Fig 1: INHBA activates YAP and induces its nuclear translocation in lung cancer cells. (A and B) Western blotting revealed that the expression levels of pYAP, pLATS1/2, and the ratio of pYAP/YAP and pLATS1/2/LATS1/2, were decreased in H1299 cells transfected with the INHBA overexpression plasmid and increased in A549 cells transfected with si-INHBA; total YAP and LATS1/2 levels were not affected. (C-G) Subcellular fractionation indicated that, after INHBA overexpression in cytoplasm, YAP accumulation in the cytoplasmic fraction decreased and YAP accumulation in the nuclear fraction increased. RT-qPCR indicated that H1299 cells transiently transfected with the INHBA plasmid exhibited increased (H) CTGF and (I) CYR61 mRNA expression levels. RT-qPCR demonstrated that A549 cells transiently transfected with si-INHBA exhibited decreased (J) CTGF and (K) CYR61 mRNA expression levels. (L-N) Western blotting indicated that H1299 cells transiently transfected with the INHBA plasmid exhibited increased CTGF and CYR61 protein expression levels. (O-Q) Western blotting demonstrated that A549 cells transiently transfected with si-INHBA exhibited decreased CTGF and CYR61 protein expression levels. *P<0.05, **P<0.01. INHBA, inhibin βA; YAP, yes-associated protein; si, small interfering; NC, negative control; LATS, large tumor suppressor kinase; p, phosphorylated; CTGF, connective tissue growth factor; CYR61, cysteine rich angiogenic inducer 61; RT-qPCR, reverse transcription-quantitative PCR.
Fig 2: Clinical significance of INHBA in non-small cell lung cancer. (A) IHC scores of INHBA in 238 NSCLC tissues were significantly higher compared with those in 30 cases of normal lung tissues. (B) Western blotting of INHBA expression levels in T tissues and in matched, adjacent NT tissues. (C) Semi-quantification of western blotting of 24 paired tissue samples. INHBA protein expression levels were significantly higher in T samples compared with those in NT samples. (D) mRNA expression levels of INHBA were significantly higher in T samples compared with those in NT samples. (E) Representative cases showed that INHBA expression was increased in MN tissues compared with in paired PT tissues (original magnification, ×40 or ×200). (F) Based on the scores from immunohistochemistry, the abundance of INHBA was elevated in MN tissues compared with in PT tissues (n=30). (G) Kaplan-Meier curve for overall survival in 238 patients. Higher INHBA expression levels in NSCLC, based on immunohistochemistry scores, were associated with poorer overall survival. (H) Kaplan-Meier curve for disease-free survival. Higher INHBA expression levels in NSCLC were associated with poorer disease-free survival. **P<0.01, ***P<0.001. INHBA, inhibin βA; IHC, immunohistochemistry; NSCLC, non-small cell lung cancer; T, tumor; NT, non-tumor; MN, metastatic nodule; PT, primary tumor.
Fig 3: INHBA negatively regulates Hippo signaling by downregulating Merlin protein expression levels in lung cancer cells. (A) Western blotting revealed that only Merlin protein expression levels were decreased in H1299 cells transfected with the INHBA overexpression plasmid. (B) Western blotting revealed the efficacy of Merlin overexpression plasmids in H1299 cells. (C) Rescue experiments showed that overexpression of Merlin reversed the effect of INHBA on activating YAP in H1299 cells. (D) Reverse transcription-quantitative PCR indicated no change in NF2 (encoding Merlin) mRNA expression levels after H1299 cells were transiently transfected with the INHBA plasmid. (E) Immunoprecipitation analysis revealed that INHBA could bind to Merlin. *P<0.05, **P<0.01, ***P<0.001. INHBA, inhibin βA; YAP, yes-associated protein; WWC1, WW and C2 domain-containing 1; FRMD6, FERM domain-containing 6; NF2, neurofibromin 2.
Fig 4: INHBA promotes lung cancer cell invasion via inhibiting the Hippo pathway. (A) Protein complex of Merlin, FRMD6 and WWC1 activates the Hippo pathway, which increases the phosphorylation of YAP and prevents its translocation and binding to TEAD in the nucleus. (B) INHBA inhibits the Hippo pathway by downregulating Merlin expression, which decreases the phosphorylation of YAP, and promotes its translocation and binding to TEAD in the nucleus. INHBA, inhibin βA; YAP, yes-associated protein; WWC1, WW and C2 domain-containing 1; FRMD6, FERM domain-containing 6; MST, mammalian sterile 20-like 1/MOB kinase; LATS, large tumor suppressor kinase; p, phosphorylated; TEAD, TEA domain; CTGF, connective tissue growth factor; Cyr61, cysteine rich angiogenic inducer 61.
Fig 5: Immunohistochemistry for detecting the expression of APOD, CTLA4, CXCR4, DKK1, INHBA, NPR1, PENK, PROC, RBP4, S100A12, and STC1 in 5 paired gastric cancer and normal tissues. Bar = 20 μm. Magnification, ×200.
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