LINC00599 role in human PASMC proliferation

Phase 1: Cell Culture and Validation -- Days 1-7
Human PASMCs (Lonza or ScienCell) cultured in SmGM-2 medium with 5% FBS, 1% P/S at 37°C, 5% CO2. Validate cell identity by immunofluorescence staining for smooth muscle markers (α-SMA, SM22α, calponin). Use cells between passages 3-6. Power calculation: n=6 wells per condition for 80% power to detect 20% difference in proliferation with α=0.05.

Phase 2: siRNA Knockdown and Overexpression -- Days 8-12
Transfect PASMCs at 70% confluency using Lipofectamine RNAiMAX. siRNA conditions: scrambled control (50 nM), LINC00599 siRNA pool (50 nM, 3 sequences). Overexpression: pcDNA3.1-LINC00599 (2 μg/well) or empty vector control using Lipofectamine 3000. Validate knockdown/overexpression at 48h by RT-qPCR using TaqMan probes.

Phase 1: Cell Culture and Validation -- Days 1-7
Human PASMCs (Lonza or ScienCell) cultured in SmGM-2 medium with 5% FBS, 1% P/S at 37°C, 5% CO2. Validate cell identity by immunofluorescence staining for smooth muscle markers (α-SMA, SM22α, calponin). Use cells between passages 3-6. Power calculation: n=6 wells per condition for 80% power to detect 20% difference in proliferation with α=0.05.

Phase 2: siRNA Knockdown and Overexpression -- Days 8-12
Transfect PASMCs at 70% confluency using Lipofectamine RNAiMAX. siRNA conditions: scrambled control (50 nM), LINC00599 siRNA pool (50 nM, 3 sequences). Overexpression: pcDNA3.1-LINC00599 (2 μg/well) or empty vector control using Lipofectamine 3000. Validate knockdown/overexpression at 48h by RT-qPCR using TaqMan probes.

Phase 3: Proliferation and Viability Assays -- Days 10-15
Proliferation measured by: (1) BrdU incorporation assay (10 μM, 4h pulse) quantified by colorimetric ELISA, (2) Cell counting using hemocytometer at 24, 48, 72h post-transfection, (3) MTT assay for metabolic activity. Cell cycle analysis by propidium iodide staining and flow cytometry. Apoptosis assessment by Annexin V/PI dual staining.

Phase 4: Stress Granule Formation Analysis -- Days 13-16
Stress granule induction by sodium arsenite (0.5 mM, 30 min) or heat shock (43°C, 45 min). Immunofluorescence staining for stress granule markers: G3BP1 (1:500, Cell Signaling), PABP (1:300, Abcam), and TIA-1 (1:400, Santa Cruz). Quantify stress granule number, size, and colocalization with LINC00599 by FISH using custom probes. Confocal microscopy with 63x objective.

Phase 5: m6A Modification and Phase Separation Studies -- Days 17-21
RNA immunoprecipitation (RIP) using m6A antibody (Synaptic Systems, 1:100) to assess LINC00599 m6A levels. MeRIP-qPCR validation with specific primers spanning predicted m6A sites. Liquid-liquid phase separation assay: recombinant MYH9 protein (2 μM) mixed with in vitro transcribed LINC00599 RNA (1 μM) in phase separation buffer (50 mM Tris pH 7.5, 150 mM KCl, 10% PEG-8000). Microscopy analysis of droplet formation and FRAP recovery kinetics.

Phase 6: Protein Interaction and Pathway Analysis -- Days 22-28
RNA pulldown assay using biotinylated LINC00599 followed by mass spectrometry to identify interacting proteins. Validate MYH9 interaction by RNA-IP and Western blot (MYH9 antibody 1:1000, Abcam). Cell cycle gene expression by RT-qPCR array (84 genes). Western blot analysis of key cell cycle proteins: cyclin D1, CDK4, p21, p27 (1:1000 dilutions, overnight 4°C).