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Fig. 4 | Journal of Biomedical Science

Fig. 4

From: N-terminal functional domain of Gasdermin A3 regulates mitochondrial homeostasis via mitochondrial targeting

Fig. 4

N-Gsdma3 partially colocalizes with mitochondria and induces mitochondrial ROS production. (a) Immunostaining of HaCaT cells transfected with the Gsdma3/V5, C-Gsdma3/mh, or N-Gsdma3/mh plasmid. Scale bar: 30 μM. (b) HaCaT cells transfected with Gsdma3/V5, N-Gsdma3/mh, Rim3/mh, and I359N/V5 plasmids were double immunostained for the tag and the mitochondrial marker Tom20. The region of interest (ROI) is determined by masking the images using the Imaris software such that the pixels selected in green and red channels closely outline the area of immunofluorescence staining. Arrows indicate partial colocalization; arrowheads point to mitochondria; Scale bar: 20 μM. (c) HaCaT cells were transfected with both the C-Gsdma3 and N-Gsdma3/mh plasmids and double immunostained for the myc tag and Gsdma3. The arrowheads indicate the cells expressing a low amount of C-Gsdma3; the arrows indicate the cells expressing a high amount of C-Gsdma3. Scale bar, 20 μM. The color coding indicates the secondary antibody labeling. DAPI counterstains the nuclei in blue. (d) Quantification of the colocalization of N-Gsdma3 and Gsdma3Rim3 with the mitochondrial marker. The bar graph shows the % ROI colocalized with Tom20 (mean + s.d., n > 10 from 5-7 z-stacked confocal images). ***, P < 0.01 (e) Immunoblot analysis of mitochondrial and cytosolic fractions prepared from transfected HEK293T cells. Cox4 (a mitochondrial marker) and GAPDH (a cytosol marker) immunoblots confirmed the purity of the fractionations. (f) Quantification of the mitochondria/cytosol distribution based on relative densitometry levels from mitochondrial and cytosolic fractions. The bar graph represents mitochondrial/cytoplasmic ratio relative to Gsd (mean + s.d., n = 3). (g) Flow cytometry analysis of the MitoSox Red staining in HEK293T cells transfected with the Gsdma3/flag (Gsdma3), N-Gsdma3/flag (N-Gsdma3), C-Gsdma3/flag (C-Gsdma3), Rim3/flag plus Gsdma3/mh in a ratio of 1:2 (Rim3 + Gsd), and control pIRES-hrGFP-1a (pIRES) plasmids. (h) Quantification of the MitoSox Red staining results by the fold increase of MitoSox Red staining in the fraction of GFP+ cells (mean + s.d., n = 3) over that of vector-transfected cells (average = 3.79 ± 1.53 %).*, P < 0.05; **, P < 0.01; n.s., not significant

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