Journal: eLife

Article Title: HIF-1α is required for disturbed flow-induced metabolic reprogramming in human and porcine vascular endothelium

doi: 10.7554/eLife.25217

Figure Lengend Snippet: HAECs were subjected to either DF or UF for 24, 48, or 72 hr prior to cell lysis, RNA purification, and qRT-PCR. Fold change of expression of HK2 , SLC2A1 , EGLN3 , and VEGFA , relative to cells not treated with flow at each time point, are given above according to the color-code legend (n = 3 for each time point and each condition). DOI: http://dx.doi.org/10.7554/eLife.25217.010

Article Snippet: Primary antibodies used were HIF-1α (1:500 in 5% non-fat milk and TBST for Western, 1:100 in 5% BSA and TBST for immunofluorescence, Cayman, Ann Arbor, MI, 10006421, RRID: AB_10099184 ), SLC2A1 (1:1000 in 5% BSA and TBST, ProteinTech, Rosemont, IL, 21829–1-AP, RRID: AB_10837075 ), HK2 (1:1000 in 5% BSA and TBST, Cell Signaling, Danvers, MA, C64G5, RRID: AB_2232946 ), β-actin (1:5000 in 5% BSA and TBST, Abcam, ab6276, RRID: AB_2223210 ), PDK1 (1:1000 in 5% non-fat milk and TBST, Cell Signaling, C47H1, RRID: AB_1904078 ), NOX4 (1:500 in 5% milk and TBST for Western, 1:50 in 5% BSA and TBST for immunofluorescence, Santa Cruz, Dallas, TX, sc-30141, RRID: AB_2151703 ), CD31 (BD, Franklin Lakes, NJ 550274, RRID: AB_393571 ), EPAS1 (1:1000 in 5% BSA and TBST, Novus Biologicals, Littleton, CO, NB100-122, RRID: AB_10002593 ).

Techniques: Lysis, Purification, Quantitative RT-PCR, Expressing

Journal: eLife

Article Title: HIF-1α is required for disturbed flow-induced metabolic reprogramming in human and porcine vascular endothelium

doi: 10.7554/eLife.25217

Figure Lengend Snippet: HAECs were subjected to either no flow (static), DF or UF for 48 hr prior to cell lysis, RNA purification, and qRT-PCR. Fold change of expression of HK2 , SLC2A1 , and PDK1 are shown (relative to static) (n = 3). *p<0.05; **p<0.005. DOI: http://dx.doi.org/10.7554/eLife.25217.013

Article Snippet: Primary antibodies used were HIF-1α (1:500 in 5% non-fat milk and TBST for Western, 1:100 in 5% BSA and TBST for immunofluorescence, Cayman, Ann Arbor, MI, 10006421, RRID: AB_10099184 ), SLC2A1 (1:1000 in 5% BSA and TBST, ProteinTech, Rosemont, IL, 21829–1-AP, RRID: AB_10837075 ), HK2 (1:1000 in 5% BSA and TBST, Cell Signaling, Danvers, MA, C64G5, RRID: AB_2232946 ), β-actin (1:5000 in 5% BSA and TBST, Abcam, ab6276, RRID: AB_2223210 ), PDK1 (1:1000 in 5% non-fat milk and TBST, Cell Signaling, C47H1, RRID: AB_1904078 ), NOX4 (1:500 in 5% milk and TBST for Western, 1:50 in 5% BSA and TBST for immunofluorescence, Santa Cruz, Dallas, TX, sc-30141, RRID: AB_2151703 ), CD31 (BD, Franklin Lakes, NJ 550274, RRID: AB_393571 ), EPAS1 (1:1000 in 5% BSA and TBST, Novus Biologicals, Littleton, CO, NB100-122, RRID: AB_10002593 ).

Techniques: Lysis, Purification, Quantitative RT-PCR, Expressing

Journal: eLife

Article Title: HIF-1α is required for disturbed flow-induced metabolic reprogramming in human and porcine vascular endothelium

doi: 10.7554/eLife.25217

Figure Lengend Snippet: ( A ) Expression profile of all significantly regulated glycolytic enzymes in the RNAseq data set of UF vs DF (flow-seq). Four biological replicates for each condition, and three averaged technical replicates. SLC2A1 and HK2 are the top two enzymes that are upregulated under DF. Relative expression is normalized to UF, last row. HAECs were treated with siRNA for HIF-1α (si HIF-1α ) or non-targeting control (SC) for 24 hr before DF for an additional 48 hr. Cell lysates were then collected and sent for total RNA sequencing (si HIF-1α /DF-seq). ( B ) Expression profile of all significantly regulated glycolytic enzymes in si HIF-1α /DF-seq. Three biological replicates for each condition, and two averaged technical replicates. Relative expression is normalized to SC, first row. ( C ) SLC2A1 and HK2 upregulated in flow-seq were confirmed with qPCR (n = 4). ( D ) Western blot and quantification of select glycolytic enzymes ( SLC2A1 and HK2 ) under differential flow after 24 hr (n = 4). HAECs were treated with si HIF-1α or SC for 24 hr before DF for an additional 48 hr before cell lysis and ( E ) qPCR for HK2 and SLC2A1 (n = 13) or ( F ) Western blotting/quantification of HIF-1α , HK2 , SLC2A1 and β-actin (n = 4). ( G ) An overexpression vector containing HIF-1α with mutated known prolyl hydroxylase binding amino acids was expressed in HAECs using in vitro transcription for 6 hr before cell lysis and analysis of HIF-1α , SLC2A1 ( β-actin serves as a loading control) and qRT-PCR analysis for glycolytic genes SLC2A1 and HK2 at 50 ng dose (n = 3). ( H ) HAECs were first treated with either SC or si HIF-1α before being subjected to DF for 48 hr. The cells then underwent a glycolysis stress test. Arrows indicate injection of glucose, oligomycin, and 2-deoxyglucose (2DG). ( I ) Glycolysis and glycolytic capacity obtained during glycolysis stress test are significantly downregulated by HIF-1α knockdown (n = 10). *p<0.05; **p<0.005; ***p<0.0005 as determined by Student’s t-test. Data represent mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.25217.023

Article Snippet: Primary antibodies used were HIF-1α (1:500 in 5% non-fat milk and TBST for Western, 1:100 in 5% BSA and TBST for immunofluorescence, Cayman, Ann Arbor, MI, 10006421, RRID: AB_10099184 ), SLC2A1 (1:1000 in 5% BSA and TBST, ProteinTech, Rosemont, IL, 21829–1-AP, RRID: AB_10837075 ), HK2 (1:1000 in 5% BSA and TBST, Cell Signaling, Danvers, MA, C64G5, RRID: AB_2232946 ), β-actin (1:5000 in 5% BSA and TBST, Abcam, ab6276, RRID: AB_2223210 ), PDK1 (1:1000 in 5% non-fat milk and TBST, Cell Signaling, C47H1, RRID: AB_1904078 ), NOX4 (1:500 in 5% milk and TBST for Western, 1:50 in 5% BSA and TBST for immunofluorescence, Santa Cruz, Dallas, TX, sc-30141, RRID: AB_2151703 ), CD31 (BD, Franklin Lakes, NJ 550274, RRID: AB_393571 ), EPAS1 (1:1000 in 5% BSA and TBST, Novus Biologicals, Littleton, CO, NB100-122, RRID: AB_10002593 ).

Techniques: Expressing, Control, RNA Sequencing, Western Blot, Lysis, Over Expression, Plasmid Preparation, Binding Assay, In Vitro, Quantitative RT-PCR, Injection, Knockdown

Journal: eLife

Article Title: HIF-1α is required for disturbed flow-induced metabolic reprogramming in human and porcine vascular endothelium

doi: 10.7554/eLife.25217

Figure Lengend Snippet: ( A ) HAECs were subjected to 24 hr of either UF or DF prior to cell lysis and Western blotting. Both HIF-1α and EPAS1 are increased under DF. HAECs were subjected to DF for 24 hr after 24 hr of treatment with either control siRNA or siRNA targeted towards EPAS1 prior to cell lysis, followed by ( B ) Western blotting or ( C ) RNA isolation and qRT-PCR. ( B ) The siRNA is able to reduce EPAS1 under disturbed flow. ( C ) SLC2A1 and HK2 are unchanged following si EPAS1 treatment and DF. EPAS1 levels are significantly lower (n = 6). *p<0.05. DOI: http://dx.doi.org/10.7554/eLife.25217.025

Article Snippet: Primary antibodies used were HIF-1α (1:500 in 5% non-fat milk and TBST for Western, 1:100 in 5% BSA and TBST for immunofluorescence, Cayman, Ann Arbor, MI, 10006421, RRID: AB_10099184 ), SLC2A1 (1:1000 in 5% BSA and TBST, ProteinTech, Rosemont, IL, 21829–1-AP, RRID: AB_10837075 ), HK2 (1:1000 in 5% BSA and TBST, Cell Signaling, Danvers, MA, C64G5, RRID: AB_2232946 ), β-actin (1:5000 in 5% BSA and TBST, Abcam, ab6276, RRID: AB_2223210 ), PDK1 (1:1000 in 5% non-fat milk and TBST, Cell Signaling, C47H1, RRID: AB_1904078 ), NOX4 (1:500 in 5% milk and TBST for Western, 1:50 in 5% BSA and TBST for immunofluorescence, Santa Cruz, Dallas, TX, sc-30141, RRID: AB_2151703 ), CD31 (BD, Franklin Lakes, NJ 550274, RRID: AB_393571 ), EPAS1 (1:1000 in 5% BSA and TBST, Novus Biologicals, Littleton, CO, NB100-122, RRID: AB_10002593 ).

Techniques: Lysis, Western Blot, Control, Isolation, Quantitative RT-PCR

Journal: eLife

Article Title: HIF-1α is required for disturbed flow-induced metabolic reprogramming in human and porcine vascular endothelium

doi: 10.7554/eLife.25217

Figure Lengend Snippet: ( A ) Aortas were harvested from pigs due for slaughter in less than 10 min after sacrifice. The inner curvature ( B ) of the aortic arch (AA) or descending thoracic (DT) aorta ( C ) was cut out and immediately stained with dihydroethidium bromide (DHE) before fixation in cold 4% paraformaldehyde. The sections were then permeabilized and stained with lectin. Other aortas were dissected out and immediately washed with PBS before a #10 scalped was passed along the inner curve of the aorta or along the descending thoracic aorta. Endothelial cells were immediately stored in cold lysis buffer. ( D ) Western blotting for NOX4 , HIF-1α , phospho-p65 , HK2 , SLC2A1 , PDK1 , and β-actin of AA and DT samples. ( E ) The Western blots are quantified for NOX4 , HIF-1α , phospho-p65 , HK2 , SLC2A1 , and PDK1 (n = 4). The AA region of the pig aortas have significantly more expression of all these enzymes. *p<0.05; **p<0.005 as determined by Student’s t-test. Data represent mean ± SEM. Bar is 10 microns. DOI: http://dx.doi.org/10.7554/eLife.25217.036

Article Snippet: Primary antibodies used were HIF-1α (1:500 in 5% non-fat milk and TBST for Western, 1:100 in 5% BSA and TBST for immunofluorescence, Cayman, Ann Arbor, MI, 10006421, RRID: AB_10099184 ), SLC2A1 (1:1000 in 5% BSA and TBST, ProteinTech, Rosemont, IL, 21829–1-AP, RRID: AB_10837075 ), HK2 (1:1000 in 5% BSA and TBST, Cell Signaling, Danvers, MA, C64G5, RRID: AB_2232946 ), β-actin (1:5000 in 5% BSA and TBST, Abcam, ab6276, RRID: AB_2223210 ), PDK1 (1:1000 in 5% non-fat milk and TBST, Cell Signaling, C47H1, RRID: AB_1904078 ), NOX4 (1:500 in 5% milk and TBST for Western, 1:50 in 5% BSA and TBST for immunofluorescence, Santa Cruz, Dallas, TX, sc-30141, RRID: AB_2151703 ), CD31 (BD, Franklin Lakes, NJ 550274, RRID: AB_393571 ), EPAS1 (1:1000 in 5% BSA and TBST, Novus Biologicals, Littleton, CO, NB100-122, RRID: AB_10002593 ).

Techniques: Staining, Lysis, Western Blot, Expressing

Journal: Cell Insight

Article Title: APC orchestrates microtubule dynamics by acting as a positive regulator of KIF2A and a negative regulator of CLASPs

doi: 10.1016/j.cellin.2024.100210

Figure Lengend Snippet: APC and KIF2A form a complex clustering at the cell edge and function in destabilizing MTs at the MT plus ends (A) Immunofluorescence staining of APC in KIF2A-GFP-Strep knock-in LLC-PK1 cells. Lower panels show enlargements of boxed areas in the upper panels. (B) TIRF microscopy time-lapse images and kymograph showing KIF2A and APC tracking the MT plus-ends in KIF2A-GFP-Strep knock-in LLC-PK1 cell line transiently transfected with mCherry-APC. Arrows indicate representative MTs. Scale bars: horizontal, 2 μm; vertical, 20 s (C) Immunofluorescence staining of α-tubulin in control KIF2A-GFP knock-in LLC-PK1 cells or APC KO/KIF2A-GFP-Strep knock-in double-engineered LLC-PK1 cell line. Insets show enlargements of boxed areas. (D) Quantification of intensities of KIF2A at the cell edge for the experiments shown in (C). n = 27–35 cells from three independent experiments. (E) TIRF live-cell imaging of control KIF2A-GFP-Strep knock-in LLC-PK1 cells or APC KO/KIF2A-GFP-Strep knock-in double-engineered LLC-PK1 cell line. The left panels show the single frame and the maximum intensity projection over 200 frames (100 s) of the GFP channel. The right panels show representative kymographs illustrating the behaviors of KIF2A at MT plus-end in control or APC KO cells. Scale bars: horizontal, 2 μm; vertical, 20 s (F) Quantification of intensities of KIF2A at the MT plus-end for the experiments shown in (E). n = 20–23 cells from three independent experiments. (G) Immunofluorescence staining of α-tubulin in control Strep-GFP-APC knock-in LLC-PK1 cells or KIF2A KO/Strep-GFP-APC knock-in double-engineered LLC-PK1 cell line. Insets show enlargements of boxed areas. (H) Quantification of intensities of APC at the cell edge for the experiments shown in (G). n.s, no significance in unpaired two-tailed t -test. n = 25–32 cells from three independent experiments. (I) TIRF live-cell imaging of control Strep-GFP-APC knock-in LLC-PK1 cells or KIF2A KO/Strep-GFP-APC knock-in double-engineered LLC-PK1 cell line. The left panels show the single frame and the maximum intensity projection over 200 frames (100 s) of the GFP channel. The right panels show representative kymographs illustrating the behaviors of APC at MT plus-end in control or KIF2A KO cells. Scale bars: horizontal, 2 μm; vertical, 20 s (J) Quantification of intensities of APC at the MT plus-end for the experiments shown in (I). n.s, no significance in unpaired two-tailed t -test. n = 13–20 cells from two independent experiments. (K) TIRF live-cell imaging of control EB3-GFP knock-in LLC-PK1 cell line, APC KO/EB3-GFP knock-in double-engineered LLC-PK1 cell line, and KIF2A KO/EB3-GFP knock-in double-engineered LLC-PK1 cell line. The left panels show the single frame and the maximum intensity projection over 200 frames (100 s) of the GFP channel. Middle panels show time-lapse images of boxed areas. The right panels show corresponding kymographs of MTs indicated by arrows in the middle panels. Scale bars: horizontal, 2 μm; vertical, 20 s (L–P) Quantification of the catastrophe frequency (L), rescue frequency (M), growth rate (N), depolymerization rate (O), and the length of the shortening excursions (P) of MT plus-ends in the vicinity of the cell edge for experiments shown in (K). n.s, no significance in unpaired two-tailed t -test. n = 11–20 cells from three independent experiments. Data information: Unless otherwise stated, data represent mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; two-tailed t -test (unpaired). Scale bars, 5 μm.

Article Snippet: The following antibodies were used for western blotting (WB) and immunofluorescence (IF): rat monoclonal antibodies against APC (Absea, 030903E07, IF-1:100), CLASP1 (Absea, 05008R01A06, IF-1:200), CLASP2 (Absea, 03020R06E03, IF-1:200), α-tubulin YL1/2 (Thermo Fisher Scientific, MA1-80017, IF-1:600); rabbit polyclonal antibodies against GFP (Proteintech, 50430-2-AP, WB-1:2500), detyrosinated α-tubulin (Abcam, ab48389, IF-1:600), KIF2A (Proteintech, 13105-1-AP, IF-1:300, WB-1:1000), paxillin (Proteintech, 10029-1-lg, IF-1:200).

Techniques: Immunofluorescence, Staining, Knock-In, Microscopy, Transfection, Control, Live Cell Imaging, Two Tailed Test

Journal: Cell Insight

Article Title: APC orchestrates microtubule dynamics by acting as a positive regulator of KIF2A and a negative regulator of CLASPs

doi: 10.1016/j.cellin.2024.100210

Figure Lengend Snippet: Biochemical characterization and mutational analysis of the APC-KIF2A complex (A) Left, schematic overview of the domain organization of APC and the deletion mutants and a summary of their interactions with KIF2A. Right, schematic overview of the domain organization of KIF2A and the deletion mutants and summary of their interactions with APC. OD, oligomerization domain; ARD, Armadillo repeat domain; CC, Coiled-coil. (B) Streptavidin pull-down assays with extracts of HEK293T cells expressing Bio-tagged KIF2A full length (FL) (bait) together with the indicated GFP-tagged APC truncations (prey), analyzed by western blotting with GFP antibody. The Bio-tagged bait proteins were detected by IRDye® 680RD Streptavidin. (C) Streptavidin pull-down assays with extracts of HEK293T cells expressing Bio-GFP-tagged KIF2A FL and the indicated truncations (bait) together with GFP-tagged APC C1 (prey), analyzed by western blotting with GFP antibody. The Bio-GFP-tagged bait proteins were detected by IRDye® 680RD Streptavidin. (D) Streptavidin pull-down assays with extracts of HEK293T cells expressing Bio-GFP-tagged KIF2A FL and the indicated truncations (bait) together with GFP-tagged APC short (prey), analyzed by western blotting with GFP antibody. The Bio-GFP-tagged bait proteins were detected by IRDye® 680RD Streptavidin (E) Streptavidin pull-down assays with extracts of HEK293T cells expressing Bio-GFP-tagged KIF2A N (bait) together with the indicated GFP-tagged APC truncations (prey), analyzed by western blotting with GFP antibody. The Bio-GFP-tagged bait proteins were detected by IRDye® 680RD Streptavidin. (F) Streptavidin pull-down assays with extracts of HEK293T cells expressing Bio-GFP-tagged KIF2A C (bait) together with the indicated GFP-tagged APC truncations (prey), analyzed by western blotting with GFP antibody. The Bio-GFP-tagged bait proteins were detected by IRDye® 680RD Streptavidin. (G) Alignment of KIF2A N-binding region in APC from five vertebrate species. The threonine and tryptophan residues critical for KIF2A N-binding were indicated with asterisks. (H) Streptavidin pull-down assay with extracts of HEK293T cells expressing Bio-GST-tagged KIF2A N (bait) together with GFP-tagged wild-type (WT) APC short or its 3TW/6A mutant (prey), analyzed by western blotting with GFP antibody. The Bio-GST-tagged bait proteins were detected by IRDye® 680RD Streptavidin. (I) Alignment of KIF2A C-binding region in APC from five vertebrate species. The threonine, methionine (or phenylalanine), and isoleucine residues critical for KIF2A C-binding were indicated with asterisks. (J) Streptavidin pull-down assay with extracts of HEK293T cells expressing Bio-GFP-tagged KIF2A C (bait) together with GFP-tagged WT APC short or its 2TxI/6A mutant (prey), analyzed by western blotting with GFP antibody. The Bio-GFP-tagged bait proteins were detected by IRDye® 680RD Streptavidin. (K) Streptavidin pull-down assay with extracts of HEK293T cells expressing Bio-tagged KIF2A FL (bait) together with GFP-tagged WT APC short or its indicated mutants (prey), analyzed by western blotting with GFP antibody. The Bio-tagged bait proteins were detected by IRDye® 680RD Streptavidin. (L) Left, images of control KIF2A-GFP-Strep knock-in LLC-PK1 cell line or APC KO/KIF2A-GFP-Strep knock-in double-engineered LLC-PK1 cell line transiently transfected with control mCherry vector. Right, images of APC KO/KIF2A-GFP-Strep knock-in double-engineered LLC-PK1 cell line transiently transfected with mCherry-tagged WT APC or its indicated mutants. (M) Quantification of intensities of KIF2A at the cell edge for the experiments shown in (L). n = 13–20 cells from three independent experiments. Data information: Data represent mean ± SD. ∗∗ p < 0.01; ∗∗∗ p < 0.001; two-tailed t -test (unpaired). Scale bars, 5 μm.

Article Snippet: The following antibodies were used for western blotting (WB) and immunofluorescence (IF): rat monoclonal antibodies against APC (Absea, 030903E07, IF-1:100), CLASP1 (Absea, 05008R01A06, IF-1:200), CLASP2 (Absea, 03020R06E03, IF-1:200), α-tubulin YL1/2 (Thermo Fisher Scientific, MA1-80017, IF-1:600); rabbit polyclonal antibodies against GFP (Proteintech, 50430-2-AP, WB-1:2500), detyrosinated α-tubulin (Abcam, ab48389, IF-1:600), KIF2A (Proteintech, 13105-1-AP, IF-1:300, WB-1:1000), paxillin (Proteintech, 10029-1-lg, IF-1:200).

Techniques: Expressing, Western Blot, Binding Assay, Pull Down Assay, Mutagenesis, Control, Knock-In, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Cell Insight

Article Title: APC orchestrates microtubule dynamics by acting as a positive regulator of KIF2A and a negative regulator of CLASPs

doi: 10.1016/j.cellin.2024.100210

Figure Lengend Snippet: APC potentiates KIF2A-mediated MT catastrophes in vitro (A) Images and corresponding kymographs showing the behavior of GFP-APC short on dynamic MTs at different concentrations. (B–D) Quantification of MT plus-end depolymerization rate (B), growth rate (C), and catastrophe frequency (D) in the presence of GFP-APC short at indicated concentrations. n = 21–31 MTs from three independent experiments. (E) Images and corresponding kymographs of dynamic MTs grown in the presence of 75 nM mCherry-EB3 or mCherry-EB3 delta Tail alone or together with GFP-APC short at indicated concentrations. (F–H) Quantification of MT plus-end depolymerization rate (F), growth rate (G), and catastrophe frequency (H) for the experiments shown in (E). n = 20–23 MTs from 2 to 3 independent experiments. (I) Images and corresponding kymographs of dynamic MTs (shown here in blue) grown in the presence of KIF2A-SNAP labeled with Alexa Fluor 647 (shown here in red) at indicated concentrations. (J) Images and corresponding kymographs of dynamic MTs grown in the presence of WT GFP-APC short and KIF2A-SNAP at indicated concentrations. (K) Images and corresponding kymographs of dynamic MTs grown in the presence of mCherry-EB3 (shown here in blue) and KIF2A-SNAP (shown here in red) at indicated concentrations. (L) Images and corresponding kymographs of dynamic MTs grown in the presence of WT GFP-APC short , KIF2A-SNAP (shown here in red), and mCherry-EB3 (shown here in blue) at indicated concentrations. (M) Images and corresponding kymographs of dynamic MTs grown in the presence of GFP-APC short mutant and KIF2A-SNAP (shown here in red) at indicated concentrations. (N) Images and corresponding kymographs of dynamic MTs grown in the presence of GFP-APC short mutant, KIF2A-SNAP (shown here in red), and mCherry-EB3 (shown here in blue) at indicated concentrations. (O–P) Quantification of KIF2A intensity on MT lattices (O) and catastrophe frequency (P) for the experiments shown in (I–N). n = 20–31 MTs from 2 to 3 independent experiments. Data information: Data represent mean ± SD. ∗∗∗ p < 0.001; two-tailed t -test (unpaired). Scale bars: horizontal, 2 μm; vertical, 2 min.

Article Snippet: The following antibodies were used for western blotting (WB) and immunofluorescence (IF): rat monoclonal antibodies against APC (Absea, 030903E07, IF-1:100), CLASP1 (Absea, 05008R01A06, IF-1:200), CLASP2 (Absea, 03020R06E03, IF-1:200), α-tubulin YL1/2 (Thermo Fisher Scientific, MA1-80017, IF-1:600); rabbit polyclonal antibodies against GFP (Proteintech, 50430-2-AP, WB-1:2500), detyrosinated α-tubulin (Abcam, ab48389, IF-1:600), KIF2A (Proteintech, 13105-1-AP, IF-1:300, WB-1:1000), paxillin (Proteintech, 10029-1-lg, IF-1:200).

Techniques: In Vitro, Labeling, Mutagenesis, Two Tailed Test

Journal: Cell Insight

Article Title: APC orchestrates microtubule dynamics by acting as a positive regulator of KIF2A and a negative regulator of CLASPs

doi: 10.1016/j.cellin.2024.100210

Figure Lengend Snippet: APC antagonizes the stabilizing effect of CLASP2 in vitro (A) Upper, representative images of GMPCPP-stabilized MTs incubated with 0.1 nM, 0.5 nM and 1 nM GFP-CLASP2 in the absence or presence of the indicated concentrations of mCherry-APC short . Lower, the line-scan intensity profiles of CLASP2 and MT as indicated by the dotted lines in the upper images. (B–D) Quantification of the intensities of APC short (B) and CLASP2 along the MT lattices and at MT ends (C and D) for the experiments shown in (A). n = 25 MTs from 3 independent experiments. (E) Images and corresponding kymographs of dynamic MTs grown in the presence of 75 nM TagBFP-EB3 alone (left), 75 nM TagBFP-EB3 together with 2.5 nM GFP-CLASP2 (middle) or 5 nM GFP-CLASP2 (right), as well as either 5 nM mCherry- APC short or 2.5 nM KIF2A-SNAP (shown here in purple), or all four proteins together. (F–I) Quantification of rescue frequency (F), catastrophe frequency (G), and the intensities of CLASP2 along the MT lattices (H) and at MT plus ends (I) for the experiments shown in (E). n = 15–20 MTs from 3 independent experiments. Data information: Data represent mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; two-tailed t -test (unpaired). Scale bars: horizontal, 2 μm; vertical, 2 min.

Article Snippet: The following antibodies were used for western blotting (WB) and immunofluorescence (IF): rat monoclonal antibodies against APC (Absea, 030903E07, IF-1:100), CLASP1 (Absea, 05008R01A06, IF-1:200), CLASP2 (Absea, 03020R06E03, IF-1:200), α-tubulin YL1/2 (Thermo Fisher Scientific, MA1-80017, IF-1:600); rabbit polyclonal antibodies against GFP (Proteintech, 50430-2-AP, WB-1:2500), detyrosinated α-tubulin (Abcam, ab48389, IF-1:600), KIF2A (Proteintech, 13105-1-AP, IF-1:300, WB-1:1000), paxillin (Proteintech, 10029-1-lg, IF-1:200).

Techniques: In Vitro, Incubation, Two Tailed Test

Journal: Cell Insight

Article Title: APC orchestrates microtubule dynamics by acting as a positive regulator of KIF2A and a negative regulator of CLASPs

doi: 10.1016/j.cellin.2024.100210

Figure Lengend Snippet: Model for how APC regulates MT plus-end dynamics CLASPs stabilize MT plus ends and promote processive MT polymerization in cell regions with low APC concentration or activity. However, APC can be locally concentrated in regions such as extending membranes due to Kinesin-2 complex-mediated transportation and EBs-mediated recruitment. In such regions, APC dissociates MT-stabilizer CLASPs from plus ends and recruits MT-destabilizer KIF2A there, leading to MT depolymerization.

Article Snippet: The following antibodies were used for western blotting (WB) and immunofluorescence (IF): rat monoclonal antibodies against APC (Absea, 030903E07, IF-1:100), CLASP1 (Absea, 05008R01A06, IF-1:200), CLASP2 (Absea, 03020R06E03, IF-1:200), α-tubulin YL1/2 (Thermo Fisher Scientific, MA1-80017, IF-1:600); rabbit polyclonal antibodies against GFP (Proteintech, 50430-2-AP, WB-1:2500), detyrosinated α-tubulin (Abcam, ab48389, IF-1:600), KIF2A (Proteintech, 13105-1-AP, IF-1:300, WB-1:1000), paxillin (Proteintech, 10029-1-lg, IF-1:200).

Techniques: Concentration Assay, Activity Assay

Journal: Science Advances

Article Title: MLX phosphorylation stabilizes the ChREBP-MLX heterotetramer on tandem E-boxes to control carbohydrate and lipid metabolism

doi: 10.1126/sciadv.adt4548

Figure Lengend Snippet: ( A ) and ( B ) MLX phosphorylation in mouse (A) and human (B) white adipose tissue (WAT) lysates treated with calf intestine phosphatase (CIP) or CIP and phosphatase (PP) inhibitors. n = 3. ( C ) MLX phosphorylation in 293T cells expressing the MLX isoforms alpha, beta, or gamma. CALX serves as a loading control. N = 3. ( D ) Amino acid sequence alignment of wild-type MLX (MLX-WT), phospho-deficient MLX-A, and phospho-mimetic MLX-D. ( E ) MLX phosphorylation in 293T cells expressing MLX-WT, MLX-A, or MLX-D. N = 3. ( F ) ChREBP-MLX luciferase reporter activity in 293T cells expressing ChREBP, HK2 and MLX-WT, MLX-A, or MLX-D. Cells were starved for glucose overnight and treated with 25 mM glucose for 3 hours. Two-way ANOVA, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. N = 3.

Article Snippet: Protein lysates from mouse WAT or 293T cells transiently expressing ChREBP, MLX, and HK2 were incubated with 5 μl of CIP (Quick CIP New England Biolabs, M0525S) and/or PhosSTOP (Roche, 4906837001) in 40 μl of 1× CutSmart buffer (New England Biolabs, B6004S).

Techniques: Expressing, Control, Sequencing, Luciferase, Activity Assay

Journal: Science Advances

Article Title: MLX phosphorylation stabilizes the ChREBP-MLX heterotetramer on tandem E-boxes to control carbohydrate and lipid metabolism

doi: 10.1126/sciadv.adt4548

Figure Lengend Snippet: ( A ) Interactors of TurboID-tagged MLX. 293T cells expressing ChREBP, HK2, and MLX-TurboID were labeled with 25 nM biotin for 10 min. N = 4. ( B ) Putative CK2 and GSK3 phosphorylation motifs on MLX. ( C ) Interaction between MLX and the CK2 catalytic site based on an AlphaFold 3 structure prediction. ( D ) MLX phosphorylation in 293T cells treated with or without the CK2 inhibitor CX-4945 (10 μM) for 60 min. CALX serves as a loading control. t test, *** P < 0.001. N = 3. ( E ) MLX phosphorylation in 293T cells treated with or without the GSK3 inhibitor CHIR99021 (5 μM) for 90 min. t test, * P < 0.05. N = 3. ( F ) In vitro CK2-mediated MLX phosphorylation (thiophosphate). Recombinant CK2, MLX-WT or MLX-A, and γ-S were incubated with or without 25 nM CX-4945 for 30 min. N = 3. ( G and H ) In vitro CK2 and GSK3-mediated MLX phosphorylation (thiophosphate). Recombinant CK2, GSK3, MLX-WT, and ATPγ-S were incubated with or without 25 nM CX-4945 or 5 μM CHIR99021 for 30 min. One-way ANOVA, * P < 0.05 and **** P < 0.0001; ns, not significant. N = 3. ( I ) Acly and Fasn mRNA levels in mouse WAT explants treated with 10 μM CX-4945 for 60 min. t test, ** P < 0.01 and *** P < 0.001. n = 5.

Article Snippet: Protein lysates from mouse WAT or 293T cells transiently expressing ChREBP, MLX, and HK2 were incubated with 5 μl of CIP (Quick CIP New England Biolabs, M0525S) and/or PhosSTOP (Roche, 4906837001) in 40 μl of 1× CutSmart buffer (New England Biolabs, B6004S).

Techniques: Expressing, Labeling, Control, In Vitro, Recombinant, Incubation

Journal: Science Advances

Article Title: MLX phosphorylation stabilizes the ChREBP-MLX heterotetramer on tandem E-boxes to control carbohydrate and lipid metabolism

doi: 10.1126/sciadv.adt4548

Figure Lengend Snippet: ( A ) MLX phosphorylation in 293T cells starved for glucose and serum. CALX serves as a loading control. t-test, **** P < 0.0001. N = 6. ( B and C ) MLX phosphorylation in 293T cells starved for glucose and serum overnight and re-fed with 25 mM glucose. One-way ANOVA, ** P < 0.01 and **** P < 0.0001. N = 4. ( D and E ) MLX phosphorylation in WAT from overnight fasted or overnight fasted and 3-hour re-fed mice. One-way ANOVA, **** P < 0.0001. n = 6. ( F and G ) MLX phosphorylation in 293T cells starved for glucose and re-fed with 25 mM 2DG. One-way ANOVA, **** P < 0.0001. N = 4. ( H ) ChREBP-MLX luciferase reporter activity in 293T cells expressing ChREBP, HK2, and MLX-WT. Cells were starved for glucose and re-fed with 25 mM glucose or 25 mM 2DG for 3 hours. One-way ANOVA, **** P < 0.0001; ns, not significant. N = 3. ( I ) In vitro CK2-mediated MLX phosphorylation (thiophosphate) in the presence of different concentrations of glucose-6-phosphate (G6P) or glucose. One-way ANOVA, ** P < 0.01 and *** P < 0.001. N = 4. ( J ) Binding of MLX and/or ChREBP to single E-box, perfect ChoRE, or PK ChoRE probes. ChREBP and MLX were purified from 293T cells treated with or without 25 mM 2DG for 1 hour. ( K and L ) Quantification of the data in (J). t test, * P < 0.05 and **** P < 0.0001. N = 3 to 4.

Article Snippet: Protein lysates from mouse WAT or 293T cells transiently expressing ChREBP, MLX, and HK2 were incubated with 5 μl of CIP (Quick CIP New England Biolabs, M0525S) and/or PhosSTOP (Roche, 4906837001) in 40 μl of 1× CutSmart buffer (New England Biolabs, B6004S).

Techniques: Control, Luciferase, Activity Assay, Expressing, In Vitro, Binding Assay, Purification

Journal: Journal of Cancer

Article Title: Large Intergenic Non-coding RNA-RoR Inhibits Aerobic Glycolysis of Glioblastoma Cells via Akt Pathway

doi: 10.7150/jca.20869

Figure Lengend Snippet: LincRNA-RoR inhibits Rictor expression, Akt phosphorylation and suppresses the expression of glycolytic molecules. ( A ) Over-expression of LincRNA-RoR in GBM cells inhibited the expression of Rictor, the phosphorylations of Akt, mTOR and S6, and down-regulate expression of Glut1, HK2, PKM2 and LDHA. ( B ) LincRNA-RoR suppressed the proliferation of GBM cells in vitro . * P <0.05, ** P <0.01.

Article Snippet: Commercial antibodies were mouse anti-human Ki67 (Kit-0005, Maixin, Fuzhou, China), rabbit anti-human Glut1 (1:200) (ab115730, Abcam), rabbit anti-human HK2 (1:200) (#8337, Cell Signal Technology), rabbit anti-human PKM2 (1:200) (#8337, Cell Signal Technology), rabbit anti-human LDHA (1:200) (#8337, Cell Signal Technology), rabbit anti-human Rictor (1:100) (#2114, Cell Signal Technology), rabbit anti-human p-Akt (1:200) (#4060, Cell Signal Technology).

Techniques: Expressing, Phospho-proteomics, Over Expression, In Vitro

Journal: Journal of Cancer

Article Title: Large Intergenic Non-coding RNA-RoR Inhibits Aerobic Glycolysis of Glioblastoma Cells via Akt Pathway

doi: 10.7150/jca.20869

Figure Lengend Snippet: LincRNA-RoR inhibited the expression of glycolytic effectors in xenograft tumors by western blot. ( A ) The mRNA level of LincRNA-RoR is higher in over-LincRNA-RoR-tumor xenografts as compared with that in control xenografts. ( B ) The expression of Glut1, HK2, PKM2 and LDHA are significantly impaired in over-LincRNA-RoR-tumor xenografts as compared with that in control xenografts. ( C ) LincRNA-RoR over-expression significantly inhibits Rictor expression, as well as the phosphorylations of Akt, mTOR and S6 in tumor xenografts.

Article Snippet: Commercial antibodies were mouse anti-human Ki67 (Kit-0005, Maixin, Fuzhou, China), rabbit anti-human Glut1 (1:200) (ab115730, Abcam), rabbit anti-human HK2 (1:200) (#8337, Cell Signal Technology), rabbit anti-human PKM2 (1:200) (#8337, Cell Signal Technology), rabbit anti-human LDHA (1:200) (#8337, Cell Signal Technology), rabbit anti-human Rictor (1:100) (#2114, Cell Signal Technology), rabbit anti-human p-Akt (1:200) (#4060, Cell Signal Technology).

Techniques: Expressing, Western Blot, Control, Over Expression

Journal: Journal of Cancer

Article Title: Large Intergenic Non-coding RNA-RoR Inhibits Aerobic Glycolysis of Glioblastoma Cells via Akt Pathway

doi: 10.7150/jca.20869

Figure Lengend Snippet: Immunostaining of Glut1, HK2, PKM2, LDHA, p-Akt and Rictor in xenograft tumors in presence of LincRNA-RoR. ( A ) The expression of Glut1, HK2, PKM2, LDHA as well as (B) p-Akt and Rictor were analyzed by immunohistochemistry. Mean density was calculated by Image Pro-plus software. Scale bar represents 100 μm. Data are represented as means ± SD of each group. * P <0.05, ** P <0.01.

Article Snippet: Commercial antibodies were mouse anti-human Ki67 (Kit-0005, Maixin, Fuzhou, China), rabbit anti-human Glut1 (1:200) (ab115730, Abcam), rabbit anti-human HK2 (1:200) (#8337, Cell Signal Technology), rabbit anti-human PKM2 (1:200) (#8337, Cell Signal Technology), rabbit anti-human LDHA (1:200) (#8337, Cell Signal Technology), rabbit anti-human Rictor (1:100) (#2114, Cell Signal Technology), rabbit anti-human p-Akt (1:200) (#4060, Cell Signal Technology).

Techniques: Immunostaining, Expressing, Immunohistochemistry, Software

Journal: International journal of biological sciences

Article Title: Purinergic receptor P2Y12 boosts autoimmune hepatitis through hexokinase 2-dependent glycolysis in T cells.

doi: 10.7150/ijbs.85133

Figure Lengend Snippet: Figure 5. P2RY12 orchestrates T cells aerobic glycolysis through HK2 in ConA-induced immune hepatitis. (A-C) GSEA results indicate that genes related to “carbohydrate metabolic process” (A), “glycolysis gluconeogenesis” (B) and “mitochondrion organization” (C) are differentially enriched in hepatic T cells gathered from WT and P2RY12-/- mice after ConA administration 12 hr. NES, normalized enrichment score. FDR, false-discovery rate. (D, E) Seahorse analysis of maximum ECAR (measured after oligomycin injection, Oligo) and baseline OCR and maximum OCR in hepatic T cells from WT mice and P2RY12-/- mice after ConA administration 12 hr. (F) Total intracellular ATP production in hepatic T cells from WT mice and P2RY12-/- mice induced by ConA was measured by ATP-dependent luminescent activity. (G) Heat map depicting changes in the expression of protein involved in “glycolysis” in hepatic T cells. (H) Relative protein expression levels of HK2, GPI, PFK2, MCT1, Tpi and β-actin in hepatic T cells gathered from WT mice and P2RY12-/- mice after ConA administration 12 hr. (I) Flow cytometric analysis of HK2+ hepatic T cells gathered from WT mice and P2RY12-/- mice after ConA administration 12 hr. Pooled data are presented in the right panel. (J) Schematic diagram of HK2 function in glycolysis metabolism. (K) Targeted metabolomics analysis the level of glucose in hepatic T cells. (L) Schematic diagram of cell adoptive transfer. T cells from WT mice splenic lymphocytes were co-culture with PBS or 3-BrPA for 8 hr, then

Article Snippet: After blocking the membrane with 5% nonfat milk to prevent nonspecific binding, immunoblotting was performed using the following antibodies against mouse proteins: GPI, PFK2, MCT1, TPI(Proteintech Inc., Rosemont, USA); JNK, p-JNK (Thr183/Tyr185), cleaved caspase3 and β-actin (Cell Signaling Technology, Danvers, MA, USA); P2RY12 (Abcam Inc., Cambridge, UK); HK2, P62 and LC3 (ABclonal, Wuhan, China); Akt, P-Akt (Ser473) (Proteintech Inc., Rosemont, USA); P-Akt (Thr308) (Affinity Biosciences, Cincinnati, OH, USA).

Techniques: Injection, Activity Assay, Expressing, Adoptive Transfer Assay, Co-Culture Assay

Journal: International journal of biological sciences

Article Title: Purinergic receptor P2Y12 boosts autoimmune hepatitis through hexokinase 2-dependent glycolysis in T cells.

doi: 10.7150/ijbs.85133

Figure Lengend Snippet: Figure 6. P2RY12 affects HK2 stability through regulating PI3K/Akt signaling pathway and inhibiting lysosomal degradation in T cells. (A) Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in WT cells and P2RY12-/- T cells after ConA administration 12 hr. (B) Immunoblot analysis of the indicated HK2,

Article Snippet: After blocking the membrane with 5% nonfat milk to prevent nonspecific binding, immunoblotting was performed using the following antibodies against mouse proteins: GPI, PFK2, MCT1, TPI(Proteintech Inc., Rosemont, USA); JNK, p-JNK (Thr183/Tyr185), cleaved caspase3 and β-actin (Cell Signaling Technology, Danvers, MA, USA); P2RY12 (Abcam Inc., Cambridge, UK); HK2, P62 and LC3 (ABclonal, Wuhan, China); Akt, P-Akt (Ser473) (Proteintech Inc., Rosemont, USA); P-Akt (Thr308) (Affinity Biosciences, Cincinnati, OH, USA).

Techniques: Western Blot

Journal: International journal of biological sciences

Article Title: Purinergic receptor P2Y12 boosts autoimmune hepatitis through hexokinase 2-dependent glycolysis in T cells.

doi: 10.7150/ijbs.85133

Figure Lengend Snippet: Figure 7. P2RY12 medicated the expression of IFN-γ and HK2 stability in AIH patients and healthy human PBMCs stimulated by ConA in vitro. (A) Healthy human lymphocytes were obtained and cultured for 24 hr in an environment containing 2 μg/ml ConA and treated with different doses of clopidogrel and ticagrelor (1, 3, 10 μM).

Article Snippet: After blocking the membrane with 5% nonfat milk to prevent nonspecific binding, immunoblotting was performed using the following antibodies against mouse proteins: GPI, PFK2, MCT1, TPI(Proteintech Inc., Rosemont, USA); JNK, p-JNK (Thr183/Tyr185), cleaved caspase3 and β-actin (Cell Signaling Technology, Danvers, MA, USA); P2RY12 (Abcam Inc., Cambridge, UK); HK2, P62 and LC3 (ABclonal, Wuhan, China); Akt, P-Akt (Ser473) (Proteintech Inc., Rosemont, USA); P-Akt (Thr308) (Affinity Biosciences, Cincinnati, OH, USA).

Techniques: Expressing, In Vitro, Cell Culture

Journal: International journal of biological sciences

Article Title: Purinergic receptor P2Y12 boosts autoimmune hepatitis through hexokinase 2-dependent glycolysis in T cells.

doi: 10.7150/ijbs.85133

Figure Lengend Snippet: Figure 8. Summary of the mechanism by purinergic receptor P2Y12 drives ConA-mediated autoimmune hepatitis through hexokinase 2-dependent glycolysis pathway. Purinergic receptor P2Y12 (P2RY12) affects T cells HK2 stability through regulating PI3K/Akt signaling pathway and inhibiting lysosomal degradation in ConA-mediated autoimmune hepatitis.

Article Snippet: After blocking the membrane with 5% nonfat milk to prevent nonspecific binding, immunoblotting was performed using the following antibodies against mouse proteins: GPI, PFK2, MCT1, TPI(Proteintech Inc., Rosemont, USA); JNK, p-JNK (Thr183/Tyr185), cleaved caspase3 and β-actin (Cell Signaling Technology, Danvers, MA, USA); P2RY12 (Abcam Inc., Cambridge, UK); HK2, P62 and LC3 (ABclonal, Wuhan, China); Akt, P-Akt (Ser473) (Proteintech Inc., Rosemont, USA); P-Akt (Thr308) (Affinity Biosciences, Cincinnati, OH, USA).

Techniques:

Journal: British Journal of Pharmacology

Article Title: Dual inhibition of cannabinoid CB 1 receptor and inducible NOS attenuates obesity‐induced chronic kidney disease

doi: 10.1111/bph.14849

Figure Lengend Snippet: MRI‐1867 reverses the fatty acid‐induced reduction in adiponectin signalling. Mice on standard diet (STD) or high‐fat diet (HFD) for 18 weeks were treated with vehicle (Veh) or MRI‐1867 (3 mg·kg −1 ) orally for 28 days. (a–c) MRI‐1867 restored the HFD‐induced reduction in the mRNA renal expression of adiponectin, and Adipo2 but not Adipo1 receptors. Similarly, exposing HK‐2 cells to O:P (0.5 mM, 2:1, respectively) resulted in reduced (d–f) mRNA and protein expression of adiponectin as well as reduced mRNA levels of (g) Adipo1 and (h) Adipo2 receptors. Pretreatment of the cells with MRI‐1867 (100 ng·ml −1 ) completely normalized these changes. (i) A proposed mechanism for the dual blockade of CB 1 receptors and inducible NOS by MRI‐1867 in reversing obesity‐induced chronic kidney disease (CKD) is shown. RQ, relative quantitation. In vivo data represent the mean ± SEM from 8 to 14 mice per group. * P < .05, significantly different from animals on STD; # P < .05, significantly different from animals on the same diet. In vitro data represent the mean ± SEM from five independent experiments. * P < .05, significantly different from Veh‐treated cells under normal conditions; # P < .05, significantly different from Veh‐treated cells under O:P conditions. Data were analysed by one‐way ANOVA, followed by a Bonferroni post hoc test

Article Snippet: HK‐2 cells were transfected with CRISPR‐CAS9 vector containing an sgRNA sequence to target all human isoforms of CNR1 (Genecopeia™, Cat# CS‐HCP263432‐CG01‐01‐B) using Lipofectamine 3000 (Invitrogen, Cat# L3000‐001) and selected with hygromycin (200‐μM; Sigma‐Aldrich, Cat# H3274).

Techniques: Expressing, Quantitation Assay, In Vivo, In Vitro