Journal: Autophagy

Article Title: Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance

doi: 10.1080/15548627.2021.1936932

Figure Lengend Snippet: Pexophagy occurs in proportion to whole-cell peroxisome levels. (A) Immunoblot validation of Fr.1 from all conditions, alongside respective whole-cell extracts (inputs). Peroxisomal proteins: CAT, ACSL1, PEX11B, PEX19 and PEX3. LAMP2 and LC3B-II are lysosome and autophagosome protein, respectively. (B) Whole-cell extract peroxisomal protein profile. Shown are acute Vor-treated U937 cells (6,12,18 h), chronically-treated B8 (Vor) and B8 cells washed-off from Vor for one week, referred to as B8 washoff (WO). All samples were treated with CQ. ACTB is a loading control. (C) Relative mRNA expression of PEX genes in vehicle and Vor (2 μM)-treated U937 cells, Vor-maintained B8 (B8 [Vor]), and vehicle-cultured B8 (WO) cells. MAP1LC3B and FOXO1 are shown as positive controls for HDACi-induced transcriptional upregulation. All samples were normalized to the housekeeping gene ACTB. (D) Schematic of pexophagy. Ubiquitinated PEX5 is attached to the outer surface of the peroxisome and binds the cargo receptors SQSTM1 or NBR1, which facilitates peroxisomal engulfment into an expanding autophagosome (AP). (E) PEX5-Ubiquitin colocalization (+CQ) in vehicle (DMSO)-treated U937, Vor-treated U937, and B8 cells chronically maintained in Vor, with quantitation show below. (F) PEX5 and SQSTM1 colocalization, and (G) PEX5 and NBR1 colocalization (+CQ) in vehicle (DMSO)-treated U937, Vor-treated U937, and B8 cells chronically maintained in Vor. Insets shown for each condition (scale bar for D and E: 10 μm). (H) Number of colocalized puncta per condition (merge) from analyses of (F) PEX5 with SQSTM1, and (G) PEX5 with NBR1 are shown below respective images. (I) Co-immunoprecipitation of PEX5 with SQSTM1 (top), and corresponding inputs (below). All Vor treatments are 2 μM (18 h U937, chronic treatment B8), and CQ 25 μM (18 h).

Article Snippet: Immunoblotting Immunoblots were performed as previously described [ 18 ], with the following primary antibodies (manufacturer, product number, dilution): ACSL1 (Cell Signaling Technology, 9189; 1:1000), ACTB/β-actin (Sigma-Aldrich, A5441; 1:10,000), CAT/catalase (Cell Signaling Technology, 12980; 1:10,000), COX4I1/COX-IV (Abcam, ab14744; 1:500), LAMP2 (Santa Cruz Biotechnology, SC-5571; 1:1000), LC3B-II (Cell Signaling Technology, 3868; 1:1000), SQSTM1/p62 (Santa Cruz Biotechnology, SC-292; 1:1000), PEX1 (BD Biosciences, 61179; 1:1000), PEX3 (Sigma-Aldrich, HPA042830; 1:1000), PEX5 (Proteintech Group, 12545-1-AP; 1:2000), PEX6 (kindly provided by Dr. N. Braverman, 1:2000), PEX7 (Abcam, ab134962; 1:1000), PEX11B (Abcam, ab110004; 1:5000), PEX13 (Proteintech Group, 26649-1-AP; 1:1000), PEX14 (Abcam, ab113286; 1:1000), PEX19 (Proteintech Group, 26649-1-AP; 1:1000).

Techniques: Western Blot, Expressing, Cell Culture, Quantitation Assay, Immunoprecipitation

Journal: PLoS ONE

Article Title: Transcriptional regulation of Acsl1 by CHREBP and NF-kappa B in macrophages during hyperglycemia and inflammation

doi: 10.1371/journal.pone.0272986

Figure Lengend Snippet: A) BMDMs were differentiated under normal glucose (NG) and high glucose (HG), and Acsl1 mRNA absolute copy number was determined by quantitative real-time PCR (qPCR) using a standard curve generated with known quantities of mouse Acsl1 cDNA. B) Western blot of total cell lysates from BMDMs cultured in NG and HG using antibodies against ACSL1 and β-actin as (loading control). C) The ratio of ACSL1/β-actin band intensities was quantified using Image studio 5.2 (n = 3). D) Nascent Acsl1 RNA expression in BMDMs under NG and HG conditions was determined by qPCR. Nascent Acsl1 expression was quantified relative to cyclophilin A and shown as fold change. The data presented are the means ± standard error (n = 3); the p-value was calculated using the student’s t-test. (*p < 0.05).

Article Snippet: Membranes were probed with rabbit anti-ACSL1 (#9189, 1:1,000; Cell Signaling or # PA5-17136, 1;1000; Thermo Scientific), rabbit anti-CHREBP Antibody (# NB400-135, 1:500; Novus Biologicals), rabbit anti-histone H3 (1:1,000; Cell Signaling), rabbit anti-actin (1:5,000; Abcam; ab8227), rabbit-anti Sodium Potassium ATPase antibody (1:5,000; Abcam, ab76020) or mouse anti-tubulin Mouse Monoclonal Antibody (HRP-66031, 1:5000, Proteintech), followed by horseradish peroxidase-conjugated anti-mouse, or anti-rabbit IgG antibody (1:5,000; Life Technologies).

Techniques: Real-time Polymerase Chain Reaction, Generated, Western Blot, Cell Culture, RNA Expression, Expressing

Journal: PLoS ONE

Article Title: Transcriptional regulation of Acsl1 by CHREBP and NF-kappa B in macrophages during hyperglycemia and inflammation

doi: 10.1371/journal.pone.0272986

Figure Lengend Snippet: A) CHREBP expression plasmid or vector only (VO) were co-transfected with Acsl1 -GLuc reporter in HEK293 cells cultured under HG conditions. Luciferase assay was performed 48 hours post-transfection and shown as relative luciferase units (RLU). Expression of transfected Myc-tagged CHREBP is shown by western blot using a c-Myc antibody, with tubulin as a loading control. B) Wild type or Chrebp -/- BMDMs were differentiated under HG conditions. Acsl1 mRNA absolute copy number was quantified by qPCR. The data presented are means ± standard error (n = 3); the p-value was calculated using student’s t-test. (*p < 0.05).

Article Snippet: Membranes were probed with rabbit anti-ACSL1 (#9189, 1:1,000; Cell Signaling or # PA5-17136, 1;1000; Thermo Scientific), rabbit anti-CHREBP Antibody (# NB400-135, 1:500; Novus Biologicals), rabbit anti-histone H3 (1:1,000; Cell Signaling), rabbit anti-actin (1:5,000; Abcam; ab8227), rabbit-anti Sodium Potassium ATPase antibody (1:5,000; Abcam, ab76020) or mouse anti-tubulin Mouse Monoclonal Antibody (HRP-66031, 1:5000, Proteintech), followed by horseradish peroxidase-conjugated anti-mouse, or anti-rabbit IgG antibody (1:5,000; Life Technologies).

Techniques: Expressing, Plasmid Preparation, Transfection, Cell Culture, Luciferase, Western Blot

Journal: PLoS ONE

Article Title: Transcriptional regulation of Acsl1 by CHREBP and NF-kappa B in macrophages during hyperglycemia and inflammation

doi: 10.1371/journal.pone.0272986

Figure Lengend Snippet: A) BMDMs were differentiated under NG and HG conditions. Cells were stimulated with LPS (10ng/mL) for 24 hours. Total RNA was isolated, and Acsl1 mRNA was determined by qPCR relative to cyclophilin A1 and shown as fold change. NG treated sample (M0) was set to 1. B) BMDMs were differentiated in HG. Cells were pretreated for 4 hours with NF-kappa B inhibitor CAPE (5 μM) and then treated with LPS for 16 hours. RNA was isolated, and Acsl1 mRNA copy number was determined by qPCR. The data presented are means ± standard error (n = 3); the p-value was calculated using one-way ANOVA (p < 0.05; **p < 0.01; and ***p < 0.001).

Article Snippet: Membranes were probed with rabbit anti-ACSL1 (#9189, 1:1,000; Cell Signaling or # PA5-17136, 1;1000; Thermo Scientific), rabbit anti-CHREBP Antibody (# NB400-135, 1:500; Novus Biologicals), rabbit anti-histone H3 (1:1,000; Cell Signaling), rabbit anti-actin (1:5,000; Abcam; ab8227), rabbit-anti Sodium Potassium ATPase antibody (1:5,000; Abcam, ab76020) or mouse anti-tubulin Mouse Monoclonal Antibody (HRP-66031, 1:5000, Proteintech), followed by horseradish peroxidase-conjugated anti-mouse, or anti-rabbit IgG antibody (1:5,000; Life Technologies).

Techniques: Isolation

Journal: PLoS ONE

Article Title: Transcriptional regulation of Acsl1 by CHREBP and NF-kappa B in macrophages during hyperglycemia and inflammation

doi: 10.1371/journal.pone.0272986

Figure Lengend Snippet: BMDMs were differentiated under NG conditions. A) Total cell lysates were prepared with (+) and without (-) LPS treatment (10 ng/ml for 24 hours). ACSL1 protein expression was determined by western blot with an anti-ACSL1 antibody. An anti-alpha tubulin antibody was used as a loading control. B) Bands were quantified using the Image studio 5.2. C) Cytoplasmic and membrane proteins were isolated to determine the localization of ACSL1 protein by western blotting as a function of LPS treatment. Alpha-tubulin (cytoplasmic protein) and Na+/K+ ATPase (membrane-associated protein) were used to confirm the fidelity of fractionation. D) Images were quantitated as in B with cytoplasmic ACSL1 protein normalized to tubulin and membrane ACSL1 protein normalized to Na+/K+ ATPase. The data presented are means ± standard error (n = 3); the p-value was calculated using student’s t-test. (*p < 0.05). E) Mitochondria were isolated from BMDMs, and ACSL1 abundance was determined by western blot with and without LPS treatment. The non-specific (ns) band serves as a loading control.

Article Snippet: Membranes were probed with rabbit anti-ACSL1 (#9189, 1:1,000; Cell Signaling or # PA5-17136, 1;1000; Thermo Scientific), rabbit anti-CHREBP Antibody (# NB400-135, 1:500; Novus Biologicals), rabbit anti-histone H3 (1:1,000; Cell Signaling), rabbit anti-actin (1:5,000; Abcam; ab8227), rabbit-anti Sodium Potassium ATPase antibody (1:5,000; Abcam, ab76020) or mouse anti-tubulin Mouse Monoclonal Antibody (HRP-66031, 1:5000, Proteintech), followed by horseradish peroxidase-conjugated anti-mouse, or anti-rabbit IgG antibody (1:5,000; Life Technologies).

Techniques: Expressing, Western Blot, Isolation, Fractionation

Journal: PLoS ONE

Article Title: Transcriptional regulation of Acsl1 by CHREBP and NF-kappa B in macrophages during hyperglycemia and inflammation

doi: 10.1371/journal.pone.0272986

Figure Lengend Snippet: Prediction of transcription factor motifs 2 kb upstream of the mouse and human ACSL1 gene.

Article Snippet: Membranes were probed with rabbit anti-ACSL1 (#9189, 1:1,000; Cell Signaling or # PA5-17136, 1;1000; Thermo Scientific), rabbit anti-CHREBP Antibody (# NB400-135, 1:500; Novus Biologicals), rabbit anti-histone H3 (1:1,000; Cell Signaling), rabbit anti-actin (1:5,000; Abcam; ab8227), rabbit-anti Sodium Potassium ATPase antibody (1:5,000; Abcam, ab76020) or mouse anti-tubulin Mouse Monoclonal Antibody (HRP-66031, 1:5000, Proteintech), followed by horseradish peroxidase-conjugated anti-mouse, or anti-rabbit IgG antibody (1:5,000; Life Technologies).

Techniques:

Journal: PLoS ONE

Article Title: Transcriptional regulation of Acsl1 by CHREBP and NF-kappa B in macrophages during hyperglycemia and inflammation

doi: 10.1371/journal.pone.0272986

Figure Lengend Snippet: HEK293 cells cultured in HG conditions were transfected with Acsl1 -GLuc reporter (250ng; columns 1–7) and individually with p65/RELA (150ng; column 2) or CHREBP (150ng; column 3). Cells were transfected with Acsl1 -GLuc reporter and a fixed amount of p65/RELA (150ng; columns 4–7), along with increasing amounts of CHREBP (100ng, 150ng, 200ng, 250ng; columns 4–7, respectively). Total DNA was adjusted to 750ng with vector only. Luciferase assay was performed 48 hours post-transfection and shown as relative luciferase units (RLU). The data presented are means ± standard errors (n = 3); the p-value was calculated using one-way ANOVA. *p < 0.05. B) Western blot of lysates from panel A with antibodies against the Myc-tag on CHREBP, FLAG-tag on p65/RELA, and tubulin as a loading control. The p65/RELA blot for lanes 1–2 was run on a different gel than lanes 3–7 and denoted by a black line between lanes 2 and 3. Exposure times were the same.

Article Snippet: Membranes were probed with rabbit anti-ACSL1 (#9189, 1:1,000; Cell Signaling or # PA5-17136, 1;1000; Thermo Scientific), rabbit anti-CHREBP Antibody (# NB400-135, 1:500; Novus Biologicals), rabbit anti-histone H3 (1:1,000; Cell Signaling), rabbit anti-actin (1:5,000; Abcam; ab8227), rabbit-anti Sodium Potassium ATPase antibody (1:5,000; Abcam, ab76020) or mouse anti-tubulin Mouse Monoclonal Antibody (HRP-66031, 1:5000, Proteintech), followed by horseradish peroxidase-conjugated anti-mouse, or anti-rabbit IgG antibody (1:5,000; Life Technologies).

Techniques: Cell Culture, Transfection, Plasmid Preparation, Luciferase, Western Blot, FLAG-tag

Journal: PLoS ONE

Article Title: Transcriptional regulation of Acsl1 by CHREBP and NF-kappa B in macrophages during hyperglycemia and inflammation

doi: 10.1371/journal.pone.0272986

Figure Lengend Snippet: A) Putative binding sites of CHREBP and p65/RELA on mouse Acsl1 . Arrows denote the region amplified by qPCR. B) Chromatin was immunoprecipitated from BMDMs differentiated in HG using an antibody against CHREBP and isotype-matched IgG control. C) Chromatin was immunoprecipitated from BMDMs differentiated in HG and stimulated with LPS (10ng/mL) for 1 hour using an antibody against p65/RELA along with isotype-matched IgG control. Percent of precipitated DNA compared to total input DNA is shown. The data are means with an error bar representing the spread of the mean from two independent experiments.

Article Snippet: Membranes were probed with rabbit anti-ACSL1 (#9189, 1:1,000; Cell Signaling or # PA5-17136, 1;1000; Thermo Scientific), rabbit anti-CHREBP Antibody (# NB400-135, 1:500; Novus Biologicals), rabbit anti-histone H3 (1:1,000; Cell Signaling), rabbit anti-actin (1:5,000; Abcam; ab8227), rabbit-anti Sodium Potassium ATPase antibody (1:5,000; Abcam, ab76020) or mouse anti-tubulin Mouse Monoclonal Antibody (HRP-66031, 1:5000, Proteintech), followed by horseradish peroxidase-conjugated anti-mouse, or anti-rabbit IgG antibody (1:5,000; Life Technologies).

Techniques: Binding Assay, Amplification, Immunoprecipitation

Journal: PLoS ONE

Article Title: Transcriptional regulation of Acsl1 by CHREBP and NF-kappa B in macrophages during hyperglycemia and inflammation

doi: 10.1371/journal.pone.0272986

Figure Lengend Snippet: A) We propose that in high glucose, CHREBP is depressed, and active nuclear CHREBP promotes the expression of Acsl1 . B) Under inflammatory conditions that activate the NF-kappa B via dismissal of the inhibitory protein I kappa B (IkB) that prevents NF-kappa B nuclear transport, Acsl1 transcription is induced. C) Acsl1 expression can be further increased by high glucose and inflammatory stimuli via CHREBP and NF-kappa B.

Article Snippet: Membranes were probed with rabbit anti-ACSL1 (#9189, 1:1,000; Cell Signaling or # PA5-17136, 1;1000; Thermo Scientific), rabbit anti-CHREBP Antibody (# NB400-135, 1:500; Novus Biologicals), rabbit anti-histone H3 (1:1,000; Cell Signaling), rabbit anti-actin (1:5,000; Abcam; ab8227), rabbit-anti Sodium Potassium ATPase antibody (1:5,000; Abcam, ab76020) or mouse anti-tubulin Mouse Monoclonal Antibody (HRP-66031, 1:5000, Proteintech), followed by horseradish peroxidase-conjugated anti-mouse, or anti-rabbit IgG antibody (1:5,000; Life Technologies).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Vitronectin-derived bioactive peptide prevents spondyloarthritis by modulating Th17/Treg imbalance in mice with curdlan-induced spondyloarthritis

doi: 10.1371/journal.pone.0262183

Figure Lengend Snippet: Splenocytes harvested from BALB/c mice were stimulated with vehicle or VnP-16, and total STAT3, p STAT3 s727, and GAPDH expression were measured by Western blotting (n = 3 per group). Data are means ± SEM. * P < 0.05. P -values are in comparison with the vehicle group.

Article Snippet: The protein levels of p -STAT3(s727) (cat: #9134, 100kDa, Cell Signaling Technology, Beverly, MA, USA), total STAT3 (cat: #9189, 100 kDa, Cell Signaling Technology) and GAPDH (#ab181602; Abcam) were measured using a Western blot system (SNAP i.d.

Techniques: Expressing, Western Blot