Journal: Oncology letters

Article Title: MTCH2 promotes the malignant progression of ovarian cancer through the upregulation of AIMP2 expression levels, mitochondrial dysfunction and by mediating energy metabolism.

doi: 10.3892/ol.2024.14625

Figure Lengend Snippet: Figure 1. Increased expression levels of MTCH2, AIMP2 and claudin‑3 in OC tissue samples. (A) Representative images of immunohistochemical staining of MTCH2, AIMP2 and claudin‑3 expression in OC and adjacent normal tissue samples. Scale bar, 40 µm; magnification, 400x. Quantification of the relative expression levels of (B) MTCH2, (C) AIMP2 and (D) claudin‑3 in OC and adjacent normal tissue samples. (E) Data from cBioPortal was used to assess the association between MTCH2 control and MTCH2 mutant groups and the overall survival time of patients with OC. *P<0.05, **P<0.01, ***P<0.001. OC, ovarian cancer; MTCH2, mitochondrial carrier homology 2; AIMP2, aminoacyl transfer RNA synthetase‑interacting multifunctional protein 2.

Article Snippet: The cells were then incu‐ bated with primary antibodies against MTCH2 (1:200; cat no. 16888‐1‐AP; Proteintech Group, Inc.) and AIMP2 (1:100; cat no. 10424‐1‐AP; Proteintech Group, Inc.) at 4 ̊C overnight.

Techniques: Expressing, Immunohistochemical staining, Staining, Control, Mutagenesis

Journal: Oncology letters

Article Title: MTCH2 promotes the malignant progression of ovarian cancer through the upregulation of AIMP2 expression levels, mitochondrial dysfunction and by mediating energy metabolism.

doi: 10.3892/ol.2024.14625

Figure Lengend Snippet: Figure 2. MTCH2 effects on cell invasion and migration of SK‑OV‑3 cells. (A) Representative western blot images and (B) quantification of the validation of MTCH2 siRNA transfection efficiency. (C) Representative western blot images and (D) quantification of the validation of MTCH2‑OE transfection efficiency. (E) Representative wound healing assay images of cell migration following knockdown or overexpression of MTCH2 in SK‑OV‑3 cells and (F) quantification or the relative migratory rate. Scale bar, 400 µm; magnification, 400x. (G) Quantification of the number of invasive cells per field of view and (H) representa‑ tive Transwell assay images of cell migration following knockdown or overexpression of MTCH2 in SK‑OV‑3 cells. Scale bar, 100 µm; magnification, 200x. ***P<0.001. MTCH2, mitochondrial carrier homology 2; AIMP2, aminoacyl transfer RNA synthetase‑interacting multifunctional protein 2; OE, overexpres‑ sion; NC, negative control; siRNA, small interfering RNA.

Article Snippet: The cells were then incu‐ bated with primary antibodies against MTCH2 (1:200; cat no. 16888‐1‐AP; Proteintech Group, Inc.) and AIMP2 (1:100; cat no. 10424‐1‐AP; Proteintech Group, Inc.) at 4 ̊C overnight.

Techniques: Migration, Western Blot, Biomarker Discovery, Transfection, Wound Healing Assay, Knockdown, Over Expression, Transwell Assay, Negative Control, Small Interfering RNA

Journal: Oncology letters

Article Title: MTCH2 promotes the malignant progression of ovarian cancer through the upregulation of AIMP2 expression levels, mitochondrial dysfunction and by mediating energy metabolism.

doi: 10.3892/ol.2024.14625

Figure Lengend Snippet: Figure 3. MTCH2 knockdown and overexpression effects mitochondrial function and ATP production in SK‑OV‑3 cells. (A) Representative images of the ATP fluorescence probe assay in SK‑OV‑3 cells, indicating ATP production of cells following MTCH2 knockdown or overexpression and (B) quantification of the relative fluorescent intensities. Scale bar, 40 µm; magnification, 400x. (C) Representative images of mitochondrial red fluorescence which indicated mitochondrial function cell following knockdown or overexpression of MTCH2 in SK‑OV‑3 cells. Scale bar, 100 µm; magnification, 200x. (D) Quantification of the mitochondrial red fluorescence intensities. **P<0.01 and ***P<0.001. MTCH2, mitochondrial carrier homology 2; AIMP2, aminoacyl transfer RNA synthetase‑interacting multifunctional protein 2; OE, overexpression; NC, negative control.

Article Snippet: The cells were then incu‐ bated with primary antibodies against MTCH2 (1:200; cat no. 16888‐1‐AP; Proteintech Group, Inc.) and AIMP2 (1:100; cat no. 10424‐1‐AP; Proteintech Group, Inc.) at 4 ̊C overnight.

Techniques: Knockdown, Over Expression, Fluorescence, Negative Control

Journal: Oncology letters

Article Title: MTCH2 promotes the malignant progression of ovarian cancer through the upregulation of AIMP2 expression levels, mitochondrial dysfunction and by mediating energy metabolism.

doi: 10.3892/ol.2024.14625

Figure Lengend Snippet: Figure 4. MTCH2 knockdown and overexpression on the proliferation and apoptosis of SK‑OV3 cells. (A) Representative images of EdU assay results, which indicated the proliferation of SK‑OV‑3 cell following knockdown or overexpression of MTCH2. Scale bar, 100 µm; magnification, 200x. (B) Quantification of the EdU-positive cells in these groups. (C) Representative images of GreenNuc™ caspase‑3 activity and annexin V cell apoptosis assay in SK‑OV‑3 cells transfected with MTCH2‑OE and MTCH2‑siRNA compared with NC‑OE and NC‑siRNA, respectively and (D) quantification of the apoptotic cells in these groups. Scale bar, 100 µm; magnification, 200x. *P<0.05 and ***P<0.001. EdU, 5‑ethynyl‑2'‑deoxyuridine; MTCH2, mitochondrial carrier homology 2; AIMP2, aminoacyl transfer RNA synthetase‑interacting multifunctional protein 2; OE, overexpression; NC, negative control; siRNA, small interfering RNA.

Article Snippet: The cells were then incu‐ bated with primary antibodies against MTCH2 (1:200; cat no. 16888‐1‐AP; Proteintech Group, Inc.) and AIMP2 (1:100; cat no. 10424‐1‐AP; Proteintech Group, Inc.) at 4 ̊C overnight.

Techniques: Knockdown, Over Expression, EdU Assay, Activity Assay, Apoptosis Assay, Transfection, Negative Control, Small Interfering RNA

Journal: Oncology letters

Article Title: MTCH2 promotes the malignant progression of ovarian cancer through the upregulation of AIMP2 expression levels, mitochondrial dysfunction and by mediating energy metabolism.

doi: 10.3892/ol.2024.14625

Figure Lengend Snippet: Figure 5. MTCH2 regulated cytoskeletal remodeling and expression levels of claudin‑3 and AIMP2. (A and B) Representative images of the cytoskeleton assay to evaluate the expression of F‑actin in SK‑OV‑3 cells following knockdown or overexpression of MTCH2. The red triangle indicates changes in pseudopodia. Scale bar, 20 µm, magnification, 400x. (C‑E) Representative immunofluorescent images to assess the expression levels of MTCH2 and AIMP2 following knockdown or overexpression of MTCH2. Scale bar, 40 µm; magnification, 400x. (F‑I) Western blots to assess the expression levels of MTCH2, claudin‑3 and AIMP2 in SK‑OV‑3 cells following knockdown or overexpression of MTCH2. **P<0.01 and ***P<0.001. MTCH2, mitochondrial carrier homology 2; AIMP2, aminoacyl transfer RNA synthetase‑interacting multifunctional protein 2; OE, overexpression; NC, negative control.

Article Snippet: The cells were then incu‐ bated with primary antibodies against MTCH2 (1:200; cat no. 16888‐1‐AP; Proteintech Group, Inc.) and AIMP2 (1:100; cat no. 10424‐1‐AP; Proteintech Group, Inc.) at 4 ̊C overnight.

Techniques: Expressing, Knockdown, Over Expression, Western Blot, Negative Control

Journal: Oncology letters

Article Title: MTCH2 promotes the malignant progression of ovarian cancer through the upregulation of AIMP2 expression levels, mitochondrial dysfunction and by mediating energy metabolism.

doi: 10.3892/ol.2024.14625

Figure Lengend Snippet: Figure 6. MTCH2 regulated claudin‑3 expression levels through AIMP2. (A and B) Co‑IP assay verified the binding between MTCH2 and AIMP2 in SK‑OV‑3 cells. (C and D) The efficiency of AIMP2 knockdown was validated using western blotting. (E) The expression levels of (F) MTCH2, (G) claudin‑3 and (H) AIMP2 were detected by western blot following the transfection of NC‑siRNA, AIMP2 siRNA, AIMP2 siRNA2, AIMP2 siRNA + MTCH2 siRNA and AIMP2 siRNA + MTCH2‑OE. *P<0.05, **P<0.01 and ***P<0.001. IP, immunoprecipitation; MTCH2, mitochondrial carrier homology 2; AIMP2, aminoacyl transfer RNA synthetase‑interacting multifunctional protein 2; OE, overexpression; NC, negative control; ns, not significant; siRNA, small interfering RNA.

Article Snippet: The cells were then incu‐ bated with primary antibodies against MTCH2 (1:200; cat no. 16888‐1‐AP; Proteintech Group, Inc.) and AIMP2 (1:100; cat no. 10424‐1‐AP; Proteintech Group, Inc.) at 4 ̊C overnight.

Techniques: Expressing, Co-Immunoprecipitation Assay, Binding Assay, Knockdown, Western Blot, Transfection, Immunoprecipitation, Over Expression, Negative Control, Small Interfering RNA

Journal: bioRxiv

Article Title: A mitochondrial SCF-FBXL4 ubiquitin E3 ligase complex restrains excessive mitophagy to prevent mitochondrial disease

doi: 10.1101/2022.11.11.516094

Figure Lengend Snippet: (A) Volcano plot of the screen result of mitophagy regulators in FBXL4-KO HeLa cells. Mitochondrial outer-membrane protein MTCH2 and mitophagy receptors BNIP3 and NIX are highlighted in red. (B) Immunoblot analysis of the indicated HeLa cells. WT and FBXL4-KO HeLa cells were rescued with vector or FBXL4-FLAG. (C) qPCR analysis of BNIP3 and NIX in the indicated HeLa cells. (D) Immunoblot analysis of the indicated HeLa cells. B: BNIP3; N: NIX. Two clones were shown for each double and triple knockout cells. (E and F) Representative live cell imaging (E) and quantitative analysis (F) of mitophagy levels in the indicated HeLa cells. n: number of cells analyzed (left); number of imaging areas analyzed (right). (G) Representative FACS analysis of mitophagy levels in the indicated HeLa cells. (H) FACS-based quantitative analysis of mitophagy levels in the indicated HeLa cells. Data are mean + SD from three biological replicates (C, H). Statistics: two-tailed unpaired Student’s t-test (C, F and H); ** P < 0.01; *** P < 0.001.

Article Snippet: BNIP3 (Abcam, ab109362; Abcam, ab10433; Cell signaling, 3769S), NIX (Cell signaling, 12396S), FUNDC1 (From Quan Chen lab), FLAG (Sigma-Aldrich, F1804), HA (Sigma-Aldrich, H6533), ACTIN (Sigma-Aldrich, A2066), TOM70 (Proteintech, 14528-1-AP), TOM20 (ABclonal, A19403), SMAC (Cell signaling, 2954S), MITOFILIN (Proteintech, 10179-1-AP), HSP60 (Cell signaling, 4870S), TIMM23(Proteintech, 11123-1-AP), SKP1 (Cell signaling, 2156S), CUL1 (Proteintech, 12895-1-AP), Total OXPHOS Cocktail (Abcam, ab110411), Cytochrome C (BD Biosciences, 556433), mCherry (Easybio, BE2026), MTCH2 (Proteintech, 16888-1-AP), BECLIN1 (Cell signaling, 3495T), FIP200 (ABclonal, A14685), TFAM (Abcam, ab131607), LONP1 (Proteintech, 15440-1-AP), VDAC (Cell signaling, 4866S), Donkey polyclonal anti-Rabbit IgG (H+L),HRP-conjugated (Jackson ImmunoResearch, 711-035-152), Donkey polyclonal anti-Mouse IgG (H+L), HRP-conjugated (Jackson ImmunoResearch, 711-035-151), Goat polyclonal anti-Mouse IgG, Fcγ fragment specific, HRP-conjugated (Jackson ImmunoResearch, 115-035-008), Goat polyclonal anti-Mouse IgG, light chain specific, HRP-conjugated (Jackson ImmunoResearch, 115-035-174).

Techniques: Membrane, Western Blot, Plasmid Preparation, Clone Assay, Triple Knockout, Live Cell Imaging, Imaging, Two Tailed Test

Journal: bioRxiv

Article Title: A mitochondrial SCF-FBXL4 ubiquitin E3 ligase complex restrains excessive mitophagy to prevent mitochondrial disease

doi: 10.1101/2022.11.11.516094

Figure Lengend Snippet: (A and B) Representative FACS analysis (A) and quantitative analysis (B) of mitophagy levels in the indicated HeLa cells. Two independent sgRNAs for MTCH2 were used. Data are mean + SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test; *** P < 0.001. (C) Immunoblot analysis of the indicated HeLa cells. *: non-specific bands.

Article Snippet: BNIP3 (Abcam, ab109362; Abcam, ab10433; Cell signaling, 3769S), NIX (Cell signaling, 12396S), FUNDC1 (From Quan Chen lab), FLAG (Sigma-Aldrich, F1804), HA (Sigma-Aldrich, H6533), ACTIN (Sigma-Aldrich, A2066), TOM70 (Proteintech, 14528-1-AP), TOM20 (ABclonal, A19403), SMAC (Cell signaling, 2954S), MITOFILIN (Proteintech, 10179-1-AP), HSP60 (Cell signaling, 4870S), TIMM23(Proteintech, 11123-1-AP), SKP1 (Cell signaling, 2156S), CUL1 (Proteintech, 12895-1-AP), Total OXPHOS Cocktail (Abcam, ab110411), Cytochrome C (BD Biosciences, 556433), mCherry (Easybio, BE2026), MTCH2 (Proteintech, 16888-1-AP), BECLIN1 (Cell signaling, 3495T), FIP200 (ABclonal, A14685), TFAM (Abcam, ab131607), LONP1 (Proteintech, 15440-1-AP), VDAC (Cell signaling, 4866S), Donkey polyclonal anti-Rabbit IgG (H+L),HRP-conjugated (Jackson ImmunoResearch, 711-035-152), Donkey polyclonal anti-Mouse IgG (H+L), HRP-conjugated (Jackson ImmunoResearch, 711-035-151), Goat polyclonal anti-Mouse IgG, Fcγ fragment specific, HRP-conjugated (Jackson ImmunoResearch, 115-035-008), Goat polyclonal anti-Mouse IgG, light chain specific, HRP-conjugated (Jackson ImmunoResearch, 115-035-174).

Techniques: Two Tailed Test, Western Blot

Journal: bioRxiv

Article Title: MTCH2 is a mitochondrial outer membrane protein insertase

doi: 10.1101/2022.09.15.508165

Figure Lengend Snippet: (A) Additional substrates were tested in parallel with the data displayed in , where the indicated TMDs and flanking residues were fused to VHP and examined for insertion into wild type (WT) and MTCH2 knockout (KO) mitochondria. As a control, a canonical IMS localized TOM substrate derived from a fusion of the cytochrome b targeting sequence to DHFR (cyt. b 2 -DHFR) was tested in parallel. (B) As in except using mitochondria isolated from K562 CRISPRi cells expressing either a non-targeting (nt) or MTCH2 targeting (kd) sgRNA. A panel of mitochondrial TAs was tested in parallel with a matrix (Su9-DHFR) and IMS (cyt. b 2 -DHFR) targeted control that rely on the TOM pathway, which were unaffected. *Denotes the folded, protease resistant, DHFR domain that migrates immediately below the mature, matrix targeted control, and is visible in the absence of mitochondria. (C) Note that because some residual ER is present in the enriched mitochondrial membranes used in these insertion reactions (see ), for those substrates that are dual localized, we cannot formally differentiate between insertion into mitochondria vs ER in this assay alone. To address this, we have tested the complete TA panel in an EMC knockdown background, which we found eliminated mistargeting of mitochondrial TAs to the ER . Therefore, we perform insertion assays as in (A) except using (see ) mitochondria isolated from either EMC2 knock down or EMC2 and MTCH2 knock down K562 CRISPRi cells. The insertion defect in the absence of MTCH2 was enhanced for some substrates (see CYB5B) when entry to the ER was blocked, suggesting these substrates may be partially dual localized under these conditions.

Article Snippet: The following antibodies were used in this study: MTCH2 (ab113707, Abcam, UK); FUNDC1 (OAAB12808, Aviva systems biology, USA); CYB5B (HPA007893, Atlas antibodies, USA); MIRO2 (RHOT2) (ab224089, Abcam, UK); CYC1 (4272, Cell signaling technology, USA); SYNJ2BP (OMP25) (15666-1-AP, Proteintech, USA); EMC3 (67205, Proteintech, USA); VDAC1 (sc-390996, Santa Cruz Biotech, USA); mitofilin (ab110329, Abcam, UK); SAMM50 (ab133709, Abcam, UK); ATP13A1 (16244-1-AP, Proteintech, USA); tubulin (T9026, Sigma-Aldrich, USA).

Techniques: Knock-Out, Control, Derivative Assay, Sequencing, Isolation, Expressing, Knockdown