TFEB Antibody - #AF7015

Fig. 4. Disorder of normal cell fate and behavior that induced by the up-regulated exogenous ROS associated with HEPES. (A) Viability of L-02 cells cultured in RPMI 1640 mediums that supplied with 25 mM of representative Good's buffers (HEPES, POPSO, MOPSO, and Tricine) and zwitterionic polybetaines (pSBMA, pMPC, and pCBMA). RPMI 1640 medium without buffer was set as the negative control. (B) Live/Dead staining of L-02 cells cultured in HEPES/RPMI 1640 and pCBMA/RPMI 1640 mediums under visible light illumination of the ordinary clean bench (1 mW/cm2, 3 h). All scale bars are 100 μm. (C) LysoTrackerRed staining of Ana-1 cells that cultured in RPMI 1640 mediums supplied with different buffers (HEPES v.s. pSBMA). All scale bars are 10 μm. (D) Immunofluorescence staining of RAW264.7 cells that cultured in RPMI 1640 mediums supplied with identical concentration of HEPES and pMPC buffers (25 mM). (E) Log-plot of the half-life of 1O2, ·OH, O2• −, and H2O2. (F) Schematic illustration of systematic disorder of cell fate and behavior that induced by the massive exogenous ROS associated with HEPES.

FIGURE 5 Gastrodin increases the phosphorylation and nuclear translocation of FoxO1 to up‐regulate TFEB expression in the foam cells. A, Gastrodin increased the phosphorylation of FoxO1 in the foam cells. B and C, Gastrodin promoted nuclear translocation of FoxO1 in the foam cells. B, Images of the subcellular locations of FoxO1. C, Representative blots of cytoplasmic and nuclear fractions of FoxO1. D and E, The determination of mRNA and protein expression levels of FoxO1 after transfecting by control shRNA and shFoxO1. D, Measurement of mRNA expression level by RT‐PCR. E, Measurement of protein expression level by Western blotting. F and G, Inhibition of FoxO1 expression abolished the effect of gastrodin on autophagy in the foam cells. The protein levels of LC3I/II and p62 F, and mRNA levels of autophagic genes G, were determined by Western blotting and RT‐PCR. H, Inhibition of FoxO1 reduced the protein level of TFEB. *P < .05; **P < .01; ***P < .001. Results are presented as mean ±SD of three independent experiments. The value represents fold of vehicle. shCont., Control shRNA. Cyto, Cytoplasm; Nucl, Nucleus

FIGURE 6 AMPK is a critical upstream regulator of FoxO1 and TFEB. A and B, Gastrodin activated AMPK in the foam cells. A, Representative blots of AMPK and p‐AMPK in macrophages. B, Immunofluorescence analysis of p‐AMPK in macrophages. C and D, The inhibition of AMPK activity decreased the phosphorylation of FoxO1 and nuclear translocation of TFEB. Macrophages were treated with CC (10μM) for 1 h. The phosphorylation level of FoxO1 was analysed by Western blotting C, and nuclear translocation of TFEB was determined by immunofluorescence D. *P < .05; **P < .01. Results are presented as mean ± SD of three independent experiments. The value represents fold of vehicle. CC, Dorsomorphin dihydrochloride

FIGURE 6 | Effect of RSV on TFEB activity in NRK-52E cells.(C) TFEB immunofluorescence assay results showed that the fluorescence intensity of TFEB antibody in nrK-52E cell nucleus increased after RSV treatment. *P < 0.05, **P < 0.01.

FIGURE 6 | Effect of RSV on TFEB activity in NRK-52E cells.(A) NRK-52E cells were treated with RSV at different concentrations (0–32 µM) for 24 h. The expressions of nuclear TFEB (n-TFEB), cytoplasmic TFEB (c-TFEB), and total TFEB (t-TFEB) proteins were detected by western blot assay. Quantitative analysis of the densities of western blot bands.

FIGURE 5 Expression and co-localization of Bax and Bcl2 in the rat dorsal CA1 region of hippocampus. (A) Protein expression of Bax in the hippocampal dorsal CA1 region of rats. (B) Protein expression of Bcl2 in the hippocampal dorsal CA1 region of rats. (C) Bax/Bcl2 ratios in the hippocampal dorsal CA1 region of rats. (D) Pur’s effects on Bax and Bcl2 co-localization in the hippocampal dorsal CA1 region of rats, assessed by immunofluorescent double staining. Representative micrographs are shown at ×200. Results were mean ± SEM (n = 6). *P < 0.05, **P < 0.01 vs. Sham; ##P < 0.01 vs. BCCAO; $P < 0.05, $$P < 0.01 vs. BCCAO + Pur (150 mg/kg/day); &P < 0.05, &&P < 0.01 vs. BCCAO + NGF (20 mg/kg/day).

Fig. 2 Changes of autophagy-related genes in RA rats. The expressions of transcription factor EB (TFEB), microtubule-associated proteins 1 light chain 3 (LC3B), and p62 in the synovial tissues by IHC (n = 3, biological replications). Original magnification ×150