Journal: PLoS ONE

Article Title: The effects of different frequency treadmill exercise on lipoxin A 4 and articular cartilage degeneration in an experimental model of monosodium iodoacetate-induced osteoarthritis in rats

doi: 10.1371/journal.pone.0179162

Figure Lengend Snippet: Increase in the frequency with same amount of treadmill exercise decreased the MIA-induced degradation of COL2A1 in articular cartilage and reduced the expression of MMP-13 and NF-κB p65. The micrographs showed the intensity of immunohistochemical staining of COL2A1, NF-κB p65, MMP-13, and TIMP-1 in the articular cartilage of each experimental group, and the figures showed the percentages of positively stained cells. Differences between CG and OAG were significant (* P <0.001, * a P = 0.001), and differences between OAG and the treadmill exercise groups were significant ( + P <0.001, +a P = 0.012). One-way ANOVA, n = 5 rats for each group, mean score with 95% confidence interval.

Article Snippet: The sections were incubated with rabbit polyclonal anti-COL2A1 antibody (sc-28887, 1:50; Santa Cruz), mouse monoclonal anti-NF-κB p65 antibody (sc-8008, 1:50; Santa Cruz), goat polyclonal anti-MMP-13 antibody (sc-31813, 1:50; Santa Cruz), or rabbit polyclonal anti-TIMP-1 antibody (sc-5538, 1:50; Santa Cruz) overnight at 4°C.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: The effects of different frequency treadmill exercise on lipoxin A 4 and articular cartilage degeneration in an experimental model of monosodium iodoacetate-induced osteoarthritis in rats

doi: 10.1371/journal.pone.0179162

Figure Lengend Snippet: Effect of different frequency with same amount of treadmill exercise on the expression of MMP-13, TIMP-1, NF-κB p65, IκB-β, and COL2A1 expression in knee joint cartilage of rats with MIA-induced OA. Protein expression was determined in western blots of total protein extracted from cartilage tissues as described in Materials and Methods. The data was obtained in three separate experiments β-actin as an internal standard. Differences between CG and the OAG group were significant (* P < 0.001), and differences between the OAG and the treadmill frequency groups were significant ( + P <0.001, +a P = 0.030, +b P = 0.001, +c P = 0.039, +d P = 0.010, +e P = 0.038, +f P = 0.002,). One-way ANOVA, n = 3 rats for each group, means with 95% confidence interval.

Article Snippet: The sections were incubated with rabbit polyclonal anti-COL2A1 antibody (sc-28887, 1:50; Santa Cruz), mouse monoclonal anti-NF-κB p65 antibody (sc-8008, 1:50; Santa Cruz), goat polyclonal anti-MMP-13 antibody (sc-31813, 1:50; Santa Cruz), or rabbit polyclonal anti-TIMP-1 antibody (sc-5538, 1:50; Santa Cruz) overnight at 4°C.

Techniques: Expressing, Western Blot

Journal: Clinical Orthopaedics and Related Research

Article Title: Development and Characterization of a Novel Bipedal Standing Mouse Model of Intervertebral Disc and Facet Joint Degeneration

doi: 10.1097/CORR.0000000000000712

Figure Lengend Snippet: Shown here are the intervertebral disc immunohistochemistry results of the novel bipedal standing mouse model. (A) The results showed that the degree of intervertebral disc degeneration in the experimental group was higher than that in the control group. (a-c) Specifically, the experimental group showed higher expression of collagen X in the cartilage endplate, (d-f) decreased expression of Col2a1 in the annulus fibrosus, and (g-i) increased expression of MMP-13 and (j-l) OCN in the annulus fibrosus. (B) The statistical results of the ratio of collagen X-positive cells in the cartilage endplate are shown. (C) The statistical results of the ratio of MMP-13-positive cells in the annulus fibrosus are shown. (D) The statistical results of the ratio of OCN-positive cells in the annulus fibrosus are shown. *p < 0.05 compared with the control group; ▲p < 0.05 compared with the 6-week posttreatment group (scale bars = 50 μm [a-l]). CON = control group; POST6W = post-6-week treatment group; POST10W = post-10-week treatment group.

Article Snippet: The sections were then incubated with rabbit antimouse osteocalcin (OCN; ABclonal, Wuhan, China), matrix metalloprotease-13 (MMP-13), collagen X, P16 IKN4A , aggrecan, and vimentin antibodies(Abcam, Cambridge, USA), and mouse antimouse Col2a1 antibody (Millipore, Burlington, MA, USA) at 4° C overnight followed by incubation in secondary antibody (ABclonal) for 1 hour at room temperature, DAB chromogenesis, H&E staining, and mounting to observe the expression of the corresponding proteins using light microscopy (Axio Scope.A1, Zeiss, Jena, Germany).

Techniques: Immunohistochemistry, Expressing

Journal: JBMR Plus

Article Title: Wnt16 Elicits a Protective Effect Against Fractures and Supports Bone Repair in Zebrafish

doi: 10.1002/jbm4.10461

Figure Lengend Snippet: Transgenic Lines as Listed on zfin.org and Abbreviations Used in Text

Article Snippet: Primary antibodies were chick α‐L‐plastin (gift from the Martin laboratory ( ) ) and Col2a1 (1:50; M3F7; DSHB).

Techniques: Transgenic Assay, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting CRABP-II overcomes pancreatic cancer drug resistance by reversing lipid raft cholesterol accumulation and AKT survival signaling

doi: 10.1186/s13046-022-02261-0

Figure Lengend Snippet: High expression of CRABP-II is associated with poor prognostics and drug resistance in human PDAC. A Kaplan–Meier overall survival curves for PDAC patients based on CRABP-II levels. Data shown here are from Kaplan–Meier Plotter database. B, C CRABP-II expression in paired primary and relapsing PDAC ( n = 12) assessed by immunochemistry. All patients received gemcitabine-based chemotherapy after surgery. Immunoreactivity in ( C ) was calculated by multiplying the percentage of positive epithelium cells and the score of staining intensity (intensity: 0, undetectable; 1, weak; 2, moderate; and 3, strong). Paired sample t-test was used to determine the difference between primary group and relapsing group. Up panel in ( B ): black arrow denotes the poorly differentiated tumor; blue arrow denotes the well differentiated tumor. D, E Establishment of gemcitabine resistant cell lines using BxPC3 as parental line. Gemcitabine sensitivities of GR2000, GR4000 and BxPC3 cells were assessed by MTT assay ( D ). CRABP-II levels in these lines were detected by western blots ( E ). F Gemcitabine sensitivity comparison of Panc-1, CRABP-II knockout (CIIKO) and CRABP-II re-expressing (CIIOE) cells by MTT assays. G Gemcitabine induced cell apoptosis assessed by cleaved caspase-3 blots

Article Snippet: 1–2 mg of total proteins in cell lysate were mixed with 10 μg of rabbit IgG isotype control or anti-CRABP-II polyclonal antibody (Proteintech) and incubated at 4 °C overnight.

Techniques: Expressing, Staining, MTT Assay, Western Blot, Comparison, Knock-Out

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting CRABP-II overcomes pancreatic cancer drug resistance by reversing lipid raft cholesterol accumulation and AKT survival signaling

doi: 10.1186/s13046-022-02261-0

Figure Lengend Snippet: CRABP-II regulates cholesterol metabolic genes expression through cooperation with HuR. ( A ) Molecular and cellular function analysis by IPA software (Qiagen) based on gene expression microarray profiling. The altered lipid synthesis and accumulation functions upon CRABP-II knockout were listed. ( B ) Heat map of altered cholesterol metabolic genes. ( C, D, E ) Cholesterol metabolic genes expression assessed by Q-PCR. ( F ) Correlation between cholesterol metabolic genes and CRABP-II expression in human pancreatic cancer specimens by Pearson’s product-moment correlation coefficient analysis (PPMCC). Data shown here are combination of Pei Pancreas and Badea Pancrease datasets ( n = 75) from Oncomine. ( G ) Interaction between CRABP-II and HuR identified by co-immuprecipitation (co-IP). GR4000 cell lysis was incubated with anti-CRABP-II rabbit polyclonal antibody and the pull down proteins were separated and blotted with anti-HuR mouse monoclonal antibody. ( H ) Half-life of SREBP-1c mRNA assessed by actinomycin D treatment following with Q-PCR. ( I ) RNA-immunoprecipitation (RIP). The down pulled SREBP-1c mRNA from flagged-CRABP-II transfected CIIKO cells and empty vector transfected cells were assessed by Q-PCR. The actin mRNA was used as control. The experiment was repeated three times and the error bars present standard deviation (SD). **, p < 0.01

Article Snippet: 1–2 mg of total proteins in cell lysate were mixed with 10 μg of rabbit IgG isotype control or anti-CRABP-II polyclonal antibody (Proteintech) and incubated at 4 °C overnight.

Techniques: Expressing, Cell Function Assay, Software, Gene Expression, Microarray, Knock-Out, Co-Immunoprecipitation Assay, Lysis, Incubation, RNA Immunoprecipitation, Transfection, Plasmid Preparation, Control, Standard Deviation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting CRABP-II overcomes pancreatic cancer drug resistance by reversing lipid raft cholesterol accumulation and AKT survival signaling

doi: 10.1186/s13046-022-02261-0

Figure Lengend Snippet: CRABP-II knockout decreased lipid raft cholesterol accumulation and AKT survival signaling. ( A ) Comparison of cholesterol content in CRABP-II knockout cells and in wild type control Panc-1 cells. The free cholesterol (FC) and esterified cholesterol (EC) were assessed by liquid chromatography with tandem mass spectrometry (LC–MS-MS) and normalized to total protein. ( B ) Comparison of lipid raft cholesterol storage in wild type cells and CRABP-II knockout cells. The membrane lipid raft fractions were isolated by sucrose gradient ultracentrifuge (up panel) and the total cholesterol was assessed using cholesterol assay kit and normalized to total protein. The green bar presents the CIIKO cells pretreated with 1 mM cholesterol in media. ( C, D ) AKT activation assessed by western blots. Panc-1, CIIKO and cholesterol pretreated CIIKO cells were incubated with 100 nM insulin for 0, 15, 30 and 60 min, cells were immediately lysed. ( E, F ) Rescuing the loss of raft-cholesterol and AKT activation in CIIKO cells by re-expression of CRABP-II. ( G, H ) Increased raft-cholesterol content and AKT activation in gemcitabine resistant cell lines. All these experiments were repeated at least three times and the error bars present SD. **, p < 0.01

Article Snippet: 1–2 mg of total proteins in cell lysate were mixed with 10 μg of rabbit IgG isotype control or anti-CRABP-II polyclonal antibody (Proteintech) and incubated at 4 °C overnight.

Techniques: Knock-Out, Comparison, Control, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Membrane, Isolation, Cholesterol Assay, Activation Assay, Western Blot, Incubation, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting CRABP-II overcomes pancreatic cancer drug resistance by reversing lipid raft cholesterol accumulation and AKT survival signaling

doi: 10.1186/s13046-022-02261-0

Figure Lengend Snippet: SINPER-11 treatment induced CRABP-II degradation and suppressed PDX tumor growth. ( A ) Molecular formula of SNIPER-11. ( B ) Degradation of CRABP-II induced by SNIPER-11 in Panc-1 and BxPC3 cell culture. ( C ) Degradation of CRABP-II by SNIPER-11 in PDX tumors. ( D ) SNIPER-11 inhibited PDX tumor growth. Limited passaged PDX tumors were digested to single cell suspension and orthotopically injected into pancreas of NSG mice (1 × 10 6 cell / injection). Tumor bearing mice were treated with 5 µM SNIPER-11 or MV1 every two days for two weeks at 7 weeks after implantation and DMSO as control. Tumor size was assessed by MRI imaging (Fig. ). Two-way ANOVA was used to compare tumor growth between different treatments and the error bars represent SD. *, p < 0.05. ( E ) H&E and Ki67 staining of SNIPER-11 or DMSO treated tumor specimens. ( F, G ) The renewal of SNIPER-11/DMSO treated tumors. SNIPER-11 or DMSO treated tumors were secondarily transplanted into subcutaneous cavity of NSG mice. Tumor size was monitored and the tumor volume = ½ (width x width x length). The tumor growth was compared using two-way ANOVA (**, p < 0.01)

Article Snippet: 1–2 mg of total proteins in cell lysate were mixed with 10 μg of rabbit IgG isotype control or anti-CRABP-II polyclonal antibody (Proteintech) and incubated at 4 °C overnight.

Techniques: Cell Culture, Suspension, Injection, Control, Imaging, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting CRABP-II overcomes pancreatic cancer drug resistance by reversing lipid raft cholesterol accumulation and AKT survival signaling

doi: 10.1186/s13046-022-02261-0

Figure Lengend Snippet: A schematic model showing CRABP-II regulating PDAC cholesterol metabolism and drug resistance. By interacting with HuR, CRABP-II stabilizes the mRNA of SREBP-1c and increases expression of this center lipid metabolic transcription factor. Elevated SREBP-1c activates a cluster of cholesterol metabolic genes such as HMGCR and LDLR, resulting in increased intracellular cholesterol biosynthesis and uptake. SREBP-1c also inhibits the major cholesterol efflux transporter ABCA1 by enhancing miR-33 expression and reduces the intracellular cholesterol removal. Both arms of this machinery lead to the cholesterol accumulation in cell membrane, especially in lipid rafts, thus promoting AKT survival signaling and cancer drug resistance. The protein eraser SNIPER-11 selectively induces CRABP-II degradation in PDAC, hence interrupts CRABP-II/SREBP-1c/raft-cholesterol/AKT axis and overcomes PDAC drug resistance

Article Snippet: 1–2 mg of total proteins in cell lysate were mixed with 10 μg of rabbit IgG isotype control or anti-CRABP-II polyclonal antibody (Proteintech) and incubated at 4 °C overnight.

Techniques: Expressing, Membrane