fig3

Figure 3. RCN1 interacts with the mitochondrial protein TFAM via the scaffold protein ATAD3A. (A) Radar chart displaying the top 30 differentially expressed proteins identified by IP-MS, ranked clockwise by the magnitude of the log₂ fold change; (B) The top 10 differentially expressed proteins were sorted by the magnitude of the log₂ fold change; (C) Protein expression levels of ATAD3A, SSBP1, and TFAM in RCN1-knockdown NB4 cells were analyzed by Western blot; (D) Co-IP assays in NB4 cells overexpressing RCN1 demonstrate that RCN1 associates with TFAM indirectly through ATAD3A; (E) Representative confocal images of an in situ PLA performed in HeLa-ATAD3A cells. The scale bar indicates 10 μm. A quantitative analysis of PLA signals is shown in the right panel corresponding to RCN1-ATAD3A, ATAD3A-TFAM, and RCN1-TFAM interactions per cell (n = 24 for RCN1-ATAD3A, n = 27 for ATAD3A-TFAM, and n = 26 for RCN1-TFAM); (F) Representative confocal microscopy of HeLa-ATAD3A cells stained for RCN1 (red), ATAD3A (red or green), TFAM (green), and nuclei (DAPI, blue). The scale bars indicate 5 μm. The insets show higher-magnification views of the indicated regions. Data are expressed as mean ± standard deviation. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test. ^**P < 0.01; ^****P < 0.0001. ANOVA: Analysis of variance; ATAD3A: ATPase family AAA domain-containing protein 3A; Co-IP: co-immunoprecipitation; DAPI: 4’,6-diamidino-2-phenylindole; HeLa: human cervical cancer cell line; IB: immunoblot; IgG: immunoglobulin G; IP: immunoprecipitation; IP-MS: immunoprecipitation-mass spectrometry; PLA: proximity ligation assay; RCN1: reticulocalbin 1; SSBP1: single-stranded DNA-binding protein 1; TFAM: mitochondrial transcription factor A.