Lacritin Stimulated Cell Proliferation Assay¶
Contributed by Gordon W. Laurie, School of Medicine, University of Virginia, United States
Lacritin Stimulated Cell Proliferation Assay with siRNA or Inhibitors
- For PKCa depletion, a pool of four PKCa-specific siRNAs or individual siRNAs (100 nM; Upstate LLC, Charlottesville VA) were transfected into HSG cells via Lipofectamin 2000 in Opti-MEMI Reduced Serum Medium (Invitrogen, Grand Island NY) in the absence of serum and antibiotics according to manufacturer’s instructions.
*PKCa depletion was confirmed by Western blotting. *
- Other cells were transfected with pools of NFATc1, TRPC1, mTOR, or STIM1-specific siRNAs. In negative controls, cells were transfected with a pool of four lamin-specific siRNAs (all 100 nM; Dharmacon Inc, Lafayette CO).
- Incubate transfection for 72 hours.
- Post-transfection, mitogenesis was assessed by co-addition of 10 nM lacritin and [3H]-thymidine (2 mCi/ml; Amersham, Piscataway NJ) for 24 hrs.
- For rapamycin and cyclosporine inhibition, cells that had been incubated in serum-free media overnight were treated with 1 µM cyclosporine (Sigma Chemical Co., St. Louis MO) for 5 hours or with 100 nM rapamycin (Calbiochem) for 15 min, or both;
- Rapamycin/cyclosporin inhibiter cells were then stimulated with 10 nM lacritin in the same medium with 2 mCi/ml 3H-thymidine for an additional 24 hours.
- In syndecan experiments, incubate cells alone with lacritin or together with increasing amount of bacterial-expressed human SDC1 ectodomain (hS1ED) as a soluble inhibitor.
- Cells depleted of heparanase-1 or SDC1 were treated with lacritin in [3H]-thymidine 48 hours after siRNA tranfection.
- To rescue heparanase depleted cells, ~1 mg heparanase enriched from HSG or HEK293 cells using heparin affinity column or 0.0001 units bacterial heparitinase (Seikagaku America) was added together with lacritin and [3H]-thymidine for 24 hours.
- [3H]-thymidine incorporation was stopped by placing on ice. Cultures were washed twice with ice-cold PBS, fixed with cold and then RT TCA (10%) for 10 min each
- Wash twice with RT PBS, collected in 1 N NaOH, neutralized with 1 N HCl, and then transferred to liquid scintillation vials for measurement.