Journal: Cell Metabolism

Article Title: Nutrient-sensing AgRP neurons relay control of liver autophagy during energy deprivation

doi: 10.1016/j.cmet.2023.03.019

Figure Lengend Snippet:

Article Snippet: Sections were then mounted with DAPI using Vectashield Antifade Mounting Medium (Vector Laboratories), covered using a coverslip and stored at 4°C in the dark.

Techniques: Virus, Plasmid Preparation, Recombinant, Protease Inhibitor, Western Blot, Blocking Assay, In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Isolation, Bicinchoninic Acid Protein Assay, Reverse Transcription, RNAscope, Multiplex Assay, Software, Microscopy, Mass Spectrometry, Liquid Chromatography, Chromatography

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: Elevated NAT10 expression correlates with poor prognosis in HCC patients. (A) Dot blot analyses of total RNA (5 µg) isolated from HCC tissues and adjacent noncancerous liver tissues using an anti‐ac4C antibody, with MB staining as loading control (left panel). Calculation of relative ac4C contents on RNA in HCC tissues and adjacent noncancerous liver tissues (right panel, N = 20). (B) Detection of ac4C levels on mRNA in the same 20 HCC tissues and adjacent noncancerous liver tissues by UPLC‐MS/MS analysis. (C) Large‐scale data mining was used to compare the expression differences in NAT10 mRNA between HCC tissues and adjacent noncancerous liver tissues. (D) Analysis of NAT10 protein levels in HCC and adjacent noncancerous liver tissues ( N = 165) using CPTAC data. (E) NAT10 protein levels in HCC tissues and adjacent noncancerous liver tissues measured by western blotting ( N = 8). (F) Representative IHC images of NAT10 expression in the Tongji cohort tissue microarray (TMA) including HCC tissues and adjacent noncancerous liver tissues (left panel); pie chart showing percentages of cases with differential NAT10 expression profiles compared to adjacent noncancerous liver tissues ( N = 103, right panel, scale bar: 200 µm or 20 µm). (G) Kaplan‐Meier plot correlating NAT10 expression with patient overall and recurrence‐free survival using CPTAC, TCGA, and Tongji datasets. (H) Analysis of NAT10 expression correlation with malignant features of HCC using Tongji TMA cohort and TCGA LIHC datasets. Statistical tests: (A, B, D) paired t test; (G) log‐rank test; (H) Pearson chi‐squared test (2‐sided). Abbreviations: ANL, adjacent noncancerous liver tissue; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; LIHC, Liver hepatocellular carcinoma; TCGA, The Cancer Genome Atlas; CPTAC, Clinical Proteomic Tumor Analysis Consortium; MB, methylene blue; ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; mRNA, messenger RNA.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Expressing, Dot Blot, Isolation, Staining, Control, Tandem Mass Spectroscopy, Western Blot, Microarray, Immunohistochemistry

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: NAT10 knockdown inhibits HCC progression in vitro and in vivo. (A) Western blot analysis of NAT10 expression post NAT10 knockdown (upper panel). Dot blot analysis of ac4C levels in total RNA and mRNA isolated from control or NAT10‐knockdown HCC cells (lower panel, MHCC‐97H and SNU449), with MB staining as loading control. (B) and (C) CCK8 assay for cell viability of NAT10‐knockdown and control cells at indicated time points. (D) Colony formation assay quantification for indicated cells. (E) Soft agar colony formation assay quantification for indicated cells. (F) Scratch wound healing assays quantification for indicated cells. (G) Cell migration and invasion assays quantification for MHCC‐97H and SNU449 cells. (H) Subcutaneous xenograft models with NAT10‐knockdown and control cells ( n = 5, scale bar: 1 cm). Tumor volume monitored and growth curves generated over 24 days (left panel); tumors weighed (right panel). (I) and (J) Representative images and quantitative data analysis of Ki67‐positive (I) and TUNEL‐positive (J) cells, (scale bar: 20 µm). (K) Representative bioluminescence imaging of liver orthotopic implantation models (left panel) and quantification of intrahepatic metastatic nodules (right panel) ( n = 5). (L) Representative bioluminescence imaging of lung metastasis models (left panel) and quantification of lung metastatic nodules (right panel) ( n = 5). (B‐G) Data shown as mean ± SD, n = 3. Statistical analysis: Unpaired t tests. ** P < 0.01, *** P < 0.001. Abbreviations: MB, methylene blue; ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick‐end labeling; CCK‐8, Cell Counting Kit‐8; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Knockdown, In Vitro, In Vivo, Western Blot, Expressing, Dot Blot, Isolation, Control, Staining, CCK-8 Assay, Colony Assay, Soft Agar Assay, Migration, Generated, TUNEL Assay, Imaging, End Labeling, Cell Counting, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: Effects of nat10 on ac4c mRNA modification and global mRNA translation. (A) Flow chart depicting acRIP‐seq. (B) Number of ac4C peaks identified in acRIP‐seq in shCtrl and shNAT10 MHCC‐97H cells. (C) Number of ac4C‐modified genes identified in acRIP‐seq. Common ac4C genes have ≥ 1 common ac4C peak, while unique ac4C genes have no common ac4C peaks. (D) Top consensus motif identified by HOMER with acRIP‐seq peaks in MHCC‐97H cells with or without NAT10 knockdown. (E) Normalized distribution of ac4C peaks on mRNA in shCtrl and shNAT10 MHCC‐97H cells. (F) Heatmaps of 125 transcripts displaying reduced ac4C peaks in shNAT10 cells. (G) Flow chart depicting RNA‐seq and Ribo‐seq. (H) Heatmaps of transcript level (RNA‐seq) and RPF abundance (Ribo‐seq). (I) Cumulative distribution of log2‐fold changes of mRNA‐normalized ribosome footprint reads (T.E) for ac4C(−) and ac4C(+) transcripts in shCtrl and shNAT10 MHCC‐97H cells (Kolmogorov‐Smirnov test or two‐tailed t‐test). (J) Polysome profiling of shCtrl and shNAT10 MHCC‐97H cells. (K) Western blot images of SUnSET assays quantifying nascent (puromycin‐labeled) peptides in shCtrl and shNAT10 MHCC‐97H cells. GAPDH used as loading control. Abbreviations: acRIP‐seq, acetylated RNA immunoprecipitation and sequencing; RNA‐seq, RNA sequencing; Ribo‐seq, ribosome profiling analyses; T.E, translation efficiency; ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; RPF, ribosome protected fragment; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Modification, Knockdown, RNA Sequencing, Two Tailed Test, Western Blot, Labeling, Control, RNA Immunoprecipitation, Sequencing, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: ac4C modification enhances translation of HMGB2. (A) Schematic diagram of the strategy for pinpointing key NAT10 targets in HCC. (B) Fold changes of transcript level (RNA‐seq) and RPF abundance (Ribo‐seq) of 4 downstream targets. (C) Heatmap of RPF abundance for downstream 4 target genes and rank in ordered NAT10 targets gene list using RPF and T.E fold changes. (D) ac4C (top) and RPF (bottom) abundances on HMGB2 mRNA transcripts in shCtrl and shNAT10 MHCC‐97H cells. (E) acRIP‐qPCR analysis for indicated cells. (F) Western blot of HMGB2 protein expression in NAT10‐deficient cells. (G) HCC cells were treated with 10 µmol/L MG132 for 12 hours. (H) NAT10‐deficient or control cells transfected with pmirGLO‐HMGB2 reporter for 24 h, and HMGB2 translation efficiency defined as reporter protein production (F‐luc/R‐luc) divided by mRNA abundance . (I) Relative mRNA distribution of HMGB2 in ribosome fractions analyzed by qRT‐PCR in shCtrl and shNAT10 MHCC‐97H cells. (J) Subcutaneous xenograft model transplanted with indicated cells ( n = 6, scale bar: 1 cm). Tumor volume monitored and growth curves generated over 24 days (left panel); tumors weighed (right panel). (K) Liver orthotopic implantation models transplanted with indicated cells (scale bar: 1,000 µm). Representative bioluminescence imaging of mice (left panel) and quantification of tumor nodules (right panel) ( n = 5). (L) Lung metastasis model transplanted with indicated cells (scale bar: 1,000 µm). Representative bioluminescence imaging of mice (left panel) and quantification of lung metastatic nodules (right panel) ( n = 5). (M) Representative images of NAT10 and HMGB2 protein expression in HCC tissue arrays ( N = 103) by IHC (upper panel). Pearson correlation analysis between NAT10 and HMGB2 protein expression (lower panel). (N) Pearson correlation analysis between NAT10 and HMGB2 protein expression using CPTAC LIHC dataset. (E) and (H) Data are shown as mean ± SD, n = 3. Statistical analysis: Unpaired t‐tests. * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; CPTAC, Clinical Proteomic Tumor Analysis Consortium; qRT‐PCR, quantitative real‐time polymerase chain reaction; RNA‐seq, RNA sequencing; Ribo‐seq, ribosome profiling analyses; T.E, translation efficiency; acRIP, acetylated RNA immunoprecipitation; HMGB2, High Mobility Group Protein B2; IHC, immunohistochemistry; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Modification, RNA Sequencing, Western Blot, Expressing, Control, Transfection, Quantitative RT-PCR, Generated, Imaging, Real-time Polymerase Chain Reaction, RNA Immunoprecipitation, Immunohistochemistry, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: CDS ac4C sites of HMGB2 mRNA enhance binding of eEF2. (A) Schematic of RNA affinity chromatography and MS analysis. (B) Dot blot showing ac4C levels (left) and MB (right, loading controls) in ac4C/C ssRNA probes. (C) GO biological process analysis of proteins identified in quantitative MS via the Database for Annotation, Visualization, and Integrated Discovery. (D) Venn diagram comparing proteins identified in ss‐ac4C and acFUS. (E) Volcano plot of identified proteins. Proteins significantly enriched in ss‐ac4C RNA are shown as red dots. Log2 fold change plotted on x‐axis; ‐log10 P value on y‐axis. (F) Western blot images of endogenous eEF2 and SRP68 proteins pulled down by biotin‐labeled C‐Oligos and ac4C‐Oligos from MHCC‐97H whole cell lysates. (G) RNA pull‐down assays showing dose‐dependent interaction between endogenous eEF2 and biotin‐labeled C‐Oligos (top) or ac4C‐Oligos (below). Gray signal of bands in Western blots (left) quantified by Image Master Total Lab (right). (H) RIP‐qPCR analysis of enrichment of HMGB2 mRNA on eEF2 relative to IgG in shCtrl and shNAT10 MHCC‐97H cells. (I) Western blot analysis of HMGB2 expressions in Huh7 cells co‐transfected with NAT10 CDS plasmid or eEF2 siRNA. (J) Schematic representation of acRIP‐qPCR with fragmented RNA from cells (left). ac4C in HMGB2 mRNA analyzed by acRIP‐qPCR using fragmented RNA in MHCC‐97H cells with shCtrl and shNAT10 (right). (K) Western blot analysis on HA‐HMGB2 in MHCC‐97H cells seeded in 6‐well plates and transfected with either wild‐type HMGB2 CDS or mutant1/2/3 HMGB2 CDS plasmid, along with control or NAT10‐deficient cells. (L) NAT10‐deficient or control cells transfected with either wild‐type HMGB2 CDS or mutant1/2/3 HMGB2 CDS reporter for 24 h, and the quotient of reporter protein production defines translation efficiency of HMGB2. (M) Binding of eEF2 with CDS or 5′ UTR in control or NAT10‐deficient cells analyzed by eEF2 RIP‐qPCR using fragmented RNA. (N) Western blotting showing endogenous eEF2 proteins pulled down by biotin‐labeled exon‐C RNA and exon‐ac4C RNA from MHCC‐97H whole cell lysates. (H, J, L‐N) Data are shown as mean ± SD, n = 3. Statistical analysis: Unpaired t‐tests. * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: MS, mass spectrometry; ac4C, N4‐acetylcytidine; MB, methylene blue; GO, Gene ontology analysis; RIP, RNA immunoprecipitates; NAT10, N‐acetyltransferase 10; HMGB2, High Mobility Group Protein B2; eEF2, eukaryotic Elongation Factor 2; CDS, coding sequence; UTR, untranslated regions; acRIP, acetylated RNA immunoprecipitation; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Binding Assay, Affinity Chromatography, Dot Blot, Western Blot, Labeling, Transfection, Plasmid Preparation, Control, Mass Spectrometry, Sequencing, RNA Immunoprecipitation, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: Characterization of the NAT10 inhibitor panobinostat. (A) Western blot and dot blot were used to detect HMGB2 expression and ac4C levels after transfection of NAT10 (WT) or G641E NAT10 mutant plasmid, respectively. (B) Flow diagram of NAT10 inhibitor screening. (C) Three‐dimensional (3D) binding model of Panobinostat in NAT10 catalytic pocket. (D) Biacore analysis revealing binding between NAT10 protein and Panobinostat. (E) Western blots for effects of Panobinostat on thermal stabilization of NAT10 protein. CETSA assayed in cell lysates. (F) Identification of direct binding between Panobinostat and NAT10 via DARTS assays. (G) Effects of various concentrations of Panobinostat on global ac4C modification in MHCC‐97H cells. Dot blot assays were conducted with total RNA. (H) Effects of various concentrations of Panobinostat on poly(A)+ RNA ac4C modification in MHCC‐97H cells. (I) Effects of Panobinostat on levels of nascent (puromycin‐labeled) peptides and expression of HMGB2. (J) Effects of Panobinostat on HMGB2 mRNA ac4C level using acRIP‐qPCR. (K) Effects of Panobinostat on HMGB2 mRNA translation efficiency. (L) RIP‐qPCR analysis was performed to determine the enrichment of HMGB2 mRNA on eEF2 relative to IgG in both control and Panobinostat‐treated cells. (J‐L) Data is shown as mean ± SD, n = 3. Statistical analysis: Unpaired t‐tests. ** P < 0.01, *** P < 0.001. Abbreviations: WT, wild type; ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; HMGB2, High Mobility Group Protein B2; eEF2, eukaryotic Elongation Factor 2; RIP, RNA immunoprecipitates; DARTS, drug affinity responsive targets stability assay; CETSA, cellular thermal shift assay; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Western Blot, Dot Blot, Expressing, Transfection, Mutagenesis, Plasmid Preparation, Binding Assay, Modification, Labeling, Control, Stability Assay, Thermal Shift Assay, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: Panobinostat exhibits promising anti‐HCC efficacy in vitro and in vivo. (A) IC50 values of Panobinostat in MHCC‐97H after 48 h of treatment. (B) Effects of various concentrations of Panobinostat on colony formation abilities of MHCC‐97H cells. (C) Effects of various concentrations of Panobinostat on cell migration and invasion abilities of MHCC‐97H cells (scale bars = 100 µm). (D) Schematic diagram of generation and treatment of HCC models in mice. (E) Effects of different concentrations of Panobinostat on tumor volume and weight of subcutaneous xenograft model ( n = 8, scale bar: 1 cm). (F) Effects of various concentrations of Panobinostat on ac4C level in subcutaneous xenograft model. (G) Effects of various concentrations of Panobinostat on HMGB2 expression level in subcutaneous xenograft model. (H) Representative IHC images of HMGB2 expression in subcutaneous xenograft model (scale bar: 20 µm). (I) Liver orthotopic implantation models transplanted with indicated cells (scale bar: 1000 µm). Representative bioluminescence imaging of mice (left panel) and quantification of tumor nodules (right panel) (0 mg, n = 8; 5‐10 mg, n = 10). (J) Lung metastasis model transplanted with indicated cells (scale bar: 1000 µm). Representative bioluminescence imaging of mice (left panel) and quantification of lung metastatic nodules (right panel) ( n = 9). (K) Graphic illustration depicts how NAT10 modulates ac4C‐mediated translation elongation to promote HCC progression. (A‐C) Data is shown as mean ± SD, n = 3. Statistical analysis: Unpaired t‐tests. ** P < 0.01, *** P < 0.001. Abbreviations: IC50, a half‐maximal inhibitory concentration; ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; HMGB2, High Mobility Group Protein B2; IHC, immunohistochemistry; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: In Vitro, In Vivo, Migration, Expressing, Imaging, Concentration Assay, Immunohistochemistry, Standard Deviation

Journal: BioMed Research International

Article Title: A Novel Hemagglutinin with Antiproliferative Activity against Tumor Cells from the Hallucinogenic Mushroom Boletus speciosus

doi: 10.1155/2014/340467

Figure Lengend Snippet: (a) Gel filtration of fraction on Superdex 75 HR 10/30 column by FPLC. Fraction SU1 represents purified hemagglutinin BSH. (b) SDS-PAGE of purified BSH. Right lane: molecular weight markers, from top downwards, phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), and lactalbumin (14.4 kDa).

Article Snippet: The adsorbed fraction S3 was further purified by gel filtration on a fast protein liquid chromatography Superdex 75 10/300 GL column (GE Healthcare).

Techniques: Filtration, Purification, SDS Page, Molecular Weight

Journal: Journal of Biological Chemistry

Article Title: Cell Growth Regulation through GM3-enriched Microdomain (Glycosynapse) in Human Lung Embryonal Fibroblast WI38 and Its Oncogenic Transformant VA13

doi: 10.1074/jbc.m403857200

Figure Lengend Snippet: FIG. 1. Growth curve of WI38 and VA13 cells. On day 0, 8.8 103 cells/cm2 were seeded in a 35-mm dish in 3 ml of medium and kept at 37 °C in 5% CO2. On subsequent days (1–8) the cells were trypsinized and counted in a Neubauer chamber. The growth curve for WI38 (f) in the upper panel was expressed with a different ordinate scale than that for VA13 (G) and WI38 (f) in the lower panel. Data from two independ- ent experiments with similar results are shown.

Article Snippet: Human embryonic lung fibroblast cell line WI38 (10, 11) and SV40transformed WI38, termed VA13 (12), were from American Type Culture Collection (ATCC, Manassas, VA).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Cell Growth Regulation through GM3-enriched Microdomain (Glycosynapse) in Human Lung Embryonal Fibroblast WI38 and Its Oncogenic Transformant VA13

doi: 10.1074/jbc.m403857200

Figure Lengend Snippet: FIG. 2. GEM components from WI38 and VA13 cells and their distribution pattern. A-1, fractions 1–3, 4–6, 7–9, and 10–12 were separated by sucrose density gradient centrifugation from the postnuclear fraction (PNF) prepared in 1% Brij98 from WI38 and VA13 cells as described under ‘‘Materials and Methods.’’ The above fractions, having the same protein concentration, were passed through a C18 column and washed with water, and the GSL fraction was eluted with C/M (2:1, v/v). This eluate was evaporated to dryness under a stream of N2 and dissolved in the appropriate volume of C/M. Aliquots equivalent to 20 g of protein were applied in each lane, and HPTLC was developed in solvent C/M/ 0.2% CaCl2 (50:40:10, v/v/v) and stained with orcinol. GM3 was not detectable in Fr. 1–3 or 7–9 from either WI38 or VA13 (data not shown). The exclusive presence of GM3 in GEM prepared in the 1% Triton X-100 postnuclear fraction was reported previously (see ‘‘Discussion’’). A-2, distribution pattern of membrane components in low density GEM (Fr. 4–6) versus the high density soluble fraction (Fr. 10–12). Cells were grown to 90% confluence, and the GEM fraction and high density soluble fraction were prepared in 1% Brij98 or 1% Triton X-100 lysis buffer as described under ‘‘GEM Preparation and Determination of GEM Components’’ of ‘‘Materials and Methods.’’ An equal protein quantity (1 g) of each fraction was analyzed. B, major components showing a clear difference in WI38 versus VA13 GEM. Data from Fr. 5 and Fr. 12 prepared in 1% Brij98 lysis buffer are shown. S, sparse growing cells; C, confluent cells. An equal protein quantity (1 g) was analyzed. C, quantitative densitometry of immunoblotted bands for FGFR, cSrc, CD81, and CD9 in Fr. 5 of WI38 versus VA13. Note that the FGFR level is 4–5 times higher in VA13 than in WI38. cSrc was 2–3 times higher in VA13 than in WI38, whereas CD9 and CD81 were 3–4 times higher in WI38 than in VA13. Results similar to those shown in A, B, and C were obtained in three separate experiments.

Article Snippet: Human embryonic lung fibroblast cell line WI38 (10, 11) and SV40transformed WI38, termed VA13 (12), were from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Gradient Centrifugation, Protein Concentration, High Performance Thin Layer Chromatography, Solvent, Staining, Membrane, Lysis

Journal: Journal of Biological Chemistry

Article Title: Cell Growth Regulation through GM3-enriched Microdomain (Glycosynapse) in Human Lung Embryonal Fibroblast WI38 and Its Oncogenic Transformant VA13

doi: 10.1074/jbc.m403857200

Figure Lengend Snippet: FIG. 3. Effect of -CD and saponin on distribution pattern of GEM components in WI38 and VA13 cells. Nearly confluent cells were harvested and pretreated with -CD or saponin as described under ‘‘Materials and Methods.’’ The effects of these treatments on distribution patterns of major components are shown by Western blot- ting pattern. Note that FGFR and cSrc were barely reduced in GEM from VA13 but were significantly reduced in GEM from WI38. Csk, CD9, and CD81 were slightly reduced in WI38, but caveolin was com- pletely resistant to -CD. All components in both WI38 and VA13 were resistant to saponin. P, postnuclear fraction.

Article Snippet: Human embryonic lung fibroblast cell line WI38 (10, 11) and SV40transformed WI38, termed VA13 (12), were from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Western Blot

Journal: Journal of Biological Chemistry

Article Title: Cell Growth Regulation through GM3-enriched Microdomain (Glycosynapse) in Human Lung Embryonal Fibroblast WI38 and Its Oncogenic Transformant VA13

doi: 10.1074/jbc.m403857200

Figure Lengend Snippet: FIG. 6. Presence of Csk in WI38 GEM fraction. GEM fractions from sparse and confluent WI38 and VA13 cultures were analyzed for the presence of the Src family inhibitor Csk. Cells were processed as described under ‘‘Materials and Methods’’ using 1% Brij98 lysis buffer. After protein quantification, 2.3 g of protein was loaded on an 8% gel and analyzed by Western blot using anti-Csk Ab (Santa Cruz Biotech- nology, Inc.). Note that Csk was present in WI38 GEM (Fr. 5 and 6) but absent in VA13 GEM. P, postnuclear fraction.

Article Snippet: Human embryonic lung fibroblast cell line WI38 (10, 11) and SV40transformed WI38, termed VA13 (12), were from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Lysis, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Cell Growth Regulation through GM3-enriched Microdomain (Glycosynapse) in Human Lung Embryonal Fibroblast WI38 and Its Oncogenic Transformant VA13

doi: 10.1074/jbc.m403857200

Figure Lengend Snippet: FIG. 4. HPTLC immunostaining of total GSLs extracted from WI38 and VA13 cells. A, cells were grown, and GSLs were extracted and stained with mAb DH2 as described under ‘‘GM3 Composition of Various Fractions’’ of ‘‘Materials and Methods.’’ B, cells were pretreated with () or without () P4, and GSLs were extracted and stained as in A. For both panels, GSLs extracted from 20 g of cell protein were loaded per lane. Note that the level of GM3 is essentially the same in WI38 as in VA13. GM3 was completely depleted by P4 treatment in both WI38 and VA13 regardless of sparse (S) versus confluent (C) condition. Similar results to those shown in A and B were obtained in three separate experiments.

Article Snippet: Human embryonic lung fibroblast cell line WI38 (10, 11) and SV40transformed WI38, termed VA13 (12), were from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: High Performance Thin Layer Chromatography, Immunostaining, Staining

Journal: Journal of Biological Chemistry

Article Title: Cell Growth Regulation through GM3-enriched Microdomain (Glycosynapse) in Human Lung Embryonal Fibroblast WI38 and Its Oncogenic Transformant VA13

doi: 10.1074/jbc.m403857200

Figure Lengend Snippet: FIG. 5. Src and MAPK status in response to FGF and depletion of GM3 in WI38 versus VA13 cells. A, sparse growing (S) and con- fluent (C) cultures of WI38 (W) and VA13 (V) cells were prepared as described under ‘‘Materials and Methods.’’ The stimulatory effect of 10 pg/ml acidic FGF and the inhibitory effect of GM3 (probed by P4 treatment) were determined as described under ‘‘Materials and Meth- ods.’’ Three major points should be noted. (i) FGF-induced MAPK acti- vation in sparse growing WI38 was inhibited by GM3 as probed by P4 treatment (W, S, row 6). This inhibitory effect was stronger in confluent cells (W, C, row 6). (ii) Inactive Src with Tyr-527 was higher in confluent (W, C, row 4) than in sparse growing (W, S, row 4) WI38. The inactive Src expression was inhibited by GM3 (in the absence of P4) (W, S, row 4, two right lanes). (iii) Both MAPK and Src activation in VA13 were observed regardless of cell population density or P4 treatment (V, S, rows 3, 4, and 6; V, C, rows 3, 4, and 6). B, [3H]thymidine incorporation of WI38 after P4 treatment and FGF stimulation. Cells were seeded and processed as described under ‘‘Materials and Methods.’’ The statistical difference for each of the comparisons indicated by a bracket (*, between P4-treated versus P4-nontreated cells; **, between FGF-treated versus FGF-non-treated cells) was significant at the p 0.001 level.

Article Snippet: Human embryonic lung fibroblast cell line WI38 (10, 11) and SV40transformed WI38, termed VA13 (12), were from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Cell Growth Regulation through GM3-enriched Microdomain (Glycosynapse) in Human Lung Embryonal Fibroblast WI38 and Its Oncogenic Transformant VA13

doi: 10.1074/jbc.m403857200

Figure Lengend Snippet: FIG. 8. Interaction of FGFR in WI38 GEM with GM3 or other GSLs probed by polystyrene beads coated with GSLs. This interaction was determined by incubation of GSL-coated polystyrene beads with GEM and determination of the relative amount of FGFR adsorbed on beads by SDS-PAGE with Western blotting as described under ‘‘Materials and Methods.’’ Upper panel, level of FGFR adsorbed on GSL-coated beads determined by Western blot. ‘‘GEM,’’ FGFR in 1 g of protein in GEM for reference. ‘‘No GSL,’’ beads without GSL coating. GM3, GM2, GM1, SPG, Gb4, and LacCer: beads coated with 10 g of the respective GSLs. ‘‘GM3 sialidase,’’ beads coated with 10 g of GM3 treated with C. perfringens sialidase; beads were incubated with a defined amount of GEM under defined conditions (see ‘‘Materials and Methods’’) and subjected to Western blot. Lower panel, densitometric analysis (by Scion imaging) of intensity of FGFR in upper panel bands. Note that the FGFR interaction with GM3-coated beads was much higher than that with beads coated with GM2, GM1, or SPG. Neutral GSLs (Gb4 and LacCer) showed no interaction.

Article Snippet: Human embryonic lung fibroblast cell line WI38 (10, 11) and SV40transformed WI38, termed VA13 (12), were from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Incubation, SDS Page, Western Blot, Imaging

Journal: Journal of Biological Chemistry

Article Title: Cell Growth Regulation through GM3-enriched Microdomain (Glycosynapse) in Human Lung Embryonal Fibroblast WI38 and Its Oncogenic Transformant VA13

doi: 10.1074/jbc.m403857200

Figure Lengend Snippet: FIG. 10. Components and their possible role in GEM of WI38 versus VA13 cells. Possible growth control mechanisms of WI38 versus VA13 cells are illustrated based on differences in components and signal transducer activity. A, in WI38 cells (i) FGF causes FGFR acti- vation and minimal signaling (2) through its association with FGFR and cSrc, and consequently minimal MAPK activation (5). This lack of response is due to the fact that the FGFR level in WI38 is low (compared with VA13), and GM3 inhibits FGFR activity (4). This inhibitory effect of GM3 may be enhanced by formation of a complex with CD9/CD81. (ii) GM3 clustering, or GM3 interaction with FGFR, may occur upon cell contact, which in turn activates cSrc (1, 2). However, activation of cSrc is minimal because Csk, the physiological cSrc inhibitor (3), is associ- ated in the same microdomain. Which molecule interacts with GM3 upon cell contact (i.e. trans interaction between interfacing GEM) to induce contact inhibition is unknown. FGFR was shown to interact preferentially with GM3 based on direct binding of FGFR to GM3- coated beads and data from confocal microscopy. Such trans interaction is stronger in WI38 than in VA13. B, in VA13 cells, because the CD9/ CD81 level is minimal and Csk is absent, signaling through FGFR to cSrc (1) and that through clustered GM3 to cSrc (2) is relayed to MAPK without interruption (3). The inhibitory effect of GM3 on FGFR is nearly absent since the CD9/CD81 level is low. Thus, FGF-dependent activation of FGFR through cSrc is relayed to MAPK (3), and both activated cSrc and MAPK are continuously present regardless of cell contact. trans interaction between FGFR and GM3, as indicated by dashed arrows, is weaker than in WI38. Levels of FGF and cSrc in VA13 GEM are much higher than in WI38 as reflected by the size of the shapes and symbols.

Article Snippet: Human embryonic lung fibroblast cell line WI38 (10, 11) and SV40transformed WI38, termed VA13 (12), were from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Control, Activity Assay, Activation Assay, Inhibition, Binding Assay, Confocal Microscopy