DEX effects on K2P channel transcriptome in mouse TM cells

Phase 1: Cell Culture and Characterization -- Days 1-5
Mouse trabecular meshwork cells isolated from C57BL/6 mice (8-12 weeks old) using established protocols. Culture in DMEM with 10% FBS, 1% P/S at 37°C, 5% CO2. Validate TM cell identity by immunostaining for myocilin, α-SMA, and fibronectin. Use cells between passages 2-4. Power calculation: n=6 wells per condition for 80% power to detect 2-fold expression changes with α=0.05.

Phase 2: Dexamethasone Treatment Protocol -- Days 6-9
Seed cells at 80% confluency in 6-well plates. Treatment groups: (1) Vehicle control (0.1% ethanol), (2) DEX 100 nM, (3) DEX 1 μM. Fresh medium with treatments replaced every 48h. Harvest at 48h and 72h timepoints. Cell viability assessed by trypan blue exclusion (>95% required).

Phase 1: Cell Culture and Characterization -- Days 1-5
Mouse trabecular meshwork cells isolated from C57BL/6 mice (8-12 weeks old) using established protocols. Culture in DMEM with 10% FBS, 1% P/S at 37°C, 5% CO2. Validate TM cell identity by immunostaining for myocilin, α-SMA, and fibronectin. Use cells between passages 2-4. Power calculation: n=6 wells per condition for 80% power to detect 2-fold expression changes with α=0.05.

Phase 2: Dexamethasone Treatment Protocol -- Days 6-9
Seed cells at 80% confluency in 6-well plates. Treatment groups: (1) Vehicle control (0.1% ethanol), (2) DEX 100 nM, (3) DEX 1 μM. Fresh medium with treatments replaced every 48h. Harvest at 48h and 72h timepoints. Cell viability assessed by trypan blue exclusion (>95% required).

Phase 3: RNA Extraction and Quality Control -- Days 10-11
RNA extraction using TRIzol reagent following manufacturer's protocol. Quality assessment by Nanodrop (A260/280 >1.8, A260/230 >2.0) and Bioanalyzer (RIN >8.0). DNase treatment with TURBO DNase. cDNA synthesis from 1 μg total RNA using High-Capacity cDNA Reverse Transcription Kit.

Phase 4: Gene Expression Analysis by RT-qPCR -- Days 12-15
TaqMan RT-qPCR performed on Applied Biosystems QuantStudio 3. Target genes: K2P channels (Kcnk2/TREK-1, Kcnk4/TRAAK, Kcnk12/THIK-2, Kcnk10/TREK-2, Kcnk7/TWIK3, Kcnk3/TASK1), mechanosensitive channels (Trpv4, Piezo1, Trpc1), fibrotic markers (Fsp1/S100a4, Col1a1, Fn1), and reference genes (Gapdh, Actb). Assays run in triplicate with no-template controls.

Phase 5: Protein Validation Studies -- Days 16-20
Western blot analysis for key targets. Protein extraction using RIPA buffer with protease inhibitors. BCA protein assay for quantification. SDS-PAGE (10-12% gels) with 20 μg protein per lane. Primary antibodies: TREK-1 (1:500, Alomone Labs), FSP1 (1:1000, Abcam), β-actin (1:5000, loading control). Chemiluminescence detection and densitometry analysis using ImageJ.

Phase 6: Functional Validation and Data Analysis -- Days 21-25
Immunofluorescence staining for TREK-1 and FSP1 localization changes. Statistical analysis using GraphPad Prism: one-way ANOVA with Tukey's post-hoc test for multiple comparisons. Gene expression calculated using ΔΔCt method with multiple reference gene normalization. Significance threshold p<0.05, with Benjamini-Hochberg correction for multiple testing.