Fig 1: Neat1 inhibits the expression of myogenic genes via Ezh2.a, b ChIP-qPCR results revealing that Neat1 knockdown significantly decreased the enrichments of Ezh2 (a) and H3k27me3 (b) at the Myog, Myh4, and Tnni2 promoters. c, d ChIP-qPCR results indicating Neat1 overexpression significantly increased the enrichments of Ezh2 (c) and H3k27me3 (d) at the Myog, Myh4, and Tnni2 promoters. e Neat1 expression vector and Ezh2 siRNA fragment were co-transfected into C2C12 cells. The indicated genes expression were measured by qPCR after Neat1 expression vector and Ezh2 siRNA fragment were co-transfected 3 days post differentiation. The results showed that overexpression of Neat1 inhibits the expression of Myog, Myhc and Tnni2, but had no significant effect when co-transfected with Ezh2 siRNA fragment. f Neat1 siRNA fragment was con-transfected with Ezh2 expression vector into C2C12 cells at 5 days post differentiation, the cells were performed with Myhc immunofluorescence staining. The quantification of Myhc immunofluorescence staining results showed Neat1 knockdown alone increased Myhc protein expression but not when co-transfected with Ezh2 expression vector. g, h ChIRP-qPCR results revealed that Neat1 binds directly to the Myog, Myh4, and Tnni2 promoters but not to the Myod promoter in C2C12 cells (g) and primary myoblasts (h). Relative RNA levels were normalized to those of ß-actin. All values represent the mean ± s.d. of three independent experiments. *p < 0.05, **p < 0.01, N.S. indicates not significant
Fig 2: Knockdown of Neat1 improves muscle growth in vivo.a The injection scheme for LV3-shNeat1 or LV3-shNC particles into the right or left hindlimb muscles of C57 mice. The injection was given every one week for one month. b qPCR results showing that Neat1 expression was reduced after LV3-shNeat1 particle injection, while the mRNA expression of Myog, Myhc, a-actin, and Tnni2 was significantly increased after LV3-shNeat1 particle injection. c Western blotting analysis showing that the protein expression of Myog, Myhc, a-actin, and Tnni2 was increased after LV3-shNeat1 particle injection. The protein levels of these genes were quantified using ImageJ software. d Representative photograph of the three muscles from the right or left hindlimbs showing that the volume of the Qu, TA, and Gas muscles of the right hindlimb were larger than those of the left hindlimb. e Quantification of the weight of three muscles from the right or left hindlimbs of 12 injected mice showing that the weight of the Qu, TA, and Gas muscles of the right hindlimb were higher than those of the left hindlimb. f Representative photograph of myosin immunofluorescence staining in Qu, TA, and Gas muscles from the right or left hindlimb following injection with LV3-shNeat1 or LV3-shNC particles. Compared with LV3-shNC particle injection, LV3-shNeat1 particle injection increased the average cross-sectional areas of the indicated muscles. The fiber sizes of the Qu, TA, and Gas muscles were quantified using ImageJ. Relative RNA and protein levels were normalized to those of Gapdh. All values represent the mean ± s.d. of three independent experiments. *p < 0.05, **p < 0.01
Fig 3: TNNI2 promotes pancreatic cancer progression by directly regulating the ERRa/SIRT1 pathway.A Representative dot plots from flow cytometric analysis of apoptosis in the indicated cells measured after Annexin-V/propidium iodide staining. B, C Representative images and quantification of colony-formation assays in BxPC-3 and Panc-1 cells expressing either siTNNI2 or TNNI2 overexpression vector, respectively. D–F Representative images and quantification of cell migration and invasion in BxPC-3 or PANC-1 cells were measured using a Transwell system 48 h post-transfection. Scale bar: 100 µm. G Densitometric quantification and representative western blot images showing TNNI2, ERRa, and SIRT1 protein expression levels in BxPC-3 and PANC-1 cells expressing either siRNA or the overexpression vector against TNNI2. Data are presented as mean ± SD with at least three independent experiments. *P < 0.05.
Fig 4: TNNI2 regulates SYT8-induced pancreatic cancer progression via the ERRa/SIRT1 signaling axis.A Densitometric quantification and representative western blot images showing SYT8, TNNI2, ERRa, and SIRT1 protein expression levels in BxPC-3 and PANC-1 cells co-expressing different siRNA or overexpression vectors against SYT8 and TNNI2. Glyceraldehyde 3-phosphate dehydrogenase was used as the loading control. B–D Quantification of cell proliferation from the colony-formation assay. The Transwell assay was used to measure cell migration and invasion in the respective samples. E In vivo tumor assay. Representative images of sections from xenograft tumors from BALB/c nude mice subcutaneously injected with BxPC-3 cells and Panc-1 cells transfected with siSYT8 or the SYT8 overexpression vector. Hematoxylin and eosin and immunohistochemical staining were used to visualize Ki-67, SYT8, and TNNI2 expression. Scale bar: 50 µm. Data are presented as mean ± SD with at least three independent experiments. *P < 0.05.
Fig 5: Schematic model of Neat1 regulation in myogenesis.In proliferating myoblasts, Neat1 guides Ezh2 to the P21 promoter and inhibits P21 expression, leading to the promotion of myoblast proliferation. Upon differentiation, Neat1 recruits Ezh2 to inhibit the expression of muscle-specific genes, such as Myog, Myh4, and Tnni2, and suppresses myogenic differentiation
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