Fig. 2: Characterization of mitochondria targeting process of InCasor.

a Confocal imaging of subcellular localization of InCasor in HepG2 cells after treated with 50 nM InCasor_{Cyt C apt+} or InCasor_{Cyt C apt-}. InCasor were labelled with Cy5 (red), mitochondrial were stained with TOMM20 antibody (green). Scale bar: 25 μm. b The Pearson’s correlation coefficient (PCC) of InCasor and mitochondrial were statistically analyzed by Fiji. Data are expressed as mean ± SD (n = 3 biologically independent experiments). c Bio-TEM of HepG2 cells treated with Au NPs labelled InCasor_{Cyt C apt+} or InCasor_{Cyt C apt-}. d Proteinase protection assay and western blots showing successful delivery of Cas12a into mitochondria. HepG2 cells were incubated with 50 nM InCasor_{Cyt C apt+} or InCasor_{Cyt C apt-} for 6 h. After incubation, 20 μg of isolated crude mitochondrial fraction was treated with 50 μg/mL proteinase K for 30 min on ice, followed by western blotting. 20 μg of crude mitochondrial fraction w/o proteinase K treatment was used as control. e Schematic (left) of InCasor for mitochondria targeting in living HepG2 cells by fluorescent ribonucleoprotein consisting of synthesized fluorescent crRNA and Cas12a. Confocal imaging (right) of subcellular localization of Cas12a RNP in HepG2 cells after treated with 50 nM InCasor_{Cyt C apt+} or InCasor_{Cyt C apt-}. Cas12a protein was labelled with Cy5 (purple), crRNA was labelled with FITC (green), and mitochondrial were stained with Mito-Tracker (red). Scale bar: 25 μm. f Fluorescence microscopy images of JC-1-labeled HepG2 cells treated with 50 nM InCasor_{Cyt C apt+} or InCasor_{Cyt C apt-} probe at 37 °C for 6 h; scale bar: 50 µm. g Fluorescence microscopy images of Calcein AM-labeled HepG2 cells treated with 50 nM InCasor_{Cyt C apt+} or InCasor_{Cyt C apt-} probe at 37 °C for 6 h; scale bar: 200 µm. h Semiquantitative statistics of mitochondrial membrane potential (Δϕm) measurements and mitochondrial permeability of HepG2 cells after different treatments. Data are analyzed by two-sided Student’s t-test and shown as mean ± SD. For a, c, d, e, and h, the experiments were repeated three times independently. Source data from (b, d, and h) are provided as a Source Data file.