Fig 2: AMPK-mediated 3-hydroxy-3-methyl-glutaryl-coA (HMGCR) downregulation is needed for nuciferine to induce pYAP(Ser127). (a) Western blot of AMPK, pAMPK(Thr172), pYAP(Ser127) and ß-actin from PANC-1 cells transfected with none-targeted siRNA(NT-siRNA) and siRNA-AMPK. (b) YAP nuclear translocation in PANC-1 cells transfected with NT-siRNA and siRNA-AMPK in the presence of nuciferine (50 µM). Scale bars: 20 µm. (c) Western blot of AMOTL1 and ß-actin (a loading control); (d) Western blot of LATS1, LATS2, pYAP(Ser127) and ß-actin from PANC-1 cells transfected with NT-siRNA and siRNA-LATS1/2. (e) Cells were treated with nuciferine or DMSO for 72 h and collected for WB. (f) Western blot of AMPK and HMGCR from cells transfected with NT-siRNA and siRNA-AMPK. (g) Cells transfected with HMGCR overexpressed plasmid were treated with nuciferine or DMSO for 72 h and collected for western blot. Nuc, nuciferine; Gem, gemcitabine.

Fig 3: LATS2 compromised the promoting effect of CRNDE on HCC cell proliferation, migration, chemoresistance, and its inhibition on the Hippo pathway.Bel-7405 cells were transfected with pcDNA3.1(+) vectors containing CRNDE and/or pcDNA3.1(+) vectors containing LATS2. a MTT assay was carried out to measure the cell viability. b Cell migration and invasive abilities were determined by transwell assay. c Huh-7/ADR cells were transfected with lentiviruses encoding CRNDE and/or LATS2, and cell viability was measured by MTT assay when ADR was added. Cells transfected with empty vectors were used as negative control. d ADR IC50 was determined in Huh-7/ADR cells overexpressing CRNDE and/or LATS2. e Sphere-forming capacity was assessed and shown. f WB was used to evaluate expression levels of Hippo pathway components in Bel-7405 cells overexpressing CRNDE and/or LATS2. The data were shown as mean ± SD based on at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 4: LIF anti-CSC effects are linked to Hippo pathway core kinases activation. (A) Hippo kinase LATS2 and p-LATS1/2Thr1079/1041 and JAK/STAT effector STAT3 and p-STAT3Tyr705 protein expression in AGS and MKN45 cell lines. Values under each band represent quantification of relative GAPDH or tubulin-normalised protein expression according to band density (whole Western Blot available in Figure S5). (B) TEAD 8xCTIIC-luciferase reporter assay showing activity of transcription factor TEAD in AGS and MKN45 cell lines. (C) Relative Hippo target genes mRNA levels in AGS (2D) and MKN45 (3D) cell lines. (D) 3D tumoursphere assays carried out on GC cell lines (AGS and MKN45, on the left side of the panel) and PDX cells (GC07, on the right side of the panel). All cells were treated with 50 ng/mL LIF (green) and/or 0.5 µM XMU-MP-1 (XMU) (emerald green) at different time intervals (0, 2, 5, 24 or 48 h). For tumoursphere assays, LIF treatment was carried out every 48 h, 1 µM JAK1 inhibitor Ruxolitinib (Ruxo) (hatched bars) and combination of both XMU and Ruxo (checked bars) was used and sphere counting was performed after 7 days. For each experiment, inhibitors were added 30 min before each LIF stimulation. * p < 0.05, ** p < 0.005, *** p < 0.0005 and **** p < 0.0001 versus untreated controls with ANOVA statistical analyses. $ p < 0.05, $$ p < 0.005, $$$ p < 0.0005 and $$$$ p < 0.0001 versus conditions treated with XMU-MP-1 and/or Ruxolitinib alone with Student t-tests.

Fig 5: MIR22HG regulates LATS2 expression via inhibition of miR-629-5p activity.a The expression correlation between MIR22HG and LATS2 based on the Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database. b Venn diagram showing the overlapping target genes of miR-629-5p predicted using the miRWalk, starBase, and TargetScan databases. c Schematic diagram of the interaction between miR-629-5p and wild-type/mutant LATS2 3'-UTR. d Dual-luciferase assays of HEK293T cells co-transfected with wild-type/mutant LATS2 3'-UTR and miR-629-5p mimics (25, 50, and 100 nM) or NC. At 48 h after transfection, the 293 T cells were collected for the detection of firefly luciferase activity, with Renilla luciferase activity as internal control. e Real-time PCR was performed to detect the changes in LATS2 expression in MDA-MB-231 and BT549 after transfection with miR-629-5p mimics for 48 h. f, g Expression of Hippo-YAP1 signals, namely LATS2, YAP1, phosphorylated YAP1 (S127), and CTGF, was assessed by Western blotting after MIR22HG knockdown or overexpression. h Western blot analysis revealed that miR-629-5p inhibited the expression of LATS2, while MIR22HG was found to slightly counteract the miR-629-5p-induced reduction in LATS2 expression. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All experiments were repeated thrice.