail vein injection (TVI) of 2 × 106 KP4 control cells (WT; top) or SPP1 KO (bottom) into NOD/SCID mice. Scale bars, 400 µm. (I) Quantification of metastatic tumor burden in the lungs as a percentage of total lung area per mouse (n = 5 CTRL; n = 10 SPP1 KO). (J) Kaplan-Meier graph depicting survival of NOD/SCID mice following TVI of KP4 WT (black) or SPP1 KO cells (red). (K) High expression of SPP1 in basal-like PDA patient tumors from TCGA predicts shorter overall survival (SPP1 high n = 17, SPP1 low n = 48). Data from 65 patients. (L) Model depicting a classical to basal-like switch mediated by a GLI2-OPN signaling axis and in response to KRASG12D ablation. The classical and basal-like subtypes are marked by GLIlo/Hhhi and GLIhi/Hhlo expression, respectively. p-Value calculated by Log-rank test and by two-tailed unpaired t test. *p<0.05; **p<0.01; ***p<0.001.10.7554/eLife.45313.022Figure 7—source data 1.OPN loss impairs growth of basal-like PDA.