Fig 2: LINC01116 recruits EZH2 to suppress TPM1 gene transcription. The binding of LINC01116 and EZH2 were predicted by RNAInter online tools (a). The potential binding of EZH2 and TPM1 was predicted by UCSC (b). TCGA database was used to assess the expression of EZH2 in CRC tissues and the association between EZH2 and TPM1 (c). qRT-PCR (d) and Western blot (e) were applied to measure the levels of EZH2 in CRC cell lines. RIP assay confirmed the interaction between LINC01116 and EZH2 (f). Binding of EZH2 to TPM1 promoter was verified by ChIP (g). The augment of TPM1 promoter enriched in EZH2 antibody following overexpression of LINC01116 was detected by ChIP (h). RNA pull-down assay was performed to measure H3K27me3 in LINC01116-complex following overexpression of LINC01116 (i). *P < 0.05, **P < 0.01, ***P < 0.001; TPM1 tropomyosin 1, CRC colorectal cancer

Fig 3: LINC01116 accelerates CRC cell proliferation and angiogenesis by mediating TPM1 Note: The expression of TPM1 in CRC tissues of TCGA dataset was searched through GEPIA database (a). The levels of TPM1 in CRC cells were tested by qRT-PCR (b) and Western blot (c). After transfection of pcDNA3.1-LINC01116 or sh-LINC01116 into SW480 and HCT116 cells, qRT-PCR (d) and Western blot (e) were applied to test the levels of TPM1. After SW480 and HCT116 cells were transfected with sh-TPM1, the levels of TPM1 were tested by qRT-PCR (f) and Western blot (g). Cell viability and proliferation were assessed by CCK-8 (h) and colony formation assays (i). Cell scratch (j) and tube formation assays (k) were used to measure migration and angiogenesis. *P < 0.05, **P < 0.01; CRC colorectal cancer, TPM1 tropomyosin 1

Fig 4: Characterization of the human iPSC line CHOPWT17_TPM1KOc28. A. Sequencing analysis of the targeted iPSC clone showing homozygous deletion at the indicated site. B. Western blot showing absence of TPM1 protein in the selected clone. C. Morphologic appearance of CHOPWT17_TPM1KOc28 iPSC clones match the parental cell line. Scale bar, 50 μm. D. Immunofluorescence staining confirms intranuclear SOX2 staining in CHOPWT17_TPM1KOc28 iPSCs and parental cells. Scale bar, 50 μm. E. Flow cytometry analysis shows appropriate stem cell marker expression in CHOPWT17_TPM1KOc28 iPSCs and parental cells. F. Representative normal karyotype for CHOPWT17_TPM1KOc28 iPSCs. G. Directed differentiation shows efficient production of mesoderm (including mesoderm-derived KDR+CD31+ endothelial cells shown in box), endoderm, and ectoderm.

Fig 5: Induction of caveolins by adenoviral transduction of MRTF-A and myocardin.(A) Adenoviral transduction of human coronary artery smooth muscle cells with MRTF-A (MKL1, 20 MOI) increased expression of caveolin-1 and caveolin-2, but not caveolin-3 mRNA compared to empty virus. (B) Transduction of myocardin (MYOCD, 100 MOI) increased mRNA expression of all three caveolins (-1 to -3). (C) A dose-response curve with increasing concentrations of myocardin (1 to 300 MOI) demonstrated a similar threshold of sensitivity of caveolin-1 mRNA to that of the prototypical myocardin target calponin (CNN1). (D) Western blotting of MYOCD-transduced cells demonstrated increases of caveolins and cavins at the protein level. The magnitude of induction was similar to that of the well-established myocardin targets tropomyosin (TPM1) and SM22α (TAGLN). Summarized data from the western blots is shown in panels E through J (n = 8). 18S was used as a house-keeping gene for the qRT-PCR and HSP90 as loading control for western blotting. Data are presented as means±S.E.M. **P<0.01, ***P<0.001.