Journal: The Journal of Headache and Pain

Article Title: S100A11 regulates microglial inflammatory response in neuropathic pain via H3K27ac-TFEB-mitochondrial autophagy axis

doi: 10.1186/s10194-026-02328-9

Figure Lengend Snippet: S100A11 knockdown alleviates microglial inflammation by inhibiting mitophagy through the TFEB/H3K27ac axis. A-B Western blot analysis of TFEB, TFEC, and TFE3 protein expression levels in the cytoplasm and nucleus of primary microglia, along with the corresponding detection and quantification plots ( D-F , for three independent experiments, statistical significance was determined by one-way ANOVA with appropriate post hoc tests). C Immunofluorescence staining of TFEB in primary microglia. G Western blot analysis of pink1, Parkin, P62, LC3II protein levels of primary microglia; H-K Relative levels normalized to COX IV were quantified (for three independent experiments, statistical significance was determined by one-way ANOVA with appropriate post hoc tests). M Labeling of mitochondria (red) in primary microglia with Tom20; immunofluorescence staining for LC3B (green). Quantification of the mean fluorescence intensity of LC3 is shown in panel L . and relative fluorescence intensity was quantified in panel L ( n = 3). N Western blot analysis of H4K16ac, H4K12ac, H3K27ac, H3K18ac, H3K9ac protein levels of microglia; O-S Relative levels normalized to histone H3 were quantified (for three independent experiments, statistical significance was determined by two-way ANOVA with appropriate post hoc tests). T Western blot analysis of Pink1, Parkin, P62, LC3II protein levels of primary microglia; U-X Relative levels normalized to COX IV were quantified (for three independent experiments, statistical significance was determined by one-way ANOVA with appropriate post hoc tests). Z Labeling of mitochondria (red) in primary microglia with Tom20; immunofluorescence staining for LC3B (green), and relative fluorescence intensity was quantified in panel Y ( n = 3). Scale bars as indicated. Data are presented as mean ± SEM. * P < 0.05 , ** P < 0.01 , *** P < 0.001

Article Snippet: Membranes were blocked with 5% skim milk for 60 min, rinsed with TBST, and incubated with primary antibodies at 4 °C overnight (β-actin: Proteintech, 66009-1-Ig, 1:10000; S100A11: Abcam, ab169530, 1:1000; NLRP3: AdipoGen, AG-20B-0014, 1:1000; GSDMD-N: Abcam, ab215203, 1:1000; ASC: Wanleibio, WL02462, 1:500; Caspase1: CST, 89332, 1:1000; Bax: Proteintech, 50599-2-Ig, 1:2000; Bcl-2: abclone, A19693, 1:2000; COXIV: Proteintech, 11242-1-AP, 1:1000; LC3: Proteintech, 14600-1-AP, 1:2000; P62: Proteintech, 18420-1-AP, 1:2000; Parkin: Proteintech, 14060-1-AP, 1:1000; Pink1: Proteintech, 23274-1-AP, 1:2000; H3: Proteintech, 17168-1-AP, 1:2000; TFEB: Proteintech, 13372-1-AP, 1:1000; TFEC: Proteintech, 13547-1-AP, 1:1000; TFE3: Proteintech, 14480-1-AP, 1:1000; H3K9ac: CST, 9649, 1:1000; H3K18ac: CST, 13998, 1:1000; H3K27ac: CST, 8173, 1:1000; H4K12ac; CST, 13944, 1:1000; H4K16ac: CST, 13534, 1:1000).

Techniques: Knockdown, Western Blot, Expressing, Immunofluorescence, Staining, Labeling, Fluorescence

Journal: Autophagy

Article Title: HSPA5 negatively regulates lysosomal activity through ubiquitination of MUL1 in head and neck cancer

doi: 10.1080/15548627.2017.1414126

Figure Lengend Snippet: HSPA5 has a pivotal role in NTS-mediated regulation of lysosomal activity. (A) FaDu cells were seeded in a 48-well plate and treated with NTS for the indicated times. Cells viability was measured by the MTT assay (n = 6). Data are means ± SD. Asterisks indicate statistically significant differences (P < 0.05). (B) FaDu cells were treated with or without NTS for 24 h in the absence of serum and lysosomal activity was measured by FACS analysis (n = 3). Data are means ± SD. Asterisks indicate statistically significant differences (P < 0.05) (*, vs O H). (C) GFP-MAP1LC3-II puncta was accumulated by inhibiting lysosomes. NH4Cl (10 mM), E-64 (10 μM), and NTS were added to GFP-MAP1LC3-II-transfected cells for 24 h in the absence of serum. GFP-MAP1LC3-II puncta were observed by fluorescence microscopy. Scale bar: 20 μm. (D) HSPA5 overexpression inhibits NTS-induced downregulation of lysosome-related proteins or gene expression. FaDu cells were transfected with or without pcDNA3.1-HSPA5 for 24 h, and then cells were treated with NTS for the indicated times. The indicated proteins or gene expression were determined by western blot assay or RT-PCR. (E to G) HSPA5 overexpression inhibits NTS-induced downregulation of lysosomal activity. HSPA5-transfected or nontransfected cells were treated with NTS for 24 h and then lysosomal activity was assessed with FACS analysis (E), TFEB transcription factor activity assay (F) or CTSD (cathepsin D) activity assay (G). (E, F and G) Data are means ± SD. Asterisks indicate statistically significant differences (n = 3, P < 0.05).

Article Snippet: Lysosomal activity was determined with 3 kinds of methods such as LysoSensor™Green DND-189 (Thermo Fisher, L-7535), TFEB transcription factor activity assay (RayBiotech, TFEH-TFEB) or CTSD activity assay (abcam, ab65302).

Techniques: Activity Assay, MTT Assay, Transfection, Fluorescence, Microscopy, Over Expression, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction