Journal: The Journal of nutritional biochemistry

Article Title: All-trans retinoic acid induces lipophagy through the activation of the AMPK-Beclin1 signaling pathway and reduces Rubicon expression in adipocytes.

doi: 10.1016/j.jnutbio.2024.109589

Figure Lengend Snippet: Fig. 1. Effects of atRA on the expression of autophagy-related proteins in the epididymal fat of mice. Thirty-five-week-old male C57BL/6J mice were treated with 0.1% DMSO (Control) or atRA (10 mg/kg) for 4 weeks. (A) Tissue weights of the epididymal fat (Epi), retroperitoneal fat (Ret), subcutaneous fat (Sub), and brown adipose tissue (BAT) were adjusted by body weight (BW). (B) Adipocytes from epididymal fat were stained with H&E for the determination of adipocyte size. Images were taken by microscope. Scale bar = 50 µm. Adipocyte area was calculated based on at least 100 adipocytes per group and was determined using ImageJ software. (C, D) Western blotting of LC3B and p62 in the epididymal fat. (E) Eight-week-old male C57BL/6J mice were treated with 0.1% DMSO (Control) or atRA (10 mg/kg) for 24 h. The Orp8 mRNA was detected by real-time PCR in epididymal fat. β-actin was used as an internal control. Values are mean ±S.E.M. ( n = 4). ∗P < .05 (two-tailed unpaired Student’s t test).

Article Snippet: Anti-MAP LC3 β (sc-271625), anti-ALDH1A1 (sc-374149), anti-phospho-EGFR (sc-57545, Tyr 1173), and antiEGFR (sc-373746) Ab were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Control, Staining, Microscopy, Software, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: The Journal of nutritional biochemistry

Article Title: All-trans retinoic acid induces lipophagy through the activation of the AMPK-Beclin1 signaling pathway and reduces Rubicon expression in adipocytes.

doi: 10.1016/j.jnutbio.2024.109589

Figure Lengend Snippet: Fig. 2. Effect of atRA on the lipolysis of LD through autophagy in 3T3-L1 adipocytes. (A) Differentiated 3T3-L1 cells were treated with 100 nM atRA, 250 nM Torin 1, or 0.1% DMSO (Control) in DMEM containing 2% BSA-FFA for 24 h ( n = 5−6). The culture medium was collected and assayed for NEFA content (mEq/L/mg protein). (B) Autophagic flux in differentiated 3T3-L1 cells stably expressing GFP-LC3-RFP-LC3 G treated with atRA, Torin 1, or DMSO (Control) for 12 h ( n = 25). GFP/RFP ratio data were expressed as the fold-value against DMSO. Scale bar = 50 µm. ∗P < .05, ∗∗P < .01 (one-way ANOVA with a Student-Newman post-hoc test). (C) After differentiated 3T3-L1 adipocytes were treated with atRA or DMSO (Control) for 12 h ( n = 7 –9), cells were fixed and stained for PLIN1 Ab conjugated to Alexa Fluor 488 (green) and LC3B Ab conjugated to Alexa Fluor 546 (red). Images were taken with a confocal laser-scanning microscope. Percentage colocalization of LC3B with PLIN1 was quantified using the ImageJ imaging software program. Scale bar = 10 µm. ∗P < .05 (two-tailed unpaired Student’s t test). (D, E) Differentiated 3T3-L1 adipocytes were treated with atRA or DMSO (Control) in DMEM containing 2% BSA-FFA for 24 h after transfection with 100 pmol Atg5 siRNA (siATG5) or negative control (siControl) ( n = 5). The culture medium was collected and assayed for NEFA content (mEq/L/mg protein). ∗P < .05, ∗∗P < .01 (one-way ANOVA with a Student-Newman post-hoc test). The comparison of atRA-induced NEFA content (mEq/L/mg protein). ∗P < .05 (two-tailed unpaired Student’s t test). (F, G) Differentiated 3T3-L1 cells were treated with atRA or DMSO (Control) for 10 d after transfection with siATG5 or siControl ( n = 6). Cells were then fixed, stained with oil red-O staining, and analyzed with a BZ-90 0 0 fluorescence microscope. LD size and numbers were quantified using the ImageJ imaging software program. Scale bar = 50 µm. ∗P < .05, ∗∗P < .01 (one-way ANOVA with a Student-Newman post-hoc test). NS, not significant.

Article Snippet: Anti-MAP LC3 β (sc-271625), anti-ALDH1A1 (sc-374149), anti-phospho-EGFR (sc-57545, Tyr 1173), and antiEGFR (sc-373746) Ab were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Control, Stable Transfection, Expressing, Staining, Laser-Scanning Microscopy, Imaging, Software, Two Tailed Test, Transfection, Negative Control, Comparison, Microscopy

Journal: The Journal of nutritional biochemistry

Article Title: All-trans retinoic acid induces lipophagy through the activation of the AMPK-Beclin1 signaling pathway and reduces Rubicon expression in adipocytes.

doi: 10.1016/j.jnutbio.2024.109589

Figure Lengend Snippet: Fig. 4. Effect of atRA on the AMPK-Beclin1 signaling pathway and Rubicon expression in the epididymal fat of mice. Eight-week-old male C57BL/6J mice were treated with 0.1% DMSO (Control) or atRA (10 mg/kg) for 24 h. (A –D) Western blotting of p-AMPK α, total AMPK α, p-Beclin1, total Beclin1, Rubicon, p62, and LC3B in the epididymal fat of mice. (E) The Rubicon mRNA in the epididymal fat of mice was detected by real-time PCR. β-actin was used as an internal control. Values are mean ±S.E.M. ( n = 3 –4). ∗P < .05 (two-tailed unpaired Student’s t test).

Article Snippet: Anti-MAP LC3 β (sc-271625), anti-ALDH1A1 (sc-374149), anti-phospho-EGFR (sc-57545, Tyr 1173), and antiEGFR (sc-373746) Ab were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Control, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: The Journal of nutritional biochemistry

Article Title: All-trans retinoic acid induces lipophagy through the activation of the AMPK-Beclin1 signaling pathway and reduces Rubicon expression in adipocytes.

doi: 10.1016/j.jnutbio.2024.109589

Figure Lengend Snippet: Fig. 5. The expression of Rubicon and ALDH1As in the epididymal fat of mice. The epididymal fat samples were from 8-week-old and 100-week-old male C57BL/6J mice. (A, B, D –G) Western blotting of Rubicon, p62, LC3B, p-AMPK α, total AMPK α, p-Beclin1, total Beclin1, p-ULK1, total ULK1, ALDH1A1, and ALDH1A2 in the epididymal fat. β-actin was used as an internal control. (C) The Rubicon mRNA was detected by real-time PCR in the epididymal fat. Values are mean ±S.E.M. ( n = 5). ∗P < .05, ∗∗P < .01 (two-tailed unpaired Student’s t test). NS, not significant.

Article Snippet: Anti-MAP LC3 β (sc-271625), anti-ALDH1A1 (sc-374149), anti-phospho-EGFR (sc-57545, Tyr 1173), and antiEGFR (sc-373746) Ab were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: American Journal of Cancer Research

Article Title: Oncolytic Newcastle disease virus induces autophagy-dependent immunogenic cell death in lung cancer cells

doi:

Figure Lengend Snippet: Effects of depletion of autophagy-related genes on NDV-triggered ICD determinants. (A) Confirmation of the depletion efficiency in A549 cells stably depleted of ATG5 (shATG5) or LC3 (shLC3) by immunoblot analysis. β-actin was used as a loading control. ATG5- or LC3-depleted A549 cells (B) and H1650 cells (C) were infected or mock-infected with NDV/FMW (MOI = 1) for 24 in A549 cells and 24 and 36 h in H1650 cells. Flow-cytometric analysis of CRT exposure was performed (Figure 2A). Data are shown as Mean ± S.D. for three independent experiments (*P<0.05, n.s = not significant). (D) ATG5- or LC3-depleted A549 cells and control cells were infected or mock-infected with NDV/FMW (MOI = 1) for 36 h. Confocal analysis of CRT exposure was carried out (Figure 2B). MTX was used as a positive control, arrowheads indicate positive area. Representative images are shown for three independent experiments. (E) IB analysis of released HMGB1 in whole cell lysates and concentrated supernatants of NDV/FMW-infected A549, ATG5- and LC3-depleted A549 cells. Blots shown are representative of two independent experiments. (F) A549 cells were treated with vehicle, Rapamycin (1 μM) or BEZ235 (1 μM), then they were infected with vehicle or NDV/FMW (1 MOI) for 24 h. Flow-cytometric analysis for CRT exposure. Representative images are shown for three independent experiments.

Article Snippet: The following lentiviral constructs were purchased from Santa Cruz: ATG5 shRNA (#sc-41445-V), MAP LC3β shRNA (#sc-43390-V) and noncoding shRNA (#sc-108080).

Techniques: Stable Transfection, Western Blot, Control, Infection, Positive Control

Journal: Frontiers in immunology

Article Title: Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome.

doi: 10.3389/fimmu.2018.01920

Figure Lengend Snippet: FIGURE 5 | CVL inhibited the NLRP3 inflammasome by enhancing autophagy. (A) J774A.1 macrophages were incubated for 0–24 h with CVL (20 µM). The levels of LC3, p62, and ATG5 in the cell lysates were measured by Western blot. (B) J774A.1 macrophages were incubated with CVL (20 µM, 12 h) or rapamycin (100 nM, 4 h). Accumulation of the fluorescent signals of MDC and AO was measured by confocal microscopy. (C) LPS-primed J774A.1 macrophages were incubated for 0.5 h with 3-MA (5 mM) and CVL (20 µM) followed by incubation with CC (100 µg/ml) for an additional 24 h. The levels of IL-1β in the culture medium were measured by ELISA. (D) LPS-primed wild-type (scramble) and LC3-knockdown J774A.1 macrophages (clones #1 and #2) were incubated for 0.5 h with CVL (20 µM) followed by incubation with CC (100 µg/ml) or MSU (100 µg/ml) for an additional 24 h. The levels of IL-1β in the culture medium were measured by ELISA. The expression levels of LC3 in the wild-type and LC3-knockdown J774A.1 macrophages were measured by Western blot. (E,F) LPS-primed wild-type and LC3-knockdown J774A.1 macrophages were incubated for 0.5 h with CVL (20 µM) followed by incubation with MSU (100 µg/ml) for an additional 24 h. Mitochondrial integrity was measured by staining with MitoTracker Deep Red and MitoTracker Green (E), and mitochondrial ROS was measured by staining with MitoSOX (F). The MFI of Mitotracker Deep Red are expressed as the means ± SD for three separate experiments. The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. * and *** indicate a significant difference at the level of p < 0.05 and p < 0.001, respectively.

Article Snippet: For generating protein knockdown J774A.1 macrophages, cells were transfected with CRISPR/Cas9 knockout plasmids targeting LC3 (cat. No.: sc-426563 and sc-417828-HDR, Santa Cruz Biotechnology) or Sirt1 (cat. No.: sc-430046 and sc-430046-HDR, Santa Cruz Biotechnology).

Techniques: Incubation, Western Blot, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Knockdown, Clone Assay, Expressing, Staining

Journal: Frontiers in immunology

Article Title: Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome.

doi: 10.3389/fimmu.2018.01920

Figure Lengend Snippet: FIGURE 6 | CVL inhibited the NLRP3 inflammasome by enhancing the Sirt1/autophagy axis. (A) J774A.1 macrophages were incubated for 0–24 h with CVL (20 µM). The Sirt1 levels in the cell lysates were measured by Western blot. (B) LPS-primed J774A.1 macrophages were incubated for 0.5 h with EX527 (10 µM) and CVL (20 µM) followed by incubation with CC (100 µg/ml) for an additional 24 h. The IL-1β levels in the culture medium were measured by ELISA. (C) LPS-primed wild-type (scramble) and Sirt1-knockdown J774A.1 macrophages (clones #1 and #2) were incubated for 0.5 h with CVL (20 µM) followed by incubation with CC (100 µg/ml) for an additional 24 h. The IL-1β levels in the culture medium were measured by ELISA. The expression levels of Sirt1 in the wild-type and Sirt1-knockdown J774A.1 macrophages were measured by Western blot. (D,E) J774A.1 macrophages were incubated for 0.5 h with EX527 (10 µM) followed by incubation with CVL (20 µM) for an additional 12 h. The levels of LC3 and p62 in the cell lysates were measured by Western blot (D), and the accumulation of the fluorescent signals of MDC and AO was measured by confocal microscopy (E). The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. *** indicates a significant difference at the level of p < 0.001.

Article Snippet: For generating protein knockdown J774A.1 macrophages, cells were transfected with CRISPR/Cas9 knockout plasmids targeting LC3 (cat. No.: sc-426563 and sc-417828-HDR, Santa Cruz Biotechnology) or Sirt1 (cat. No.: sc-430046 and sc-430046-HDR, Santa Cruz Biotechnology).

Techniques: Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown, Clone Assay, Expressing, Confocal Microscopy