Journal: Nature Communications

Article Title: ABHD5 inhibits YAP-induced c-Met overexpression and colon cancer cell stemness via suppressing YAP methylation

doi: 10.1038/s41467-021-26967-5

Figure Lengend Snippet: a , b Immunoblotting of ABHD5 levels of control (shCTRL) and ABHD5 -knockdown (shABHD5) HCT116 cells (left). The representative pictures of spheres are shown (right) ( a ). The number and the size ( n = 5, 10 biologically independent samples) of the spheres were quantified ( b ). AU arbitrary units. c The number and the size ( n = 5, 10 biologically independent samples) of the colonies were quantified. d Flow cytometry of ALDH + proportion in the absence or presence of the ALDH inhibitor diethylaminobenzaldehyde (DEAB; 15 μmol/L) ( n = 3 biologically independent samples). e Flow cytometry of CD133 + /CD44 + cells ( n = 3 biologically independent samples). f Representative images of immunohistochemical staining of Lgr5 of intestinal tumour sections from Apc Min/+ /Abhd5 +/+/Cre+ and Apc Min/+ /Abhd5 f/f/Cre+ mice. The positive cell number is quantified (right) (r.u. relative units) ( n = 12 mice/group). g Schematic of limiting dilution assay. ABHD5 levels of cells injected were assessed by immunoblotting prior to the experiment (top). h Stem cell frequencies were determined using L-Calc software. The upper table shows the number of tumours with a positive response (response = tumour > 0.1 cm 3 at 20 days post injection)/total number of tumours and is depicted in the pie chart diagram below. i Images of resected xenografts from the group injected with 1 × 10 5 shCTRL or sh ABHD5 HCT116 cells at 15 days after injection. The incidence of tumourigenicity and tumour volume at different time points is quantified (below). ( n = 9 mice/group, ns no significance ( p > 0.05), **** p < 0.0001). j Dot density plot of final tumour weight ( n = 6 mice/group). k The analyses of the relative primary and second sphere-forming unit of shABHD5 or shCTRL HCT116 cells digested from the xenografts ( n = 3 biologically independent samples, *** p = 0.0002, **** p < 0.0001) (Data are shown as mean ± s.e.m. Unpaired two-sided Student’s t test was used in panels ( b ) (right), ( c ), ( e ) and ( f ). Panels ( b ) (left), ( d ) and ( i − k ) were analysed using two-way ANOVA and Sidak’s multiple comparison test. Source data are provided as a Source Data file).

Article Snippet: The recombinant GST-ABHD5 fusion proteins were labelled with Biotin (Full Moon Biosystems) and used to probe the ProtoArray Human Protein Microarray (Wayen Biotechnologies).

Techniques: Western Blot, Flow Cytometry, Immunohistochemical staining, Staining, Limiting Dilution Assay, Injection, Software

Journal: Nature Communications

Article Title: ABHD5 inhibits YAP-induced c-Met overexpression and colon cancer cell stemness via suppressing YAP methylation

doi: 10.1038/s41467-021-26967-5

Figure Lengend Snippet: a , b Pathway enrichment analysis ( a ) and target analysis ( b ) of the FDA-approved compounds that reduced cell viability (>90% viability) of ABHD5 -knockdown cells (>90%) with little to no effect on control cells. c The inhibitory rate analysis of shCTRL or shABHD5 HCT116 cells treated with FDA-approved c-Met inhibitors ( n = 5 biologically independent samples). d Sphere formation assay of HCT116 shCTRL or shABHD5 cells treated with 1 μM savolitinib or vehicle control (DMSO) ( n = 5 biologically independent samples). e Savolitinib dose–response curves in HCT116 shCTRL or shABHD5 cells ( n = 5 biologically independent samples). f Caspase-3 activation in shCTRL or shABHD5 HCT116 cells treated with savolitinib from at least three independent experiments ( n = 5 biologically independent samples). g Subcutaneuos xenografts were established by shCTRL and shABHD5 HCT116 cells in NOD/SCID mice. The tumour-bearing mice were randomized into groups ( n = 6 mice/group) and dosed by oral gavage as follows: 10 ml/kg vehicle (acidic CMC-Na 0.5%, pH = 2.1) once daily or 2.5 mg/kg savolitinib once daily for 21 days. The representative images of the xenografts were shown. h Statistical analyses of the tumour volume and tumour weight in the indicated groups in ( g ) ( n = 6 mice/group). i Lung metastatic xenografts were established by shCTRL and shABHD5 HCT116 cells in NOD/SCID mice. The tumour-bearing mice were randomized into groups ( n = 6 mice/group) and dosed by oral gavage as follows: 10 ml/kg vehicle (acidic CMC-Na 0.5%, pH = 2.1) once daily or 2.5 mg/kg savolitinib once daily for 21 days. The representative bioluminescent and gross images of lung metastases in NOD/SCID mice were shown. j The representative images of HE staining of lung tissues in NOD/SCID mice of ( i ). k The quantifications of bioluminescent images of lung metastases in the indicated groups in ( i ) ( n = 6 mice/group) (Data are shown as mean ± s.e.m. Panels ( c − f ), ( h ) and ( k ) were analysed using two-way ANOVA and Sidak’s multiple comparison test. ns no significance, **** p < 0.0001. Source data are provided as a Source Data file).

Article Snippet: The recombinant GST-ABHD5 fusion proteins were labelled with Biotin (Full Moon Biosystems) and used to probe the ProtoArray Human Protein Microarray (Wayen Biotechnologies).

Techniques: Tube Formation Assay, Activation Assay, Staining

Journal: Nature Communications

Article Title: ABHD5 inhibits YAP-induced c-Met overexpression and colon cancer cell stemness via suppressing YAP methylation

doi: 10.1038/s41467-021-26967-5

Figure Lengend Snippet: a Immunoblotting of β-Catenin target gene levels in shCTRL or shABHD5 HCT116 cells. b Statistical analyses of sphere formation assay and anchorage-independent growth assay of shCTRL or shABHD5 HCT116 cells transfected with either the control shRNA (Control) or MET- knockdown shRNA ( MET KD) ( n = 3 biologically independent samples). c Representative immunohistochemistry images of c-Met in the intestine tumours and intestine mucosa of Apc Min/+ /Abhd5 +/+/Cre+ or Apc Min/+ /Abhd5 f/f/Cre+ male mice at 100 days of age. Scale bar, 500 μm and 200 μm. d Immunoblotting of GFP and c-Met expression levels in the intestinal mucosa of Apc Min/+ /Abhd5 f/f/Cre+ mice injected with GFP-labelled control (AAV-shScrmbl) or Met- silencing-AAV (AAV-shMet) via the tail vein (iv1–2: tail vein injection in 2 mice). e Statistical analysis of the number of total and locally invasive tumours in the entire small intestine of control and Apc Min/+ /Abhd5 f/f/Cre+ mice injected with control (AAV-shScrmbl) or Met- silencing-AAV (AAV-shMet) via the tail vein ( n = 6 mice/group). f Immunofluorescent staining of β-Catenin in shCTRL or shABHD5 HCT116 treated with either Wnt3a (250 ng/ml) or vehicle (PBS). g Subcellular fractionation immunoblotting of cytoplasmic and nuclear β-Catenin in shCTRL or shABHD5 HCT116 cells treated with either Wnt3a (250 ng/ml) or vehicle (PBS). h β-Catenin immune complexes were immunoprecipitated from shCTRL or shABHD5 HCT116 cells and subjected to immunoblotting of TCF4. i Top-flash (Wnt/β-Catenin pathway-responsive firefly luciferase plasmid) reporter gene assay in shCTRL or shABHD5 HCT116 cells ( n = 6 biologically independent samples). j β-Catenin or TCF4 immune complexes were isolated from shCTRL or shABHD5 HCT116 cells and subjected to ChIP-qPCR analysis with primers derived from the promoter regions of MET ( n = 9 (left) or 8 (right) biologically independent samples). k Immunoblotting of ABHD5 and c-Met expression levels in shCTRL or shABHD5 HCT116 cells treated with either XAV-939 (10 μM) or vehicle (DMSO) (Data are shown as mean ± s.e.m. Unpaired two-sided Student’s t test was used in panels ( i ) and ( j ), whereas panels ( b ) and ( e ) were analysed using two-way ANOVA and Sidak’s multiple comparison test. Source data are provided as a Source Data file).

Article Snippet: The recombinant GST-ABHD5 fusion proteins were labelled with Biotin (Full Moon Biosystems) and used to probe the ProtoArray Human Protein Microarray (Wayen Biotechnologies).

Techniques: Western Blot, Tube Formation Assay, Growth Assay, Transfection, shRNA, Immunohistochemistry, Expressing, Injection, Staining, Fractionation, Immunoprecipitation, Luciferase, Plasmid Preparation, Reporter Gene Assay, Isolation, Derivative Assay

Journal: Nature Communications

Article Title: ABHD5 inhibits YAP-induced c-Met overexpression and colon cancer cell stemness via suppressing YAP methylation

doi: 10.1038/s41467-021-26967-5

Figure Lengend Snippet: a Heatmap showing the expression patterns of β-Catenin or YAP target genes in shCTRL or shABHD5 HCT116 cells screened by Microarray. b KEGG pathway enrichment analysis of differentially expressed genes between shCTRL or shABHD5 HCT116 cells screened by Microarray. The top 20 positively and negatively enriched pathways are shown in the bar plot. c Representative immunofluorescent staining of YAP in shCTRL or shABHD5 HCT116 cells. d Subcellular fractionation immunoblotting of cytosolic and nucleic YAP in shCTRL or shABHD5 HCT116 cells treated with either EGF (200 ng/ml) or vehicle (PBS). e TEAD immune complexes were immunoprecipitated from shCTRL or shABHD5 HCT116 cells and subjected to immunoblotting of YAP. f TEAD luciferase reporter assay in shCTRL or shABHD5 HCT116 cells ( n = 3 biologically independent samples). g Real-time PCR assay of the indicated target gene mRNA levels in shCTRL or shABHD5 HCT116 cells ( n = 3 biologically independent samples). h Immunoblotting of ABHD5 and c-Met expression levels in shCTRL or shABHD5 HCT116 cells treated with YAP knockdown or YAP inhibitor verteporfin (15 μM), DMSO as a vehicle control. i Representative immunohistochemistry staining of YAP expression in paraffin sections of the intestinal tumours of 100-day-old male mice. Apc Min/+ /Abhd5 +/+/Cre+ , Apc Min/+ /Abhd5 f/+/Cre+ and Apc Min/+ /Abhd5 f/f/Cre+ . Scale bar, 200 μm and 50 μm. j Immunoblotting of YAP and c-Met expression levels in the intestinal tumours of Apc Min/+ /Abhd5 +/+/Cre+ and Apc Min/+ /Abhd5 f/f/Cre+ mice injected with AAV-Scrmbl or AAV-shYap via the tail vein. H2A as a loading control for nuclear protein, and tubulin as a loading control for cytoplasmic protein. k Statistical analysis of the number of total and locally invasive tumours in the entire small intestine of Apc Min/+ /Abhd5 +/+/Cre+ and Apc Min/+ /Abhd5 f/f/Cre+ mice injected with AAV-Scrmbl or AAV-shYap via the tail vein ( n = 6 mice/group) (Data are shown as mean ± s.e.m. Unpaired two-sided Student’s t test was used in panel ( f ), whereas panels ( g ) and ( k ) were analysed using two-way ANOVA and Sidak’s multiple comparison test. Source data are provided as a Source Data file).

Article Snippet: The recombinant GST-ABHD5 fusion proteins were labelled with Biotin (Full Moon Biosystems) and used to probe the ProtoArray Human Protein Microarray (Wayen Biotechnologies).

Techniques: Expressing, Microarray, Staining, Fractionation, Western Blot, Immunoprecipitation, Luciferase, Reporter Assay, Real-time Polymerase Chain Reaction, Immunohistochemistry, Injection

Journal: Nature Communications

Article Title: ABHD5 inhibits YAP-induced c-Met overexpression and colon cancer cell stemness via suppressing YAP methylation

doi: 10.1038/s41467-021-26967-5

Figure Lengend Snippet: a Immunoblotting of YAP K342me levels in shCTRL or shABHD5 HCT116 cells transfected with control or siRNA plasmids silencing the indicated SET1A subunits. b Immunoblotting of DPY30. c Immunoblotting of ABHD5, YAP K342me and c-Met in shCTRL and shABHD5 HCT116 cells transfected with control vector or DPY30 shRNA plasmids ( DPY30 KD). d TEAD luciferase reporter assay in shCTRL and shABHD5 HCT116 cells transfected with control vector or DPY30 shRNA plasmids ( DPY30 KD) ( n = 3 biologically independent samples). e Representative immunohistochemistry staining of DPY30 in the intestinal tumours of Apc Min/+ /Abhd5 +/+/Cre+ and Apc Min/+ /Abhd5 f/f/Cre+ . f Immunoblotting of YAP K342me and c-Met in the intestinal tumours of Apc Min/+ /Abhd5 +/+/Cre+ and Apc Min/+ /Abhd5 f/f/Cre+ mice injected with AAV-Scrmbl or AAV-shDpy30 via tail vein. g Statistical analysis of intestinal tumours in Apc Min/+ /Abhd5 +/+/Cre+ and Apc Min/+ /Abhd5 f/f/Cre+ mice injected with AAV-Scrmbl or AAV-shDpy30 ( n = 6 mice/group). h Real-time PCR assay of DPY30 mRNA levels ( n = 3 biologically independent samples). i Immunoblotting of cytoplasmic and nuclear DPY30. j Representative immunofluorescent staining of ABHD5, YAP and DPY30. k DPY30 immune complexes were immunoprecipitated from shCTRL or shABHD5 HCT116 cells and subjected to immunoblotting of ABHD5, YAP and DPY30. l The interaction complex model between ABHD5 and DPY30 predicted by docking methods. m A direct interaction between GST-labelled-ABHD5 and DPY30 based on Human Proteome Microarray assay. n Ubiquitin levels of DPY30 immune complexes. o Immunoblotting of Flag-DPY30 expression using Flag antibody in ABHD5 -knockdown HCT116 cells transfected with Flag- DPY30 . p Immunoblotting of the indicated proteins in vector control or ABHD5 -overexpressing HCT116 cells in the presence of MG132 (5 mM) for 16 h. q Immunoblotting showing the stability of DPY30 protein in HCT116 cells transfected with hemagglutinin-tagged DPY30 (HA- DPY30 ) and WT Flag- ABHD5 or mutant Flag- ABHD5 deleted the binding domain to DPY30. Cells were treated with 20 mM cycloheximide (CHX) at the indicated intervals (Data are shown as mean ± s.e.m. Unpaired two-sided Student’s t test was used in panel ( h ), whereas panels ( d ) and ( g ) were analysed using two-way ANOVA and Sidak’s multiple comparison test. Source data are provided as a Source Data file).

Article Snippet: The recombinant GST-ABHD5 fusion proteins were labelled with Biotin (Full Moon Biosystems) and used to probe the ProtoArray Human Protein Microarray (Wayen Biotechnologies).

Techniques: Western Blot, Transfection, Plasmid Preparation, shRNA, Luciferase, Reporter Assay, Immunohistochemistry, Staining, Injection, Real-time Polymerase Chain Reaction, Immunoprecipitation, Microarray, Expressing, Mutagenesis, Binding Assay

Journal: Nature Communications

Article Title: ABHD5 inhibits YAP-induced c-Met overexpression and colon cancer cell stemness via suppressing YAP methylation

doi: 10.1038/s41467-021-26967-5

Figure Lengend Snippet: a Represent immunohistochemistry stainings of ABHD5/DPY30/c-Met in CRC tissue microarray. Score = 0–1 (low) and score = 2–3 (high) indicate ABHD5, DPY30, and c-Met levels in representative tumour tissues. Scale bars, 500 μm (main micrographs) and 100 μm (insets). b Correlation analyses for the correlation between ABHD5, DPY30 and c-Met in CRC tissues. Plotted data are the expression index calculated from immunostaining. Spearman correlation analysis ( n = 76 biologically independent samples). c Disease-free survival (DFS) of CRC patients with ABHD5 low /DPY30 high / c-Met high ( n = 53) or ABHD5 high /DPY30 low / c-Met low CRC tumours ( n = 54 biologically independent samples). d Represent immunohistochemistry stainings of ABHD5/DPY30/c-Met in surgically resected CRC tissues. Scale bars, 500 μm (main micrographs) and 100 μm (insets). e Schematic of the experiment. Surgically resected ABHD5 high /DPY30 low /c-Met low or ABHD5 low /DPY30 high /c-Met high CRC tumours were dissociated into single cells. Ex vivo sphere formation assays of tumour-derived cells were performed in the presence of 1 μM savolitinib or DMSO (vehicle). f Representative images and statistical analyses of cancer cell spheres derived from surgically resected ABHD5 high /DPY30 low /c-Met low or ABHD5 low /DPY30 high /c-Met high CRC tumours ( n = 5 biologically independent samples) (Data are shown as mean ± s.e.m. The correlation analysis was determined by Spearman correlation coefficient in panel ( b ). Kaplan−Meier survival method was used in panel ( c ). Panel ( f ) was analysed using two-way ANOVA and Sidak’s multiple comparison test. **** p < 0.0001. Source data are provided as a Source Data file).

Article Snippet: The recombinant GST-ABHD5 fusion proteins were labelled with Biotin (Full Moon Biosystems) and used to probe the ProtoArray Human Protein Microarray (Wayen Biotechnologies).

Techniques: Immunohistochemistry, Microarray, Expressing, Immunostaining, Ex Vivo, Derivative Assay

Journal: Nature Communications

Article Title: ABHD5 inhibits YAP-induced c-Met overexpression and colon cancer cell stemness via suppressing YAP methylation

doi: 10.1038/s41467-021-26967-5

Figure Lengend Snippet: ABHD5 interacts in the cytoplasm with DPY30, the core subunit of the SET1A complex, to sequester DPY30 in cytoplasm and prevent its nuclear translocation. In ABHD5-deficient CRC cells, loss of ABHD5 releases DPY30 to translocate into the nucleus and achieve SET1-mediated mono-methylation of YAP at K342, which increases the nuclear location of YAP and YAP-induced transcription of c-Met for increasing and sustaining cancer cell stemness, consequently promoting the development and progression of CRCs.

Article Snippet: The recombinant GST-ABHD5 fusion proteins were labelled with Biotin (Full Moon Biosystems) and used to probe the ProtoArray Human Protein Microarray (Wayen Biotechnologies).

Techniques: Translocation Assay, Methylation

Journal: Diabetes

Article Title: Characterization and Clinical Association of Autoantibodies Against Perilipin 1 in Patients With Acquired Generalized Lipodystrophy.

doi: 10.2337/db21-1086

Figure Lengend Snippet: Figure 3—Blocking activity of anti-PLIN1 autoantibodies. A: Detection of blocking activity of anti-PLIN1 autoantibodies using serum sam- ples at 1/250 dilution incubated with a fixed amount (100 ng) of recombinant PLIN1. The unspecific blocking activity (8.31%) was estab- lished using the mean value obtained for 50 healthy donors. B: Dose-dependent inhibition of the interaction of PLIN1 and ABHD5 in the presence of sera samples from anti-PLIN1–positive patients (AGL3, AGL13, AGL33, and AGL39). Serum from AGL19 and pooled serum from five healthy donors were negative for anti-PLIN1 autoantibodies. Serially diluted serum samples were mixed with a fixed amount (100 ng) of recombinant PLIN1. For both images, results were expressed as mean ± SD from two independent experiments.

Article Snippet: For blocking assays, ELISA plates (Costar Medium binding) were coated with 100 ng/well of recombinant ABHD5 (OriGene) in carbonate/bicarbonate (pH 9.3) overnight at 4 C. The plates were blocked with assay buffer (PBS supplemented with 0.1% Tween 20 and 5% BSA) for 1 h at 37 C and washed five times with PBS-0.1% Tween 20.

Techniques: Blocking Assay, Activity Assay, Incubation, Recombinant, Inhibition

Journal: Diabetes

Article Title: Characterization and Clinical Association of Autoantibodies Against Perilipin 1 in Patients With Acquired Generalized Lipodystrophy.

doi: 10.2337/db21-1086

Figure Lengend Snippet: Figure 4—Functional effects of anti-PLIN1 autoantibodies on lipolysis and lipase activity. Preadipocytes (3T3-L1) were incubated for 180 min with human serum at 1/10 dilution from four patients with AGL (AGL3, AGL13, AGL33, and AGL39) with anti-PLIN1 autoantibodies, one patient without antibodies (AGL19), and a pool of healthy donors. A: Results of the radiometric assessment of basal and stimulated lipolysis. Data rep- resent mean ± SD for triplicates of two independent experiments for each sample. Statistical significance was assessed with one-way ANOVA assay comparing the mean of each column with the mean from untreated cells. B: Results of lipase activity (nanomoles of glycerol), represented as the percentage over maximum activity from cells treated with healthy donor serum. Data represent mean ± SD for triplicates of two indepen- dent experiments for each sample. C: Confocal microscopic analysis of mouse preadipocytes revealed colocalization of PLIN1 and IgG from patient AGL39 on the lipid droplet surface (merged image, in yellow). However, no colocalization was observed when using serum from healthy donors (HD). ABHD5 was localized mainly on lipid droplets under treatment with serum from HD or untreated (basal conditions). Under stimu- lated conditions or in the presence of serum from AGL39, ABHD5 staining was localized on cytosol. DNA was stained with DAPI (blue); IgG binding was detected using FITC-conjugated rabbit anti-human IgG (green); and PLIN1 or ABHD5 was detected with biotin-labeled rabbit IgG followed by Texas Red–labeled streptavidin (red). Scale bars, 20 mm. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Article Snippet: For blocking assays, ELISA plates (Costar Medium binding) were coated with 100 ng/well of recombinant ABHD5 (OriGene) in carbonate/bicarbonate (pH 9.3) overnight at 4 C. The plates were blocked with assay buffer (PBS supplemented with 0.1% Tween 20 and 5% BSA) for 1 h at 37 C and washed five times with PBS-0.1% Tween 20.

Techniques: Functional Assay, Activity Assay, Incubation, Staining, Binding Assay, Labeling

Journal: Molecular Metabolism

Article Title: G0/G1 Switch Gene 2 controls adipose triglyceride lipase activity and lipid metabolism in skeletal muscle

doi: 10.1016/j.molmet.2016.04.004

Figure Lengend Snippet: G0S2 inhibits ATGL activity in mouse and human skeletal muscle. (A) Representative blots of G0S2 and CGI-58 protein content in mouse extensor digitorum longus (EDL), gastrocnemius (Gast), and soleus skeletal muscles. Triacylglycerol hydrolase activity (TAGH) was measured in absence (control) or presence of recombinant human G0S2 (rhG0S2) in (B) mice soleus and EDL (n = 7) and (C) human vastus lateralis muscle (n = 5). (D) TAGH activity was measured in absence (control) or presence of recombinant human G0S2 (rhG0S2), CGI-58 (rhCGI-58), or both (rhG0S2+rhCGI-58) in COS7 cell extracts overexpressing human ATGL (n = 4). *p < 0.05 versus control.

Article Snippet: ATGL activity was measured in the presence of 1 μg of human recombinant G0S2 protein (rhG0S2) (OriGene), CGI-58 (rhCGI-58) (Abnova), or both.

Techniques: Activity Assay, Muscles, Control, Recombinant

Journal: Nature communications

Article Title: ABHD5 blunts the sensitivity of colorectal cancer to fluorouracil via promoting autophagic uracil yield.

doi: 10.1038/s41467-019-08902-x

Figure Lengend Snippet: Fig. 1 ABHD5 impedes the sensitivity of pMMR CRCs to FU. a Analyses from the GDSC (Genomics of Drug Sensitivity in Cancer) dataset revealed the correlation between ABHD5 proficiency and the sensitivity to FU in pMMR or dMMR CRC cell lines (Pearson’s correlations). b MTT assay determining the IC50 of ABHD5 knockdown (ABHD5 KD) and control SW480 cells (n = 3, Student’s t-test). c MTT assay determining the cell viability at different time points during FU (25 μM) treatment (n = 4, Student’s t-test). d Fluorescence activated cell sorting (FACS) showing the apoptotic rate of cells stained with Annexin V-PE/7AAD following 24 h of treatment with PBS or FU (25 μM) (n = 4, Student’s t-test). e, f ABHD5 KD and control SW480 cells were inoculated intra-abdominally into NOD-SCID mice, and intraperitoneal injection (i.p.) of PBS or FU (50 mg per kg) + calcium folinate (80 mg per kg) was administrated once per week for 3 weeks. Tumor burden was measured by bioluminescent imaging (n = 5, Two-way ANOVA) (e), and the xenografts were stained with TUNEL to calculate apoptotic cells (n = 5, Student’s t-test) (f). Scale bar: 200 μm. g–j pMMR/ABHD5high or pMMR/ABHD5low xenografts were established in PDX mice, and the mice were treated with PBS or FU (50 mg per kg) + calcium folinate (80 mg per kg) (i.p., once per week for 3 weeks) when tumor volume reached about 100 mm3. Representative macroscopic views showing the xenografts in the indicated subgroups (g); plot representing the evolution of the body weight and tumor volume of xenografts in the indicated groups (n = 5, Two-way ANOVA) (h); the proliferating and apoptotic cells in the dissected xenografts were immunostained with Ki67 or caspase-3 (i) and analyzed (Horizontal lines indicate arithmetic mean values, and error bars show the 95% CI. r.u., relative units. n = 12, Student’s t-test) (j). The quantitative data were presented as mean ± S.D (error bar) (N.S. no significant, *p < 0.05, **p < 0.01, ***p < 0.001)

Article Snippet: Plasmids for overexpression of mutant ABHD5 and wild type ABHD5, lentiviruses for human RNASET2 knockdown, the recombinant proteins of human wild type His-ABHD5, HisRNASET2, His-PDIA5, GST-PDIA5 and mutant GST-PDIA5 were from Wuhan GeneCreate Biological Engineering Co., Ltd. ABHD5 (Human) recombinant protein (H00051099-P01) and RNASET2 (Human) recombinant protein (H00008635P01) were purchased from Novus.

Techniques: MTT Assay, Knockdown, Control, Fluorescence, FACS, Staining, Injection, Imaging, TUNEL Assay

Journal: Nature communications

Article Title: ABHD5 blunts the sensitivity of colorectal cancer to fluorouracil via promoting autophagic uracil yield.

doi: 10.1038/s41467-019-08902-x

Figure Lengend Snippet: Fig. 2 ABHD5 expression and the benefit from FU-based adjuvant chemotherapy. a–d The StepMiner algorithm was used to stratify the population of 361 pMMR CRC patients (stage II/III) in the NCBI-GEO dataset into ABHD5high and ABHD5low subgroups, and the association of ABHD5 with prognosis and benefit from FU-based adjuvant chemotherapy was evaluated. a Three hundred and sixty-one pMMR CRC patients were stratified into ABHD5high and ABHD5low subgroups based on mRNA levels (StepMiner). b ABHD5high tumors were significantly more likely to be BRAF mutant (BRAF MT) and p53 wild type (p53 WT) and CIN negative (CIN−), and ABHD5low tumors were significantly more likely to be BRAF wild type (BRAF WT) and p53 mutant (p53 MT) and CIN positive (CIN+) (*p < 0.05, ***p < 0.001, χ2-test). c DFS in the subgroup of pMMR/ABHD5low or pMMR/ABHD5high who received surgery alone (Log-rank test). d DFS in pMMR/ABHD5low or pMMR/ABHD5high subgroup treated with or without FU-based adjuvant chemotherapy (Log-rank test, Cox proportional regression). e, f A human CRC tissue microarray containing 432 pMMR patient tumor samples from our hospital was analyzed. The samples were stratified into pMMR/ABHD5high or pMMR/ABHD5low subgroups based on the immunostaining score for MMR status and ABHD5 proficiency. e DFS in the subgroup of pMMR/ABHD5low or pMMR/ABHD5high who received surgery alone (Log-rank test). f DFS in the pMMR/ABHD5low or pMMR/ ABHD5high subgroup treated with or without FU-based adjuvant chemotherapy (Log-rank test, Cox proportional regression)

Article Snippet: Plasmids for overexpression of mutant ABHD5 and wild type ABHD5, lentiviruses for human RNASET2 knockdown, the recombinant proteins of human wild type His-ABHD5, HisRNASET2, His-PDIA5, GST-PDIA5 and mutant GST-PDIA5 were from Wuhan GeneCreate Biological Engineering Co., Ltd. ABHD5 (Human) recombinant protein (H00051099-P01) and RNASET2 (Human) recombinant protein (H00008635P01) were purchased from Novus.

Techniques: Expressing, Adjuvant, Mutagenesis, Microarray, Immunostaining

Journal: Nature communications

Article Title: ABHD5 blunts the sensitivity of colorectal cancer to fluorouracil via promoting autophagic uracil yield.

doi: 10.1038/s41467-019-08902-x

Figure Lengend Snippet: Fig. 3 ABHD5 proficiency determines the FU uptake of CRC cells. a The cells were exposed to PBS or FU (25 μM) for 6 h, and the intracellular concentrations of FU were measured by HPLC (n = 3, Student’s t-test). b Western blots of indicated proteins in the cells 24 h following PBS or FU (25 μM) treatment. c The cells were exposed to FU (25 μM) for 6 h, and the concentrations of cellular uracil was analyzed by LC/MS (n = 6, Student’s t-test). d Western blots of indicated proteins in the cells 24 h after exposure to PBS or FU (25 μM). e GSEA analysis of the lysosome pathway. f The hierarchical clustering analysis showing the different expression pattern of the genes involved in the lysosome pathway. g HCS images showing RFP/GFP labeled LC3 staining in the cells at different time points. h Statistical analysis of autophagosome and autolysosome in the cells at different time points, and the corresponding intracellular FU levels are shown (n = 3, Two-way ANOVA);. i, j The cells were treated with FU (25 μM) alone or FU (25 μM) + CQ (50 μM) for 6 h. The concentrations of cellular uracil (i) and the intracellular FU (j) were measured (n = 6, Student’s t-test). k ABHD5 overexpression (ABHD5 OE) and control cells were inoculated subcutaneously in nude mice, and the mice were treated with PBS, FU (50 mg per kg) + calcium folinate (80 mg per kg) or FU (50 mg per kg) + calcium folinate (80 mg per kg) + CQ (150 mg per kg) (i.p., once per week for 3 weeks). Tumor burden was measured every 3 days (n = 5, Two-way ANOVA). l, m The cells were treated with FU (25 μM) alone or FU (25 μM) + BECN1 activator (BA) (10 μM) for 6 h (l) or 24 h (m). The concentrations of intracellular uracil (l) and the apoptotic rate (m) were analyzed (n = 6, Student’s t-test). The quantitative data were presented as mean ± S.D (error bar) (N.S. no significance, *p < 0.05, **p < 0.01, ***p < 0.001)

Article Snippet: Plasmids for overexpression of mutant ABHD5 and wild type ABHD5, lentiviruses for human RNASET2 knockdown, the recombinant proteins of human wild type His-ABHD5, HisRNASET2, His-PDIA5, GST-PDIA5 and mutant GST-PDIA5 were from Wuhan GeneCreate Biological Engineering Co., Ltd. ABHD5 (Human) recombinant protein (H00051099-P01) and RNASET2 (Human) recombinant protein (H00008635P01) were purchased from Novus.

Techniques: Western Blot, Liquid Chromatography with Mass Spectroscopy, Expressing, Labeling, Staining, Over Expression, Control

Journal: Nature communications

Article Title: ABHD5 blunts the sensitivity of colorectal cancer to fluorouracil via promoting autophagic uracil yield.

doi: 10.1038/s41467-019-08902-x

Figure Lengend Snippet: Fig. 4 RNASET2 mediates ABHD5-induced autophagic uracil yield. a Schematic representation of reactions of the uracil-related nucleotide degradation pathway. b Nucleosides and nucleobases measurement at the different time points in the cells treated with FU (25 μM). The results are presented as normalized intensities on the basis of the peak height of each metabolite in control cells (n = 6, Student’s t-test). c Schematic representation of the pathway for autophagy-dependent RNA degradation in mammalian cells. d RNASET2 knockdown (RNASET2 KD), ABHD5 KD, and control SW480 cells were treated with FU (25 μM). Nucleosides and 3’-NMPs were analyzed at different time points. The results are presented as normalized intensities on the basis of the peak height of each metabolite in control cells (n = 3, Two-way ANOVA). e RNASET2-silenced ABHD5 OE and control SW480 cells were treated with FU (25 μM) for 6 h, and the concentrations of cellular uracil were analyzed (n = 6, Student’s t-test). f RNASET2-silenced ABHD5 OE and control SW480 cells were treated with FU (25 μM) for 24 h, and the apoptotic rates were analyzed (n = 5, Student’s t-test). g RNASET2-silenced ABHD5 OE and control SW480 were subcutaneously inoculated in nude mice, and the mice were treated with PBS or FU (50 mg per kg) + calcium folinate (80 mg per kg) (i.p., once per week for 3 weeks). Tumor volume was measured every 3 days (n = 5, Two-way ANOVA). The quantitative data were presented as mean ± S.D (error bar) (N.S. no significance, *p < 0.05, **p < 0.01, ***p < 0.001)

Article Snippet: Plasmids for overexpression of mutant ABHD5 and wild type ABHD5, lentiviruses for human RNASET2 knockdown, the recombinant proteins of human wild type His-ABHD5, HisRNASET2, His-PDIA5, GST-PDIA5 and mutant GST-PDIA5 were from Wuhan GeneCreate Biological Engineering Co., Ltd. ABHD5 (Human) recombinant protein (H00051099-P01) and RNASET2 (Human) recombinant protein (H00008635P01) were purchased from Novus.

Techniques: Control, Knockdown

Journal: Nature communications

Article Title: ABHD5 blunts the sensitivity of colorectal cancer to fluorouracil via promoting autophagic uracil yield.

doi: 10.1038/s41467-019-08902-x

Figure Lengend Snippet: Fig. 5 ABHD5 sustains the activity of RNASET2. a Representative images of immunofluorescent stainings of lysosome (stained by Lysotracker, red) and ABHD5 (green) in SW480 cells. Scale bar: 10 μm. b Western blots showing ABHD5 expression in the lysosome lysates of ABHD5 KD and control cells. LAMP1 was used as a reference. c Immunoprecipitation showing an interaction between ABHD5 and RNASET2. SUCLG2 was used as a positive control. d Yeast two-hybrid assay showing a negative result for the direct interaction between ABHD5 and RNASET2. e Western blots of RNASET2 expression in the lysosome lysates of ABHD5 KD and control cells. f A direct interaction between ABHD5 and PDIA5 was shown based on HuProtTM human protein chip. g Immunoprecipitation showing an interaction between ABHD5 and PDIA5. CALR was used as a positive control. h The interaction complex model between ABHD5, PDIA5, and RNASET2 predicted by protein-protein docking methods. i Competitive binding assays of ABHD5 and RNASET2 to PDIA5. j In vitro binding assay with wild type PDIA5 (W) or mutant PDIA5 (T) and His-tagged RNASET2 or ABHD5 as indicated. k ABHD5 knockout (ABHD5 KO) and control SW480 cells were transfected with wild type ABHD5 or mutant ABHD5 plasmid, and subjected to FU treatment (25 μM) for 6 h. The intracellular uracil was analyzed (n = 6, Student’s t-test). l ABHD5 KO and control SW480 cells were transfected with wild type ABHD5 or mutant ABHD5 plasmid, and subjected to FU treatment (25 μM) for 24 h. The apoptotic rates were analyzed (n = 5, Student’s t-test). m ABHD5 KO and control SW480 cells transfected with wild type ABHD5 or mutant ABHD5 plasmid were subcutaneously inoculated in nude mice, and intraperitoneal injection of PBS or FU (50 mg per kg) + calcium folinate (80 mg per kg) (i.p., once per week for 3 weeks). Tumor volume was measured every 3 days (n = 5, Two-way ANOVA). The quantitative data were presented as mean ± S.D (error bar) (N.S. no significance, **p < 0.01, ***p < 0.001)

Article Snippet: Plasmids for overexpression of mutant ABHD5 and wild type ABHD5, lentiviruses for human RNASET2 knockdown, the recombinant proteins of human wild type His-ABHD5, HisRNASET2, His-PDIA5, GST-PDIA5 and mutant GST-PDIA5 were from Wuhan GeneCreate Biological Engineering Co., Ltd. ABHD5 (Human) recombinant protein (H00051099-P01) and RNASET2 (Human) recombinant protein (H00008635P01) were purchased from Novus.

Techniques: Activity Assay, Staining, Western Blot, Expressing, Control, Immunoprecipitation, Positive Control, Y2H Assay, Binding Assay, In Vitro, Mutagenesis, Knock-Out, Transfection, Plasmid Preparation, Injection

Journal: Nature communications

Article Title: ABHD5 blunts the sensitivity of colorectal cancer to fluorouracil via promoting autophagic uracil yield.

doi: 10.1038/s41467-019-08902-x

Figure Lengend Snippet: Fig. 6 The proposed mechanism underlying ABHD5-induced FU resistance. ABHD5 localizes to lysosome and shares share a common interaction domain in PDIA5 with RNASET2. ABHD5 directly interacts with PDIA5 to prevent PDIA5 from directly interacting with RNASET2 and inactivating RNASET2. ABHD5 deficiency releases PDIA5 to directly interact with RNASET2 and leave RNASET2 in an inactivate state, which impairs RNASET2-induced autophagic uracil yield. ABHD5 deficiency promotes CRC cells to uptake FU as an exogenous uracil, thus increasing their sensitivity to FU. In contrast, ABHD5 proficient CRC cells showed an inherent resistance to FU due to an increased autophagic uracil yield

Article Snippet: Plasmids for overexpression of mutant ABHD5 and wild type ABHD5, lentiviruses for human RNASET2 knockdown, the recombinant proteins of human wild type His-ABHD5, HisRNASET2, His-PDIA5, GST-PDIA5 and mutant GST-PDIA5 were from Wuhan GeneCreate Biological Engineering Co., Ltd. ABHD5 (Human) recombinant protein (H00051099-P01) and RNASET2 (Human) recombinant protein (H00008635P01) were purchased from Novus.

Techniques: