Journal: BMC Immunology

Article Title: SIRT5 affects lipid accumulation in cells of a metabolic-associated fatty liver disease model by regulating the succinylation status of HSPD1

doi: 10.1186/s12865-025-00776-7

Figure Lengend Snippet: SIRT5 leads to HSPD1 desuccinylation. A - B Western blot was carried out to evaluate the HSPD1 protein levels and the succinylation level of HSPD1. C - D co-IP was performed to verify the interaction between SIRT5 and HSPD1. E The co-locationof SIRT5 and HSPD1 obtained by the immunofluorescence method. F SPR analysis of the direct binding between SIRT5 and HSPD1. G - H Representative protein bands and quantitative analysis of protein degradation of HSPD1 when SIRT5 was overexpressed. *** p < 0.001

Article Snippet: Rabbit anti-KAT2A monoclonal antibody (#3305, 1:1000), rabbit anti-KAT3B monoclonal antibody (#4771, 1:1000), anti-CPT1A antibody (#97361, 1:1000), anti-SIRT5 antibody (#8779, 1:1000), anti-SIRT7 antibody (#5360, 1:1000), rabbit anti-HSPD1 monoclonal antibody (#12165, 1:1000), and anti-GAPDH antibody (#2118, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Immunofluorescence, Binding Assay

Journal: BMC Immunology

Article Title: SIRT5 affects lipid accumulation in cells of a metabolic-associated fatty liver disease model by regulating the succinylation status of HSPD1

doi: 10.1186/s12865-025-00776-7

Figure Lengend Snippet: SIRT5-mediated hepatic steatosis function is reversed by the elevation of HSPD1. A PCR analysis was carried out to evaluate the mRNA levels of HSPD1 in HepG2 and Huh7 cells after transfection. *** p < 0.001. B - C TG and TC content of HepG2 and Huh7 cells of each group were accessed by ELISA. *** p < 0.001 (vs. control), # p < 0.05, ## p < 0.01, ### p < 0.001 (vs. FFA + oe-NC). D - G Images and quantitative analysis of Oil red O staining and BODIPY staining of HepG2 and Huh7 cells. *** p < 0.001 (vs. control), # p < 0.05, ### p < 0.001 (vs. FFA + oe-NC)

Article Snippet: Rabbit anti-KAT2A monoclonal antibody (#3305, 1:1000), rabbit anti-KAT3B monoclonal antibody (#4771, 1:1000), anti-CPT1A antibody (#97361, 1:1000), anti-SIRT5 antibody (#8779, 1:1000), anti-SIRT7 antibody (#5360, 1:1000), rabbit anti-HSPD1 monoclonal antibody (#12165, 1:1000), and anti-GAPDH antibody (#2118, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Control, Staining

Journal: bioRxiv

Article Title: Hypoxia-induced mitoROS triggers M1 linear ubiquitin chains and activates NF-κB signaling

doi: 10.1101/2025.11.06.686916

Figure Lengend Snippet: A . Representative images of HeLa cells stably expressing matrix-targeted-Keima-XL (mitoKeima), grown under normoxic or hypoxic conditions. Functional mitophagy was monitored by dual excitation of mitokeima with 440 nm and 555 nm lasers and evaluating the relative shift in mitoKeima excitation. B . Quantification of mitophagy by the average number of red puncta per cell in ten independent microscopic fields treated as in panel A. The data is represented as mean±SEM of triplicate experiments. C . Immunoblots of total ubiquitin, BNIP3, and HSP60, in crude mitochondria fractions isolated from HeLa cells that were exposed to hypoxia as per the indicated time points. D . Representative images of WT and 5KO (deleted for p62/SQSTM1, NDP52, Optineurine, NBR1 and TAX1BP1) cells stably expressing mitoKeima exposed to hypoxia or normoxia. Functional mitophagy was evaluated by the relative shift in mitoKeima excitation. Scale bar: 10 µm. E . Quantification of mitophagy by the average number of red puncta per cell in ten independent microscopic fields treated as in panel D. The data is represented as mean±SEM of triplicate experiments. F . Immunoblots of clarified whole cell extracts (WCE) from cells stably expressing mitoKeima and exposed to hypoxia for the indicated time points. Mitophagy was estimated by immunoblotting the mitoKeima cleavage using anti-Keima Red antibody: P, Precursor; C, Cleaved. G . Crude mitochondria were isolated from HeLa cells following exposure to hypoxic and Normoxic conditions, and proteins were quantified through Tandem mass tag (TMT) Mass Spectrometry. Quantified levels (average from quadruple samples) of relevant proteins are displayed in the bar graph as mean±SEM. H . Representative images of WT and DKO (deleted for BNIP3 and NIX) HeLa cells stably expressing mitoKeima, grown under normoxic or hypoxic conditions. Functional mitophagy was evaluated by the mitoKeima assay as mentioned above. Scale bar: 10 µm. I . Quantification of mitophagy by the average number of red puncta per cell in ten independent microscopic fields treated as in panel H and the data is represented as mean±SEM of triplicate experiments.

Article Snippet: Cleaved caspase-3 (Cat#9664) and HSP60 Rb (Cat#12165) antibodies were purchased from Cell Signaling.

Techniques: Stable Transfection, Expressing, Functional Assay, Western Blot, Ubiquitin Proteomics, Isolation, Mass Spectrometry

Journal: bioRxiv

Article Title: Hypoxia-induced mitoROS triggers M1 linear ubiquitin chains and activates NF-κB signaling

doi: 10.1101/2025.11.06.686916

Figure Lengend Snippet: A . A schematic representation of mitochondrial KεGG enrichment. Isolated crude mitochondria are differentially isotopically labelled (Tandem Mass Tagged), and then subject to tryptic digestion. Peptides with residual diglycine modifications on Lysine side chains (KεGG) are selectively enriched with a specific antibody for KεGG. Peptides are analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify different types of isopeptide linkages on ubiquitin-derived peptides. Note that the peptide products of trypsin digestion of M1 linear ubiquitin cannot be detected with KεGG antibody. B . Changes in ubiquitin landscape at mitochondria following hypoxia. Crude mitochondria isolated from hypoxic and normoxic cells were differently labeled and analyzed as in Scheme A. Ratios of linkage abundance between hypoxic and normoxic conditions are plotted for each linkage type. K6, K11, K27, K48, and K63 represent ubiquitination on a single lysine of ubiquitin, whereas K11;K27, K29;K33 represent dual modifications on a single peptide. M1/K6 reflects a KεGG at the sixth lysine of the ubiquitin sequence, to which two glycine’s are appended to the N-terminus. C . Crude mitochondria were isolated from WT HeLa cells following exposure to hypoxia at 1% for 24 hours. Normoxic (N) and hypoxic (H) crude mitochondrial extracts were immunoblotted with ubiquitin linkage-specific antibodies. HSP60 serves as a loading control for mitochondria. D . Clarified whole cell extract (WCE) from cells treated as in C was fractionated into cytosolic and crude mitochondria. The samples were immunoblotted for M1 Linear-Ub. HSP60 serves as a loading control for mitochondria.

Article Snippet: Cleaved caspase-3 (Cat#9664) and HSP60 Rb (Cat#12165) antibodies were purchased from Cell Signaling.

Techniques: Isolation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Ubiquitin Proteomics, Derivative Assay, Labeling, Sequencing, Control

Journal: bioRxiv

Article Title: Hypoxia-induced mitoROS triggers M1 linear ubiquitin chains and activates NF-κB signaling

doi: 10.1101/2025.11.06.686916

Figure Lengend Snippet: A . The cells either grown under normoxia or exposed to hypoxia (1% for 24H) were stained with MitoSOX Red dye (detecting superoxide in the mitochondrial context) and analyzed by flow cytometry. Histogram analysis was performed to ascertain the percentage of cell population with MitoSOX Red staining. B . HeLa cells were grown under normoxia, hypoxia (1% for 24H) or hypoxia+MQ (1 µM MQ) were stained with MitoTracker Green and MitoSOX Red dyes and imaged by confocal microscope. C . Histograms of (B) showing the MitoSOX intensity fold change compare to normoxia. ** p<0.01. D . Crude mitochondria were isolated from WT HeLa cells grown in normoxia, hypoxia, hypoxia + MQ (1 µM) or Hypoxia + Gliotoxin (1 µM). The extracts were analyzed by immunoblotting for the detection of total ubiquitin linkages. HSP60 confirms equal amount of loaded mitochondrial fraction. E . HeLa cells growing in normoxia, hypoxia, hypoxia + MQ (1 µM), hypoxia + Hoipin-8 (30 µM), normoxia + TNF (20 ng/mL) or normoxia +TNF + Hoipin-8. TNF treatment serves as a positive control for the detection of M1-Ub. All samples were stained with DAPI (nucleus), linear-ubiquitin and mitochondria and imaged by confocal microscope.

Article Snippet: Cleaved caspase-3 (Cat#9664) and HSP60 Rb (Cat#12165) antibodies were purchased from Cell Signaling.

Techniques: Staining, Flow Cytometry, Microscopy, Isolation, Western Blot, Ubiquitin Proteomics, Positive Control