Fig 1: Silencing CDK16 abolishes the effects of TPT1-AS1 overexpression in thermally injured HDFs. (A) The silencing efficiency of CDK16 after the transfection of sh-CDK16#1/#2 in HDFs alone was detected by RT-qPCR analysis. (B) RT-qPCR was used to measure CDK16 expression in HDFs after the indicated treatment. (C) MTT confirmed the viability of the HDFs under the indicated treatment. (D) Flow cytometry was used to analyze cell cycle progression in each group. (E) Expression levels of cell cycle-associated markers were validated by western blot analysis after the indicated stimulation. (F and G) The apoptotic ability of HDFs under each stimulation was assessed via flow cytometric analysis. (H) Levels of apoptosis-associated proteins under the indicated treatments were evaluated by western blotting. (I) Western blot analysis was used to measure the levels of extracellular matrix synthesis-associated markers in each group. *P<0.05; **P<0.01; #P<0.05. TPT1-AS1, TPT1-antisense RNA 1; HDFs, human dermal fibroblasts; RT-qPCR, reverse transcription-quantitative PCR; sh-, short hairpin RNA-; NC, negative control; CDK16, cyclin-dependent kinase 16.
Fig 2: CDK16 is a target of miR-324-5p. (A) Putative mRNAs were obtained via bioinformatics analysis, and RT-qPCR was used to measure the expression of candidate mRNAs in response to miR-324-5p mimics. (B) Western blot analysis of the protein level of CDK16 after miR-324-5p upregulation. (C) The target sequence of miR-324-5p in the 3’UTR of CDK16 was obtained from TargetScan. (D) A luciferase reporter experiment verified the binding ability of CDK16 and miR-324-5p. (E) RT-qPCR analysis of CDK16 expression levels in HDFs after thermal injury. (F) A luciferase reporter assay was used to detect the luciferase activity of the CDK16 promoter after HDF transfection with miR-324-5p mimics. **P<0.01. CDK16, cyclin-dependent kinase 16; miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; UTR, untranslated region; NC, negative control; HDFs, human dermal fibroblasts; Mut, mutant; Wt, wild-type.
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