DEX effects on K2P channel transcriptome in mouse TM cells

PRIMARY OUTCOME

gene expression changes following DEX treatment

EXPECTED OUTCOMES

- 1. TREK-1 (Kcnk2) will be the most highly expressed K2P channel in mouse TM cells, with Ct values 5-8 cycles lower than other K2P channels - 2. DEX treatment (100 nM, 72h) will cause 50-70% reduction in Kcnk2 mRNA expression compared to vehicle control (p<0.01) - 3. Fibrotic marker Fsp1 will show 3-5 fold upregulation following DEX treatment in a dose-dependent manner - 4. Other K2P channels (TRAAK, THIK-2, TREK-2, TWIK3, TASK1) will show minimal expression changes (<2-fold) with DEX treatment - 5. Mechanosensitive channels (TRPV4, Piezo1, TRPC1) will remain unchanged by DEX treatment, with fold-changes between 0.8-1.2 - 6. Western blot analysis will confirm TREK-1 protein reduction (40-60% decrease) and FSP1 protein increase (2-4 fold) matching mRNA changes - 7. Immunofluorescence will show reduced TREK-1 membrane localization and increased cytoplasmic FSP1 staining in DEX-treated cells

SUCCESS CRITERIA

- • RNA quality metrics must meet standards (RIN >8.0, A260/280 >1.8) for all samples with consistent cDNA synthesis efficiency - • RT-qPCR amplification efficiency between 90-110% for all primer sets with R² >0.99 in standard curves - • Detect statistically significant Kcnk2 suppression (p<0.05) and Fsp1 upregulation (p<0.05) in at least 2 independent experiments - • Demonstrate dose-response relationship for DEX effects with 100 nM and 1 μM concentrations showing progressive changes - • Protein-level validation must confirm mRNA findings with consistent directional changes and statistical significance - • No significant changes in reference gene expression (Gapdh, Actb) across treatment conditions (CV <15%) - • Successful completion requires data from 3 independent cell isolations with reproducible expression patterns

PROTOCOL

**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.

LINKED HYPOTHESES

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[DEX effects on K2P channel transcriptome in mouse TM cells](http://scidex.ai/artifact/exp-b63db77f-7772-437a-a677-a83a2c161986)

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