Journal: bioRxiv

Article Title: Dysregulated myogenesis and autophagy in genetically induced pulmonary emphysema

doi: 10.1101/2021.07.08.450201

Figure Lengend Snippet: A1: Double-injury repair assay reveals suboptimal and delayed muscle repair of IL13 TG over time when compared with WT littermates. A2: IL13 TG TA muscle recovery at day 14 as determined by fibers CSA remains significantly below baseline muscle CSA when compared to the individuals counter-lateral leg TA muscle CSA (n=3). B: Fully induced IL13 TG animals have significantly reduced myogenic cell proliferation capacity as evaluated by 5-Ethynyl-2′-deoxyuridine (EdU) assay (n=4). C: Emphysematous MMP1TG mice show a significant reduction in myogenic cell proliferation (n=4). D: Integrin α 7 positive cells isolated using MACS cell separation technique showed similar counts between IL13 TG and WT animals (n=4). E: Pax7-GFP auto-fluorescent muscle stems cells counted in situ using automatic cell counting showed no significant difference between IL13TG and WT mice (n=4); * P < 0.05; *** P < 0.001.

Article Snippet: Mouse skeletal muscle satellite cells were first enriched using a Mouse Satellite Cell Isolation Kit (MACS Miltenyi Biotech, 130104268) and then selected for using anti-integrin α-7 microbeads (MACS Miltenyi Biotech, 130104261) to insure population purity as previously established [ ].

Techniques: EdU Assay, Isolation, In Situ, Cell Counting

Journal: bioRxiv

Article Title: Dysregulated myogenesis and autophagy in genetically induced pulmonary emphysema

doi: 10.1101/2021.07.08.450201

Figure Lengend Snippet: Example of GFP-Pax7/IL13 TG reporter mouse used for quantification of satellite cells per muscle section.

Article Snippet: Mouse skeletal muscle satellite cells were first enriched using a Mouse Satellite Cell Isolation Kit (MACS Miltenyi Biotech, 130104268) and then selected for using anti-integrin α-7 microbeads (MACS Miltenyi Biotech, 130104261) to insure population purity as previously established [ ].

Techniques:

Journal: bioRxiv

Article Title: Dysregulated myogenesis and autophagy in genetically induced pulmonary emphysema

doi: 10.1101/2021.07.08.450201

Figure Lengend Snippet: Post injury isolated muscle satellite cells’ RNA sequencing shows dysregulation of multiple transcripts related to cell cycle and autophagy in the IL13 TG animal as determined by gene ontology (GO) enrichment. Heat map of gene expression shows direction of differential gene expression in IL13 TG versus WT cells (n=5). Red indicates up-regulated gene expression and blue indicated down-regulated gene expression.

Article Snippet: Mouse skeletal muscle satellite cells were first enriched using a Mouse Satellite Cell Isolation Kit (MACS Miltenyi Biotech, 130104268) and then selected for using anti-integrin α-7 microbeads (MACS Miltenyi Biotech, 130104261) to insure population purity as previously established [ ].

Techniques: Isolation, RNA Sequencing, Gene Expression

Journal: bioRxiv

Article Title: Dysregulated myogenesis and autophagy in genetically induced pulmonary emphysema

doi: 10.1101/2021.07.08.450201

Figure Lengend Snippet: A: LC3-GFP/IL13 TG reporter mice show normalization of puncta buildup and MFI in isolated satellite cells after treatment with spermidine (n=4). B: EdU proliferation assay shows no difference between IL13 TG and WT animals after treatment with spermidine (n=4). C: Transplantation experiments in IL13 WT recipients conducted as previously described show no difference in number of recovered red fibers when both IL13 TG and IL13 WT donors were treated with spermidine (n=4).

Article Snippet: Mouse skeletal muscle satellite cells were first enriched using a Mouse Satellite Cell Isolation Kit (MACS Miltenyi Biotech, 130104268) and then selected for using anti-integrin α-7 microbeads (MACS Miltenyi Biotech, 130104261) to insure population purity as previously established [ ].

Techniques: Isolation, Proliferation Assay, Transplantation Assay

Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: Exploring downstream gene regulated by IMUP. a Heatmap and volcano plot analyzed using RNA-seq of BxPC-3 cells infected with shNC, IMUP-sh1, sh2, and sh3. b Gene ontology analysis of IMUP-correlated genes from TCGA database by LinkedOmics. c The intersection of negatively correlated genes (NCGs) from RNA-seq (fold-change > 2), NCGs from GeneChip, and NCGs from TCGA. d , e Pearson correlation of FHL1 and IMUP expression according to d the results of GeneChip and e TCGA data. f Kaplan–Meier analysis of OS by FHL1 expression data from TCGA

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: RNA Sequencing, Infection, Expressing

Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: IMUP regulates cyclin A2/cyclin E1/CDK2 proteins via FHL1. a WB analysis of BxPC-3 and SW1990 cells infected with lentivirus carrying IMUP-sh1 or sh2. GAPDH and tubulin were used as a loading control. b WB analysis of BxPC-3 and SW1990 cells infected with lentivirus carrying IMUP-sh or co-infected with IMUP-sh and FHL1-siRNA. c Representative IHC staining of IMUP and FHL1 in 57 human PDAC samples. Case 1 and case 2 were two representative specimens analyzed as high and low expression of IMUP, respectively. (case 1, high IMUP and low FHL1; Case 2, low IMUP and high FHL1). Scale bars: 100 μm. Correlation between IMUP and FHL1 analyzed by Pearson correlation tests

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: Infection, Control, Immunohistochemistry, Expressing

Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: Knockdown of FHL1 rescues the phenotype inhibited by IMUP depletion. a , b , c , g BxPC-3 and SW1990 cells infected with shNC or IMUP-sh, or co-infected with IMUP-sh and FHL1-sh were used to analyze the a proliferation by cell Counting Kit-8, b tumorigenicity by mice xenograft ( n = 5/group; male), c colony formation capability by colony formation assay, and g cell cycle by flow cytometry. d Xenograft tumor volume, e colony formation, and h cell cycle were analyzed by two-way ANOVA test. f Tumor weight was analyzed by unpaired Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.0001

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: Knockdown, Infection, Cell Counting, Colony Assay, Flow Cytometry

Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: IMUP inhibits FHL1 transcription by NPM1-induced promoter methylation. a BxPC-3 cells were transfected with GFP-tagged IMUP. Exogenous co-immunoprecipitation was performed by GFP beads (left). Endogenous interaction was tested in BxPC-3 cells by anti-NPM1 (right). b WB analysis of proteins extracted from BxPC-3 infected with control shRNAs, IMUP sh1, or sh2. c Immunofluorescence double-staining of BxPC-3 cells showed the location of IMUP and NLM1. Cells were stained with anti-IMUP (green) and anti-NPM1 (red). The nuclei were stained with DAPI (blue). Scale bars: 50 μm. d BxPC-3 cells transfected with IMUP or control siRNAs were treated with 100 μg/mL CHX at the indicated time. Proteins were analyzed with anti-IMUP, anti-NPM1, and anti-tubulin. e The densitometric quantitation of NPM1 protein at the indicated time was normalized by tubulin. Statistical differences were analyzed by two-tailed Student’s t test (*** P < 0.0001). f Structure of the human FHL1 gene. Dotted lines indicate two exon fragments containing CpG islands and corresponding sequences. g The pyrosequencing maps of FHL1 promoter CpG islands. DNA was collected from BxPC-3 cells infected with control shRNAs, IMUP-shRNAs, or co-transfected with IMUP-shRNAs and NPM1 vectors. The methylation rates of FHL1 promoter CpG islands. Fragment 1 (left histogram) and fragment 2 (right histogram). Statistical differences were analyzed using two-way ANOVA test. *** P < 0.0001. h Translational factor SP1-binding motif and putative SP1-binding sequences of FHL1 promoter. i ChIP-qPCR revealed SP1 enrichment on the FHL1 promoter in BxPC-3 cells infected with either control shRNAs, or IMUP-shRNAs or co-transfected with IMUP-shRNAs and NPM1 vectors. The upstream of FHL1 promoter and actin promoter were used as negative controls. Statistical differences were analyzed by two-tailed Student’s t test (** P < 0.001, *** P < 0.0001)

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: Methylation, Transfection, Immunoprecipitation, Infection, Control, Immunofluorescence, Double Staining, Staining, Quantitation Assay, Two Tailed Test, Binding Assay, ChIP-qPCR

Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: FHL1 interacts with CHK1/CDC25A/14–3-3ξ and promotes the phosphorylation of CDC25A via CHK1. a WB analysis of input and anti-Flag IP derived from BxPC-3 cells transfected with Flag-FHL1 vectors. b Endogenous proteins from BxPC-3 were immunoprecipitated using anti-CDC25A, CDC25C, CHK1, 14–3-3ξ antibody, or rabbit IgG as a negative control. c WB analysis of BxPC-3 and SW1990 cells treated with control vectors, Flag-FHL1 vectors, or/and treated with 25 μmol CHK1 inhibitor (GDC-0575 analog). d BxPC-3 cells were treated with control vectors or Flag-FHL1 vectors. WB analysis of IP by anti-CHK1 or IgG

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: Phospho-proteomics, Derivative Assay, Transfection, Immunoprecipitation, Negative Control, Control

Journal: Cell Biology and Toxicology

Article Title: Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

doi: 10.1007/s10565-022-09695-4

Figure Lengend Snippet: FHL1 causes CDC25A to become sequestered in the cytoplasm via binding to 14–3-3ξ. a BxPC-3 and SW1990 cells were treated with control vectors, Flag-FHL1 vectors, or co-transfected with Flag-FHL1 vectors and 14–3-3ξ siRNAs. Nuclear-cytoplasmic isolated proteins were used for WB analysis. C, cytoplasm; N, nucleus. b BxPC-3 and SW1990 cells were treated with control siRNAs, IMUP-siRNAs, or co-transfected with IMUP siRNAs and 14–3-3ξ siRNAs. Nuclear-cytoplasmic isolated proteins were used for WB analysis. Right panels show the densitometric analysis of CDC25A distributed in the cytoplasm or nucleus in three independent experiments. Statistical differences (the ratio of nucleus to cytoplasm) were analyzed by unpaired Student’s t test. *** P < 0.0001. c BxPC-3 cells were transfected with control vectors or Flag-FHL1 vectors. WB analysis of IP by anti-14–3-3ξ or IgG. d Representative immunofluorescence analysis of xenograft mouse tumors infected with control shRNA and IMUP-sh1. Cells were stained with anti-CDC25A (red). Nuclei are stained with DAPI (blue). Scale bars: 100 μm. e Schematic of the mechanism underlying IMUP-regulated S phase progression through NPM1/FHL1-mediated cell-cycle kinase protein activation. IMUP enhances the stability of NPM1 by direct binding. NPM1 indirectly facilitates promoter CpG island methylation of FHL1 and inhibits the transcription of FHL1 . FHL1 promotes the phosphorylation of CDC25A by CHK1 and sequesters CDC25A in the cytoplasm by forming CDC25A/14–3-3ξ complexes

Article Snippet: Antibodies against IMUP (1:100; #ab221063) from Abcam (Cambridge, UK) and FHL1 (1:50; #10,991–1-AP) from Proteintech (Wuhan, China) were used for IHC to verify their expression in PDAC and xenograft tumor tissues.

Techniques: Binding Assay, Control, Transfection, Isolation, Immunofluorescence, Infection, shRNA, Staining, Activation Assay, Methylation, Phospho-proteomics

Journal: The Journal of Physiological Sciences : JPS

Article Title: 17β-Estradiol-induced enhancement of estrogen receptor biosynthesis via MAPK pathway in mouse skeletal muscle myoblasts

doi: 10.1007/s12576-009-0023-0

Figure Lengend Snippet: Distribution of estrogen receptors in various organs and tissues of adult mouse. a Western blot analysis of whole-cell fractions with a specific monoclonal estrogen receptor (ER) antibody. Upper panel immunoreactive bands corresponding to the ER protein (66 kDa). The histogram (lower) indicates the results of densitometric analysis for a 66-kDa band (mean ± SE, n = 4). Relative expression level with respect to the ovary is shown in %. From left to right: skeletal muscle, uterus, lung, adipose tissue, kidney, and myocardium. b Relative expression (to ovary) of whole cell and nonnuclear ER proteins obtained from C2C12 mouse myoblasts (n = 3)

Article Snippet: C2C12 murine skeletal muscle cell line [ 20 ], obtained from Dainippon Pharmaceutical Ltd., was routinely cultured in Dulbecco’s Modified Eagle’s Medium F12 (DMEM/F12) containing 10% heat-inactivated (30 min, 56°C) fetal bovine serum (FBS).

Techniques: Western Blot, Expressing

Journal: The Journal of Physiological Sciences : JPS

Article Title: 17β-Estradiol-induced enhancement of estrogen receptor biosynthesis via MAPK pathway in mouse skeletal muscle myoblasts

doi: 10.1007/s12576-009-0023-0

Figure Lengend Snippet: Time course of ER mRNA and protein expression during E2 exposure. a Representative of C2C12 mRNA transcripts for ER and GAPDH at several time points after exposure to E2 (10−8 M) (upper). The mRNA level of ER relative to GAPDH is averaged from five individual experiments and displayed as mean ± SE (statistically significant). At 0, 0.5, 1, 3, and 5 h (lower). b Proteins were extracted from nucleus-free C2C12 cells exposed to E2 (10−8 M) for varying periods of time (from left: 0, 1, 3, 4, 6, 12, and 24 h) and immunoblotted with the ER antibody. Immunodensity in a 66-kDa band is quantified by densitometry and presented as the % increase of control (time 0). Symbols and bars represent mean ± SE (n = 5); statistically significant

Article Snippet: C2C12 murine skeletal muscle cell line [ 20 ], obtained from Dainippon Pharmaceutical Ltd., was routinely cultured in Dulbecco’s Modified Eagle’s Medium F12 (DMEM/F12) containing 10% heat-inactivated (30 min, 56°C) fetal bovine serum (FBS).

Techniques: Expressing, Control

Journal: The Journal of Physiological Sciences : JPS

Article Title: 17β-Estradiol-induced enhancement of estrogen receptor biosynthesis via MAPK pathway in mouse skeletal muscle myoblasts

doi: 10.1007/s12576-009-0023-0

Figure Lengend Snippet: Dose–dependent effects of 17β-estradiol on the expression of total and nonnuclear ER. Representative immunoreactive bands for total ER in C2C12 myoblasts (upper panel) and the relative expression of total ER protein (a) or nonnuclear ER protein (b) quantified by densitometric analysis (lower panel); after treatment with either vehicle (control), different concentrations of 17β-estradiol (10−12–10−5 M) in a and (10−10–10−6 M) in b for 15 h. c Shows the relative expression of total ER protein after treatment with vehicle (control), 17β-estradiol (10−8 M), and BSA-conjugated E2 (10−8 M) for 15 h. Each bar is normalized to the control (far left) and expressed as %. Columns and bars indicate mean ± SE (n = 3 in a, n = 4 in b, and n = 5 in c). *P < 0.01, ☆ P < 0.05 compared with control values, determined using Student’s t test

Article Snippet: C2C12 murine skeletal muscle cell line [ 20 ], obtained from Dainippon Pharmaceutical Ltd., was routinely cultured in Dulbecco’s Modified Eagle’s Medium F12 (DMEM/F12) containing 10% heat-inactivated (30 min, 56°C) fetal bovine serum (FBS).

Techniques: Expressing, Control

Journal: The Journal of Physiological Sciences : JPS

Article Title: 17β-Estradiol-induced enhancement of estrogen receptor biosynthesis via MAPK pathway in mouse skeletal muscle myoblasts

doi: 10.1007/s12576-009-0023-0

Figure Lengend Snippet: Influence of 17-βestradiol on C2C12 proliferation. C2C12 cells were grown in the serum-free medium for 48 h, which was then changed to ones with or without E2 (10−8 M) in 1% FBS (open triangle, closed triangle) or 10% FBS (open circle, closed circle). The rate of cell growth is shown as the cell number at every 24 h relative to 0 h. Symbols and bars represent the mean ± SE (n = 5)

Article Snippet: C2C12 murine skeletal muscle cell line [ 20 ], obtained from Dainippon Pharmaceutical Ltd., was routinely cultured in Dulbecco’s Modified Eagle’s Medium F12 (DMEM/F12) containing 10% heat-inactivated (30 min, 56°C) fetal bovine serum (FBS).

Techniques:

Journal: The Journal of Physiological Sciences : JPS

Article Title: 17β-Estradiol-induced enhancement of estrogen receptor biosynthesis via MAPK pathway in mouse skeletal muscle myoblasts

doi: 10.1007/s12576-009-0023-0

Figure Lengend Snippet: Effects of ER antagonists on E2-induced whole-cell and non-nuclear ER increase. C2C12 cells were treated with E2 (10−8 M) in the absence or presence of either tamoxifen or ICI 182,780 (10−7 M; a total ER) or (10−5 M; b non-nuclear fractions) for 15 h. Tamoxifen or ICI 182,780 was added to the culture medium 30 min prior to the administration of E2. Total and nonnuclear fractions of ER were prepared as described in “Materials and methods” and subjected to Western blotting. Histograms represent the extent of inhibition of E2-induced ER increase in total a and nonnuclear b fractions, which are expressed as the percentage of control (no stimulation), averaged from five separate experiments (mean ± SE). *P < 0.01, compared with control values, determined using Student’s t test

Article Snippet: C2C12 murine skeletal muscle cell line [ 20 ], obtained from Dainippon Pharmaceutical Ltd., was routinely cultured in Dulbecco’s Modified Eagle’s Medium F12 (DMEM/F12) containing 10% heat-inactivated (30 min, 56°C) fetal bovine serum (FBS).

Techniques: Western Blot, Inhibition, Control

Journal: The Journal of Physiological Sciences : JPS

Article Title: 17β-Estradiol-induced enhancement of estrogen receptor biosynthesis via MAPK pathway in mouse skeletal muscle myoblasts

doi: 10.1007/s12576-009-0023-0

Figure Lengend Snippet: Effects of 17β-estradiol and/or TPA on ER de novo synthesis. In the absence (control) or presence of 17β-estradiol (10−8 M) and/or TPA (10−6 M), C2C12 cells were labeled with [35S] methionine. At the time points indicated in the figure, the total ER fractions were prepared and subjected to selective immunoprecipitation for ER. The immunoprecipitates were analyzed by SDS-PAGE and image scanning. Data are the mean ± SE from three separate experiments. Left panel time course of 35methionine incorporation in ER. Right panel rates of the increase at 5 h by the relative ratio of control. *P < 0.01, compared with control values, determined using Student’s t test. ☆ P < 0.01, compared with E2 values, determined using Student’s t test

Article Snippet: C2C12 murine skeletal muscle cell line [ 20 ], obtained from Dainippon Pharmaceutical Ltd., was routinely cultured in Dulbecco’s Modified Eagle’s Medium F12 (DMEM/F12) containing 10% heat-inactivated (30 min, 56°C) fetal bovine serum (FBS).

Techniques: Control, Labeling, Immunoprecipitation, SDS Page