Journal: Molecular Neurobiology

Article Title: Oxymatrine Alleviates Cerebral Ischemia/Reperfusion Injury By Targeting HDAC1 to Regulate Mitochondria-Related Autophagy and Oxidative Stress

doi: 10.1007/s12035-025-05423-1

Figure Lengend Snippet: Oxymatrine ameliorates excessive autophagy induced by cerebral I/R in mice. A Western blotting results. B – G Western blotting analyses of LC3-II/I, PINK1, Parkin, NBR1, Beclin-1 and P62 in different groups when normalized to GAPDH. H – K Representative images showing double immunofluorescence staining for autophagy-related proteins (LC3, PINK1, Beclin1, NBR1) co-localized with neurons in different groups. Scale bar = 20 μm. # p < 0.05 vs. Sham, * p < 0.05 vs. I/R

Article Snippet: Protein extracts were separated on 10%–12% SDS-PAGE gels, transferred to PVDF membranes (Millipore), and probed with the following primary antibodies: Apaf-1 (1:1000, ABclonal, Wuhan, China), cleaved-caspase3 (1:500, Proteintech, Wuhan, China), Fis1 (1:2000, Proteintech, Wuhan, China), NBR1 (1:1000, Proteintech, Wuhan, China), Mfn2 (1:4000, Proteintech, Wuhan, China), LC3 (1:1000, ABclonal, Wuhan, China), HDAC1(1:2000, ABclonal, Wuhan, China), PINK1 (1:800, ABclonal, Wuhan, China), Parkin (1:2000, Proteintech, Wuhan, China), Beclin1 (1:2000, Proteintech, Wuhan, China), p62 (1:2000,Proteintech, Wuhan, China).

Techniques: Western Blot, Double Immunofluorescence Staining

Journal: Molecular Neurobiology

Article Title: Oxymatrine Alleviates Cerebral Ischemia/Reperfusion Injury By Targeting HDAC1 to Regulate Mitochondria-Related Autophagy and Oxidative Stress

doi: 10.1007/s12035-025-05423-1

Figure Lengend Snippet: Oxymatrine restores mitochondrial homeostasis via coordinated modulation of fission/fusion balance and autophagy in glutamate-treated HT22 cells. A Western blotting for Fis1 and Mfn2. B , C Quantification of western blotting. D Immunofluorescence analysis showing the expression of LC3, PINK1, Beclin-1, NBR1 and P62. Scale bar = 20 μm. E Western blotting for LC3-II/I, PINK1, Beclin-1, NBR1 and P62. F – K Quantification of western blotting. # p < 0.05 vs. Control, * p < 0.05 vs. Glu

Article Snippet: Protein extracts were separated on 10%–12% SDS-PAGE gels, transferred to PVDF membranes (Millipore), and probed with the following primary antibodies: Apaf-1 (1:1000, ABclonal, Wuhan, China), cleaved-caspase3 (1:500, Proteintech, Wuhan, China), Fis1 (1:2000, Proteintech, Wuhan, China), NBR1 (1:1000, Proteintech, Wuhan, China), Mfn2 (1:4000, Proteintech, Wuhan, China), LC3 (1:1000, ABclonal, Wuhan, China), HDAC1(1:2000, ABclonal, Wuhan, China), PINK1 (1:800, ABclonal, Wuhan, China), Parkin (1:2000, Proteintech, Wuhan, China), Beclin1 (1:2000, Proteintech, Wuhan, China), p62 (1:2000,Proteintech, Wuhan, China).

Techniques: Western Blot, Immunofluorescence, Expressing, Control

Journal: Autophagy

Article Title: The BAX-binding protein MOAP1 associates with LC3 and promotes closure of the phagophore

doi: 10.1080/15548627.2021.1896157

Figure Lengend Snippet: MOAP1 is dispensable during the early steps of autophagy signaling involving initiation and elongation of phagophore. (A and B) Formation of ATG5 or p-ATG16L1 puncta is unaffected in the absence of MOAP1 in cells treated with EBSS. The MOAP1 +/+ and MOAP1 -/- cells were subjected to EBSS treatment for the indicated time periods. The cells were then harvested for immunofluorescence staining by detecting the presence of GFP-ATG5 (A) or p-ATG16L1 puncta (labeled with Cy3) (B) to monitor the formation process of autophagosome during autophagy signaling. Scale bar: 10μm. ns, not significant (Student’s t test). (C) Loss of MOAP1 does not affect conjugation of ATG12–ATG5-ATG16L1 complex. The MOAP1 +/+ and MOAP1 -/- HeLa cells were harvested under basal condition or after 2 h of EBSS treatment before being subjected to immunofluorescence staining. GFP-ATG5 or Cy3 labeled-p-ATG16L1 puncta were used as markers for detecting the formation of ATG5-12-16 L1 complex. Scale bar: 5μm. (D) Loss of MOAP1 does not affect the interaction between ATG5-12 and ATG16L1 complex. Plasmid encoding MYC-ATG5 or ATG12 was transfected into the MOAP1 +/+ or MOAP1 -/- HeLa cells. After 24 h, the transfected cells were subjected to immunoprecipitation to evaluate the interaction between ATG16L1 and ATG12–ATG5. Arrows: indicated protein signal; Asterisk: IgG signal. (E) LC3 conjugation process induced by EBSS treatment is unaffected by the absence of MOAP1 . Total cell lysates from the MOAP1 +/+ and MOAP1 -/- HeLa cells were harvested after 6 h of EBSS treatment. BECN1, ATG7, ATG5-12 and LC3 are the markers for the upstream signaling events during autophagic induction whereas SQSTM1 and NBR1 are the autophagy substrates. (F) Low number of LC3 puncta in the MOAP1-deficient EBSS-treated cells. The MOAP1 +/+ and MOAP1 -/- HeLa cells were subjected to EBSS treatment for 30 or 60 min as indicated. At the indicated time points, the treated and untreated MOAP1 +/+ or MOAP1 -/- cells were subjected to immunofluorescence staining. The number of LC3 puncta was then quantified by ImageJ analysis. Scale bar: 10μm. ***p < 0.001, ns, not significant (Student’s t test). (G) Low level of autophagy flux in the absence of MOAP1 during starvation. The MOAP1 +/+ and MOAP1 -/- HeLa cells were transfected with plasmid encoding the GFP-LC3-RFP-LC3G reporter prior to EBSS treatment for 2 h. The level of fluorescence signals was then measured and the ratio of GFP:RFP was then calculated by FACS analysis. Scale bar: 5μm. **p < 0.01, ns, not significant (Student’s t test)

Article Snippet: The primary antibodies used were as follows: ACTB (Sigma-Aldrich, SAB5500001), ATG16L1 (Abcam, ab187671), ATG16L1 (phospho-S278; Abcam, ab192542), ATG5 (nanoTools, 0262–100), ATG7 (Abcam, ab133528), BECN1 (Cell Signaling Technology, 3495), cl-CASP3 (Cleaved-Asp175; Cell Signaling Technology, 9661), FLAG (Sigma-Aldrich, F3165), GST (Santa Cruz Biotechnology, sc-138), HA (Abcam, ab9110), His (Santa Cruz Biotechnology, clone H-3), HSPD1/HSP60 (Santa Cruz Biotechnology, sc-13115), LMNA/lamin A/C (Santa Cruz Biotechnology, sc-376248), LAMP1 (Cell Signaling Technology, 9091), LC3B (Cell Signaling Technology, 2775), MOAP1 (Sigma-Aldrich, HPA000939), MYC (Santa Cruz Biotechnology, sc-40), NBR1 (Novus Biologicals, H00004077-M01), SQSTM1/p62 (Abcam, ab56416), ABCD3/PMP70 (Abcam, ab109448), SEC22B (Abcam, 181076), STX17 (MBL International, PM076), TFR2 (Abcam, 185550), TOMM20 (Santa Cruz Biotechnology, sc-11415), TUBA/tubulin (Santa Cruz Biotechnology, sc-8035), ULK1 (Cell Signaling Technology, 8054), ULK1 (phospho-S317; Cell Signaling Technology, 12753), WIPI2 (Abcam, ab105459).

Techniques: Immunofluorescence, Staining, Labeling, Conjugation Assay, Plasmid Preparation, Transfection, Immunoprecipitation, Fluorescence

Journal: Cancer genetics

Article Title: Germline mutation in the NBR1 gene involved in autophagy detected in a family with renal tumors.

doi: 10.1016/j.cancergen.2021.07.003

Figure Lengend Snippet: Fig. 1. Identification of a germline mutation in the NBR1 gene within a particular family (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.). (A). Pedigree of a French family with familial renal tumors, followed by PREDIR center. Affected patients are in black. Birth date and age of diagnosis are indicated below symbols, with NBR1 status after sequencing of blood DNA: wild-type (WT) or mutated (MUT). (B). Chromatograms showing NBR1 sequence of affected individuals from pedigree showed in A. The arrows show the frameshift mutation (c.1581dupA; p.E528fs ∗17) in patient samples. The chromatograms correspond to germline (left panel) and tumor (right panel) DNA Sanger sequencing. (C). NBR1 immunostaining observed by immunohistochemistry for the three patient tumors and one normal kidney tissue. In the II.2 patient, we observed a weak NBR1 cytoplasmic staining of proximal tubules and podocytes in the normal kidney and a cytoplasmic expression in tumor oncocytic cells in the oncocytic papillary Renal Cell Carcinoma. Immunohistochemistry was similar for all normal kidney tissue analyzed. In the II.1 patient angiomyolipoma, NBR1 expression is present in smooth muscle cells and adipocytes. No expression was noted in the clear-cell Renal Cell Carcinoma in the patient I.2. (D). Schematic representation of NBR1 protein modified from [13] . The 966 amino acids isoform contains Phox and Bem1 domain (PB1), zinc finger (ZZ), 4 tryptophan (FW), glutamic acid rich region (polyE), LC3-interacting motif (LIR in red) and ubiquitin-binding domain (UBA in blue). The arrow indicates the location of the frameshift mutation leading to a truncated isoform of 544 amino acids lacking the polyE, LIR and UBA domains. (E). Chromatograms showing NBR1 sequence of tumor cDNA (after reverse transcription of tumor mRNA) by Sanger sequencing from frozen oncocytic pRCC diagnosed in II.2 patient.

Article Snippet: Then, samples were incubated with the NBR1 antiody (Atlas Antibodies) for 30 min at RT.

Techniques: Mutagenesis, Biomarker Discovery, Sequencing, Immunostaining, Immunohistochemistry, Staining, Expressing, Ubiquitin Proteomics, Binding Assay, Reverse Transcription

Journal: Cancer genetics

Article Title: Germline mutation in the NBR1 gene involved in autophagy detected in a family with renal tumors.

doi: 10.1016/j.cancergen.2021.07.003

Figure Lengend Snippet: Fig. 2. Functional study of the NBR1 mutation. (A). Co-immunoprecipitation of mCherry-NBR1 WT, mCherry-NBR1 E528fs ∗17 and endogenous p62 with GFP-NBR1 or GFP-NBR1 E528fs ∗17. Protein extracts are from HKC8 transfected with the indicated plasmids above immunoblot for 24 h. (B). Overexpression of NBR1 protein carrying the E528fs ∗17 mutation in HKC8 normal renal cell line leads to a delay in p62 degradation induced by starvation. Cells were transfected with empty vector, HA-NBR1 or HA-NBR1 E528fs ∗17. After 24 h of transfection, cells were treated with DMEM (control condition), HBSS (Hanks’ Balanced Salt Solution) for different times. (C). p62 turnover quantification. p62 turnover was analysed by electrophoresis and quantified using Image Quant TL 8.2 software. Relative quantity of p62 was normalized to ß-actin (mean ± s.e.m., n = 3). ∗∗∗p < 0.005. ∗∗p < 0.01. ∗p < 0.05. compared to wild-type NBR1. (D). Cell proliferation experiments using tetrazolium-based MTT assay with cells expressing the empty vector, HA-NBR1 or HA-NBR1 E528fs ∗17. The experiment was per- formed in 786-O (left panel) and ACHN (right panel) cell lines (mean ± s.d., n = 3). ∗∗∗∗p < 0.0 0 05. ∗∗∗p < 0.005. (E). Colony Formation Assay for the growth of 786-O cells transfected with empty vector, HA-NBR1 or HA-NBR1 E528fs ∗17. The colonies (left panel) were counted and compared between groups (right panel) (mean ± s.d., n = 3). ∗∗∗∗p < 0.0 0 05.

Article Snippet: Then, samples were incubated with the NBR1 antiody (Atlas Antibodies) for 30 min at RT.

Techniques: Functional Assay, Mutagenesis, Immunoprecipitation, Transfection, Western Blot, Over Expression, Plasmid Preparation, Control, Electrophoresis, Software, MTT Assay, Expressing, Colony Assay