Journal: PLoS ONE

Article Title: Role of IL13RA2 in Sunitinib Resistance in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0130980

Figure Lengend Snippet: mRNA changes after sunitinib treatment in KURC1 and KURC2 xenograft tumors.

Article Snippet: Antibodies were purchased commercially as follows: human IL13RA2 (MAB614 for immunoblotting and AF146 for immunohistochemistry, R&D Systems, Minneapolis, MN), human STAT6, human phospho-STAT6 Y641 (Cell Signaling Technology, Beverly, MA), human ssDNA (IBL, Tokyo, Japan), human β-actin, human N-cadherin and mouse CD31 (Abcam, Cambridge, MA).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Role of IL13RA2 in Sunitinib Resistance in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0130980

Figure Lengend Snippet: (A) Evaluation of IL13RA2 mRNA expression in KURC1 and KURC2 xenograft tumors treated with sunitinib or vehicle by qPCR. All samples were prepared in triplicate and data are presented as the mean ± SE from indicated number of samples. Columns, mean; bar, SE. The difference in the mRNA expression levels between the sunitinib-treated group and control or sensitive group in KURC1 was statistically significant (* P < 0.01; Students’ t -test). There was no significant difference in KURC2 groups. (B) Immunohistochemical staining of IL13RA2 in KURC1 xenograft tumors. Scale bar, 50 μm. (C) IL13RA2 expression in human ccRCC tumors with the response to sunitinib treatment evaluated by immunohistochemistry. ccRCC tumor samples were collected from patients prior to sunitinib treatment. Left: representative pictures of immunohistochemistry sections of tumors showing none, weak, or strong staining for IL13RA2. Right: ratio of IL13RA2 expression pattern and correlation of the response to sunitinib treatment. Scale bar, 100 μm.

Article Snippet: Antibodies were purchased commercially as follows: human IL13RA2 (MAB614 for immunoblotting and AF146 for immunohistochemistry, R&D Systems, Minneapolis, MN), human STAT6, human phospho-STAT6 Y641 (Cell Signaling Technology, Beverly, MA), human ssDNA (IBL, Tokyo, Japan), human β-actin, human N-cadherin and mouse CD31 (Abcam, Cambridge, MA).

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

Journal: PLoS ONE

Article Title: Role of IL13RA2 in Sunitinib Resistance in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0130980

Figure Lengend Snippet: (A) Immunoblot analysis of 786-O subclones infected with retrovirus encoding mock or WT IL13RA2. Whole cell extracts were immunoblotted using the indicated antibodies. Sequential changes in subcutaneous xenograft tumors from 786-O subclones infected with (B) mock or (C) WT IL13RA2 treated with sunitinib and vehicle (control). Each time point represents the mean ± SE of tumor volume in each group. The difference in tumor size between the treatment group and control was statistically significant in 786-O-mock cells but not statistically significant in 786-O-IL13RA2 cells (* P < 0.05, n.s.: not significant; two-way repeated ANOVA). The horizontal arrow bars indicate the periods of sunitinib administration. (D) Immunoblot analysis of Caki-1 subclones infected with lentivirus encoding scrambled or IL13RA2 shRNA. Whole cell extracts were immunoblotted using the indicated antibodies. Sequential changes of subcutaneous xenograft tumors from a Caki-1 subclone infected with (E) scrambled or (F) IL13RA2 shRNA treated with sunitinib and vehicle (control). Each time point represents the mean ± SE of tumor volume in each group. Day 0 is the first day of sunitinib administration 4 weeks after transplantation. The difference in tumor size between the treatment group and control was not significant in Caki-1-sh-scrambled cells but statistically significant in Caki-1-sh-IL13RA2 cells (n.s.: not significant, * P < 0.05; two-way repeated ANOVA). The arrow bars indicate the period of sunitinib administration.

Article Snippet: Antibodies were purchased commercially as follows: human IL13RA2 (MAB614 for immunoblotting and AF146 for immunohistochemistry, R&D Systems, Minneapolis, MN), human STAT6, human phospho-STAT6 Y641 (Cell Signaling Technology, Beverly, MA), human ssDNA (IBL, Tokyo, Japan), human β-actin, human N-cadherin and mouse CD31 (Abcam, Cambridge, MA).

Techniques: Western Blot, Infection, Control, shRNA, Transplantation Assay

Journal: PLoS ONE

Article Title: Role of IL13RA2 in Sunitinib Resistance in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0130980

Figure Lengend Snippet: MVD was decreased by sunitinib treatment of each xenograft tumor derived from (A) 786-O or (B) Caki-1 subclones regardless of IL13RA2 expression level. MVD was determined from CD31 staining using Image J software. Statistical analysis was performed using the Students’ t -test (* P < 0.01). Immunoblot analysis of (C) 786-O subclones and (D) Caki-1 subclones. IL13RA2 expression was negatively correlated with the phosphorylation of STAT6. Whole cell extracts were immunoblotted using the indicated antibodies. ssDNA staining of xenograft tumors derived from (E) 786-O subclones and (B) Caki-1 subclones treated with sunitinib or vehicle only. Apoptosis was assessed by calculating the ssDNA positivity rate. Statistical analysis was performed using the Students’ t -test (* P < 0.05, ** P < 0.01).

Article Snippet: Antibodies were purchased commercially as follows: human IL13RA2 (MAB614 for immunoblotting and AF146 for immunohistochemistry, R&D Systems, Minneapolis, MN), human STAT6, human phospho-STAT6 Y641 (Cell Signaling Technology, Beverly, MA), human ssDNA (IBL, Tokyo, Japan), human β-actin, human N-cadherin and mouse CD31 (Abcam, Cambridge, MA).

Techniques: Derivative Assay, Expressing, Staining, Software, Western Blot, Phospho-proteomics

Journal: Biomaterials Research

Article Title: Insulin-Like Growth Factor 2 Secreted from Mesenchymal Stem Cells with High Glutathione Levels Alleviates Osteoarthritis via Paracrine Rejuvenation of Senescent Chondrocytes

doi: 10.34133/bmr.0152

Figure Lengend Snippet: Intra-articular injection of primed MSCs suppresses OA progression without evidence of direct regeneration. (A) Representative histological analysis using Safranin-O and immunohistochemistry (IHC) (collagen type II, MMP13, collagen type X, DPP4, and human β2-microglobulin) in sham control and DMM-induced rabbits injected with PBS and naïve or primed MSCs. (B) Scoring of OA using OARSI grading system in sham control and DMM-induced OA rabbits that had undergone intra-articular injection of PBS and naïve or primed MSCs. (C to F) Quantification of collagen type II, MMP13, collagen type X, and DPP4 using scoring system. (G) Representative histological analysis using Safranin-O and DPP4 IHC staining in DMM-induced OA rabbits injected with PBS, primed MSCs, low-dose, mid-dose, or high-dose secretome, or triple high-dose secretome isolated from primed MSCs. (H and I) Scoring of OA using OARSI grading system and quantification of DPP4. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Sections were then incubated with primary antibodies against collagen type II (Thermo Fisher Scientific, MA1-37493), MMP13 (R&D Systems, MAB511), collagen type X (Abcam, ab49945), DPP4 (Thermo Fisher Scientific, MA2607), and human β2 microglobulin (Abcam, ab759) diluted in 4% bovine serum albumin (BSA; Biosesang, AC1025-100-00) for 1 h at 37 °C.

Techniques: Injection, Immunohistochemistry, Control, Isolation

Journal: Biomaterials Research

Article Title: Insulin-Like Growth Factor 2 Secreted from Mesenchymal Stem Cells with High Glutathione Levels Alleviates Osteoarthritis via Paracrine Rejuvenation of Senescent Chondrocytes

doi: 10.34133/bmr.0152

Figure Lengend Snippet: Paracrine activity of primed MSC suppresses senescence phenotypes of OA chondrocytes. (A) Left: Representative immunofluorescence staining of p16 and p21 (red) in OA chondrocytes (OA), OA + naïve MSC media, OA + MSC priming media, and direct coculture of OA and naïve or primed MSCs. OA chondrocytes were distinguished with CellTracker Dil staining (green), and nuclei were counterstained with DAPI (blue). Right: Bar graphs showing quantification of p16 + OA chondrocytes (top) and quantification of p21 + OA chondrocytes ( n = 3 per group) (bottom). (B) Schematic illustration of the experiments. (C) mRNA levels of p16 and p21 in OA, OA + naïve CM, and OA + primed CM measured by RT-qPCR. (D) Representative SA-β-Gal staining and quantification for OA, OA + naïve CM, and OA + primed CM ( n = 3 per group). (E) Cell counts of OA, OA + naïve CM, and OA + primed CM for 14 d to evaluate proliferation ( n = 3 per group). (F) mRNA expression of matrix degradation factors (ADAMTS5 and MMP13) in OA, OA + naïve CM, and OA + primed CM ( n = 3 per group). (G) Schematic illustration of indirect coculture system using transwell. (H) mRNA levels of p16 and p21 in OA, OA + naïve MSCs, and OA + primed MSCs measured by RT-qPCR. (I) Representative SA-β-Gal staining and quantification for OA, OA + naïve MSCs, and OA + primed MSCs ( n = 3 per group). (J) Cell counts of OA, OA + naïve MSCs, and OA + primed MSCs for 14 d to evaluate proliferation ( n = 3 per group). (K) mRNA expression of matrix degradation factors (ADAMTS5 and MMP13) in OA, OA + naïve MSCs, and OA + primed MSCs ( n = 3 per group). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Sections were then incubated with primary antibodies against collagen type II (Thermo Fisher Scientific, MA1-37493), MMP13 (R&D Systems, MAB511), collagen type X (Abcam, ab49945), DPP4 (Thermo Fisher Scientific, MA2607), and human β2 microglobulin (Abcam, ab759) diluted in 4% bovine serum albumin (BSA; Biosesang, AC1025-100-00) for 1 h at 37 °C.

Techniques: Activity Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing

Journal: Biomaterials Research

Article Title: Insulin-Like Growth Factor 2 Secreted from Mesenchymal Stem Cells with High Glutathione Levels Alleviates Osteoarthritis via Paracrine Rejuvenation of Senescent Chondrocytes

doi: 10.34133/bmr.0152

Figure Lengend Snippet: Knockdown of IGF2 expression in primed MSCs using siRNA inhibits therapeutic efficacy in vivo. (A) Representative histological analysis using Safranin-O and IHC (collagen type II, MMP13, collagen type X, and DPP4) in sham control and DMM-induced rabbits injected with PBS, primed MSCs, or primed MSCs with IGF2 knockdown (IGF2KD MSCs). (B) Scoring of OA using OARSI grading system in sham control and DMM-induced OA rabbits that had undergone intra-articular injection of PBS, primed MSCs, or IGF2KD MSCs. (C to F) Quantification of collagen type II, MMP13, collagen type X, and DPP4 using scoring system. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Sections were then incubated with primary antibodies against collagen type II (Thermo Fisher Scientific, MA1-37493), MMP13 (R&D Systems, MAB511), collagen type X (Abcam, ab49945), DPP4 (Thermo Fisher Scientific, MA2607), and human β2 microglobulin (Abcam, ab759) diluted in 4% bovine serum albumin (BSA; Biosesang, AC1025-100-00) for 1 h at 37 °C.

Techniques: Knockdown, Expressing, Drug discovery, In Vivo, Control, Injection

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: a Western blot analysis and semiquantitative evaluation of DLL4, VEGFA, and CD31 expression in PDX mice tissues by densitometry analysis of protein bands reveals a downregulation of DLL4, VEGFA, and CD31 protein expression in PDX mice treated with GSI. The bands were measured compared with the housekeeping GAPDH protein band, for each tissue. Average value of DLL4, VEGFA, and CD31 expression levels among all mouse treated with LY3039478 or vehicle is reported in the graph. P value showed versus vehicle treatment. Tissues PDX mice n = 10 for vehicle treatment in gray, n = 10 for LY3039478 treatment in black. b Representative images with immunofluorescence staining show DLL4 and CD31 downregulation in representative images of PDX tissues treated with LY30349478. DLL4 (green) and CD31 (red) and overlapping staining (yellow) were immunolocalized in PDX tissues. The yellow arrows highlight the detail of the co-localization of DLL4 and CD31 in PDX tissues (#4, #14, #24) not treated with LY339478. DAPI, 4′,6‐diamidino‐2‐phenylindole. c Immunofluorescence staining with MMP13 in red and nucleus in DAPI shown a significantly reduction of MMP13 in iCCA PDX tissues treated with LY3039478. Magnifications: ×20; inset ×60. d Representative images demonstrate a significant ( P < 0.001) destruction of the network created by the HUVECs following the treatment with LY3039478 (1 µM). The concomitant administration of MMP13 counteracts significantly ( P < 0.01) drug effectiveness.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: a Analysis of 31 primary tumors from iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database (GSE107943). Mean expression data were expressed in RPKM (Reads Per Kilobase Million). *** P < 0.001 calculated with Student’s t test. b NOTCH1 gene and its pro-angiogenic targets are overexpressed in human intrahepatic cholangiocarcinoma (iCCA). Levels of NOTCH1, DLL4, VEGFA, and MMP13 mRNA were significantly more elevated in iCCA ( n = 42) than corresponding nontumorous surrounding livers (SL; n = 42), as detected by quantitative reverse-transcription PCR. Number target (NT) = 2 −ΔCt , wherein ΔCt value of each sample was calculated by subtracting the average Ct value of the gene of interest from the average Ct value of the β-actin gene. Mann–Whitney test: vs SL, P < 0.0001. c Expression of the NOTCH1 gene correlates with mRNA levels of putative target genes (HES1, DLL4, VEGFA, and MMP13) in a collection of human intrahepatic cholangiocarcinoma (CCA) samples ( n = 42). Linear regression analysis was used. d Representative expression patterns of CK19, NOTCH1, HES1, DDL4, and MMP13 in human intrahepatic cholangiocarcinoma (iCCA) as detected by immunohistochemistry. Upper panels: CCA case (CCA1) showing strong, concomitant immunoreactivity for NOTCH1, HES1, DDL4, and MMP13. Lower panels: CCA specimens (CCA2) exhibiting low levels of NOTCH1, HES1, DDL4, and MMP13. As expected, both iCCA display robust immunolabeling for CK19 (a biliary marker). Magnification: ×200; scale bar = 100 μm. H&E hematoxylin and eosin staining.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Expressing, Reverse Transcription, MANN-WHITNEY, Immunohistochemistry, Immunolabeling, Marker, Staining

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: Levels of tumor microvessel density (MVD) correlate with mRNA expression of NOTCH1 ( a ), HES1 ( b ), DLL4 ( c ), and MMP13 ( e ), but not with those of VEGFA ( d ), in a collection of human intrahepatic cholangiocarcinoma (iCCA) samples ( n = 42). Linear regression analysis was used. f Representative examples of human iCCA specimens with high and low MVD.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Expressing

Journal: Journal of Biological Chemistry

Article Title: Modulation of matrix metabolism by ATP-citrate lyase in articular chondrocytes

doi: 10.1074/jbc.ra118.002261

Figure Lengend Snippet: Figure 5. Inhibition of ACLY by HCA attenuated IL-1β-induced augmentation of acetylation H3K9 and H3K27 both globally and specifically in the iNOS, MMP3 and MMP13 promoters.

Article Snippet: Assays of nitric oxide (NO), MMP3, and MMP13 release, and SOX9 acetylation, distribution, and expression Conditioned media were tested for NO generation by Griess reaction assay to quantify nitrite, and MMP3 and MMP13 release measured using Total MMP3 and MMP13 Quantikine ELISA Kits (R&D Systems).

Techniques: Inhibition