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: BMC Gastroenterology

Article Title: Senescence marker protein 30 in acute liver failure: validation of a mass spectrometry proteomics assay

doi: 10.1186/1471-230X-8-17

Figure Lengend Snippet: Plasma SMP30 levels were only elevated in mice with ALF . A: 2-D gel of murine plasma proteins. B: Magnified regions of 2-D gel of murine plasma proteins. The results shown are representative of three independent experiments. The protein spot corresponding to SMP30 is indicated with an arrow. C: Spectrum from LC-ESI-MS/MS analysis of the trypsin digestion products of senescence marker protein 30 (SMP30). D: WB analysis of plasma SMP30 levels in GalN/LPS-treated mice. Lane and gel 1: 5 h after saline injection; lane and gel 2: 5 h after LPS injection; lane and gel 3: 5 h after GalN injection; lane and gel 4: 5 h after GalN/LPS injection; lane and gel 5: 24 h after saline injection; lane and gel 6: 24 h after LPS injection; lane and gel 7: 24 h after GalN injection; lane and gel 8: 24 h after GalN/LPS injection.

Article Snippet: Membranes were then incubated overnight at 4°C with goat polyclonal anti-SMP30 antibody (diluted 1: 300 for plasma and 1:500 for tissues; Santa Cruz Biotechnology, USA) or rabbit polyclonal anti-β-actin antibody (diluted 1:1000; Santa Cruz Biotechnology).

Techniques: Clinical Proteomics, Tandem Mass Spectroscopy, Marker, Saline, Injection

Journal: BMC Gastroenterology

Article Title: Senescence marker protein 30 in acute liver failure: validation of a mass spectrometry proteomics assay

doi: 10.1186/1471-230X-8-17

Figure Lengend Snippet: WB analysis of liver tissue SMP30 levels in GalN/LPS-treated mice . Lane 1: 5 h after saline injection; lane 2: 5 h after LPS injection; lane 3: 5 h after GalN injection; lane 4: 5 h after GalN/LPS injection; lane 5: 24 h after saline injection; lane 6: 24 h after LPS injection; lane 7: 24 h after GalN injection; lane 8: 24 h after GalN/LPS injection. SMP30 levels were significantly reduced in the liver tissues of mice treated with GalN/LPS compared with those of the control groups ( P < 0.01). There was no significant difference between the SMP30 protein levels of groups 4 and 8 ( P > 0.05).

Article Snippet: Membranes were then incubated overnight at 4°C with goat polyclonal anti-SMP30 antibody (diluted 1: 300 for plasma and 1:500 for tissues; Santa Cruz Biotechnology, USA) or rabbit polyclonal anti-β-actin antibody (diluted 1:1000; Santa Cruz Biotechnology).

Techniques: Saline, Injection, Control

Journal: BMC Gastroenterology

Article Title: Senescence marker protein 30 in acute liver failure: validation of a mass spectrometry proteomics assay

doi: 10.1186/1471-230X-8-17

Figure Lengend Snippet: RT-PCR analysis of liver tissue SMP30 mRNA levels in GalN/LPS-treated mice . Lane 1: 5 h after saline injection; lane 2: 5 h after LPS injection; lane 3: 5 h after GalN injection; lane 4: 5 h after GalN/LPS injection; lane 5: 24 h after saline injection; lane 6: 24 h after LPS injection; lane 7: 24 h after GalN injection group; lane 8: 24 h after GalN/LPS injection. SMP30 mRNA levels were significantly reduced in liver tissues only at 24 h after GalN/LPS administration ( P 8 < 0.01 vs other control groups).

Article Snippet: Membranes were then incubated overnight at 4°C with goat polyclonal anti-SMP30 antibody (diluted 1: 300 for plasma and 1:500 for tissues; Santa Cruz Biotechnology, USA) or rabbit polyclonal anti-β-actin antibody (diluted 1:1000; Santa Cruz Biotechnology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Saline, Injection, Control

Journal: BMC Gastroenterology

Article Title: Senescence marker protein 30 in acute liver failure: validation of a mass spectrometry proteomics assay

doi: 10.1186/1471-230X-8-17

Figure Lengend Snippet: WB analysis of plasma SMP30 levels in ALF patients . Plasma from healthy volunteers was used as the control. n = 4 for both groups. SMP30 protein levels in ALF patients were significantly elevated, about 3.65 ± 0.34 times, compared with those in healthy volunteers ( P < 0.01).

Article Snippet: Membranes were then incubated overnight at 4°C with goat polyclonal anti-SMP30 antibody (diluted 1: 300 for plasma and 1:500 for tissues; Santa Cruz Biotechnology, USA) or rabbit polyclonal anti-β-actin antibody (diluted 1:1000; Santa Cruz Biotechnology).

Techniques: Clinical Proteomics, Control

Journal: Molecular Neurodegeneration

Article Title: CCR5 antagonist reduces HIV-induced amyloidogenesis, tau pathology, neurodegeneration, and blood-brain barrier alterations in HIV-infected hu-PBL-NSG mice

doi: 10.1186/s13024-021-00500-0

Figure Lengend Snippet: Schematic representation of animals’ engraftment, infection, treatment, samples collection, and analyses. Abbreviations: NSG: NOD/ scid –IL-2Rγ c null ; hPBL: human peripheral blood lymphocytes; FACS: fluorescence activated cell sorting; h: hours; MVC: maraviroc; DMSO: Dimethyl sulfoxide; PBS: phosphate-buffered saline; UPLC-MS/MS: ultraperformance liquid chromatography-tandem mass spectrometry; IHC: immunohistochemistry; IF: immunofluorescence; qPCR: quantitative (real-time) polymerase chain reaction; ELISA: enzyme-linked immunosorbent assay

Article Snippet: Levels of Aβ-42 in animals plasma samples (100 μl) were quantified using the mouse Aβ (1–42) ELISA kit (Novus Biologicals, Centennial, CO); Aβ-42 levels in human MDM and HBMEC culture supernatants and cell lysates (100 μl) were quantified using the human Aβ (1–42) Quantikine ELISA kit (R&D Systems), according to the manufacturers’ protocols.

Techniques: Infection, Fluorescence, FACS, Saline, Tandem Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Immunohistochemistry, Immunofluorescence, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Molecular Neurodegeneration

Article Title: CCR5 antagonist reduces HIV-induced amyloidogenesis, tau pathology, neurodegeneration, and blood-brain barrier alterations in HIV-infected hu-PBL-NSG mice

doi: 10.1186/s13024-021-00500-0

Figure Lengend Snippet: MVC decreased viremia and abrogated HIV-induced cellular infiltration in the brain of infected animals. Brain HIV RNA copy (yellow arrows) numbers were quantified by RNAscope and, for each experimental group, representative images from the somatosensory cortex ( a ) and meningeal / somatosensory area layer 1 ( b ) are shown. c : Metamorph software was used to quantify HIV RNA copies numbers in all samples. For each animal brain sample, 10 random fields-of-view (FOV) were analyzed (5 FOV from the somatosensory cortex and 5 FOV from the meningeal / somatosensory area layer 1). d : Levels of HIV-1 gag, pol, LTR, and tat genes in brain tissues were quantified by qPCR and normalized to samples’ hCD45+ cells levels. e : HIV-1 p24 antigen levels in plasma samples were quantified by ELISA. f, g : HLA-DR expression in brain tissues was analyzed by immunohistochemistry and, for each experimental group, representative images from the somatosensory cortex ( f ) and meningeal / somatosensory area layer 1 ( g ) are shown. h : Metamorph was used to quantify HLA-DR+ cells in all brain samples and for each sample, 10 random FOV (5 FOV from the somatosensory cortex and 5 FOV from the meningeal / somatosensory area layer 1) were analyzed. For panels a, b, f, and g , images were at 40X. The four animal groups included PBS, HIV, HIV + MVC, and MVC; 9 to 12 animals in each group. # P < 0.0001. Error bars represent SD

Article Snippet: Levels of Aβ-42 in animals plasma samples (100 μl) were quantified using the mouse Aβ (1–42) ELISA kit (Novus Biologicals, Centennial, CO); Aβ-42 levels in human MDM and HBMEC culture supernatants and cell lysates (100 μl) were quantified using the human Aβ (1–42) Quantikine ELISA kit (R&D Systems), according to the manufacturers’ protocols.

Techniques: Infection, RNAscope, Software, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemistry

Journal: Molecular Neurodegeneration

Article Title: CCR5 antagonist reduces HIV-induced amyloidogenesis, tau pathology, neurodegeneration, and blood-brain barrier alterations in HIV-infected hu-PBL-NSG mice

doi: 10.1186/s13024-021-00500-0

Figure Lengend Snippet: MVC abrogated HIV-induced increased formation and accumulation of CNS GSAP and amyloid-β (Aβ), but increased plasma Aβ levels. Aβ formation in the somatosensory cortex ( a ) and hypothalamus region ( b ) were quantified by immunohistochemistry using antibodies to Aβ1–42, followed by densitometry quantification of Aβ levels in all samples: 10 random FOV (5 FOV from the somatosensory cortex and 5 FOV from the hypothalamus region) analyzed for each sample ( c ). Aβ levels in brain tissues were also analyzed by Western blot ( d ) and densitometry quantification normalized to sample’s β-actin levels ( e ). Both immunohistochemistry and Western blot showed no Aβ in brain tissues of animals in the PBS or MVC groups. mRNA levels of GSAP (the enzyme that catalyzes Aβ formation) in brain tissues were quantified by real-time PCR ( f ). Plasma Aβ1–42 were quantified by ELISA ( g ). For panels a , and b , images were at 40X . The four animal groups analyzed included PBS, HIV, HIV + MVC, and MVC; 9 to 11 animals in each group. # P < 0.0001, ***[( e ) P = 0.0009, ( f ) P = 0.0008, ( g ) P = 0.0005]. Error bars represent SD

Article Snippet: Levels of Aβ-42 in animals plasma samples (100 μl) were quantified using the mouse Aβ (1–42) ELISA kit (Novus Biologicals, Centennial, CO); Aβ-42 levels in human MDM and HBMEC culture supernatants and cell lysates (100 μl) were quantified using the human Aβ (1–42) Quantikine ELISA kit (R&D Systems), according to the manufacturers’ protocols.

Techniques: Clinical Proteomics, Immunohistochemistry, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: The aldehyde dehydrogenase 2 rs671 variant enhances amyloid β pathology

doi: 10.1038/s41467-024-46899-0

Figure Lengend Snippet: a Association of risk factors with ALDH2 rs671 polymorphism and Alzheimer’s disease (AD)-related neuropathologic changes after adjustment for age in Chinese populations. Odds ratios and P values were calculated by ordinal logistic regression with adjustment of age using SPSS software. n = 469. b Representative image of the anatomy of the right human brain with formalin fixation. Eight brain regions were immunostained for amyloid-β (Aβ) plaque deposition to assess Aβ pathology according to National Institute on Aging/Alzheimer Association guidelines for the neuropathologic assessment of AD. c Representative images of Aβ deposits labeled with 6E10 antibody (anti-β-amyloid 1–16 antibody) in six subregions from postmortem brains with pathological AD with different rs671 genotypes. rs671 GG genotype ( n = 18, 82.89 ± 7.76 yr), GA ( n = 18, 84.78 ± 4.40 yr), AA ( n = 8, 84.62 ± 9.76 yr). Percentage of amyloid plaque area was determined. Scale bar, 500 μm. d–f Quantification of Aβ42 and Aβ40 peptides in frozen ( d ) MFG homogenates, ( e ) STG homogenates, and ( f ) Hipp homogenates from the above-mentioned 44 postmortem brains with pathological AD, determined by enzyme-linked immunosorbent assay (ELISA). rs671 GG genotype ( n = 18, 82.89 ± 7.76 yr), GA ( n = 18, 84.78 ± 4.40 yr), AA ( n = 8, 84.62 ± 9.76 y). One datapoint represents one sample per genotype. All box plots include the median line, the box indicates the interquartile range, and whiskers indicate minima and maxima. g Western blot (WB) with anti-ALDH2 in frozen MFG, STG, and Hipp subregions from postmortem brains with pathological AD with rs671 GG (81.50 ± 1.91 yr), GA (84.25 ± 7.85 yr), and AA (90.40 ± 2.79 yr) genotypes. n = 4 or 5. IPL inferior parietal lobule, MFG middle frontal gyrus, STG superior temporal gyrus, Hipp hippocampus, BG basal ganglia, VC visual cortex. Data are presented as mean values ± SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) with least significant difference (LSD) post-hoc test for multiple groups. Source data are provided as a Source Data file.

Article Snippet: The levels of Aβ42 and Aβ40 peptides were measured using a specific human amyloid-β peptide 1−42 ELISA Kit (Cusabio, CSB-E10684h) and a human amyloid-β peptide 1−40 ELISA Kit (Cusabio, CSB-E08299h), respectively.

Techniques: Software, Labeling, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Nature Communications

Article Title: The aldehyde dehydrogenase 2 rs671 variant enhances amyloid β pathology

doi: 10.1038/s41467-024-46899-0

Figure Lengend Snippet: a Schematic image of the endogenous loci to generate Aldh2 -knockout mice in a C57BL/6 background. (Image was created using Photoshop CS6, with elements from ChemBioDraw Ultra software 14.0.) b WB confirmed the Aldh2 expression in Aldh2 −/− mouse brains. All mice with Aldh2 −/− showed similar results. c Levels of Aβ42 and Aβ40 peptides in cortex (COR) homogenates from 3-month-old Aldh2 −/− mice ( n = 5 mice) and age-matched wild-type mice ( n = 4 mice) by ELISA. d Scheme illustrating intragastric administration of daidzin (150 mg/kg/day) in 2-month-old APP/PS1 mice for 2 months. Aβ pathology was assessed in 7-month-old mice. (Image was created using Photoshop CS6 and ChemBioDraw Ultra software 14.0.) e Immunohistochemical staining of Aβ plaque deposits in COR and Hipp and f quantification of Aβ40 and Aβ42 levels in COR from APP/PS1 transgenic mice with phosphate-buffered saline (PBS, n = 4 mice) or daidzin administration ( n = 4 mice). g and h ALDH2 knockdown by siRNA or daidzin (30–100 μM) treatment for 48 h in SH-SY5Y cells. g Enzymatic activity of ALDH2 in cell lysates, measured over 120 min by using a mitochondrial aldehyde dehydrogenase (ALDH2) Activity Assay Kit (Abcam, ab115348). n = 3 biologically independent samples. h Levels of intracellular Aβ42 and Aβ40 peptides. n = 3 biologically independent samples. i – k ALDH2 functional deficiency caused by ALDH2 silencing or combined with daidzin co-treatment for 48 h in HEK293T cells. i RT-qPCR of relative ALDH2 mRNA levels ( n = 3 biologically independent experiments) and j WB detection of ALDH2, APP, and PS1 expression levels. k Levels of intracellular Aβ42 and Aβ40 peptides. n = 3 or 4 biologically independent samples. siNC negative control small interfering RNA. Data are presented as mean values ± SD. All box plots include the median line, the box indicates the interquartile range, and whiskers indicate minima and maxima. Statistical analysis was performed using a two-tailed Student’s t -test for two groups ( c , e , f , h , i , k ) and one-way ANOVA with LSD post-hoc test for multiple groups ( h ). Source data are provided as a Source Data file.

Article Snippet: The levels of Aβ42 and Aβ40 peptides were measured using a specific human amyloid-β peptide 1−42 ELISA Kit (Cusabio, CSB-E10684h) and a human amyloid-β peptide 1−40 ELISA Kit (Cusabio, CSB-E08299h), respectively.

Techniques: Knock-Out, Software, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Transgenic Assay, Saline, Knockdown, Activity Assay, Functional Assay, Quantitative RT-PCR, Negative Control, Small Interfering RNA, Two Tailed Test

Journal: Nature Communications

Article Title: The aldehyde dehydrogenase 2 rs671 variant enhances amyloid β pathology

doi: 10.1038/s41467-024-46899-0

Figure Lengend Snippet: a Proteomic profiling was carried out using N2a-APPswe cells pretreated with PBS or 60 μM daidzin for 48 h. KEGG pathways analysis of the differentially expressed proteins (DEPs). b Representative images of 4-HNE immunohistochemical staining of paraffin sections of the human hippocampus with pathological AD. n = 3 per group. rs671 GG (86.33 ± 7.02 yr), GA/AA (82.33 ± 1.15 yr). Scale bar, 50 μm. c Representative images of 4-HNE immunostaining of frozen brain sections of 3-month-old male Aldh2 −/− mice ( n = 5) and wild-type mice ( n = 4). Scale bar, 50 μm. d Representative images of immunocytochemical staining with 4-HNE antibody (green) in SH-SY5Y cells after ALDH2 silencing. Three independent biological replicates were performed. Scale bar, 25 μm. e Separation of ( R )−4-HNE and ( S )−4-HNE from (±)−4-HNE by high-performance liquid chromatography (Shimadzu) with a chiral column (4.6 × 150 mm, CHIRALPAK AS-H). f and g SH-SY5Y cells were pretreated with PBS, or (±)−4-HNE, ( R )−4-HNE, or ( S )−4-HNE at 2 μM for 72 h or 10 μM for 4 h. f Autophagy was assessed by WB for LC3A-I/LC3A-II, the typical marker of autophagy. g Apoptosis was assessed by flow cytometry. The flow-cytometry gating strategies were shown in Supplementary Fig. . h and i Levels of intracellular Aβ40 and Aβ42 were determined by quantitative ELISA. h SH-SY5Y cells were treated with 0, 5, or 10 μM (±)−4-HNE, ( R )−4-HNE, or ( S )−4-HNE for 4 h. i SH-SY5Y cells were treated with 2 μM (±)−4-HNE, ( R )−4-HNE, or ( S )−4-HNE for 24 h. n = 3 biologically independent samples. Data are presented as mean values ± SD. All box plots include the median line, the box indicates the interquartile range, whiskers indicate minima and maxima. Statistical analysis was performed using two-tailed Student’s t -test for two groups ( b and c ) and one-way ANOVA with LSD post-hoc test for multiple groups ( h and i ). Source data are provided as a Source Data file.

Article Snippet: The levels of Aβ42 and Aβ40 peptides were measured using a specific human amyloid-β peptide 1−42 ELISA Kit (Cusabio, CSB-E10684h) and a human amyloid-β peptide 1−40 ELISA Kit (Cusabio, CSB-E08299h), respectively.

Techniques: Immunohistochemical staining, Staining, Immunostaining, High Performance Liquid Chromatography, Marker, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: The aldehyde dehydrogenase 2 rs671 variant enhances amyloid β pathology

doi: 10.1038/s41467-024-46899-0

Figure Lengend Snippet: a The Schiff-base formation reaction of Ac-Leu–Lys–Lys–Lys–Gln (residues 52-56 of C99) with ( R )−4-HNE. Matrix-assisted laser desorption/ionization-time of flight MS (MALDI-TOF MS) detection of natural peptide Ac-Leu–Lys–Lys–Lys–Gln (left, upper) and 4-HNE-modified peptides (right, upper). Representative MS/MS spectra of natural peptide Ac-Leu–Lys–Lys–Lys–Gln (left, lower) and modified peptide (right, lower) with 4-HNE adduction on the first Lys residue (Lys53 of C99) via Schiff-base formation, the main product of this reaction. b WB verification of recombinant C99, Lys53Arg mutant C99 (C99 Lys53Arg ), and 4-HNE-modified HNE-C99 and HNE-C99 Lys53Arg . C99 was detected by C1/6.1 antibody. 4-HNE-modified proteins were detected by 4-HNE antibody. c Levels of generated Aβ40 and Aβ42 in in vitro γ-secretase cleavage assays from recombinant C99, C99 Lys53Arg , and their 4-HNE-modified forms, determined by ELISA assay. n = 3 biologically independent samples. d The predicted docking structures of human γ-secretase with peptide Val46–Lys55 of C99 (purple) or with modified peptide Val46–Lys53(HNE)–Lys55 (green) by ZDOCK and sequence alignment was done in PyMOL. The structures of PS1 (the main γ-secretase component) and natural Val46–Lys55 peptide were derived from the Cryo-EM structure ( PDB: 6IYC ). The 4-HNE modification on Lys53 (after Schiff-base formation) was manually added in PyMOL based on the natural peptide structure from 6IYC. The dashed lines and the numbers represent the distance between Asp385 or Asp257 of PS1 and –NH or C=O groups of residue Leu49 of C99 [purple, natural Val46-Lys53; green, modified Val46-Lys53(HNE)-Lys55]. Data are presented as mean values ± SD. Statistical analysis was performed using one-way ANOVA with LSD post-hoc test for multiple groups. Source data are provided as a Source Data file.

Article Snippet: The levels of Aβ42 and Aβ40 peptides were measured using a specific human amyloid-β peptide 1−42 ELISA Kit (Cusabio, CSB-E10684h) and a human amyloid-β peptide 1−40 ELISA Kit (Cusabio, CSB-E08299h), respectively.

Techniques: Modification, Tandem Mass Spectroscopy, Residue, Recombinant, Mutagenesis, Generated, In Vitro, Enzyme-linked Immunosorbent Assay, Sequencing, Derivative Assay, Cryo-EM Sample Prep

Journal: Scientific Reports

Article Title: Dihydrotestosterone and 17β-estradiol modulate TMJ osteoarthritis development and reveal sex-specific differences in pathogenesis

doi: 10.1038/s41598-025-03475-w

Figure Lengend Snippet: Effects of DHT reduction and mechanical stress on TMJ cartilage. (a) Histochemical staining of the mandibular head of a male mouse is shown. Cartilage thickness was confirmed by H-E staining (scale bar = 100 μm). Positive areas were measured by safranin-O staining (scale bar = 100 μm). The number of osteoclasts per mm 2 was measured by TRAP staining (scale bar = 100 μm).(b) Cartilage thickness measured on H-E stained sections. Proteoglycan area in Safranin-O stained sections. mean number of osteoclasts per mm 2 ( N > 3 nuclei) in TRAP stained sections. Results of the Modified Mankin score for OA severity assessment. (c) Immunohistochemical staining of the mouse mandibular head for MMP13 (scale bar = 100 μm; MMP13: red, nuclei: blue) and ADAMTS5 (scale bar = 100 μm; ADAMTS5: red, nuclei: blue). (d) Semi-quantitative analysis of MMP13 and ADAMTS5 expression comparing the control, MS, ORX, and MS + ORX groups. * P < 0.033, ** P < 0.002, *** P < 0.001, ns = no significance.

Article Snippet: Sections were incubated with the primary antibody (diluted in PBS supplemented with 1% BSA) according to procedures recommended by the manufacturer, as follows: ADAMTS5 rabbit polyclonal antibody as primary antibody (ab182795, Abcam, Cambridge, MA, USA) and MMP13 rabbit polyclonal antibody (LS-C352547, Proteintech, Chicago, IL, USA) as primary antibodies for immunohistochemical staining.

Techniques: Staining, Modification, Immunohistochemical staining, Expressing, Control

Journal: Scientific Reports

Article Title: Dihydrotestosterone and 17β-estradiol modulate TMJ osteoarthritis development and reveal sex-specific differences in pathogenesis

doi: 10.1038/s41598-025-03475-w

Figure Lengend Snippet: Impact of 17β-estradiol deficiency on TMJ degeneration under mechanical stress. (a) Histochemical staining of the mandibular head of a male mouse is shown. Cartilage thickness was confirmed by H-E staining (scale bar = 100 μm). Positive areas were measured by safranin-O staining (scale bar = 100 μm). The number of osteoclasts per mm 2 was measured by TRAP staining (scale bar = 100 μm).(b) Cartilage thickness measured on H-E stained sections. Proteoglycan area in Safranin-O stained sections. mean number of osteoclasts per mm 2 ( N > 3 nuclei) in TRAP stained sections. Results of the Modified Mankin score for OA severity assessment. ** P < 0.002, *** P < 0.001, ns = no significance. (c) Immunohistochemical staining of the mouse mandibular head for MMP13 (scale bar = 100 μm; MMP13: red, nuclei: blue) and ADAMTS5 (scale bar = 100 μm; ADAMTS5: red, nuclei: blue). ADMTS5 were clearly expressed in the subchondral bone in MS and MS + ORX group. (d) Semi-quantitative analysis of MMP13 and ADAMTS5 expression comparing the control, MS, Ai, and MS + Ai groups. * P < 0.033, ** P < 0.002, *** P < 0.001, ns = no significance.

Article Snippet: Sections were incubated with the primary antibody (diluted in PBS supplemented with 1% BSA) according to procedures recommended by the manufacturer, as follows: ADAMTS5 rabbit polyclonal antibody as primary antibody (ab182795, Abcam, Cambridge, MA, USA) and MMP13 rabbit polyclonal antibody (LS-C352547, Proteintech, Chicago, IL, USA) as primary antibodies for immunohistochemical staining.

Techniques: Staining, Modification, Immunohistochemical staining, Expressing, Control

Journal: Scientific Reports

Article Title: Dihydrotestosterone and 17β-estradiol modulate TMJ osteoarthritis development and reveal sex-specific differences in pathogenesis

doi: 10.1038/s41598-025-03475-w

Figure Lengend Snippet: Combined effects of DHT and 17β-estradiol deficiency with mechanical stress. (a) Histochemical staining of the mandibular head of a male mouse is shown. Cartilage thickness was confirmed by H-E staining (scale bar = 100 μm). Positive areas were measured by safranin-O staining (scale bar = 100 μm). The number of osteoclasts per mm 2 was measured by TRAP staining (scale bar = 100 μm).(b) Cartilage thickness measured on H-E stained sections. Proteoglycan area in Safranin-O stained sections. mean number of osteoclasts per mm2 ( N > 3 nuclei) in TRAP stained sections. Results of the Modified Mankin score for OA severity assessment. * P < 0.033, ** P < 0.002, *** P < 0.001, ns = no significance. (c) Immunohistochemical staining of the mouse mandibular head for MMP13 (scale bar = 100 μm; MMP13: red, nuclei: blue) and ADAMTS5 (scale bar = 100 μm; ADAMTS5: red, nuclei: blue). ADMTS5 were clearly expressed in the subchondral bone in MS, MS + Ai, MS + ORX and MS + ORX + Ai group. (d) Semi-quantitative analysis of MMP13 and ADAMTS5 expression comparing the control, MS, MS + Ai, MS + ORX, and MS + ORX + Ai groups. * P < 0.033, ** P < 0.002, *** P < 0.001, ns = no significance.

Article Snippet: Sections were incubated with the primary antibody (diluted in PBS supplemented with 1% BSA) according to procedures recommended by the manufacturer, as follows: ADAMTS5 rabbit polyclonal antibody as primary antibody (ab182795, Abcam, Cambridge, MA, USA) and MMP13 rabbit polyclonal antibody (LS-C352547, Proteintech, Chicago, IL, USA) as primary antibodies for immunohistochemical staining.

Techniques: Staining, Modification, Immunohistochemical staining, Tandem Mass Spectroscopy, Expressing, Control

Journal: Scientific Reports

Article Title: Dihydrotestosterone and 17β-estradiol modulate TMJ osteoarthritis development and reveal sex-specific differences in pathogenesis

doi: 10.1038/s41598-025-03475-w

Figure Lengend Snippet: Protective role of 17β-estradiol supplementation in TMJ-OA. Histochemical staining of the mandibular head of male mice is shown as control, E2− (MS + ORX + Ai) and E2+ (MS + ORX + Ai + E2). Cartilage thickness was confirmed by H-E staining (scale bar = 100 μm). Positive areas were measured by safranin-O staining (scale bar = 100 μm). The number of osteoclasts per mm 2 was measured by TRAP staining (scale bar = 100 μm).(b) Cartilage thickness measured in H-E stained sections, proteoglycan area in Safranin-O stained sections was thicker with E2 + than E2−; average number of osteoclasts per mm 2 ( N > 3 nuclei) in TRAP stained sections was reduced with E2 + Modified for OA severity assessment Mankin score for assessing OA severity was greater with E2+. ** P < 0.002, *** P < 0.001, ns = no significance. (c) Immunohistochemical staining of MMP13 (scale bar = 100 μm; MMP13: red, nuclei: blue) and ADAMTS5 (scale bar = 100 μm; ADAMTS5: red, nuclei: blue) in the mouse mandible head showed that expression was suppressed by E2+. (d) Semi-quantitative analysis of MMP13 and ADAMTS5 expression comparing the control, E2 + and E2 − groups. * P < 0.033, ** P < 0.002, *** P < 0.001, ns = no significance. (e) Quantitative evaluation of NFκβ , BMP7 , Gremlin1 , and Rela , mRNAs involved in TMJ-OA deterioration, by RT-qPCR. Bars indicate each mRNA level relative to GAPDH mRNA level ( n = 5) Data are shown as mean ± SD. * P < 0.033, ** P < 0.002, *** P < 0.001, ns = no significance.

Article Snippet: Sections were incubated with the primary antibody (diluted in PBS supplemented with 1% BSA) according to procedures recommended by the manufacturer, as follows: ADAMTS5 rabbit polyclonal antibody as primary antibody (ab182795, Abcam, Cambridge, MA, USA) and MMP13 rabbit polyclonal antibody (LS-C352547, Proteintech, Chicago, IL, USA) as primary antibodies for immunohistochemical staining.

Techniques: Staining, Control, Modification, Immunohistochemical staining, Expressing, Quantitative RT-PCR

Journal: PLOS Genetics

Article Title: Restoration of energy homeostasis under oxidative stress: Duo synergistic AMPK pathways regulating arginine kinases

doi: 10.1371/journal.pgen.1010843

Figure Lengend Snippet: (a) Paraquat-induced mRNA and protein expression of TcFOXO at different times post-treatment. mRNA expression was determined by qPCR using ribosomal protein S3 (rps3) as an internal reference. Protein expression was determined by western blot using the anti-α-tubulin antibody as a loading control. These experiments were repeated three times with similar results. (b) The effects of knockdown of TcFOXO mRNA expression on tolerance of beetles to paraquat-induced oxidative stress. The larvae were collected for mRNA expression analysis and tolerance assay on the fourth day after dsTcFOXO treatment. The dsTcFOXO-injected beetles were treated with 20 mM paraquat for 12 h, and the survival rates of larvae were recorded. (c) Paraquat-induced nuclear translocation of TcFOXO. Beetles were treated with paraquat for 8 h and the midguts were dissected and stained with an anti-TcFOXO antibody (red) and DAPI (blue). Merged images showed the subcellular localization of TcFOXO. The protein levels of TcFOXO in nuclear and cytoplasmic proteins were detected with an anti-TcFOXO antibody and normalized with an anti-α-tubulin or anti-Histone H3 antibody. Representative blots from three independent experiments were shown. (d) Total protein and phosphoprotein stains of immunoprecipitated TcFOXO from paraquat-treatment and control samples. The gels were stained by coomassie blue (left panel) and Pro-Q Diamond (right panel). Arrows point to bands identified by lane labels. (e) Quantification of phosphorylation of immunoprecipitated TcFOXO from paraquat-treatment and control samples using phosphoprotein phosphate estimation kit. Numbers of moles of phosphorus per mole of TcFOXO were analyzed to detect the phosphorylation level of TcFOXO after treatment with paraquat. Data were expressed as mean ± SEM (n = 3 biologically independent replicates). Asterisks indicate differences statistically significant at * P < 0.05 and ** P < 0.01 (student’s t-test).

Article Snippet: The anti-α-tubulin mouse monoclonal antibody (Proteintech Group, Inc., Chicago, IL, USA) was used as a loading control for the cytoplasmic and total proteins, and the anti-Histone H3 Mouse Monoclonal Antibody (Beyotime, Jiangsu, China) was used as a loading control for the nuclear protein.

Techniques: Expressing, Western Blot, Control, Knockdown, Injection, Translocation Assay, Staining, Immunoprecipitation, Phospho-proteomics

Journal: PLOS Genetics

Article Title: Restoration of energy homeostasis under oxidative stress: Duo synergistic AMPK pathways regulating arginine kinases

doi: 10.1371/journal.pgen.1010843

Figure Lengend Snippet: (a) Knockdown of TcFOXO reduced the basal mRNA and protein expression of TcAK1 and TcAK2 . The relative mRNA levels of TcAK1 and TcAK2 were determined by RT-qPCR using ribosomal protein S3 (rps3) as an internal reference. The relative protein levels of TcAK1 and TcAK2 were determined by Western blot using the anti-α-tubulin tubulin antibody as a loading control. (b) The knockdown of TcFOXO attenuated the paraquat-induced mRNA expression of TcAK1 and TcAK2 . (c) Over-expression of TcFOXO increased the WT promoter activities of TcAK1 and TcAK2 in the Sf9 cells and the mutation in the promoters (Mut) attenuate the induction. Sf9 cells were transfected with luciferase vectors carrying the wild-type or mutant promoter regions of TcAKs as well as the overexpression vector of TcFOXO or a control vector. Cells were harvested for the dual-luciferase reporter assay at 24 h after transfection. The relative firefly luciferase activities were measured and normalized to Renilla luciferase from the same cell lysates. (d) Diagram of the wild-type (WT) and mutant (Mut) TcAK1 and TcAK2 promoters cloned into dual luciferase reporter vectors. Predicted FOXO binding element (FBE) is indicated in red, and randomly mutated FBE is indicated in blue. (e) ChIP-qPCR analysis revealed increased bindings of TcFOXO to the TcAK1 and TcAK2 promoter regions after exposure to paraquat. The beetles were treated with paraquat or 5% sucrose for 8 h. TcFOXO-DNA complexes were cross-linked by formaldehyde and immunoprecipitated with the anti-TcFOXO antibody. The amounts of TcAK1 and TcAK2 promoter regions binding with TcFOXO was determined by qPCR and normalized with input DNA. Data were expressed as mean ± SEM (n = 3 biologically independent replicates). Asterisks indicate differences statistically significant at * P < 0.05 and ** P < 0.01 (student’s t-test).

Article Snippet: The anti-α-tubulin mouse monoclonal antibody (Proteintech Group, Inc., Chicago, IL, USA) was used as a loading control for the cytoplasmic and total proteins, and the anti-Histone H3 Mouse Monoclonal Antibody (Beyotime, Jiangsu, China) was used as a loading control for the nuclear protein.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot, Control, Over Expression, Mutagenesis, Transfection, Luciferase, Plasmid Preparation, Reporter Assay, Clone Assay, Binding Assay, ChIP-qPCR, Immunoprecipitation

Journal: PLOS Genetics

Article Title: Restoration of energy homeostasis under oxidative stress: Duo synergistic AMPK pathways regulating arginine kinases

doi: 10.1371/journal.pgen.1010843

Figure Lengend Snippet: (a) The relative mRNA expression of TcFOXO after treatment with dsTcAMPKα and AICAR. (b) Knockdown of TcAMPKα mRNA expression attenuated the paraquat-induced nuclear translocation of TcFOXO. (c) AICAR treatment induced the nuclear translocation of TcFOXO in beetles. Midguts from the treated and control groups were dissected and stained with an anti-TcFOXO antibody (red) and DAPI (blue). Merged images showed the subcellular localization of TcFOXO. Representative images from three independent experiments were shown. The protein level of TcFOXO in nuclear and cytoplasmic proteins was examined with an anti-TcFOXO antibody and normalized with the anti-α-tubulin or anti-Histone H3 antibody. Representative blots from three independent experiments were shown. (d) Total protein and phosphoprotein stains of immunoprecipitated TcFOXO from dsTcAMPKα-treatment, dsTcAMPKα and paraquat-treatment as well as AICAR-treatment and control samples. The same gel was stained by Pro-Q Diamond (left panel) and coomassie blue (right panel). Arrows point to bands identified by lane labels. (e) Quantification of phosphorylation of immunoprecipitated TcFOXO from AICAR-treatment as well as dsTcAMPKα and paraquat-treatment and corresponding control samples using phosphoprotein phosphate estimation kit. Numbers of moles of phosphorus per mole of TcFOXO were analyzed to detect the phosphorylation level of TcFOXO after each treatment. (f) Co-IP assay for TcAMPK–TcFOXO interaction. Co-IP assays were performed using the anti-TcFOXO antibody, then the immune complex with agarose was subjected to western blot analysis to detect the presence of TcAMPKα protein with the anti-TcAMPKα antibody. The whole-cell lysates (input) were also tested with either anti-TcFOXO (WB: TcFOXO) or anti-TcAMPKα (WB: TcAMPKα) antibodies to verify the protein expression, and α-tubulin was used as a loading control. For control experiments, the cell lysates were precipitated with nonimmune rabbit IgG and subsequently analyzed by western blot. (g) The BiFC assay for TcAMPK–TcFOXO interaction. Nt-GFP -TcFOXO and Ct-GFP-TcAMPKα plasmids were transfected into Sf9 cells. Cells were fixed and scanned at 48 h post-transfection. Western blot with anti-6×His antibody was conducted to confirm the protein expression of TcFOXO and TcAMPKα. (h) Knockdown of TcAMPKα mRNA expression attenuated the paraquat-induced recruitment of TcFOXO to the TcAK1 and TcAK2 promoter regions. (i) AICAR treatment enhanced the paraquat-induced recruitment of TcFOXO to the TcAK1 and TcAK2 promoter regions. Data were expressed as mean ± SEM (n = 3 biologically independent replicates). Asterisks indicate differences statistically significant at * P < 0.05 and ** P < 0.01 (student’s t-test).

Article Snippet: The anti-α-tubulin mouse monoclonal antibody (Proteintech Group, Inc., Chicago, IL, USA) was used as a loading control for the cytoplasmic and total proteins, and the anti-Histone H3 Mouse Monoclonal Antibody (Beyotime, Jiangsu, China) was used as a loading control for the nuclear protein.

Techniques: Expressing, Knockdown, Translocation Assay, Control, Staining, Immunoprecipitation, Phospho-proteomics, Co-Immunoprecipitation Assay, Western Blot, Bimolecular Fluorescence Complementation Assay, Transfection

Journal: PLOS Genetics

Article Title: Restoration of energy homeostasis under oxidative stress: Duo synergistic AMPK pathways regulating arginine kinases

doi: 10.1371/journal.pgen.1010843

Figure Lengend Snippet: (a) Yeast-two-hybrid assay for TcAMPK–TcAKs interaction. The wild-type (WT) and constitutively active mutant (T172D) TcAMPKα were used to analyze the interactions with TcAK1 and TcAK2. Yeasts were grown on media for 72 h at 30°C. The grown spots represent the interactions of TcAMPKαT172D with TcAKs, while no interaction was found between TcAMPKα (WT) and TcAK1 or TcAK2. Yeast cells were grown on SD-WL media lacking tryptophan and leucine and SD-AHWL media lacking adenine, histidine, tryptophan, and leucine to allow verification of the incorporation of bait and prey plasmids (growth) and bait/prey interaction (selection). (b) GST-pull down assay for TcAMPK–TcAKs interaction. Whole lysates from T . castaneum cells were incubated with the GST-TcAK1 or GST-TcAK2 fusion proteins as indicated as well as GST alone (negative control) and were analyzed by western blot with an anti-AMPK α antibody (lower panel) and an anti-GST antibody as protein controls (upper panel), respectively. (c) Co-IP assay for TcAMPK–TcAKs interaction. Co-IP assays were performed using an anti-TcAK1 or TcAK2 antibody, then the immune complex with agarose was subjected to western blot analysis to detect the presence of TcAMPKα protein with the anti-TcAMPKα antibody. The whole-cell lysates (input) were also tested with anti-TcAK1 (WB: TcAK1), anti-TcAK2 (WB: TcAK2), or anti-TcAMPKα (WB: TcAMPKα) antibody to verify the protein expression, and α-tubulin was used as a loading control. For control experiments, the cell lysates were precipitated with nonimmune rabbit IgG and subsequently analyzed by western blot. (d, e, f) In vitro AMPK-dependent phosphorylation of TcAK1 at S129 and T247 as well as TcAK2 at S145. The phosphorylated TcAK1 and TcAK2 were excised by trypsin and analyzed by MALDI-TOF/TOF-MS/MS or LC-MS/MS. MS/MS spectrum showed fragmentation of SLEGYPFNPCLTEEQY (m/z = 518.9872, z = +2, RT = 100.77) and RLVTGVNDIEK (m/z = 644.6982, z = +3, RT = 30.76) in TcAK1 and SLAGYPFNPCLTEAQYK (m/z = 435.7658, z = +3, RT = 103.47) in TcAK2. (g) The enzymatic constants of in vitro expressed wild-type proteins (6×His-TcAK1 and 6×His-TcAK2) and mutant proteins (6×His-TcAK1 S129D, 6×His-TcAK1 T247D, and 6×His-TcAK2 S145D), as well as TcAK1 and TcAK2, purified from AICAR-treated groups (p-TcAK1 and p-TcAK2) and the control groups (TcAK1 and TcAK2). (h) The detection of the effect of paraquat, AICAR treatment on TcAK1 and TcAK2 phosphorylation levels, and the effect of TcAMPKα knockdown on induced-TcAK1 or TcAK2 phosphorylation levels by paraquat treatment using Pro-Q Diamond phosphoprotein-stained kit. The gels were shown stained by coomassie blue (left panel) and Pro-Q Diamond (right panel). Arrows point to bands identified by lane labels. (i) Compared to the control group, paraquat treatment significantly increased the phosphorylation levels of TcAK1 and TcAK2, which were detected by phosphoprotein phosphate estimation kit. (j) Knockdown of TcAMPKα mRNA expression significantly attenuated the paraquat-induced up-regulation of the phosphorylation levels of TcAK1 and TcAK2. (k) AICAR treatment significantly up-regulated the phosphorylation levels of TcAK1 and TcAK2 compared to the control group. Data are expressed as mean ± SEM (n = 3 biologically independent replicates). Asterisk indicates differences statistically significant at *P< 0.05 (student’s t-test).

Article Snippet: The anti-α-tubulin mouse monoclonal antibody (Proteintech Group, Inc., Chicago, IL, USA) was used as a loading control for the cytoplasmic and total proteins, and the anti-Histone H3 Mouse Monoclonal Antibody (Beyotime, Jiangsu, China) was used as a loading control for the nuclear protein.

Techniques: Y2H Assay, Mutagenesis, Selection, Pull Down Assay, Incubation, Negative Control, Western Blot, Co-Immunoprecipitation Assay, Expressing, Control, In Vitro, Phospho-proteomics, Tandem Mass Spectroscopy, Liquid Chromatography with Mass Spectroscopy, Purification, Knockdown, Staining

Journal: Nucleic Acids Research

Article Title: Hypoxia increases methylated histones to prevent histone clipping and heterochromatin redistribution during Raf -induced senescence

doi: 10.1093/nar/gkae1210

Figure Lengend Snippet: Hypoxic inhibition of histone clipping in 4-OHT-treated IMR90-ΔB-RAF:ER cells. IMR90-ΔB-RAF:ER cells were treated (+) with 4-OHT (100 nM) for six days under normoxic (21% O 2 ) or hypoxic (0.5% O 2 ) conditions. ( A ) Confocal microscopic images were obtained following ERα antibody and Hoechst staining. ( B ) (i) Western blot analysis with the indicated antibodies. β-actin was used as a loading control. (ii) The means and S.E. of the band intensities of HIF-1α normalized by β-actin intensity from eight independent experiments. (iii) Quantitative RT-PCR of Bnip3 mRNA. ( C ) (i) Representative images of the SA-β-gal assays. (ii) Mean and standard deviation of SA-β-gal-positive cells from six randomly selected microscopic fields from three independent experiments. SA-β-gal-positive cells among 180–890 cells per field were counted. ( D ) (i) Confocal microscopic images of nuclei stained with the H3K9me3 antibody and Hoechst. (ii–iii) SAHFs were measured using the coefficient of variation (CV) of nuclear images stained with H3K9me3 (ii) or Hoechst (iii) (details in the Methods section). The values on the y-axis represent the CV of 55–60 nuclear images from two independent experiments. The midline of the boxplot represents the median, and the bottom and top of the box represent the 25th and 75th percentiles, respectively. Whiskers represent the minimum and maximum values. (E–F) Confocal microscopic images of nuclei stained with the HP1β ( E ) or H3K27me3 antibodies ( F ) and Hoechst. The intensities of the images on the yellow straight lines were quantified using ImageJ software. Hoechst (purple line), HP1β (green line), and H3K27me3 (green line). ( G ) Western blot analyses of histone acid extracts derived from IMR90-ΔB-RAF:ER cells, treated as outlined. H2A served as the loading control for the histone acid extracts. (H-I) Doxorubicin-induced senescence. IMR90 cells were treated with 100 nM doxorubicin for 2 days and cultured in fresh media for 6 days. ( H ) Western blot analysis using the indicated antibodies. ( I ) Representative images of the SA-β-gal assay. (J–K) Replicative senescence induced by serial passaging of IMR90 cells as indicated. ( J ) Western blot analysis with the indicated antibodies. ( K ) Representative images of the SA-β-gal assay.

Article Snippet: Human lung fibroblast IMR90 cells (CCL-186, ATCC, Manassas, VA, USA) were cultured in Eagle's minimum essential medium (10–009, Corning, Glendale, AZ, USA) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Inhibition, Staining, Western Blot, Control, Quantitative RT-PCR, Standard Deviation, Software, Derivative Assay, Cell Culture, Passaging

Journal: Nucleic Acids Research

Article Title: Hypoxia increases methylated histones to prevent histone clipping and heterochromatin redistribution during Raf -induced senescence

doi: 10.1093/nar/gkae1210

Figure Lengend Snippet: Top-down proteomics of histone extracts. ( A ) Base peak chromatogram reconstructed from the mass spectra of LC-MS/MS data using a histone sample from IMR90-ΔB-RAF:ER cells treated with 4-OHT under normoxic conditions. (i) A 2D display (monoisotopic masses vs. elution time) of intact histones measured in LC-MS/MS experiments (upper left). An annotated MS/MS spectrum labeled for cleaved N-terminal (Δb ions, blue) and C-terminal (y-ions, red) fragments showing a truncated H3 (upper right). (ii) H2B (lower left) and (iii) H4 (lower right). ( B ) Comparisons of the 2D display of measured histones using samples obtained from IMR90-ΔB-RAF:ER cells treated with four different conditions (normoxia without OHT, normoxia with OHT, hypoxia without OHT and hypoxia with OHT). ( C ) Western blot analyses of histone acid extracts derived from IMR90-ΔB-RAF:ER cells, treated as outlined. H2A served as the loading control for the histone acid extracts.

Article Snippet: Human lung fibroblast IMR90 cells (CCL-186, ATCC, Manassas, VA, USA) were cultured in Eagle's minimum essential medium (10–009, Corning, Glendale, AZ, USA) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Labeling, Western Blot, Derivative Assay, Control

Journal: Nucleic Acids Research

Article Title: Hypoxia increases methylated histones to prevent histone clipping and heterochromatin redistribution during Raf -induced senescence

doi: 10.1093/nar/gkae1210

Figure Lengend Snippet: Cathepsin L-dependent histone clipping. ( A ) Western blot analyses of histone acid extracts derived from IMR90-ΔB-RAF:ER cells, treated with E-64 (10 μM) and/or 4-OHT (100 nM). H2A served as the loading control for the histone acid extracts. ( B ) IMR90-ΔB-RAF:ER-shScramble cells and IMR90-ΔB-RAF:ER-shCTSL cells, IMR90-ΔB-RAF:ER cells infected with lentivirus encoding shScramble or shCTSL, respectively. Histones were identified using histone acid extracts, whereas CTSL, ER, HIF-1α and β-actin were detected using whole cell lysates. β-actin was served as a loading control. ( C ) Western blots of the cytosolic and nuclear fractions of IMR90-ΔB-RAF:ER cells treated as indicated. The 14–3-3γ and H3 were used as loading controls for cytosolic and nuclear fractions, respectively. Schematic representation of processed CTSL forms. The full-length CTSL comprises distinct domains: a signal peptide ( S ), pro-domain ( P ), heavy chain ( H ) and light chain ( L ) . (D-G) Western blot analyses were performed on histone acid extracts derived from IMR90 cells treated with the indicated drugs: JIB-04 (0.5 μM) CPX (10 μM), DFP (250 μM), DMOG (0.5 mM) and hypoxia for 6 days. The numbers represent the relative band intensities ( D ). ( H to J ) In vitro proteolytic reactions were performed using recombinant histones: rH3 (200 ng), rH2B (200 ng) along with human recombinant cathepsin L (rCTSL) (50 pg), in the presence of E64 (10, 100 μM), or JIB-04 (0.5, 5 μM). Visualization of both intact and cleaved histones was achieved through western blot analysis. ( J ) In vitro proteolytic reactions involved human rCTSL (0, 10, 50, 100, 200 pg) with histone acid extract (500 ng) derived from normoxic and hypoxic IMR90 cells.

Article Snippet: Human lung fibroblast IMR90 cells (CCL-186, ATCC, Manassas, VA, USA) were cultured in Eagle's minimum essential medium (10–009, Corning, Glendale, AZ, USA) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Western Blot, Derivative Assay, Control, Infection, In Vitro, Recombinant

Journal: Nucleic Acids Research

Article Title: Hypoxia increases methylated histones to prevent histone clipping and heterochromatin redistribution during Raf -induced senescence

doi: 10.1093/nar/gkae1210

Figure Lengend Snippet: Intracellular CTSL activity. ( A ) Intracellular CTSL enzyme activity quantification in IMR90-ΔB-RAF:ER cells incubated with the membrane-permeable CTSL substrate MR-FR 2 (3.6 μM) for 1 h (details in the ‘Materials and methods’ section). The values indicate the means and standard deviation of the reaction velocity (Relative fluorescence unit (RFU)/min) from three independent assays. ( B ) The deletion mutant M56-CTSL-Myc-His fusion protein translated from internal AUG at the 56th methionine. IMR90-ΔB-RAF:ER-M56CTSL cells that are IMR90-ΔB-RAF:ER cells, additionally infected with M56-CTSL-Myc-His or an empty vector (IMR90-ΔB-RAF:ER -EV). Confocal microscopic images of cells stained with Myc antibody and Hoechst. ( C ) Western blot analysis of whole cell lysates derived from the indicated cells using the designated antibodies. Closed triangles represent endogenous CTSL bands, and open triangles indicate M56CTSL bands. ( D ) Intracellular CTSL enzyme activity quantification in IMR90-ΔB-RAF:ER-M56CTSL cells incubated with the membrane-permeable CTSL substrate MR-FR 2 (3.6 μM) for 36 min (details in the ‘Materials and methods’ section). The values indicate the means and standard deviation of the reaction velocity (Relative fluorescence Unit (RFU)/min) from three independent assays. The specific velocities for each reaction are detailed in . ( E ) Western blot analyses of H3 and H2B were performed on histone acid extracts derived from IMR90-ΔB-RAF:ER cells which were infected with M56-CTSL-Myc-His or an empty vector and treated as outlined, using the indicated histone antibodies. H2A was used as a loading control. 2 × indicates that twice the amount of cell lysates was loaded compared to 1 ×. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Student's t-test; ns, not significant.

Article Snippet: Human lung fibroblast IMR90 cells (CCL-186, ATCC, Manassas, VA, USA) were cultured in Eagle's minimum essential medium (10–009, Corning, Glendale, AZ, USA) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Activity Assay, Incubation, Membrane, Standard Deviation, Fluorescence, Mutagenesis, Infection, Plasmid Preparation, Staining, Western Blot, Derivative Assay, Control

Journal: Nucleic Acids Research

Article Title: Hypoxia increases methylated histones to prevent histone clipping and heterochromatin redistribution during Raf -induced senescence

doi: 10.1093/nar/gkae1210

Figure Lengend Snippet: CTSL proteolysis inhibition by H3K23 and H3K18 methylation. ( A ) (i) Lysine residues and CTSL cleavage sites of H3. (ii) Western blot analyses of histone acid extracts derived from IMR90 cells treated with hypoxia (0.5% O 2 ) or JIB-04 (0.5 μM) for 4 days using the indicated histone antibodies. (iii) The y-axis indicates the mean and standard deviation of the band intensities of the indicated methylated H3 normalized by H3 intensity. n = 3–7, * P < 0.05, ** P < 0.01 and *** P < 0.001 by Student's t-test; ns, not significant. ( B ) Western blot analyses of histone acid extracts derived from IMR90-ΔB-RAF:ER cells treated as indicated. H2A was used as a loading control for histone extracts. ( C ) Schematic diagram of the competitive rCTSL enzyme kinetic analyses with the histone peptides as competitive inhibitors. H3 peptides with various modifications (100 nM) were used as inhibitors. Michaëlis–Menten plots of H3 peptides were fitted using an initial velocity (RFU/min) of rCTSL (96 pM). The values on the y-axis indicate the mean and standard deviation of the initial velocity (RFU/min) of three replicates at the Z-LR-AMC concentrations shown in the x-axis. The Km and Ki values were determined using initial velocity and nonlinear regression fit with GraphPad Prism software. ( D ) Ki values of naïve or methylated H3 peptides for the substrate Z-LR-AMC by rCTSL. The Ki values were determined using the initial velocity of three replicates and nonlinear regression fit with GraphPad Prism software from a single representative experiment among two independent experiments. ( E ) MDs simulations. (i) Representative simulation snapshots of CTSL D162 and H319-24. C, O, H and N atoms are shown as cyan, red, white and blue, respectively. The red lines represent the hydrogen bonds between the hydrogen donors (N in A21, T22 and K23 of H319-24) and the hydrogen acceptors (O in D162 of CTSL): (left) A21:N-D162:O, (middle) T22:N-D162:O, and (right) K23:N-D162:O. (ii) The hydrogen bonding percentage between the three hydrogen bonding pairs for the H319-24 and H319-24K23me3 (H3K23me3) systems. The hydrogen bonding percentage was calculated by counting the number of simulation snapshots in which the distance between the corresponding hydrogen donor and acceptor was within 3.5 Å from the equilibrated simulation trajectory (85 ns).

Article Snippet: Human lung fibroblast IMR90 cells (CCL-186, ATCC, Manassas, VA, USA) were cultured in Eagle's minimum essential medium (10–009, Corning, Glendale, AZ, USA) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Inhibition, Methylation, Western Blot, Derivative Assay, Standard Deviation, Control, Software

Journal: Nucleic Acids Research

Article Title: Hypoxia increases methylated histones to prevent histone clipping and heterochromatin redistribution during Raf -induced senescence

doi: 10.1093/nar/gkae1210

Figure Lengend Snippet: Inhibiting methyltransferase and OIS under hypoxia. IMR90-ΔB-RAF:ER cells were treated with 4-OHT and the indicated inhibitors for six days. ( A - B ) Confocal microscopic images of nuclei stained with antibodies against H3K23me3 and H3K23me2 ( A ), H3K18me3 ( B ), and Hoechst. ( C – E ) IMR90-ΔB-RAF:ER cells were treated with the pan-inhibitors of methyltransferase AdOx (50 μM) and DZNep (10 μM) for 6 days. ( C ) Western blot analyses of histone acid extracts derived from IMR90-ΔB-RAF:ER cells treated as indicated. The numbers represent the relative band intensities of methylated H3 normalized to the total H3 band intensities. H2A was used as a loading control for histone extracts. ( D ) (i) Confocal microscopic images of nuclei stained with the H3K9me3 antibody and Hoechst (ii) CV boxplots of images stained with H3K9me3. H3K9me3 intensities of 30–40 nuclear images were counted. ( E ) (i) Representative images of the SA-β-gal assays. (ii) The mean and standard deviation of SA-β-gal-positive cells from 3 to 5 randomly selected microscopic fields. SA-β-gal-positive cells among 180–600 cells per field were counted. * P < 0.05, ** P < 0.01 and *** P < 0.001 by Student's t-test; ns, not significant.

Article Snippet: Human lung fibroblast IMR90 cells (CCL-186, ATCC, Manassas, VA, USA) were cultured in Eagle's minimum essential medium (10–009, Corning, Glendale, AZ, USA) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Staining, Western Blot, Derivative Assay, Methylation, Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Hypoxia increases methylated histones to prevent histone clipping and heterochromatin redistribution during Raf -induced senescence

doi: 10.1093/nar/gkae1210

Figure Lengend Snippet: Lamin Associated Domains and NFR. ( A ) Bar plots of average LAD coverage of Ras -activated IMR90 cells (OIS+) and Ras -inactivated IMR90 cells (OIS-) in each group. *** P < 0.001 by Student's t-test. ( B ) Box plots show the distribution of average RPKM of NFRs for each condition in each group. The black line in the box is the median. *** P < 0.001 by Student's t-test; ns, not significant. ( C ) Heatmap of differential NFRs from Raf -activated (+) IMR90 cells cultured under normoxia or hypoxia. LAD coverage in these regions is described in Figure . ( D ) A graphic abstract.

Article Snippet: Human lung fibroblast IMR90 cells (CCL-186, ATCC, Manassas, VA, USA) were cultured in Eagle's minimum essential medium (10–009, Corning, Glendale, AZ, USA) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Cell Culture

Journal: Arthritis Research & Therapy

Article Title: KIAA1199 as a potential diagnostic biomarker of rheumatoid arthritis related to angiogenesis

doi: 10.1186/s13075-015-0637-y

Figure Lengend Snippet: Identification and verification of KIAA1199 expression in rheumatoid arthritis (RA) patients and normal subjects. (A) Identification the over-expression of KIAA1199 in RA fibroblast-like synoviocyte (FLS) cells than in normal FLS cells from synovial membrane by automated 2D-Nano-LC-ESI-MS/MS. The left panel shows amino acid coverage of the identified KIAA1199 sequence; yellow, appraisal sequence of amino acids. The right panel is the normalized spectrum count in RA and normal subjects; * P <0.001. (B) Verification of the expression of KIAA1199 was performed in synovial tissues from 19 patients with inactive RA, 25 with active RA and 15 normal subjects by qPCR. Results were normalized for the amount of β-actin as internal control. Experiments were performed at least in triplicates; bars represents mean ± SD; * P <0.05 compared with normal. (C) Immunohistochemical staining of KIAA1199 was performed on synovial tissues from patients with inactive or active RA and normal subjects. The KIAA1199 protein was over-expressed in vascular endothelium in active and inactive RA synovial membrane compared to normal synovial membrane. (D) Immunofluorescence microscopy of KIAA1199 and CTGF was performed in FLS cells from RA patients. Result shows KIAA1199 and CTGF were co-expressed in FLS cells, were either not expressed or only very weakly expressed in the nucleus, and were strongly expressed in the cytoplasm and membrane of FLS cells.

Article Snippet: The homogenate was centrifuged in a micro-centrifuge for 5 minutes at 3,000 g, and 100 μl diluted supernatant (1:50 with incubation buffer) was incubated at room temperature in a microtiter plate coated with KIAA1199 monoclonal antibody (sc-164775, Santa Cruz, CA, USA).

Techniques: Expressing, Over Expression, Membrane, Tandem Mass Spectroscopy, Sequencing, Control, Immunohistochemical staining, Staining, Immunofluorescence, Microscopy

Journal: Arthritis Research & Therapy

Article Title: KIAA1199 as a potential diagnostic biomarker of rheumatoid arthritis related to angiogenesis

doi: 10.1186/s13075-015-0637-y

Figure Lengend Snippet: Diagnostic value of evaluation of KIAA1199 in rheumatoid arthritis (RA) determined by receiver operator characteristic (ROC) curve analysis. ( A ) ROC curve analysis of KIAA1199 in serum for diagnosis of RA. The area under the curve (AUC) for inactive RA-healthy controls (HC) and active RA-HC was 0.74 and 0.83, respectively. A cutoff value of 90.13 μg/ml diagnosed active RA at the sensitivity of 72% and the specificity of 80%, respectively. ( B ) ROC curve analysis of KIAA1199 in synovial fluids for the diagnosis of RA. The AUC for inactive RA-HC and active RA-HC was 0.78 and 0.92, respectively. A cutoff value of 129.1 μg/ml diagnosed active RA at the sensitivity of 84% and the specificity of 93.3%, respectively. ( C ) ROC curve analysis of KIAA1199 in the synovium for the diagnosis of RA. The AUC for inactive RA-HC and active RA-HC was 0.79 and 0.91, respectively. A cutoff value of 198.7 μg/ml diagnosed active RA at the sensitivity of 80% and the specificity of 93.3%, respectively. All experiments were performed at least in triplicates; * P <0.05 compared with control. OA, osteoarthritis; AS, ankylosing spondylitis.

Article Snippet: The homogenate was centrifuged in a micro-centrifuge for 5 minutes at 3,000 g, and 100 μl diluted supernatant (1:50 with incubation buffer) was incubated at room temperature in a microtiter plate coated with KIAA1199 monoclonal antibody (sc-164775, Santa Cruz, CA, USA).

Techniques: Diagnostic Assay, Biomarker Discovery, Control

Journal: Arthritis Research & Therapy

Article Title: KIAA1199 as a potential diagnostic biomarker of rheumatoid arthritis related to angiogenesis

doi: 10.1186/s13075-015-0637-y

Figure Lengend Snippet: The effect of KIAA1199 expression on fibroblast-like synoviocyte (FLS) cell proliferation detected by MTT assay. FLS cells isolated from synovial tissue were transfected with scrambled siRNA, KIAA1199 siRNA duplex, pcDNA3.2DEST and pcDNA3.2DEST-KIAA1199 for 24 h, 48 h, 72 h and 96 h at 37 °C. Cell viability was determined by MTT. All experiments were performed at least in triplicates.

Article Snippet: The homogenate was centrifuged in a micro-centrifuge for 5 minutes at 3,000 g, and 100 μl diluted supernatant (1:50 with incubation buffer) was incubated at room temperature in a microtiter plate coated with KIAA1199 monoclonal antibody (sc-164775, Santa Cruz, CA, USA).

Techniques: Expressing, MTT Assay, Isolation, Transfection

Journal: Arthritis Research & Therapy

Article Title: KIAA1199 as a potential diagnostic biomarker of rheumatoid arthritis related to angiogenesis

doi: 10.1186/s13075-015-0637-y

Figure Lengend Snippet: Effect of KIAA1199 expression on angiogenesis. ( A ) Cell migration assay. Human umbilical vein endothelial cells (HUVEC) transfected with scrambled siRNA, KIAA1199 siRNA duplex, pcDNA3.2DEST and pcDNA3.2DEST-KIAA1199 were plated onto the top of the transwell, allowed to migrate for 4 h, then rinsed, fixed, stained, and counted. Left panel: HUVEC on the undersurface of a filter, magnification × 40; right panel: number of HUVEC per field. (B) Tube formation assay: 24-well plates were pre-coated with Matrigel (diluted 1:2 in H2O) and incubated at 37°C to promote gelling. HUVEC were seeded on Matrigel-coated plates, treated with scrambled siRNA, KIAA1199 siRNA duplex, pcDNA3.2DEST and pcDNA3.2DEST-KIAA1199 for 6-8 h, tube formation observed using a phase-contrast inverted microscope, and photographs were taken from each well. The number of intersections between branches of assembled endothelial cell networks was counted in the whole field. ( C) . Chorioallantoic membrane (CAM) assay, embryonic CAM was treated on day 7 with scrambled siRNA, KIAA1199 siRNA duplex, pcDNA3.2DEST and pcDNA3.2DEST-KIAA1199. After incubation for 3 days CAM was examined under a stereomicroscope, and photographs taken (left panel). ImageJ 2.43 s was used to calculate the vascular and CAM areas. Bar graph: ratio of vascular area to CAM area (right panel). All experiments were performed at least in triplicates, values are presented as mean ± SD; * P <0.05 compared with control (*significant differences).

Article Snippet: The homogenate was centrifuged in a micro-centrifuge for 5 minutes at 3,000 g, and 100 μl diluted supernatant (1:50 with incubation buffer) was incubated at room temperature in a microtiter plate coated with KIAA1199 monoclonal antibody (sc-164775, Santa Cruz, CA, USA).

Techniques: Expressing, Cell Migration Assay, Transfection, Staining, Tube Formation Assay, Incubation, Inverted Microscopy, Membrane, Chick Chorioallantoic Membrane Assay, Control

Journal: Arthritis Research & Therapy

Article Title: KIAA1199 as a potential diagnostic biomarker of rheumatoid arthritis related to angiogenesis

doi: 10.1186/s13075-015-0637-y

Figure Lengend Snippet: Effect of KIAA1199 knock-down and over-expression on the expression of PLXNB3, SEMA5A and CTGF in fibroblast-like synoviocyte (FLS) cells. ( A) . Real-time PCR of KIAA1199, PLXNB3, SEMA5A and CTGF transfected with scrambled siRNA, KIAA1199 siRNA duplex, pcDNA3.2DEST and pcDNA3.2DEST-KIAA1199 for 24 h in FLS cells; β-actin was used as a loading control. ( B) . Western blotting of KIAA1199, PLXNB3, SEMA5A and CTGF transfected with scrambled siRNA, KIAA1199 siRNA duplex, pcDNA3.2DEST and pcDNA3.2DEST-KIAA1199 for 48 h in FLS cells; β-actin was used as a loading control. All experiments were performed at least in triplicates, the data are presented as mean ± SD; * P <0.05, compared with control (*significant differences).

Article Snippet: The homogenate was centrifuged in a micro-centrifuge for 5 minutes at 3,000 g, and 100 μl diluted supernatant (1:50 with incubation buffer) was incubated at room temperature in a microtiter plate coated with KIAA1199 monoclonal antibody (sc-164775, Santa Cruz, CA, USA).

Techniques: Knockdown, Over Expression, Expressing, Real-time Polymerase Chain Reaction, Transfection, Control, Western Blot