Journal: Journal of cell science

Article Title: Rab11FIP1 maintains Rab35 at the intercellular bridge to promote actin removal and abscission.

doi: 10.1242/jcs.244384

Figure Lengend Snippet: Fig. 5. FIP1 is required for maintaining Rab35 at the midbody. (A) Top: representative images of cells progressing through mitosis that stably express either GFP-FIP1B full length (green) or mCherry-Rab35 (yellow). Cells are stained with SiR-tubulin and tubulin signal is shown in magenta. Time zero is set as the frame corresponding to telophase. Arrowheads denote the ICB linking daughter cells or the remnants of the spindle after abscission. Bottom left: normalized average fluorescence intensity of either GFP-FIP1B (green) or mCherry-Rab35 (red) over time at the cell-cell interface between daughter cells, where time point 0 is the first observance of telophase. mCherry-Rab35, n=10; GFP-FIP1B, n=18. Bottom right: normalized average fluorescence intensity of either GFP-FIP1B (green) or mCherry-Rab35 (red) over time at the midbody, where time point 0 is the first observance of telophase. mCherry-Rab35, n=10; GFP-FIP1B, n=18. (B) Representative western blot showing the depletion of FIP1 or Rab35 in the subsequent experiment. FIP1 antibody has non-specific bands and a single asterisk denotes FIP1B, and a double asterisk denotes FIP1C. (C) Top: representative images of cells progressing through mitosis that stably express mCherry-Rab35 (shown in yellow) that were treated with either siNT or siFIP1. Arrowheads denote the location of abscission. Bottom left: normalized average fluorescence intensity of mCherry-Rab35 over time, following either siNT or siFIP1 treatment, at the cell-cell interface between daughter cells, where time point 0 is the first observance of telophase. siNT, n=10; siFIP1, n=8. Bottom right: normalized average fluorescence intensity of mCherry- Rab35 over time, following either siNT or siFIP1 treatment, at the midbody, where time point 0 is the first observance of telophase. siNT, n=10; siFIP1, n=8. Data in A and C are presented as mean±s.e.m. *P<0.05; ***P<0.001; ****P<0.0001 (unpaired two-tailed Student’s t-test). a.u., arbitrary units.

Article Snippet: Antibodies and other staining reagents Primary antibodies used were as follows: Rab11FIP1 (Sigma-Aldrich, HPA025960, used at 1/1000); Rab11FIP2 (Sigma-Aldrich, HPA037726, used at 1/1000); Rab11FIP5 (Sigma-Aldrich, HPA036407, used at 1/1000); Rab11FIP3 (Proteintech, 25843-1-AP, used at 1/100 for immunofluorescence); actin [Millipore (C4), MAB1501, used at 1/10,000]; GFP [Santa Cruz Biotechnology (B-2), sc-9996, used at 1/2000]; GAPDH [Santa Cruz Biotechnology (FL-335), sc-25778, used at 1/1000]; mCherry for immunoprecipitation (Abcam, ab183628, 1 μg used per immunoprecipitation condition); mCherry for western blotting (Abcam, ab167453, used at 1/2000); MICAL1 (Proteintech, 14818-1-AP, used at 1/100 for immunofluorescence); Rab35 (Proteintech, 11329-2-AP, used at 1/1000); MKLP1 (Abcam, ab168964, used at 1/100 for immunofluorescence); CHMP4B (Proteintech, 13683-1-AP, used at 1/100 for immunofluorescence); His-probe [Santa Cruz Biotechnology (H-15), sc-803, used at 1/1000]; β-Tubulin (Abcam, ab6046, used at 1/2000); and α-Tubulin [Cell Signaling Technology (DM1A), 3873, used at 1/1000 for immunofluorescence].

Techniques: Stable Transfection, Staining, Fluorescence, Western Blot, Two Tailed Test

Journal: International Journal of Medical Sciences

Article Title: Macrophagic HDAC3 inhibition ameliorates Dextran Sulfate Sodium induced inflammatory bowel disease through GBP5-NLRP3 pathway

doi: 10.7150/ijms.94592

Figure Lengend Snippet: HDAC3 might upregulate the expression of GBP5 to potentiate NLRP3 inflammasome activation in CX3CR1-positive monocytes in colitis. ( A ) The number of significantly upregulated and downregulated genes in the colonic mucosa or primary microglia from Hdac3 cKO mice identified by RNA sequencing (n=3 for each group). ( B ) Heatmap represents the mRNA levels of the shared genes in the colonic mucosa or primary microglia of each group. ( C ) The protein levels of GBP5, IL-1β, NLRP3 and β-actin in the colonic mucosa from WT (n=4) and Hdac3 cKO (n=3) mice administered with 2.5% DSS/water solution or water solely (n=3) were detected by western blot. ( D ) The protein levels of GBP5, IL-1β, NLRP3 and β-actin in the colonic mucosa from WT mice administered with 2.5% DSS/water solution with or without administration of RGFP966 and from the WT mice administered with water solely were detected by western blot (n=4 for each group).

Article Snippet: Antigen retrieval was performed in 10 mM sodium citrate (pH6.0) at 95 °C for 15 min. GBP5 (#13220-1-AP, 1:100, Proteintech, China) was stained with VECTASTAIN ABC Kit (Vector labratories, USA) according to the protocol of the manufacturer.

Techniques: Expressing, Activation Assay, RNA Sequencing, Western Blot

Journal: International Journal of Medical Sciences

Article Title: Macrophagic HDAC3 inhibition ameliorates Dextran Sulfate Sodium induced inflammatory bowel disease through GBP5-NLRP3 pathway

doi: 10.7150/ijms.94592

Figure Lengend Snippet: HDAC3 potentiated the activation of NLRP3 inflammasomes by promoting IFN-γ induced expression of GBP5. ( A and B ) The protein levels of GBP5 and β-actin or β-tubulin in primary macrophages ( A ) or BV2 microglia ( B ) treated with IFN-γ (100 ng/ml) for 6 h with or without RGFP966 (10 μM) pretreatment were detected by western blot. ( C and D ) The mRNA levels of GBP5 in primary macrophages ( C ) or BV2 microglia ( D ) treated with IFN-γ (100 ng/ml) for 6 h with or without RGFP966 (10 μM) pretreatment were detected by real-time quantitative PCR and normalized to the mRNA levels of β-actin (n=3). ( E ) Schematic diagram of the GBP5 reporter. ( F ) Cells were co-transfected with GBP5 reporter plasmid and TK-Renilla plasmids, treated with RGFP966 (10 μM), RGFP109 (10 μM) or vehicle, and harvested 24 h post-transfection for the dual-luciferase assay (n=3). ( G ) Plasmids encoding Flag-HDAC3 or empty vector were transfected into HEK293T cells with the GBP5 reporter and TK-Renilla reference plasmids, and the cells were harvested for the dual-luciferase assay 24 h post-transfection (n=3). ( H and I ) Primary microglia pretreated with or without RGFP966 (10 μM) for 30 min, were then exposed to IFN-γ (100 ng/ml) for 6 h, and followed by treated with 1 μg/ml LPS for 3.5 h, then stimulated with nigericin for another 30 min and the supernatant and cell were harvested for analysis the protein levels of cleaved IL-1β, cleaved casp1, pro-IL-1β, pro-Casp1, NLRP3, GBP5, ASC and β-actin by western blot ( H ) or enzyme-linked immunosorbent assay (n=3) ( I ). Annotations: the data represent at least two-independent experiments, and * indicates p < 0.05, ** indicates p < 0.01 by ANOVA (for 3 groups comparation) or Student's t-test (for 2 groups comparation).

Article Snippet: Antigen retrieval was performed in 10 mM sodium citrate (pH6.0) at 95 °C for 15 min. GBP5 (#13220-1-AP, 1:100, Proteintech, China) was stained with VECTASTAIN ABC Kit (Vector labratories, USA) according to the protocol of the manufacturer.

Techniques: Activation Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Luciferase, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Medical Sciences

Article Title: Macrophagic HDAC3 inhibition ameliorates Dextran Sulfate Sodium induced inflammatory bowel disease through GBP5-NLRP3 pathway

doi: 10.7150/ijms.94592

Figure Lengend Snippet: Conditional knockout of Hdac3 in macrophages attenuated DSS-induced colitis. ( A ) The bodyweight change ratio of WT (n=10) and Hdac3 mKO (n=10) mice administered with 2.5% DSS/water solution. ( B and C ) The length of colon from WT (n=7) and Hdac3 mKO (n=7) mice administered with 2.5% DSS were determined and analyzed. ( D and E ) Typical H&E staining of colon tissue sections from WT (n=7) and Hdac3 mKO (n=7) mice administered with 2.5% DSS/water solution and the percentage of the inflammatory cell infiltrated and structure damaged area were analyzed. ( F and G ) Typical AB-PAS staining of colon tissue sections from WT (n=7) and Hdac3 mKO (n=7) mice administered with 2.5% DSS/water solution. ( H ) The expression of pro-IL-1β, GBP5 and β-actin in the colon from WT (n=7) and Hdac3 mKO (n=7) mice administered with 2.5% DSS/water solution were determined by western blot. Annotations: * indicates p < 0.05, ** indicates p < 0.01 by Student's t-test (for 2 groups comparation).

Article Snippet: Antigen retrieval was performed in 10 mM sodium citrate (pH6.0) at 95 °C for 15 min. GBP5 (#13220-1-AP, 1:100, Proteintech, China) was stained with VECTASTAIN ABC Kit (Vector labratories, USA) according to the protocol of the manufacturer.

Techniques: Knock-Out, Staining, Expressing, Western Blot

Journal: International Journal of Medical Sciences

Article Title: Macrophagic HDAC3 inhibition ameliorates Dextran Sulfate Sodium induced inflammatory bowel disease through GBP5-NLRP3 pathway

doi: 10.7150/ijms.94592

Figure Lengend Snippet: Schematic graph of mechanism of Hdac3 deficiency in macrophages that alleviates the disease severity of colitis. During the development of inflammatory bowel disease (IBD), microbiota activate the expression of IFN-γ via toll-like receptors, then the IFN-γ is secreted to induce the expression of anti-infection genes. Gbp5 is one of its downstream genes that was found to be regulated by HDAC3 in our study. Conditional knockout of Hdac3 in CX3CR1-positive monocytes (including macrophages) or inhibition of HDAC3 by RGFP966 attenuated the disease severity and inflammation of DSS-induced colitis. In addition, inhibition of HDAC3 resulted in decreased transcriptional activity of IFN-γ induced expression of GBP5, and restrained GBP5-dependent activation of NLRP3 inflammasome. In conclusion, inhibition of HDAC3 in macrophages could ameliorate the disease severity and inflammatory response in colitis by regulating GBP5-NLRP3 axis, identifying a new therapeutic avenue for the treatment of colitis.

Article Snippet: Antigen retrieval was performed in 10 mM sodium citrate (pH6.0) at 95 °C for 15 min. GBP5 (#13220-1-AP, 1:100, Proteintech, China) was stained with VECTASTAIN ABC Kit (Vector labratories, USA) according to the protocol of the manufacturer.

Techniques: Expressing, Infection, Knock-Out, Inhibition, Activity Assay, Activation Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Structural Domain Analysis of Heparin and Heparan Sulfate Combined With Label-Free Quantitative Proteomics to Elucidate Their Functional Diversity in Liver Cancer and APAP-Induced Liver Injury

doi: 10.1016/j.mcpro.2025.101090

Figure Lengend Snippet: Functional enrichment and interaction analysis of differential proteins. A , functional enrichment and interaction network of differential proteins in HP group. B , functional enrichment and interaction network of differential proteins in HS group. C , Western blotting measurement of DIRAS2 expression in control, HP, and HS groups. D , BLI characterization of HP and HS interactions with DIRAS2 protein. Data statistic analysis: ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 (mean ± SD, n = 3). BLI, biolayer interferometry; DIRAS2, DIRAS family GTPase 2; HP, heparin; HS, heparin sulfate.

Article Snippet: DIRAS2 polyclonal antibody, GAPDH monoclonal antibody, horseradish peroxidase (HRP)–conjugated AffiniPure goat anti-rabbit IgG (H + L), HRP-conjugated AffiniPure goat anti-Mouse IgG (H + L), and other chemicals and reagents for Western blotting were purchased from Proteintech.

Techniques: Functional Assay, Western Blot, Expressing, Control

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Structural Domain Analysis of Heparin and Heparan Sulfate Combined With Label-Free Quantitative Proteomics to Elucidate Their Functional Diversity in Liver Cancer and APAP-Induced Liver Injury

doi: 10.1016/j.mcpro.2025.101090

Figure Lengend Snippet: Characterization of the binding sites involved in the interaction between HP–HS decasaccharide (derived from PDB: 1HPN ) and DIRAS2 protein (derived from PDB: 2ERX ) using molecular docking. A , the conformation of the DIRAS2 protein. B , the same binding region of DIRAS2 with HP and HS chains. C , the interactional contribution of DIRAS2 and HP binding motif. D , the interactional contribution of DIRAS2 and HS binding motif. Notes: Orange represented attractive charge, and green represented conventional hydrogen bond. DIRAS2, DIRAS family GTPase 2; HP, heparin; HS, heparan sulfate; PDB, Protein Data Bank.

Article Snippet: DIRAS2 polyclonal antibody, GAPDH monoclonal antibody, horseradish peroxidase (HRP)–conjugated AffiniPure goat anti-rabbit IgG (H + L), HRP-conjugated AffiniPure goat anti-Mouse IgG (H + L), and other chemicals and reagents for Western blotting were purchased from Proteintech.

Techniques: Binding Assay, Derivative Assay