Journal: EMBO Reports

Article Title: Trichoplein binds PCM 1 and controls endothelial cell function by regulating autophagy

doi: 10.15252/embr.201948192

Figure Lengend Snippet: Representative pictures from transmission electron microscopy analysis. Scale bars, 500 nm. Right panel: quantification of autophagic vacuoles ( n = 16 cells, unpaired t ‐test; ** P = 0.0010 vs. control). Representative pictures and quantification of HUVECs after Alexa 488 Click‐iT ® l ‐azidohomoalanine (AHA) labelling. Right panel: quantification ( n = 50 cells, one‐way ANOVA; ** P = 0.0037 vs. control, ## P = 0.0005 vs. shTCHP). Scale bars, 25 μm. TCHP knock‐down or control HUVECs were transduced with TCHP‐V5 or control vectors and were stained for anti‐PCM1 antibody. Panel below: quantification ( n = 50 cells; unpaired t ‐test; ** P = 0.0003 vs. control; # P = 0.0166 vs. shTCHP). Scale bars, 25 μm. Western blot for anti‐V5 and anti‐p62 antibody (one‐way ANOVA; ** P = 0.0044; # P = 0.0439 vs. TCHP‐V5). Data information: Statistical analyses were performed on at least three independent experiments. Data are mean ± SD.

Article Snippet: The packing plasmids used were pCMV‐dR8.2 dvpr, plasmid #8455 and pCMV‐VSVG, plasmid #8454 from Addgene. p62‐V5 (HsCD00434166), TCHP‐V5 (HsCD00444989) and empty vector (pLX304) are from DNASU plasmid collection, and pBABE‐puro mCherry‐EGFP‐LC3B (Addgene plasmid # 22418) and pBABEpuro GFP‐LC3 (Addgene plasmid # 22405) were a gift from Jayanta Debnath.

Techniques: Transmission Assay, Electron Microscopy, Control, Knockdown, Transduction, Staining, Western Blot

Journal: EMBO Reports

Article Title: Trichoplein binds PCM 1 and controls endothelial cell function by regulating autophagy

doi: 10.15252/embr.201948192

Figure Lengend Snippet: Immunofluorescent staining for LC3 and p62 in TCHP knock‐down and control cells. Scale bars, 25 μm. Lower panel: quantification ( n = 90 cells, unpaired t ‐test; LC3: ** P < 0.0001 vs. control; p62: ** P < 0.0001 vs. control). Western blot of p62 and LC3 during under normal culture condition or starved condition (HBSS) or the presence of BafA1 (2 h) in TCHP knock‐down or control cells. Lower panels: p62 quantification (one‐way ANOVA; ** P = 0.0003 vs. control, ## P = 0.0093 vs. shTCHP) and LC3 quantification (one‐way ANOVA; ** P = 0.0008 vs. control, ## P = 0.0011 vs. shTCHP). HUVECs were transduced with the tandem mCherry‐EGFP‐LC3 and with shRNA TCHP or control vectors. Left panels : representative picture of mCherrry‐EGFP‐LC3 reporters. Scale bars, 25 μm. Right panel : quantification of the number of mCherry‐only (red bars, autolysosomes) or double‐positive (mCherry + /EGFP + ; yellow bars, autophagosomes) ( n = 80 cells, one‐way ANOVA; * P = 0.0447 vs. control; ## P = 0.0007 vs. shTCHP). Ratiometric flow cytometric analysis of mCherrry‐EGFP‐LC3 reporters as in C (one‐way ANOVA; * P = 0.0247 vs. control; ## P = 0.0088 vs. shTCHP). Quantification of long‐lived protein degradation assay in TCHP knock‐down and control cells by flow cytometry (one‐way ANOVA; ** P = 0.0018 vs. control, ## P < 0.0001 vs. shTCHP). HUVECs were transduced with TCHP‐V5 and control vectors. Western blot for anti‐V5 and anti‐p62 antibody during under normal culture condition or HBSS or BafA1 or torin‐1 treatment. Lower panel : quantification (one‐way ANOVA; ** P = 0.0044; # P = 0.0117 vs. TCHP‐V5). Data information: Statistical analyses were performed on at least three independent experiments. Data are represented as mean ± SD.

Article Snippet: The packing plasmids used were pCMV‐dR8.2 dvpr, plasmid #8455 and pCMV‐VSVG, plasmid #8454 from Addgene. p62‐V5 (HsCD00434166), TCHP‐V5 (HsCD00444989) and empty vector (pLX304) are from DNASU plasmid collection, and pBABE‐puro mCherry‐EGFP‐LC3B (Addgene plasmid # 22418) and pBABEpuro GFP‐LC3 (Addgene plasmid # 22405) were a gift from Jayanta Debnath.

Techniques: Staining, Knockdown, Control, Western Blot, Transduction, shRNA, Degradation Assay, Flow Cytometry

Journal: EMBO Reports

Article Title: Trichoplein binds PCM 1 and controls endothelial cell function by regulating autophagy

doi: 10.15252/embr.201948192

Figure Lengend Snippet: The postnuclear fraction (PNS) from TCHP knock‐down and control HUVECs in the presence or absence of BafA1 was separated into (low‐speed pellet) LSP and (high‐speed pellet) HSP fractions and then analysed by immunoblots using anti p62, LC3 and GABARAP antibodies. The sub‐fractions were treated with proteinase K (Prot. K) with or without Triton X‐100 (TX‐100). Quantification of the Western blot: the p62, LC3 and GABARAP levels relative to respective GAPDH were quantified using densitometry analysis and normalized to the value of non‐treated samples. Representative TEM images of autophagosomes in control and TCHP knock‐down HUVECs in the complete medium after 3‐h incubation with BafA1 (scale bars, 600 nm): (i), quantification of mean autophagic vesicles (AVs) and autophagosome (APs) area and (ii), distribution of the cross‐section areas of the analysed vesicles expressed in percentage ( n = 7 cells; > 100 vesicles per sample; AVs: unpaired t ‐test; ** P < 0.0001 vs. control; APs: unpaired t ‐test ** P < 0.0001 vs. control). Left panels: representative single‐channel and merged images of HUVECs expressing GFP‐LC3 and immunostained for STX17. Scale bars, 25 and 2 μm in the inset. Right panels : quantification of LC3/STX17‐positive cells in the presence or absence of BafA1 (one‐way ANOVA; ** P = 0.0060 vs. control; # P = 0.0136 vs. control BafA1) and quantification LC3/STX17‐positive puncta per cells ( n = 50 cells; one‐way ANOVA; ** P < 0.0001 vs. shTCHP BafA1). Data information: Statistical analyses were performed on at least three independent experiments. Data are represented as mean ± SD.

Article Snippet: The packing plasmids used were pCMV‐dR8.2 dvpr, plasmid #8455 and pCMV‐VSVG, plasmid #8454 from Addgene. p62‐V5 (HsCD00434166), TCHP‐V5 (HsCD00444989) and empty vector (pLX304) are from DNASU plasmid collection, and pBABE‐puro mCherry‐EGFP‐LC3B (Addgene plasmid # 22418) and pBABEpuro GFP‐LC3 (Addgene plasmid # 22405) were a gift from Jayanta Debnath.

Techniques: Knockdown, Control, Western Blot, Incubation, Expressing

Journal: EMBO Reports

Article Title: Trichoplein binds PCM 1 and controls endothelial cell function by regulating autophagy

doi: 10.15252/embr.201948192

Figure Lengend Snippet: A–C Co‐localization between (A), LAMP2 and p62, (B), aggregates and p62 and (C), ubiquitinated proteins and p62 in TCHP knock‐down and control cells. Scale bars, 25 and 2 μm in the inset. Lower panels: quantification of the above panels ( n = 80 cells, unpaired‐test; A: ** P = 0.0038 vs. control; B: ** P = 0.0005 vs. control; C: ** P = 0.0017 vs. control). Data information: Statistical analyses were performed on at least three independent experiments. Data are represented as mean ± SD.

Article Snippet: The packing plasmids used were pCMV‐dR8.2 dvpr, plasmid #8455 and pCMV‐VSVG, plasmid #8454 from Addgene. p62‐V5 (HsCD00434166), TCHP‐V5 (HsCD00444989) and empty vector (pLX304) are from DNASU plasmid collection, and pBABE‐puro mCherry‐EGFP‐LC3B (Addgene plasmid # 22418) and pBABEpuro GFP‐LC3 (Addgene plasmid # 22405) were a gift from Jayanta Debnath.

Techniques: Knockdown, Control

Journal: EMBO Reports

Article Title: Trichoplein binds PCM 1 and controls endothelial cell function by regulating autophagy

doi: 10.15252/embr.201948192

Figure Lengend Snippet: Immunofluorescence for recycling (RAB11), early (EEA1), late (RAB7) endosomes and lysosome (LAMP1/2); scale bars, 25 μm. Quantitative analysis of LysoTracker Red by flow cytometry (unpaired t ‐test; ** P = 0.0007 vs. control). Representative Western blot showing epidermal growth factor receptor (EGFR) levels during a pulse‐chase experiment with EGF (20 nM) for the indicated times. Right panel: quantification, ( n = 3, one‐way ANOVA; EGF 60 min. ** P = 0.0007 EGF 90 and 120 min. ** P < 0.0001 vs. control time 0). Representative images showed TCHP knock‐down and control cells stained for LAMP2 and αTubulin. Cells were analysed in growing medium (FM), after 2‐h starvation (Starv.) or after 2‐h starvation and replenishment of growing medium for 2 h (FM/Starv.) Scale bars, 10 μm. MT network was quantified as α‐Tubulin intensity. LAMP2 radial integrated fluorescence intensity was used to quantify the distribution of lysosomes. The graph shows the percentage of cells showing cytoplasmic spread or perinuclear lysosome distribution ( n = 20 cells, one‐way ANOVA; nuclear: ** P = 0.0031 vs. control FM, ** P = 0.0026 vs. control FM/Starv. Spread: * P = 0.0195 vs. control FM, ** P = 0.0001 vs. control FM/Starv) and MT intensity ( n = 20 cells, one‐way ANOVA; ** P = 0.0056 vs. control FM). Western blot for anti‐p62 in TCHP knock‐down and control cells after serum starvation. Data information: Statistical analyses were performed on at least three independent experiments. Data are represented as mean ± SD.

Article Snippet: The packing plasmids used were pCMV‐dR8.2 dvpr, plasmid #8455 and pCMV‐VSVG, plasmid #8454 from Addgene. p62‐V5 (HsCD00434166), TCHP‐V5 (HsCD00444989) and empty vector (pLX304) are from DNASU plasmid collection, and pBABE‐puro mCherry‐EGFP‐LC3B (Addgene plasmid # 22418) and pBABEpuro GFP‐LC3 (Addgene plasmid # 22405) were a gift from Jayanta Debnath.

Techniques: Immunofluorescence, Flow Cytometry, Control, Western Blot, Pulse Chase, Knockdown, Staining, Fluorescence

Journal: EMBO Reports

Article Title: Trichoplein binds PCM 1 and controls endothelial cell function by regulating autophagy

doi: 10.15252/embr.201948192

Figure Lengend Snippet: A Representative images for p62 of control and TCHP knock‐down HUVECs treated with BAY11‐7082 (300 nM) or TYRPHOSTIN AG1288 (300 nM) or SB202190 (300 nM) or vehicle (DMSO) for 48 h. Scale bars, 50 μm. B, C (B) Expression of IL‐6, IL‐8 and IL‐1β (one‐way ANOVA; IL‐8: ** P = 0.008 vs. control DMSO; # P = 0.0198 (BAY) # P = 0.01 (AG) vs. shTCHP DMSO; IL‐6: ** P = 0.0023 vs. control DMSO; # P = 0.0063 (BAY) # P = 0.0108 (AG) vs. shTCHP DMSO; IL‐1β: ** P = 0.0011 vs. control DMSO; # P = 0.0004 (BAY) # P = 0.0015 (AG) vs. shTCHP DMSO) and (C), migration speed was measured (one‐way ANOVA; ** P = 0.0003 vs. control DMSO; # P = 0.0451 vs. shTCHP DMSO). C Western blot analysis for anti‐phosphor‐NF‐κB (S536), total NF‐κB, in TCHP knock‐down and control cells treated with BAY or vehicle. Below panel: quantification (one‐way ANOVA; ** P = 0.0014 vs. control DMSO; # P = 0.0293 vs. shTCHP DMSO). D p62 expression in TCHP knock‐down and control cells treated with BAY or vehicle (one‐way ANOVA; ** P = 0.0004 vs. control DMSO; # P = 0.0243 vs. shTCHP DMSO). E p62 expression in TCHP knock‐down and control cells treated with siRELA or control (one‐way ANOVA; ** P = 0.0023 vs. control siRNA; # P = 0.0127 vs. shTCHP siRELA). F ChIP‐qPCR analysis confirms the of NF‐κB p65 enrichment to IκBα and p62 promoter in TCHP knock‐down cells (unpaired t ‐test; ** P = 0.0057 and ** P = 0.0037 vs. control, respectively). Data information: Statistical analyses were performed on at least three independent experiments. Data are represented as mean ± SD.

Article Snippet: The packing plasmids used were pCMV‐dR8.2 dvpr, plasmid #8455 and pCMV‐VSVG, plasmid #8454 from Addgene. p62‐V5 (HsCD00434166), TCHP‐V5 (HsCD00444989) and empty vector (pLX304) are from DNASU plasmid collection, and pBABE‐puro mCherry‐EGFP‐LC3B (Addgene plasmid # 22418) and pBABEpuro GFP‐LC3 (Addgene plasmid # 22405) were a gift from Jayanta Debnath.

Techniques: Control, Knockdown, Expressing, Migration, Western Blot, ChIP-qPCR

Journal: EMBO Reports

Article Title: Trichoplein binds PCM 1 and controls endothelial cell function by regulating autophagy

doi: 10.15252/embr.201948192

Figure Lengend Snippet: Expression of TCHP, p62, IL‐1β, IL‐8 and IL‐6 in ECs from vessels wall from patients with CAD ( n = 8 patients per group, unpaired t ‐test; TCHP ** P < 0.0001; p62 ** P = 0.0007; IL‐1β ** P < 0.0001; IL‐8 ** P < 0.0001; and IL‐6 ** P < 0.0001 vs. healthy subject). Staining and quantification of p62 in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY. Scale bars, 50 μm; ( n = 3 patients per group; n = 80 cells; one‐way ANOVA; ** P < 0.0001 vs. healthy DMSO; ## P = 0.003 vs. patients DMSO). Expression of IL‐6, IL‐8 and IL‐1β ( n = 3 patients per group; one‐way ANOVA; IL‐6: * P = 0.0135 vs. healthy DMSO; # P = 0.0180 vs. patients DMSO; IL‐8: * P = 0.0139 vs. healthy DMSO; ## P = 0.0024 vs. patients DMSO; IL‐1β: * P = 0.0104 vs. healthy DMSO; ## P = 0.0069 vs. patients DMSO). Migration speed, in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY ( n = 3 patients per group; one‐way ANOVA; * P = 0.0325 vs. healthy DMSO; # P = 0.0336 vs. patients DMSO). Left panels : representative images of the heart of wild‐type and Tchp knock‐out mice stained for CD31 (green) and p62 (magenta). Scale bars, 100 and 50 μm for the inset. Right panels : quantification of the percentage of cardiac area ( n = 4 mice per group; unpaired t ‐test; ** P = 0.083 vs. wild‐type) or vessels positive for p62 ( n = 4 mice per group; unpaired t ‐test; ** P = 0.058 vs. wild‐type) and vessel density ( n = 4 mice per group; unpaired t ‐test; ** P = 0.0097 vs. wild‐type). Data information: Data are represented as mean ± SD.

Article Snippet: The packing plasmids used were pCMV‐dR8.2 dvpr, plasmid #8455 and pCMV‐VSVG, plasmid #8454 from Addgene. p62‐V5 (HsCD00434166), TCHP‐V5 (HsCD00444989) and empty vector (pLX304) are from DNASU plasmid collection, and pBABE‐puro mCherry‐EGFP‐LC3B (Addgene plasmid # 22418) and pBABEpuro GFP‐LC3 (Addgene plasmid # 22405) were a gift from Jayanta Debnath.

Techniques: Expressing, Staining, Migration, Knock-Out