Fig 2: SKIL induced autophagy through inhibition on TAZ phosphorylation by LATS2 and degradation.a Western blot analysis showed that SKIL silencing decreased expression levels of autophagy markers (LC3, p62, and Beclin-1). b, c Immunofluorescence staining indicated significant decrease of autophagosome after SKIL silencing in both CALU-3 and NCI-H520 cell lines. Percentage of cells with LC3 vacuoles were counted and calculated from 100 cells in 20 fields d Western blot analysis showed that SKIL silencing decreased expression of TAZ and its downstream signaling factors (CTGF, CYR61). e, f Cycloheximide treatment resulted in faster TAZ protein degradation in SKIL-silenced CALU-3 (shSKIL) compared to control (shNC). g Co-immunoprecipitation assay showed that SKIL could bind to elements of Hippo complex (LATS2, Sav), but not TAZ. h Western blot analysis showed that overexpression of LATS2 led to increased phosphorylation of TAZ (p-TAZ) and decreased levels of TAZ. Further overexpression of SKIL reversed the effect of LATS2 overexpression on levels of p-TAZ and TAZ, without influencing LATS2 level. *P < 0.05, **P < 0.01. Experiments were performed in triplicate.

Fig 3: KIRREL localizes in membrane and activates the Hippo pathway through SAV1. (A) Immunofluorescence staining shows membrane localization of KIRREL. The indicated cells at different densities were stained with the indicated antibodies. ZO-1 marks the cell membrane and cell–cell junction. Arrows indicate cell membrane as revealed by anti-ZO-1 staining (Scale bar, 5 µM). (B) KIRREL localized SAV1 to the membrane fraction. HEK293T cells were transfected with plasmids encoding the Myc-SAV1 and the indicated KIRREL-WT or truncated form. Then 48 h after transfection, cells were harvested and different cell fractions were separated. Western blots were then performed with the indicated antibodies. (C) The HEK293A WT cells, KIRREL KO cells and KIRREL KO cells expressing the KIRREL-WT or Δ600–757 form were harvested. The cell lysates were blotted with the indicated antibodies. (D) RT-PCR analysis of the Hippo pathway target genes AMOTL2 and CYR61 mRNA levels from the same cells described in (C). Three replicates are shown, with the bars indicating mean ± SD; n.s. = not significant; **P < 0.01, Student’s t test. (E) The 3D matrigel culture of the same cells described in (C). We plated 2 × 104 cells/well in 48-well plates with a mixture of medium and growth factor reduced matrigel (ratio 1:1). Photos were taken after 10 d of culture. The regions of the dashed boxes were enlarged and are shown in the lower panel. Representative results are shown (Scale bar, 100 µM). (F) Quantification of 3D colony sizes of each group in (F); n.s.= not significant; ***P < 0.001, Student’s t test.

Fig 4: FGF6 inhibits activation of the Hippo pathway in CMs after MI. (A) Western blot was performed to determine the protein levels of p‐MST1, MST1, p‐LATS1, LATS1, p‐YAP, YAP, CTGF and CYR61 in the heart from different group mice. n >3 per group. (B) Western blot analysis the subcellular localization of YAP in the heart from different group mice. n = 3 per group. Quantitative analysis of expressions of p‐MST1, MST1, p‐LATS1, LATS1, p‐YAP, YAP, CTGF, and CYR61 in (A), quantitative analysis of expressions of YAP in nuclear extracts. (C) Representative immunofluorescence (green for YAP, blue for DAPI) analysis subcellular localization of YAP in the heart section from different group mice. (D) RT‐PCR analysis of the mRNA levels of YAP and CTGF in the heart from different group. n >3 per group; n = 5 per group. Data represent means ± SEM. Two‐tailed Student's t test. *p <0.05, **p <0.01, ***p <0.001

Fig 5: FGF6 inhibits activation of the Hippo pathway in NRCMs after OGD. (A) Western blot was performed to determine the protein levels of p‐MST1, MST1, p‐LATS1, LATS1, p‐YAP, YAP, CTGF and CYR61 in NRCMs in different group. n >4 per group. (B) Western blot analysis the subcellular localization of YAP in NRCMs in different group. Quantitative analysis of expressions of p‐MST1, MST1, p‐LATS1, LATS1, p‐YAP, YAP, CTGF and CYR61 in (A), quantitative analysis of expressions of YAP in nuclear extracts. (C) Representative immunofluorescence (green for YAP, blue for DAPI) analysis subcellular localization of YAP in NRCMs in different group. n = 5 per group. (D) RT‐PCR analysis of the mRNA levels of YAP and CTGF in NRCMs in different group. n = 3 per group. Data represent means ± SEM. Two‐tailed Student's t test. *p <0.05, **p <0.01, ***p <0.001