Journal: Aging Cell

Article Title: Single‐cell RNA sequencing reveals the CRTAC1 + population actively contributes to the pathogenesis of spinal ligament degeneration by SPP1 + macrophage

doi: 10.1111/acel.14320

Figure Lengend Snippet: Characterization of ligament immune compartment and possible pathogenic role of SPP1 + macrophages. (a) UMAP plot of immune cells in spinal ligament tissues. (b) Inflammation scores of the main immune cell lineages in ligament. (c) UMAP plot of monocytes and macrophages in spinal ligament tissues. (d) Hierarchical clustering of DEGs in monocytes and macrophages. (e) Pseudo‐time analysis of monocyte and macrophage subpopulations. (f) Pathway enrichment analysis for DEGs of macrophages from ligament tissues. (g) Ligand–receptor interactions between macrophages and other cell types. (h–j) Quantitative PCR assays (h) and western blot analysis (i, j) of control cultured primary ligament cells and those treated with short interfering RNA targeting ATF3 and recombinant SPP1. *, p ‐value < 0.05; **, p ‐value < 0.01; ***, p ‐value < 0.001.

Article Snippet: For recombinant protein stimulation experiments, primary human ligament cells from patients with traumatic injury were incubated with 200 ng/mL recombinant SPP1 protein (1433‐OP‐050, R&D Systems, USA).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Control, Cell Culture, Small Interfering RNA, Recombinant

Journal: The Journal of Clinical Investigation

Article Title: EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer

doi: 10.1172/JCI165863

Figure Lengend Snippet: ( A ) Single-channel and merged confocal micrographs of total and surface VSV-G in H1299 cells cotransfected with siRNAs and EGFP–VSV-G and imaged 30 minutes after transfer to the permissive temperature. Plot shows the ratio of surface VSV-G to total VSV-G in each cell (dot) 30 or 60 minutes after transfer to 32°C. Scale bar: 20 μm. ( B ) BRET measurement of PI4P in RAB6A + vesicles in siRNA-transfected H1299 cells. Results are expressed as a ratio of the values from GSK-A1–treated and vehicle-treated (DMSO) cells at each time point ( n = 5 replicates per group). ( C ) Confocal micrographs of RAB6A + vesicles (blue arrows) and unfissioned RAB6A + tubules (red arrows) emerging from the Golgi. Scale bar: 20 μm. Dotted lines indicate the cell boundaries. Results were quantified per cell (dot plots). ( D ) Venn diagram of PI42KA-interacting proteins identified by TurboID and IP approaches. Overlapping proteins are listed on the right. Reported PI4K2A-interacting proteins ( , ) are shown in bold. ( E ) Schematic illustration of full-length and truncated PI4K2A constructs. WB assays on whole-cell lysates (WCLs) (input) or IP proteins isolated from H1299 cells transfected with MYC-tagged PI4K2A constructs (gel). Full-length (1–479) and truncated constructs are indicated under the gels. IgG was used as the control IP. ( F ) WB analysis of WCLs (WCL) and Golgi-enriched fractions (Golgi) from siRNA-transfected H1299 cells. ( G ) Confocal micrographs of SPP1 + vesicles (arrows) in siRNA-transfected H1299 cells costained with anti-SPP1 and anti–Golgin 97 antibodies. Scale bars: 50 μm. Dot plot shows the vesicle numbers per cell. ( H ) WB analysis of WCLs or enriched subcellular fractions from siRNA-transfected H1299 cells. Densitometric values were normalized to siCTL (graph). Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. *** P < 0.001, by 2-tailed Student’s t test for 2-group comparisons ( B ); 1-way ANOVA test for multiple comparisons ( A , C , G , and H ).

Article Snippet: 3724 and 2367), β-actin (no. 4970), GM130 (no. 12480), Golgin-97 (no. 13192), AXL (no. 8661), and EGFR (no. 2256) from Cell Signaling Technology; antibodies against ACBD3 (sc-101277), ZEB1 (sc-25388), and PI4K2A (sc-390026) from Santa Cruz Biotechnology; antibodies against SPP1 (22952-1-AP), SEMA7A (18070-1-AP), HSP90 (11405-1-AP), ZFP36L1 (12306-1-AP), and CD44 (15675-1-AP) from Proteintech; antibodies against ITGB1 (GTX636657) from GeneTex; antibodies against RNA polymerase II (AB_2732926) from Active Motif; recombinant SPP1 proteins (ab92964) from Abcam; recombinant Gas6 (885-GSB) and SPP1 (1433-OP-050) proteins from R&D Systems; PI-273 (HY-103489) from MedChemExpress; NC03 (AOB17420) from Aobious; and miRNA mimics (HMI0508, HMI0275, and HMI0280) and control mimics (HMC0002) from MilliporeSigma.

Techniques: Transfection, Construct, Isolation, Control

Journal: The Journal of Clinical Investigation

Article Title: EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer

doi: 10.1172/JCI165863

Figure Lengend Snippet: ( A ) Intracellular levels of biotinylated Tfn in siRNA-transfected H1299 cells were quantified at the indicated time points after a 30-minute pulse. The percentage of the internalized Tfn pool was calculated relative to the initial loading ( n = 4 samples per condition). ( B ) WB analysis of CD44, ITGB1, and AXL protein levels in WCLs (input) or streptavidin bead–enriched protein samples from H1299 cells that were transfected with PI4K2A/TurboID construct and treated with biotin. Controls included the PI4KB/TurboID construct (PI4KB) and Turbo alone (CTL). ( C ) WB analysis of WCLs (input) or anti-HA immunoprecipitates from H1299 cells transfected with HA-tagged PI4K2A. IgG was used as the control IP. ( D ) SPP1 protein-protein interaction network (STRING-db.org). ( E ) WB analysis of proteins isolated by streptavidin bead–based pulldowns carried out on H1299 cells treated with biotin-labeled recombinant SPP1. ( F ) Confocal micrographs of plasma membrane–bound ITGB1 (arrows, upper panels) and CD44 (arrows, lower panels) in nonpermeabilized siRNA-transfected H1299 cells stained with antibodies against endogenous ITGB1 or CD44. Scale bars: 10 μm. ( G ) WB analysis of cell membrane–enriched fractions (Mem.) and WCL. Densitometric values are shown under the gels. ( H ) WB analysis of cleaved PARP1 (gel) and flow cytometric analysis of annexin V/PI–stained cells (graph) to quantify apoptosis in siRNA-transfected H1299 cells. ( I ) Boyden chamber migration and invasion assays on siRNA-transfected cells. ( J ) Schematic illustration of the working model. PI4K2A coordinates exocytic and endocytic vesicular trafficking to activate an SPP1-dependent autocrine loop. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. P values were determined by 1-way ANOVA test for multiple comparisons ( A , H , and I ).

Article Snippet: 3724 and 2367), β-actin (no. 4970), GM130 (no. 12480), Golgin-97 (no. 13192), AXL (no. 8661), and EGFR (no. 2256) from Cell Signaling Technology; antibodies against ACBD3 (sc-101277), ZEB1 (sc-25388), and PI4K2A (sc-390026) from Santa Cruz Biotechnology; antibodies against SPP1 (22952-1-AP), SEMA7A (18070-1-AP), HSP90 (11405-1-AP), ZFP36L1 (12306-1-AP), and CD44 (15675-1-AP) from Proteintech; antibodies against ITGB1 (GTX636657) from GeneTex; antibodies against RNA polymerase II (AB_2732926) from Active Motif; recombinant SPP1 proteins (ab92964) from Abcam; recombinant Gas6 (885-GSB) and SPP1 (1433-OP-050) proteins from R&D Systems; PI-273 (HY-103489) from MedChemExpress; NC03 (AOB17420) from Aobious; and miRNA mimics (HMI0508, HMI0275, and HMI0280) and control mimics (HMC0002) from MilliporeSigma.

Techniques: Transfection, Construct, Control, Isolation, Labeling, Recombinant, Membrane, Staining, Migration