Fig 1: Effect of PI3K inhibitor LY294002 on the proliferation and migration of HaCaT keratinocytes. HaCaT keratinocytes were treated with 50 µmol/l PI3K inhibitor LY294002 for 48 h. (A) The phosphorylation level of p-AKT at S473 and T308 and the p-AKT/AKT ratio was determined via western blot analysis. (B) Proliferation of HaCaT keratinocytes was assessed using Cell Counting Kit-8 assay. (C) Colony formation assay was employed to assess the number of colonies in HaCaT keratinocytes. (D) The number of migrated HaCaT keratinocytes was examined using Transwell assay. (E) The protein level of cyclin D1, CDK6, CDK4, MMP-2 and MMP-9 in HaCaT keratinocytes was determined via western blot analysis. *P<0.05 vs. control. p, phosphorylated.
Fig 2: Effect of miR-185 expression on the proliferation of HaCaT keratinocytes. HaCaT keratinocytes were transfected with miR-185 and anti-miR-185 or their NCs. (A) Reverse transcription-quantitative PCR was performed to examine the expression of miR-185 and evaluate the transfection efficiency. (B) Cell Counting Kit-8 assay was conducted to assess the proliferation of HaCaT keratinocytes. (C) Colony formation assay was performed to examine the colony forming ability of HaCaT keratinocytes. (D) Western blot analysis was performed to evaluate the protein level of cyclin D1, CDK6 and CDK4 in HaCaT keratinocytes. *P<0.05. miR, microRNA; NC, negative control; OD, optical density.
Fig 3: CDK6 is a promising downstream molecule of miR-138-5p. (A) The binding sites of miR-138-5p and CDK6 transcripts were predicted via bioinformatic analysis. (B) The association between miR-138-5p and CDK6 was confirmed via the dual-luciferase activity assay. (C) The mRNA levels of CDK6 were downregulated in keloid fibroblasts following transfection with miR-138-5p mimics. (D) The protein levels of CDK6 were reduced in cells transfected with miR-138-5p mimics. (E) In comparison with normal skin, CDK6 expression was significantly upregulated in keloid tissues. (F) The levels of miR-138-5p and CDK6 were inversely correlated in keloid samples (r=-0.2193; P=0.00732). All experiments were performed in triplicate. *P<0.05 vs. normal samples, $P<0.05 vs. miR-NC. CDK6, cyclin-dependent kinase 6; miR, microRNA; WT, wild type; Mut, mutant; NC, negative control.
Fig 4: Depletion of ALPK2 curbed esophageal cancer tumorigenesis. (a) ALPK2 knockdown model based on nude mice was constructed. (b) The evaluation of tumor volume was carried out from feeding to sacrifice. (c) The measurement of the tumor weight was carried out after scarifying the mice. (d) Tumors were photographed following removal. (e) Prior to sacrificing the mice, the intensity of fluorescence was obtained via D-Luciferase injection. (f) The detection of Ki-67 value was carried out in tumor sections using IHC. Magnification times: 200x. (g) Detection of the expression of Akt, P-Akt, mTOR, P-mTOR, CDK6, and PIK3CA was made by western blot. The findings were represented as mean ± SD. **P < 0.01 and ***P < 0.001.
Fig 5: The role of JAG2/Notch2 in the regulation of IVDD in vivo. a A flow diagram of the in vivo experiments. A total of 32 rats were randomly divided into 4 groups: normal control, non-injection with puncture group (non-injection), recombinant JAG2 injection with puncture group (JAG2), and lentiviral sh-Notch2 injection with puncture group (sh-Notch2). b MRIs of the indicated groups were obtained 0/3/9 weeks after needle puncture. L4/5 (yellow arrow) was punctured, and the others were left intact. c Intraoperative anatomical images: the red arrow points to the renal iliolumbar vein, and the blue arrow points to the L4/5 intervertebral disc, which is a little higher than the iliolumbar vein. d TUNEL staining of the IVDs in the indicated groups at 9 weeks after needle puncture. DAPI indicating total cells; green fluorescence indicating TUNEL-positive cells. Scale bar = 100 μm. e The MRI grade in the indicated groups at 9 weeks after needle puncture. The degree of disc degeneration according to MRI grade was significantly lower in the JAG2 group than in the non-injection group; *P < 0.05. f A significant decrease in the apoptosis rate was noted in the JAG2 group compared with that in the puncture group, and there was an increased apoptosis rate in sh-Notch2 group compared with that in the puncture group; *P < 0.05. g Western blot analysis of the expression of CDK6, cyclin D1, p21, apoptotic effector caspases (caspase-3, caspase-8, and caspase-9), PI3K/Akt and Wnt/β-catenin signaling markers, catabolic enzymes (MMP-3, MMP-13, and Adamts-5), and extracellular matrix (ECM) components (collagen II, aggrecan) in the rat NP tissues. The injection of JAG2 alleviated the degenerative changes in the NP such as an enhanced apoptotic and catabolic response and reduced the expression of ECM components in the rat model of IVDD, while sh-Notch2 aggravated the degenerative changes in the NP. h Safranin O/fast green staining of the IVDs at 9 weeks after needle puncture showed signifcant IVDD in the sh-Notch2 group, while injection of JAG2 alleviated the degeneration of IVD
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