Journal: Frontiers in Endocrinology

Article Title: Transcriptome Analysis Reveal Candidate Genes and Pathways Responses to Lactate Dehydrogenase Inhibition (Oxamate) in Hyperglycemic Human Renal Proximal Epithelial Tubular Cells

doi: 10.3389/fendo.2022.785605

Figure Lengend Snippet: The protein expression levels of peroxisome proliferator-activated receptor-g coactivator 1α (PGC1α), caspase 3 (CASP3), B-cell lymphoma 2 apoptosis regulator (BCL2), BCL2 associated X apoptosis regulator (BAX), beclin1 (BECN1) and microtubule-associated proteins 1A/1B light chain 3 (MAP1LC3) in HK-2 cells treated with low D-glucose (LG) with different concentrations of oxamate (LGOXA-0mM, LGOXA-20mM, LGOXA-40mM and LGOXA-80mM) and high D-glucose (HG) with different concentrations of oxamate (HGOXA-0mM, HGOXA-20mM, HGOXA-40mM and HGOXA-80mM) for 24 hours. (A) Western blot gels. (B) Relative grey values. * indicates P -value < 0.05 after student’s t -test.

Article Snippet: The primary antibodies [Anti-PGC1α (ab54481), Anti-CASP3 (9665), Anti-BCL2 (ab59348), Anti-BAX (GB11690), Anti-BECN1 (bs-1353R), Anti-MAP1LC3 (14600-1-ap) and Anti-β-actin (GB12001)] and all secondary antibodies were purchased from abcom (Cambridge, MA, USA), Proteintech (Wuhan, China), BIOSS (Boston, Massachusetts, USA) and Servicebio (Wuhan, China), respectively.

Techniques: Expressing, Western Blot

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: Toxicology

Article Title: A protective role of autophagy in fine airborne particulate matter-induced apoptosis in LN-229 cells

doi: 10.1016/j.tox.2022.153271

Figure Lengend Snippet: (A-B) LN-229 cells were treated with 10, 50, and 100 μg/mL of PM2.5 for 24 h. (C-D) Cells were treated with 50 μg/mL of PM2.5 for 6, 12, 24, and 48 h. Cells without treatment were used as control. A and C are single Western blot results of expression of autophagic markers (LC3B-1, LC3B-2, and Beclin 1), while B and D are normalized band densitometry readings of Western blot results from three independent experiments (mean ± SE). (E) Immunofluorescence staining of LC3B in LN-229 cells. (F-I) LN-229 cells were pre-treated with 10 nM bafilomycin A1 (Baf A1) (F-G) or 10 nM rapamycin (Rapa) (H-I) for 4 h prior to treatment with 50 μg/mL of PM2.5 for another 24 h. F and H are single Western blot results, while G and I are normalized band densitometry readings of Western blot results from three independent experiments (mean ± SE). * Significant difference as compared with the control, p < 0.05; # Significant difference as compared with the PM2.5 group, p < 0.05.

Article Snippet: Anti-LC3B (ab51520) and anti-Beclin 1 (ab62557) antibodies were supplied by Abcam (Cambridge, UK).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining

Journal: Toxicology

Article Title: A protective role of autophagy in fine airborne particulate matter-induced apoptosis in LN-229 cells

doi: 10.1016/j.tox.2022.153271

Figure Lengend Snippet: (A-B) LN-229 cells were transfected with 100 nM of Beclin 1 siRNA or control siRNA for 24 h prior to treatment with 50 μg/mL of PM2.5 for another 24 h. (C-D) LN-229 cells were pre-treated with 10 nM bafilomycin A1 (Baf A1) for 4 h prior to treatment with 50 μg/mL of PM2.5 for another 24 h. A and C are single Western blot results, while B and D are normalized band densitometry readings of Western blot results from three independent experiments (mean ± SE). (E-F) Hoechst 33342 staining was performed to identify the apoptotic cells after cells were transfected with Beclin 1 siRNA (E) or treated with Baf A1 (F) prior to 50 μg/mL of PM2.5 exposure for 24 h. * Significant difference as compared with the control that was without any treatments, p < 0.05; # Significant difference as compared with the group with only PM2.5 treatment, p < 0.05.

Article Snippet: Anti-LC3B (ab51520) and anti-Beclin 1 (ab62557) antibodies were supplied by Abcam (Cambridge, UK).

Techniques: Transfection, Western Blot, Staining

Journal: Toxicology

Article Title: A protective role of autophagy in fine airborne particulate matter-induced apoptosis in LN-229 cells

doi: 10.1016/j.tox.2022.153271

Figure Lengend Snippet: LN-229 cells were transfected with 100 nM of Beclin 1 siRNA or control siRNA for 24 h. Western blot was performed to determine the expression of Beclin 1, LC3B-1, and LC3B-2. A is a single Western blot result, while B is normalized band densitometry readings of Western blot results from three independent experiments (mean ± SE). * Significant difference as compared with the control that is without any treat-ments, p < 0.05.

Article Snippet: Anti-LC3B (ab51520) and anti-Beclin 1 (ab62557) antibodies were supplied by Abcam (Cambridge, UK).

Techniques: Transfection, Western Blot, Expressing