Journal: Journal of the American Society of Nephrology : JASN

Article Title: Macrophage-stimulating protein is produced by tubular cells and activates mesangial cells.

doi: 10.1681/ASN.V133649

Figure Lengend Snippet: Figure 1. Western blot performed with anti–macrophage-stimulating protein (MSP) antibody (Ab) in culture supernatant of the human tubular cell line HK2, human mesangial cells (HMC), and the hepa- toma cell line HepG2. A 50 ml volume of supernatant was immuno- precipitated with anti-MSP polyclonal Ab adsorbed to protein A–sepharose 4B packed beads. Immunoprecipitates were washed with an ice-cold buffer, boiled after addition of 2 sample buffer, and the proteins were loaded onto 10% sodium dodecyl sulfate–polyacrylam- ide gel electrophoresis. Proteins were analyzed by Western blot that used a polyclonal anti-MSP Ab. The 85-kD bands represent mono- meric MSP (pro-MSP), and the 55-kD bands represent the -chain of dimeric (active) MSP. Both HK2 and HepG2 cells (used as controls) release pro-MSP that is cleaved into the dimeric form in the super- natant. No band is visible in the HMC lane.

Article Snippet: In some experiments, HMC were incubated with HK2 supernatant or with HK2 supernatant preincubated for 2 h with neutralizing goat polyclonal anti-human MSP antibody (Ab; 2 g/ml) (R&D Systems, Minneapolis, MN) without fresh medium.

Techniques: Western Blot, Nucleic Acid Electrophoresis

Journal: BMC Cancer

Article Title: The DNA methylation profile of liver tumors in C3H mice and identification of differentially methylated regions involved in the regulation of tumorigenic genes

doi: 10.1186/s12885-018-4221-0

Figure Lengend Snippet: Shortlist of the selected genes a

Article Snippet: After attaching to the dish, cells were incubated in a culture medium containing 5-aza-dC for 72 h. For overexpression study, mouse ORF clones Mst1r (NM_001287261) and Slpi (NM_011414), and pCMV6-Kan/Neo (pCMV6KN) as an empty vector were purchased from ORIGENE Inc. (MD, USA).

Techniques: Methylation, Gene Expression, Protease Inhibitor, Translocation Assay, Membrane, Binding Assay, Derivative Assay

Journal: BMC Cancer

Article Title: The DNA methylation profile of liver tumors in C3H mice and identification of differentially methylated regions involved in the regulation of tumorigenic genes

doi: 10.1186/s12885-018-4221-0

Figure Lengend Snippet: Mst1r associated with hypo-DMR is up-regulated in the liver tumor tissues of C3H mice. a Schematic view of the Mst1r locus. Methylation levels of CpG sites were visualized using Integrative Genomics Viewer (IGV) software. The position of TSS, DMR and the positions assessed by bisulfite sequencing (BS-1 and BS-2) were indicated. The magnified view indicates 8 CpG positions in the DMR and predicted transcription factor binding sites. b The average methylation values of the CpG sites in normal and tumor tissues. The CpG sites with ≥10 reads on both strands (≥20 reads in total) are indicated. Statistical significance between normal and tumor tissue was analyzed by Student’s t-test. **, *** Significantly different at p < 0.01 and 0.001, respectively. c Validation of RRBS data for normal and tumor liver tissues of C3H mice by bisulfite sequencing. Methylated and unmethylated cytosine are shown as ● and ○ respectively. The numbers above the circles indicate the position of CpG shown in Fig. . d Validation of gene expression levels of Mst1r by real-time PCR in the normal ( n = 6) and tumor tissues ( n = 11) of C3H mice. The expression of Mst1r is normalized to the expression of rRNA . Statistical significance between the two groups was analyzed by Student’s t-test. *** Significantly different at p < 0.001

Article Snippet: After attaching to the dish, cells were incubated in a culture medium containing 5-aza-dC for 72 h. For overexpression study, mouse ORF clones Mst1r (NM_001287261) and Slpi (NM_011414), and pCMV6-Kan/Neo (pCMV6KN) as an empty vector were purchased from ORIGENE Inc. (MD, USA).

Techniques: Methylation, Software, Methylation Sequencing, Binding Assay, Biomarker Discovery, Gene Expression, Real-time Polymerase Chain Reaction, Expressing

Journal: BMC Cancer

Article Title: The DNA methylation profile of liver tumors in C3H mice and identification of differentially methylated regions involved in the regulation of tumorigenic genes

doi: 10.1186/s12885-018-4221-0

Figure Lengend Snippet: Reduced DNA methylation of DMRs of Mst1r, Slpi, and Extl1 after 5-aza-dC treatment are associated with up-regulation of these genes in hepa1c1c7 cells and Hepa1-6 cells. Hepa1-6 cells ( b , d , e ) and Hepa1c1c7 cells ( a , c ) were cultured with 0, 0.1 or 1 μM of 5-aza-dC, and 0, 50 or 100 μM of 5-aza-dC for 72 h, respectively. Left figures: the results of bisulfite sequencing of CpGs detected in the DMRs of liver tumors in C3H mice. ●: methylated cytosine, ○: unmethylated cytosine. Right figures: the expressions of Mst1r, Slpi, and Extl were measured by real-time PCR and normalized to the expression of β-actin or rRNA ( n = 3). Statistical significance was analyzed by one-way ANOVA followed by Turkey-Kramer test as a post hoc comparison. * , ** , *** Significantly different at p < 0.05, 0.01, and 0.001, respectively

Article Snippet: After attaching to the dish, cells were incubated in a culture medium containing 5-aza-dC for 72 h. For overexpression study, mouse ORF clones Mst1r (NM_001287261) and Slpi (NM_011414), and pCMV6-Kan/Neo (pCMV6KN) as an empty vector were purchased from ORIGENE Inc. (MD, USA).

Techniques: DNA Methylation Assay, Cell Culture, Methylation Sequencing, Methylation, Real-time Polymerase Chain Reaction, Expressing, Comparison

Journal: BMC Cancer

Article Title: The DNA methylation profile of liver tumors in C3H mice and identification of differentially methylated regions involved in the regulation of tumorigenic genes

doi: 10.1186/s12885-018-4221-0

Figure Lengend Snippet: Overexpression of Mst1r induces IL33 in Hepa1c1c7 cells. Overexpression of Mst1r ( a ) and upregulation of IL33 by Mst1r overexpression ( b ) in Hepa1c1c7 cells was confirmed by real-time PCR. *** Significantly different at p < 0.001 ( n = 3). ( c ) Confirmation by real-time PCR of upregulation of IL33 in the tumor tissues ( n = 11) compared to the normal tissues ( n = 6) of C3H mice. Statistical significance between the two groups was analyzed by the Student’s t-test. *** Significantly different at p < 0.001

Article Snippet: After attaching to the dish, cells were incubated in a culture medium containing 5-aza-dC for 72 h. For overexpression study, mouse ORF clones Mst1r (NM_001287261) and Slpi (NM_011414), and pCMV6-Kan/Neo (pCMV6KN) as an empty vector were purchased from ORIGENE Inc. (MD, USA).

Techniques: Over Expression, Real-time Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: The DNA methylation profile of liver tumors in C3H mice and identification of differentially methylated regions involved in the regulation of tumorigenic genes

doi: 10.1186/s12885-018-4221-0

Figure Lengend Snippet: Human database searches showed hypomethylation of downstream regions of MST1R and SLPI with upregulation of their expressions. RNA-seq dataset and DNA methylation dataset of 41 paired normal and tumor tissues of human livers were downloaded from TCGA. a , b Average gene expression ratio (tumor tissues/normal tissues) was calculated using 41 paired data from TCGA. * , ** Significantly different at P < 0.05, and 0.01, respectively. c The difference in DNA methylation β-value (tumor – normal) for each CpG around TSS was calculated using 41 paired data from TCGA. d The correlation between expressions of MST1R and IL33 in the HCC tissues highly expressing MST1R

Article Snippet: After attaching to the dish, cells were incubated in a culture medium containing 5-aza-dC for 72 h. For overexpression study, mouse ORF clones Mst1r (NM_001287261) and Slpi (NM_011414), and pCMV6-Kan/Neo (pCMV6KN) as an empty vector were purchased from ORIGENE Inc. (MD, USA).

Techniques: RNA Sequencing, DNA Methylation Assay, Gene Expression, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Crizotinib Has Preclinical Efficacy in Philadelphia-Negative Myeloproliferative Neoplasms.

doi: 10.1158/1078-0432.CCR-22-1763

Figure Lengend Snippet: Figure 4: RON kinase is an important target of crizotinib in MPN (A) Western analysis of RON in SET2 cells treated with crizotinib and stimulated with erythropoietin (EPO), stem cell factor (SCF), and hepatocyte growth factor (HGF). Representative data from one of at least three independent experiments is shown (B) qPCR of MST1R (RON) expression in HEL cells transduced with 4 different MST1R shRNA constructs (C) Colony formation assay of transduced HEL cells in methylcellulose with myeloid growth factors. Experiments were performed at least three times, with similar results. ANOVA with Tukey’s multiple-comparison test was used. (D) Western analysis of RON, STAT3 and STAT5 phosphorylation in shRNA-transduced HEL cells stimulated with EPO, SCF, and HGF. Representative data from one of three independent experiments are shown. (E) Western analysis of JAK2 in shRNA-transduced HEL cells stimulated with EPO, SCF, and HGF (F) Flow cytometry analysis of RON phosphorylation in MPN patient or control donor peripheral blood CD45lo population with ex vivo EPO and SCF stimulation (G) Schematic for JAK2-V617F-GFP retroviral bone marrow transplantation using bone marrow from Stk−/− (RON−/−) mice (H) Peripheral blood analysis of WT (N=8) or Stk−/− (N=9) JAK2-V617F mice over time. WBC= white blood cells; Hb=hemoglobin; PLT=platelets (I) Percentage of GFP in the peripheral blood over time in WT (N=8) or

Article Snippet: Lentiviral Production: Human shRNA plasmid kits for RON (Cat # TL320425) and MET (Cat #TL320418) were purchased from Origene.

Techniques: Western Blot, Expressing, Transduction, shRNA, Construct, Colony Assay, Comparison, Phospho-proteomics, Flow Cytometry, Control, Ex Vivo, Retroviral, Transplantation Assay

Journal: Oncology reports

Article Title: Small interfering RNA-directed targeting of RON alters invasive and oncogenic phenotypes of human hepatocellular carcinoma cells.

doi: 10.3892/or.2011.1435

Figure Lengend Snippet: Figure 1. Knockdown of RON suppresses tumor cell migration and invasion of HCC cells. (A) Effect of RON knockdown on cell migration. The wound healing assay using the siRNA-transfected cells was performed and graphs of cell migration are displayed as relative healing distances (mean ± SE, n=3; *P<0.05). The artificial wound gap in plates of scramble siRNA-transfected chang, HepG2 and Huh7 cells became significantly narrower than that in RON siRNA-transfected cells at 6, 12 and 24 h (*P<0.05). (B) Invasion assay of cells under RON knockdown. The number of invading RON siRNA-transfected chang, HepG2 and Huh7 cells were significantly lower than that of scramble siRNA-transfected cells (mean ± SE, n=6; **P<0.005, *P<0.05). SS, scramble siRNA; RS, RON siRNA.

Article Snippet: RON siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and scramble siRNA (Qiagen, MD, USA) were transfected with lipofectamineTM RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations and then incubated up to 48 h. Cell migration assay.

Techniques: Knockdown, Migration, Wound Healing Assay, Transfection, Invasion Assay

Journal: Oncology reports

Article Title: Small interfering RNA-directed targeting of RON alters invasive and oncogenic phenotypes of human hepatocellular carcinoma cells.

doi: 10.3892/or.2011.1435

Figure Lengend Snippet: Figure 3. RON knockdown-induced apoptosis is associated with the modula tion of apoptotic regulatory proteins in HCC cells. (A) An increase in cleaved caspase-3 and PARP expression was detected in the chang, HepG2 and Huh7 cells after knockdown of RON. (B) RON knockdown led to a decrease of anti- apoptotic proteins, Bcl-2, Bcl-xL and survivin in chang, HepG2 and Huh7 cells. PARP, Poly (ADP-ribose) polymerase; SS, scramble siRNA; RS, RON siRNA.

Article Snippet: RON siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and scramble siRNA (Qiagen, MD, USA) were transfected with lipofectamineTM RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations and then incubated up to 48 h. Cell migration assay.

Techniques: Knockdown, Expressing

Journal: Oncology reports

Article Title: Small interfering RNA-directed targeting of RON alters invasive and oncogenic phenotypes of human hepatocellular carcinoma cells.

doi: 10.3892/or.2011.1435

Figure Lengend Snippet: Figure 2. Knockdown of RON induces apoptosis and cell cycle arrest in HCC cells. (A) The proportion of early apoptotic cells induced by transfection of RON siRNA was greater than that induced by transfection of the scramble siRNA (18.27 vs. 6.47, 16.07 vs. 5.75, and 16.34 vs. 10.77%, respectively) in chang, HepG2 and Huh7 cells. (B) Knockdown of RON induced cell cycle arrest of the G2/M phase in chang and Huh7 cells and G0/G1 phase in HepG2 cells. One representative experiment of the three independent experiments is shown. SS, scramble siRNA; RS, RON siRNA.

Article Snippet: RON siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and scramble siRNA (Qiagen, MD, USA) were transfected with lipofectamineTM RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations and then incubated up to 48 h. Cell migration assay.

Techniques: Knockdown, Transfection

Journal: Oncology reports

Article Title: Small interfering RNA-directed targeting of RON alters invasive and oncogenic phenotypes of human hepatocellular carcinoma cells.

doi: 10.3892/or.2011.1435

Figure Lengend Snippet: Figure 4. Knockdown of RON induces cell cycle arrest through the modula tion of cell cycle regulators in HCC cells. The cyclin D1 and D3 levels were significantly decreased by RON knockdown in all tested cells. The p21 and p27 protein level was significantly increased by RON knockdown in all tested cells. CDK4 protein level was not altered in response to RON knockdown. SS, scramble siRNA; RS, RON siRNA.

Article Snippet: RON siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and scramble siRNA (Qiagen, MD, USA) were transfected with lipofectamineTM RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations and then incubated up to 48 h. Cell migration assay.

Techniques: Knockdown

Journal: Oncology reports

Article Title: Small interfering RNA-directed targeting of RON alters invasive and oncogenic phenotypes of human hepatocellular carcinoma cells.

doi: 10.3892/or.2011.1435

Figure Lengend Snippet: Figure 5. Knockdown of RON decreases the phosphorylation of Akt, c-Raf and ERK signaling proteins in HCC cells. The phosphorylation levels on Ser473 of Akt were decreased by RON knockdown in all tested cells. The phosphorylation levels on Thr308 of Akt were not altered by RON knock down in all tested cells. The phosphorylation level of c-Raf was decreased by RON knockdown in all tested cells. The phosphorylation level of ERK1/2 was decreased by RON knockdown in all tested cells. SS, scramble siRNA; RS, RON siRNA.

Article Snippet: RON siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and scramble siRNA (Qiagen, MD, USA) were transfected with lipofectamineTM RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations and then incubated up to 48 h. Cell migration assay.

Techniques: Knockdown, Phospho-proteomics