Fig 1: MRPL52 resists the caspase-dependent mitochondrial apoptosis of BC cells exposed to hypoxia. (A) Cellular apoptosis examined by TUNEL staining (mean ± SD, n = 3). *P < 0.05. Scale bars, 100 µm. (B) MMP detected by JC-1 probe. JC-1 red (JC-1 aggregate) shows healthy MMP, whereas JC-1 green (JC-1 monomer) shows decreased MMP. ΔΨm was represented as the red/green ratio (mean ± SD, n = 3). *P < 0.05. Scale bars, 100 µm. (C) Alterations in mitochondrial permeability transition pore opening were detected by calcein AM staining. The weaker the green fluorescence in cells, the higher the degree of opening of mPTP (mean ± SD, n = 3). *P < 0.05. Scale bars, 100 µm. The proteins expression of (D) Cytochrome c in cytoplasm and mitochondria, (E) cleaved PARP, cleaved Caspase-3, GPX4 and Caspase-1 were analyzed using WB. Cyto, cytosolic protein; Mito, mitochondrial protein. (F-G) MDA-MB-231 cells were incubated with Z-VAD-FMK (50 μM), a pan-caspase inhibitor or ferrostatin-1 (20 nM), a ferroptosis inhibitor or necrosulfonamide (20 nM), a necroptosis inhibitor for 24 h. (F) Cell viability, (G) cleaved PARP and cleaved Caspase-3 levels were measured using CCK-8 and WB analysis, respectively (mean ± SD, n = 3). *P < 0.05.
Fig 2: MRPL52 promotes mitophagy in hypoxic BC cells. (A) Mitochondrial ultrastructure in MDA-MB-231 and MCF-7 cells under TEM. Black arrows point to mitophagy and white arrows point to vacuolar deformation of mitochondria. Scale bars, 2 µm. (B) Mitochondria were stained using MitoTracker probe. Scale bars, 5 µm. (C) Autophagy induction was investigated by examining the protein levels of p62 and LC3B by WB. The LC3BII protein level on mitochondrial fraction was assessed by WB in order to detect the mitophagy. Mito, mitochondrial protein. (D) MDA-MB-231 cells were co-transfected with GFP-LC3B and the indicated plasmid, cultured under hypoxia for 24 h, and stained with MitoTracker probe. The colocalization of LC3B puncta (green) with mitochondria puncta (red) marked by yellow fluorescent intensity was analyzed by confocal fluorescence microscopy (mean ± SD, n = 3). *P < 0.05. Scale bars, 10 µm.
Fig 3: MRPL52 promotes the survival and metastasis of BC cells in vivo. (A) Representative IF images of MRPL52 (red) and pimonidazole (green) in 4T1 xenograft tumors. Yellow color in the merged image represents co-localization of MRPL52 and pimonidazole. (B) Images, (C) growth curves and (D) weights of xenograft tumors (mean ± SD, n = 5). *P < 0.05. Scale bars, 1 cm. (E) TUNEL staining of xenografts (mean ± SD, n = 5). *P < 0.05. Scale bars, 100 µm. H&E analysis of (F) lung and (G) liver metastasis in mice. Scale bars, 100 µm. The relative metastatic regions (tumor area/organ area) were calculated according to H&E analysis (mean ± SD, n = 5). The microscopically visible metastatic nodules on the surface of lung and liver were counted (mean, n = 5). *P < 0.05.
Fig 4: MRPL52 is upregulated in human BC. (A) Screening rationale of the RNA-seq assay. The screened genes were upregulated by more than 2-fold in breast cancer (BC) tissues and BC tissues with > 3 lymph nodes metastasis (TM) compared with adjacent normal tissues (ANTs) and BC tissues without lymph node metastasis (TnM), respectively. (B)The Bubble Chart of the results of GSEA was generated using the R package 'ggplot2'. Red represents a P value < 0.05 and redder colors indicate lower P values. (C) RT-qPCR of MRPL34, MRPL4, MRPL52, MRPL55, MRPL43 and MRPS24 expression in 30 human BC tissues and their paired ANTs. Data are shown as mean ± SD. (D) RT-qPCR of MRPL52 expression in 102 human BC tissues and their paired ANTs. Data are shown as mean ± SD. (E) MRPL52 expression in unpaired BC tissues and ANTs from clinical samples. Higher Ct value indicated lower gene expression. Data are shown as mean ± SD. (F) MRPL52 expression in TnM and TM tissues from clinical samples. Data are shown as mean ± SD.
Fig 5: MRPL52 mediates hypoxia-induced EMT, migration and invasion of BC cell. (A) Ability of cell migration and invasion tested by Transwell assay (mean ± SD, n = 3). *P < 0.05. Scale bars, 50 µm. (B) The morphological changes of cells undergoing EMT include the acquisition of spindle-liked phenotype and the loss of polarity and cell-cell adhesion, which are observed by an optical microscope. Scale bars, 50 µm. (C) The expression levels of Snail, Slug, ZEB-1 and ZEB-2 under hypoxia assessed by RT-qPCR (mean ± SD, n = 3). *P < 0.05. (D) The protein expression of MRPL52 and Snail was evaluated by WB. (E) WB and (F) IF were performed to assess the protein expression levels of Snail, E-cadherin, N-cadherin, ZO-1 and Vimentin. Scale bars, 50 µm.
Supplier Page from Abcam for Anti-MRPL52 antibody