Figure 3
From: ESX1 gene as a potential candidate responsible for male infertility in nonobstructive azoospermia
Activation of the ESX1 gene using the CRISPRa system. (A) TCam-2 cells at 72 h after transfection under a Juli FL fluorescence microscope; scale bar: 100 μm. (B) Real-time PCR analysis of ESX1 gene expression. (C) Representative Western blot analysis of ESX1 protein isolated from TCam-2 cells, exposure time 300 s; and its graph of the relative quantity of the ESX1 protein normalized with reference to HPRT1 analysed by Image Lab 6.1 tools. (D) Immunofluorescence analysis of TCam-2 cells. A Leica DMi8 with a proper filter set (DAPI/TxR/Triple) was used; objectives: 40x, scale bar: 50 μm; software: LASX. Arrows indicate examples of ESX1-positive staining. The transfection efficiency after antibiotic selection and induction with doxycycline was estimated for approximately 40% for both TCam-2—cells with the activated ESX1 gene and the negative control—cells with nonspecific gRNAs (guide RNAs) for the human genome. Activation of the ESX1 gene in TCam-2 cells was successfully achieved at the mRNA level with significant upregulation (p < 0.001) of the ESX1 gene in comparison to both applied control samples—WT and NC; and that was also observed at the protein level, where the presence of the ESX1 protein was observed in TCam-2 cells with active ESX1, while in WT and NC, the protein was absent. WT wild type, NC negative control with nonspecific gRNAs for the human genome, ESX1 cells with the activated ESX1 gene using specific gRNAs for the ESX1 sequence, BR brightfield, FLU fluorescence.
