Fig 2: Overexpression of NDUFA4 Promoted the Proliferation and Migration of Human Lung Cancer Cells(A) The sketch map of an eukaryotic expression vector encoding NDUFA4 (termed as p-NDUFA4). (B) Human lung cancer cell line 95D cells were transiently transfected with p-NDUFA4 (10 μg) or p-Cont (10 μg) in vitro. 48 hr later, the morphology of cells were observed by microscopy (magnification 100×). (C) The relative expression of NDUFA4 was determined by real-time PCR. (D) The proliferation of cells was detected by CCK-8 assay. (E) The migration of 95D cells was performed by scratch assay, and the cell number was also calculated. The yellow line indicates the border of scratch wound (magnification 100×). (F) The colon-formation assay also was performed and calculated at indicated time points. Human lung cancer cell line 95D cells were transiently transfected with p-NDUFA4 (10 μg) or p-Cont (10 μg) in vitro. 48 hr later, the expression of indicated cell-growth-related molecules and metastasis-related molecules was detected by real-time PCR assay (G). (H) The protein level of NDUFA4, Akt, p-Akt, Erk, and p-Erk was also detected by western blotting and calculated, respectively. Representative data of three independent experiments are shown.*p < 0.05, **p < 0.01.

Fig 3: MOCCI is a subunit of Complex IV (cytochrome C oxidase).a The MOCCI ORF is conserved across vertebrates and contains a predicted transmembrane (TM) domain and the B12D domain found only in NADH-ubiquinone reductase complex 1 MLRQ subunits. The MOCCI transcript is also the host gene for hsa-miR-147b (right), located in the 3'UTR of the transcript. The letters in blue are the sequence of the mature miRNA. b MOCCI is a paralog of NDUFA4 which also contains both the B12D and TM domains. The 3'UTR of the NDUFA4 transcript contains top target site of miR-147b-3p as predicted by TargetScan114, miRDB115, and miRBase116. It is not clear if the binding site in chicken Ndufa4 is functional, as it is not as well conserved. c MOCCI was found in mitochondria-enriched fractions of HEK293T transfected with MOCCI ORF-expressing plasmid (MOCCI-HEK293T). Values given indicates percentage of the total fraction loaded onto the gel. n = 2 biological replicates. Source data are provided as a Source Data file. d Differential solubilization of MOCCI-HEK293T mitochondria indicated that MOCCI was located in the inner membrane (IMM). TOMM70 and MTCO-1 serve as control for outer (OMM) and inner (IMM) mitochondrial membrane proteins, respectively. n = 1 biological replicate. Source data are provided as a Source Data file. e Proteinase K protection assay of MOCCI-HEK293T mitochondria further confirmed IMM localization of MOCCI. n = 1 biological replicate. Source data are provided as a Source Data file. f Blue native PAGE (BN-PAGE) of mouse heart mitochondria with AAV9-mediated MOCCI-FLAG expression (AAV-MOCCI). Probing with a-MOCCI and a-MTCO-1 antibodies, the core enzymatic subunit of CIV, reveal co-migration of MOCCI signal with CIV. AAV-GFP was used as a negative control. Coomassie blue stain of the gel is shown on the right. n = 4 biological replicates. g Two-dimensional BN-SDS-PAGE to confirm co-migration of MOCCI and CIV. The lanes correspond to the bands annotated on the Coomassie blue-stained gel in (f). n = 2 biological replicates. Source data are provided as a Source Data file.

Fig 4: Overexpression of NDUFA4 Abrogated the Suppressive Effect of TTF-1-Promoter-Operating miR-7 ExpressionHuman lung cancer cell line 95D cells were transiently co-transfected with p-T-miR-7 (10 μg) and p-NDUFA4 (10 μg) in vitro. 48 hr later, (A) the morphology of cells were observed by microscopy (magnification 100×). (B) The proliferation of 95D cells was detected by CCK-8 assay. (C) The migration of 95D cells was performed by scratch assay, and the cell number was also calculated. The yellow line indicates the border of scratch wound (magnification 100×). (D) The colony-formation assay was also performed, and the colon numbers were calculated. (E) The relative expression of NDUFA4 was determined by real-time PCR. (F) The relative expression of indicated cell-growth-related molecules and metastasis-related molecules was also detected by real-time PCR assay. (G) The protein level of NDUFA4, Akt, p-Akt, Erk, and p-Erk was detected by western blotting and calculated, respectively. Representative data of three independent experiments are shown.*p < 0.05, **p < 0.01.

Fig 5: MOCCI and miR-147b reduce NDUFA4 expression during inflammation.a Intracellular flow cytometry staining for MOCCI in untreated (gray line) and IL-1ß-treated (red line) human aortic endothelial cells (HAECs). Percentage of MOCCI-positive HAECs is 25.7 ± 5.2% (mean ± se), n = 4 biological replicates per condition. b Co-immunofluorescent staining for MOCCI and TOMM20, a mitochondrial protein in HAECs. Scale bar = 100 µm, n = 3 biological replicates. c C15ORF48 mRNA and miR-147b expression in untreated and IL-1ß-treated HAECs. Data are presented as mean ± SEM, n = 3 biological replicates per condition. d Intracellular flow cytometry staining for NDUFA4 and MOCCI in untreated or IL-1ß-treated HAECs. e Intracellular flow cytometry staining for NDUFA4 and MOCCI in untreated or IL-1ß-treated A549 cells. f Intracellular flow cytometry staining for NDUFA4 and MOCCI in HAECs overexpressing MOCCI, C15ORF48 WT-mRNA (both MOCCI and miR-147b), and C15ORF48 ATGmut-mRNA (only miR-147b), n = 3 biological replicates per condition. g Levels of C15ORF48 mRNA (primers binding to the 5'UTR of C15ORF48) or miR-147b by qPCR in HAECs with the indicated overexpression. Data were presented as mean ± SEM, n = 3 biological replicates per condition. h Western blot of NDUFA4 in HAECs with the indicated overexpression. n = 3 biological replicates. Source data are provided as a Source Data file.