Fig 1: InCasor for imaging of mtDNA mutations in live cells.a Sequencing identification of an SNV (12084 C > T) in mtDNA (ND4 gene) in MDA-MB-231 cells (MT). HepG2 cells were used as the wild-type control (WT). b, c InCasorND4-2-MT was capable of sensitive detection of the 12084 C > T mtDNA mutation in live cells. Confocal imaging (b) and flow cytometry (c) were used to analyze MDA-MB-231 and HepG2 cells incubated with InCasorND4-2-MT. The experiments were repeated three times independently. Scale bar: 10 μm. d, e Confocal imaging were used to analyze MDA-MB-231 and HepG2 cells incubated with InCasorND4-2-MT. DiI stained HepG2 cells, purple; DiD stained MDA-MB-231 cells, red; Hoechst: nucleus, blue; FAM: InCasorND4-2-MT, green. Semi-quantitative statistics are performed on the three types of fluorescence intensities (DiI, DiD and FAM) of each cell in the co-culture group. (n = 50). Scale bar: 10 μm. f Confocal imaging of mitochondria extracted from MDA-MB-231 cells and HepG2 cells using InCasorND4-2-MT to detect mutant mtDNA (12084 C > T). Scale bar: 25 μm. The experiments were repeated three times independently. g Sequences of crRNA0 (fully matched with the 13105 A > G mutant ND5 gene) and crRNA1-crRNA4 (with different numbers of nucleotide mismatches). Each SNV is indicated by a bold red letter. Each mismatched position is indicated by an underlined bold blue letter. An in vitro fluorescence assay was used to estimate the ability of different InCasor probes to detect the 13105 A > G mutation in the ND5 gene. n = 3 biologically independent experiments, data show mean ± SD. The sequences of all crRNAs used are listed in Supplementary Table 1. h Confocal imaging of HepG2 cells (WT) and MDA-MB-231 cells (MT) treated with InCasor probe with different crRNA. Scale bar: 10 μm. i Semi-quantitative statistics of three fluorescence intensities (DiI, DiD and FAM) in co-cultured cells under different treatments. (n = 50 biologically independent cells). Source data from (c, e, g, and i) are provided as a Source Data file.
Fig 2: InCasorND4 for imaging ND4 gene in mtDNA in live cells.a Confocal imaging of HepG2 cells treated with DNF/CLR, InCasorNT and InCasorND4. Scale bar: 10 μm. The spatial co-localization of InCasor and mito were statically analyzed by Fiji. b Semi-quantitative analysis of fluorescent intensity in each cell is shown. n = 50 biologically independent cells. P < 0.0001 determined by via one-way ANOVA with Tukey’s post-test. c Flow cytometry analysis of groups corresponding to (b). d Analysis of ND4 expression in HepG2 cells treated with InCasorND4-Mg2+ or InCasorND4-Co2+ using qPCR. n = 3 biologically independent experiments, data show mean ± SD. e, f Confocal imaging (e) and flow cytometry analysis (f) of HepG2 cells treated with InCasorND4-Mg2+ or InCasorND4-Co2+. Scale bar: 10 μm. The experiments were repeated three times independently. g Confocal imaging was used to analyze the fluorescence signal of Mg-Fluo-4 AM in HepG2 cells cultured in DPBS with different concentrations of Mg2+ for 12 h. Scale bar: 10 μm. h Semi-quantitative statistics of Mg-Fluo-4 AM in HepG2 cells after different treatments. n = 5 biologically independent experiments. i Confocal imaging of HepG2 cells treated with InCasorND4-Co2+ in DPBS containing different concentrations of Mg2+. Scale bar: 10 μm. j Semi-quantitative analysis of fluorescent intensity in panel i is shown. h. j Box-and-whisker plots: the boxes extend from the first to the third quartile, the middle lines denote the median and the whiskers indicate the minimum and the maximum value range. n = 5 biologically independent experiments. k Correlation analysis of the fluorescence intensity of InCasorND4-Co2+and the fluorescence intensity of Mg-Fluo-4 AM. n = 5 biologically independent experiments, data show mean ± SD. Source data from (a, b, d, h, j, and k) are provided as a Source Data file.
Supplier Page from Abcam for Recombinant Human ND4 protein (GST tag N-Terminus)