Journal: Experimental & molecular medicine

Article Title: PEX5 regulates autophagy via the mTORC1-TFEB axis during starvation.

doi: 10.1038/s12276-017-0007-8

Figure Lengend Snippet: Fig. 1 PEX5 depletion suppresses starvation-induced autophagy. a HepG2 cells were treated with CTL siRNA (100 nM) and PEX5 siRNA #1 or #2 (100 nM) for 24 h. Then, cells were incubated in the presence or absence of serum. After 24 h, cells were harvested and immunoblotted with antibodies against PEX5, LC3I/II, p62, and β-actin. b Immunoblot analysis of p-70S6K, p-S6R, and p-4E-BP-1, and total 70S6K, S6R, and 4E-BP-1. c Cells were transfected with GFP-LC3 plasmid. After 6 h of transfection, cells were treated with CTL and PEX5 siRNAs. Visualization and quantification of GFP- LC3 puncta were performed by fluorescence microscopy after 48 h of incubation. Representative images from three independent determinations are shown (*P < 0.05). Scale bar: 10 μm. d Immunoblot analysis of TSC2 and PEX5 protein levels. e Transcript levels of TSC2 were measured by Q-PCR (**P < 0.01)

Article Snippet: Cell culture and drug treatments HepG2 (human hepatocyte carcinoma cell line) and HeLa (human epithelial carcinoma cell line) cells were originally purchased from the American Type Culture Collection (ATCC, Manassa, VA, USA).

Techniques: Incubation, Western Blot, Transfection, Plasmid Preparation, Microscopy

Journal: Experimental & molecular medicine

Article Title: PEX5 regulates autophagy via the mTORC1-TFEB axis during starvation.

doi: 10.1038/s12276-017-0007-8

Figure Lengend Snippet: Fig. 3 PEX5 depletion inhibits nuclear translocation of TFEB in response to starvation. a Cells were treated with CTL and PEX5 siRNAs (100 nM) for 24 h. Then, cells were incubated in the presence or absence of serum. After 24 h, cells were stained with an antibody against TFEB and analyzed by fluorescence microscopy. Scale bar: 10 μm. b After HepG2 cells were treated as indicated, cells were subjected to nuclear and cytosol fractionation and immunoblotted with antibodies against TFEB, PCNA, and β-actin. PCNA and β-actin were used as nuclear and cytosolic markers, respectively. c, d Cells were transfected with CTL or TFEB siRNA (100 nM) for 24 h, and then incubated in the presence or absence of serum for 24 h. Protein expression of TFEB, LC3 and SQSTM1/p62 (autophagy proteins), and PMP70, DBP, and catalase (peroxisomal proteins) were measured by immunoblotting. e Q-PCR analysis of peroxisomal genes was performed (**P < 0.01, *P < 0.05)

Article Snippet: Cell culture and drug treatments HepG2 (human hepatocyte carcinoma cell line) and HeLa (human epithelial carcinoma cell line) cells were originally purchased from the American Type Culture Collection (ATCC, Manassa, VA, USA).

Techniques: Translocation Assay, Incubation, Staining, Microscopy, Fractionation, Transfection, Expressing, Western Blot

Journal: Experimental & molecular medicine

Article Title: PEX5 regulates autophagy via the mTORC1-TFEB axis during starvation.

doi: 10.1038/s12276-017-0007-8

Figure Lengend Snippet: Fig. 5 Rapamycin restored TFEB localization and autophagy in PEX5 depletion during serum starvation. Cells transfected with CTL siRNA (100 nM) and PEX5 siRNA (100 nM) for 24 h were pretreated with DMSO or rapamycin (1 μM) for 6 h, and then incubated in the absence of serum for 24 h. (a) After HepG2 cells were treated as indicated, cells were stained with an antibody against TFEB and analyzed by fluorescence microscopy. Scale bar: 10 μm. Data are presented as mean ± SD from three independent experiments (**P < 0.01). (b) After HepG2 cells were treated as indicated, cells were subjected to nuclear and cytosol fractionation and immunoblotted with antibodies against TFEB, PCNA, and β-actin. PCNA and β-actin were used as nuclear and cytosolic markers, respectively. (c) Immunoblot analysis of LC3 and SQSTM1/p62. (d) Immunoblot analysis of PEX5, p-70S6K, p-S6R, and p-4E-BP-1

Article Snippet: Cell culture and drug treatments HepG2 (human hepatocyte carcinoma cell line) and HeLa (human epithelial carcinoma cell line) cells were originally purchased from the American Type Culture Collection (ATCC, Manassa, VA, USA).

Techniques: Transfection, Incubation, Staining, Microscopy, Fractionation, Western Blot

Journal: Cell Reports Medicine

Article Title: SERSomes for metabolic phenotyping and prostate cancer diagnosis

doi: 10.1016/j.xcrm.2024.101579

Figure Lengend Snippet:

Article Snippet: Human hepatocyte carcinoma cells , ATCC , HB-8065.

Techniques: Recombinant, Software

Journal: PLoS ONE

Article Title: Tocilizumab does not block interleukin-6 (IL-6) signaling in murine cells

doi: 10.1371/journal.pone.0232612

Figure Lengend Snippet: (A) Human U-937 cells were pretreated with tocilizumab as indicated for 30 min and then stimulated with 10 ng/ml hIL-6 for 15 min. Phosphorylation of STAT3 was determined by western blot. Quantification of three independent experiments and one representative western blot are shown. (B-D) The experiments were performed as described under (A) but with murine RAW264.7 cells and mIL-6 (B), with human HepG2 cells and hIL-6 (C) and with murine AML-12 cells and mIL-6 (D). Statistical significance was analyzed with one-way ANOVA followed by Dunnett's multiple comparisons test (**: p<0.01; ***: p < 0.001; ns: not significant).

Article Snippet: The human monocyte cell line U-937 (CRL-1593.2) and the murine macrophage cell line RAW264.7 (SC-6003) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and the human hepatocyte cell line HepG2 (ACC 180) was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).

Techniques: Phospho-proteomics, Western Blot

Journal: PLoS ONE

Article Title: Khellin and Visnagin Differentially Modulate AHR Signaling and Downstream CYP1A Activity in Human Liver Cells

doi: 10.1371/journal.pone.0074917

Figure Lengend Snippet: A) HepG2 cells were treated with 10 µM visnagin, 10 µM khellin, and/or DMSO for 4 h, 8 h, 16 h, and 24 h. The results from PCR are shown as fold of DMSO-treated control cells. The data are mean from three independent experiments and were normalized to beta-actin transcription. * - value is significantly different from DMSO-treated cells ( p < 0.05). B) HepG2 cells were pre-treated with 20 µM MNF for 1 h and then exposed to 10 µM visnagin or 10 µM khellin for additional 16 h. The results from PCR are shown as fold of DMSO-treated control cells. The data are mean from three independent experiments and were normalized to beta-actin transcription. * - value is significantly different from DMSO-treated cells ( p < 0.05). # - value is significantly reduced in comparison to cells treated with VIS and KHEL, respectively; ( p < 0.05). C) HepG2 cells were treated with visnagin (VIS; 1 µM-20 µM), khellin (KHEL; 1 µM-20 µM), 1 µM 3MC, 5 nM TCDD, and/or vehicle (DMSO; 0.1% v/v) for 48 h. Thereafter, western blotting analyses for detection of CYP1A1 and actin were performed as described in Materials and Methods section. The representative western blot analysis of two independent experiments (passages) is presented. D) HepG2 cells were treated with visnagin (VIS; 1 µM-20 µM), khellin (KHEL; 1 µM-20 µM), 1 µM 3MC, 5 nM TCDD, and/or vehicle (DMSO; 0.1% v/v) for either 16 h (upper panel) or 48 h (lower panel). EROD activity was determined as described in Materials and Method section. Analyses were performed in three independent experiments and are shown as fold induction over untreated cells. * - value is significantly different from DMSO-treated cells ( p < 0.05). E) HepG2 cells were treated with TCDD (5 nM) for 48 h. Thereafter, substrate mixture was supplemented with increasing doses of visnagin (VIS 1 nM -20 µM) or khellin (KHEL; 1 nM -20 µM) and EROD activity was determined as described in Materials and Methods section. Data are mean from three independent experiments and are expressed as percentage (%) of TCDD-mediated induction (i.e. induction by TCDD = 100%). * - value is significantly different from TCDD-treated cells ( p < 0.005).

Article Snippet: Blots were probed with primary antibodies against CYP1A1 (goat polyclonal, sc-9828, G-18, diluted 1:500 – for detection in human hepatocytes; rabbit polyclonal, sc-20772, H-70, diluted 1:500 – for detection in HepG2 cells), CYP1B1 (mouse monoclonal, sc-374228, G-4, 1:1000), actin (goat polyclonal; sc-1616, 1-19, diluted 1:2000), all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or GAPDH (rabbit monoclonal, 2118, 14C10, diluted 1:1000) purchased from Cell Signaling Technology, overnight at 4°C.

Techniques: Control, Comparison, Western Blot, Activity Assay

Journal: PLoS ONE

Article Title: Khellin and Visnagin Differentially Modulate AHR Signaling and Downstream CYP1A Activity in Human Liver Cells

doi: 10.1371/journal.pone.0074917

Figure Lengend Snippet: Influence of visnagin and khellin treatment on CYP1A1 mRNA expression in human primary hepatocytes.

Article Snippet: Blots were probed with primary antibodies against CYP1A1 (goat polyclonal, sc-9828, G-18, diluted 1:500 – for detection in human hepatocytes; rabbit polyclonal, sc-20772, H-70, diluted 1:500 – for detection in HepG2 cells), CYP1B1 (mouse monoclonal, sc-374228, G-4, 1:1000), actin (goat polyclonal; sc-1616, 1-19, diluted 1:2000), all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or GAPDH (rabbit monoclonal, 2118, 14C10, diluted 1:1000) purchased from Cell Signaling Technology, overnight at 4°C.

Techniques: Expressing