Journal: Cell

Article Title: A membrane-less organelle associated with the endoplasmic reticulum enables 3′UTR-mediated protein-protein interactions

doi: 10.1016/j.cell.2018.10.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pLKO.1-shRNA2 Control (scramble) , Sarbassov et al., 2005 , Addgene, Cat# 1864.

Techniques: Recombinant, Blocking Assay, Protease Inhibitor, Reverse Transcription, Mutagenesis, Plasmid Preparation, Control, Luciferase, shRNA, Software

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