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. 2022 Jun 27;12:10877. doi: 10.1038/s41598-022-14860-0

Figure 2.

Figure 2

A fraction of Lonp1 localizes in the nuclei of human cells. (A) Representative confocal microscopy images of SW620 cells after immunostaining with anti-Lonp1 and anti-human mitochondria (hMit) Abs. Nuclei were counterstained with DAPI. Bars: 10 mm. (B) Left panel: representative immunoblot of cytosolic (C) and nuclear (N) fractions obtained from SW620 cells. Lamin B1 is the nuclear fraction loading control, whereas β-actin is the cytosolic fraction loading control. Immunoblots of Sirtuin-3 (SIRT3) and TOM20 are also reported to indicate that mitochondrial contamination is not present. Blots were cut prior to hybridisation with antibodies during blotting. Right panel: representative immunoblot of cytosolic (C), mitochondrial (M), nuclear (N) fractions obtained from SW620 cells. β-actin is the cytosolic fraction loading control, TOM20 is the mitochondrial fraction loading control and lamin B1 is the nuclear fraction loading control. (C) Left histogram: quantification of the ratio of nuclear and cytosolic Lonp1 (n = 3 independent experiments). Right histogram: quantification of the ratio of cytosolic, mitochondrial and nuclear Lonp1 (n = 3 independent experiments). (D) Representative confocal microscopy images of SW620 cells after immunostaining with anti-Lonp1 antibody. Cells were treated or not with leptomycin B (LMB) to inhibit nuclear export of proteins. Staining of p62 was reported as positive control for LBM treatment. Nuclei were counterstained with DAPI. (E) Representative immunoblot of cytosolic (cytosol) and nuclear (N) fractions obtained from SW620 cells, treated or not with LBM. Lamin B1 is the nuclear fraction loading control, whereas β-actin is the cytosolic fraction loading control. Immunoblot for p62 is also reported as positive control for LMB treatment. Blots were cut prior to hybridisation with antibodies during blotting. (F) Alignment of Lonp1 protein sequences (amino acids 218 to 256) from the indicated mammalian species. HS, Homo sapiens; CL, Canis lupus familiaris; MM, Mus musculus; RN, Rattus norvegicus. The nuclear targeting sequence is highlighted in red. (G) Representative immunoblot of cytosolic (C), mitochondrial (M), nuclear (N) fractions obtained from SW620 cells expressing the R237A-R241A mutant of Lonp1. β-actin is the cytosolic fraction loading control, TOM20 is the mitochondrial fraction loading control and lamin B1 is the nuclear fraction loading control. Blots were cut prior to hybridisation with antibodies during blotting. (H) Representative confocal microscopy images of SW620 cells expressing the R237A-R241A mutant of eGFP-Lonp1 after immunostaining with anti-Lonp1 antibody. Nuclei were counterstained with DAPI. Bar: 10 mm.