Journal: The Journal of biological chemistry

Article Title: Membrane type 4 matrix metalloproteinase (MT4-MMP, MMP-17) is a glycosylphosphatidylinositol-anchored proteinase.

doi: 10.1074/jbc.274.48.34260

Figure Lengend Snippet: FIG. 6. Co-localization of MT4-MMP and uPAR on the cell surface of CHO-K1 cells. CHO-K1 cells were co- transfected with the expression vectors for hMT4-F1 or hMT1-F1 and uPAR or vector alone (Mock). After 48 h, cells were fixed and stained with anti-FLAG M1 and rabbit anti-uPAR antibodies. Cy3-labeled anti-mouse IgG antibody (orange) and Al- exa488-labeled anti-rabbit IgG antibody (green) were used to detect M1 and anti- uPAR antibody signals, respectively. Cells were analyzed by confocal laser mi- croscopy (3 180).

Article Snippet: Construction of the Expression Vector for Human uPAR—The fulllength human uPAR cDNA clone was obtained from ATCC (ATCC number 65768).

Techniques: Transfection, Expressing, Plasmid Preparation, Staining, Labeling

Journal: Scientific Reports

Article Title: HOXA10 promotes Gdf5 expression in articular chondrocytes

doi: 10.1038/s41598-023-50318-7

Figure Lengend Snippet: Role of HOXA10 in Gdf5 expression in articular cartilage cells. ( A ) SFZ cells isolated from WT mice were infected with empty (control) or indicated lentiviruses. Indicated gene expression was analyzed by RT-qPCR in duplicate. ( B ) Schematic diagram of Gdf5 -HiBiT screening system. ( C ) SFZ cells were isolated from Gdf5- HiBiT KI mice and were plated on a 96-well plate. Gdf5- HiBiT KI SFZ cells were infected with the indicated lentiviruses. One day later, the medium was changed. The cells were cultured for 2 days and then the supernatants were collected. The supernatants were subjected to HiBiT measurement (n = 3). RLU: relative light unit. ( D ) SFZ cells isolated from WT mice were infected with empty (control) or FLAG- Hoxa10 lentiviruses. Hoxa10 , Gdf5 , and Prg4 expression was analyzed by RT-qPCR (n = 3). ( E ) SFZ cells isolated from WT mice were infected with empty (control), shHoxa10-1, or shHoxa10-2 lentiviruses. Hoxa10 , Gdf5 , and Prg4 expression was analyzed by RT-qPCR (n = 3). Data are the mean ± SEM (*: P < 0.05, ****: P < 0.0001).

Article Snippet: For knockdown experiments, lentiviral pLKO.1 plasmids targeting Hoxa10 (Hoxa10 shRNA-1 target sequence, 5′-ATCCTTCATTCACCTTTGAG-3′ and Hoxa10 shRNA-2 target sequence, 5′-AAGCAAATGCATTCTATCGTT-3′, were generated in accordance with the manufacturer’s protocol (Addgene, Watertown, MA, USA).

Techniques: Expressing, Isolation, Infection, Control, Gene Expression, Quantitative RT-PCR, Cell Culture

Journal: Scientific Reports

Article Title: HOXA10 promotes Gdf5 expression in articular chondrocytes

doi: 10.1038/s41598-023-50318-7

Figure Lengend Snippet: Role of HOXA10 in Gdf5 expression in LB cells. ( A ) LB cells were isolated from Gdf5- HiBiT KI mice and plated on a 96-well plate. The cells were infected with empty (control), Venus , or FLAG- Hoxa10 lentiviruses. One day after infection, the medium was changed. The cells were cultured for 2 days and then the supernatants were collected. The supernatants were subjected to HiBiT measurement (n = 3). RLU: relative light unit. ( B ) LB cells isolated from WT mice were infected with empty (control), Venus , or FLAG- Hoxa10 lentiviruses. Hoxa10 , Gdf5 , and Prg4 expression was analyzed by RT-qPCR (n = 3). Data are the mean ± SEM (*: P < 0.05, **: P < 0.01).

Article Snippet: For knockdown experiments, lentiviral pLKO.1 plasmids targeting Hoxa10 (Hoxa10 shRNA-1 target sequence, 5′-ATCCTTCATTCACCTTTGAG-3′ and Hoxa10 shRNA-2 target sequence, 5′-AAGCAAATGCATTCTATCGTT-3′, were generated in accordance with the manufacturer’s protocol (Addgene, Watertown, MA, USA).

Techniques: Expressing, Isolation, Infection, Control, Cell Culture, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: HOXA10 promotes Gdf5 expression in articular chondrocytes

doi: 10.1038/s41598-023-50318-7

Figure Lengend Snippet: Effect of HOXA10 on the Gdf5 gene promoter. ( A ) The ATAC-Seq database (articular chondrocytes: SRX13791211, costal chondrocytes: SRX11156876) from ChIP-Atlas was analyzed by integrative genomics viewer IGV2.8.6. A Gdf5 gene promoter (1393 bp) exhibits an open chromatin region on the Gdf5 gene in articular chondrocytes. ( B ) Schematic diagram of luciferase reporter construction with mouse Gdf5 gene promoter (− 1081 to + 312). A putative HOXA10 binding motif (− 538 to − 529) is shown with reference to previous study . ( C ) HEK293T cells were transfected with empty or FLAG- Hoxa10 plasmids as well as luciferase reporter plasmids with mouse Gdf5 gene promoter. Cell lysates were subjected to luciferase measurement (n = 4). RLU: relative light unit. ( D ) SFZ cells isolated from WT mice were infected with empty (control) or FLAG- Hoxa10 lentiviruses. Gdf5 gene promoter fragments collected by ChIP using anti-FLAG antibody were analyzed by real-time qPCR (n = 3). Data are the mean ± SEM (*: P < 0.05, ****: P < 0.0001).

Article Snippet: For knockdown experiments, lentiviral pLKO.1 plasmids targeting Hoxa10 (Hoxa10 shRNA-1 target sequence, 5′-ATCCTTCATTCACCTTTGAG-3′ and Hoxa10 shRNA-2 target sequence, 5′-AAGCAAATGCATTCTATCGTT-3′, were generated in accordance with the manufacturer’s protocol (Addgene, Watertown, MA, USA).

Techniques: Luciferase, Binding Assay, Transfection, Isolation, Infection, Control

Journal: Scientific Reports

Article Title: HOXA10 promotes Gdf5 expression in articular chondrocytes

doi: 10.1038/s41598-023-50318-7

Figure Lengend Snippet: Immunofluorescent analysis of HOXA10 and GDF5 in articular cartilage. Tibial sections from 3-month-old mice were subjected to co-immunostaining with anti-HOXA10 and anti-GDF5 antibodies. DAPI indicates nucleus. Scale bar, 50 μm.

Article Snippet: For knockdown experiments, lentiviral pLKO.1 plasmids targeting Hoxa10 (Hoxa10 shRNA-1 target sequence, 5′-ATCCTTCATTCACCTTTGAG-3′ and Hoxa10 shRNA-2 target sequence, 5′-AAGCAAATGCATTCTATCGTT-3′, were generated in accordance with the manufacturer’s protocol (Addgene, Watertown, MA, USA).

Techniques: Immunostaining

Journal: Scientific Reports

Article Title: HOXA10 promotes Gdf5 expression in articular chondrocytes

doi: 10.1038/s41598-023-50318-7

Figure Lengend Snippet: List of transcription factor genes predominantly expressed in articular chondrocytes by microarray analysis.

Article Snippet: For knockdown experiments, lentiviral pLKO.1 plasmids targeting Hoxa10 (Hoxa10 shRNA-1 target sequence, 5′-ATCCTTCATTCACCTTTGAG-3′ and Hoxa10 shRNA-2 target sequence, 5′-AAGCAAATGCATTCTATCGTT-3′, were generated in accordance with the manufacturer’s protocol (Addgene, Watertown, MA, USA).

Techniques: Microarray

Journal:

Article Title: The von Hippel-Lindau Tumor Suppressor Gene Inhibits Hepatocyte Growth Factor/Scatter Factor-Induced Invasion and Branching Morphogenesis in Renal Carcinoma Cells

doi:

Figure Lengend Snippet: uPA and uPAR induction in 786-0 RCC cells. Subconfluent cultures of cells were incubated for 7 h at 37°C in DMEM–10% FBS supplemented with 200 ng of HGF/SF per ml or unsupplemented. Total RNA extraction and Northern hybridization were performed as described in Materials and Methods. The blots were probed or reprobed with a 32P-labeled human uPA probe, a human uPAR probe, and a human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe (52). Mut, VHL-negative 786-0 RCC cells; WT, VHL-positive 786-0 RCC cells.

Article Snippet: The probes included a uPA probe isolated as a 1.5-kb fragment (ATCC 57328), a uPAR probe isolated as a 1.1-kb fragment (ATCC 65768), a 1-kb Pst I fragment of pFos-1 ( 2 ), and the human cDNA probes for TIMP-1 (ATCC 59666), TIMP-2 (ATCC 79069), MMP-2 (ATCC 79067), and MMP-9 (ATCC 1196364).

Techniques: Incubation, RNA Extraction, Northern Blot, Hybridization, Labeling