20 25 nt sirna

CAV1 was depleted using a pool of three target-specific 20–25 nt siRNA (Santa Cruz Biotechnology). Cancer cells were transfected in 6-well culture plates, at 60–80% confluence, with siRNA CAV1. Cells were also transfected with a scr siRNA in parallel as controls. For each transfection, cells were treated for 5 h with 2.4 μM of siRNA in transfection medium (Santa Cruz) containing 0.5 μL cm⁻² of transfection reagent (Santa Cruz).
CAV1 was amplified in cancer cells using CRISPR activation plasmid (h, Santa Cruz). Cancer cells were transfected in 6-well culture plates, at 60–80% confluence, with CRISPR Activation Plasmid CAV1. For each transfection, cells were treated for 5 h with 0.9 ng μL⁻¹ of CRISPR Activation Plasmid CAV1 in transfection medium (Santa Cruz) containing 0.5 μL cm⁻² of transfection reagent (Santa Cruz).
After incubation with siRNA CAV1 or CRISPR Activation Plasmid CAV1, complete media was added and the cells were incubated for 48 h. CAV1 downregulation or upregulation was evaluated 48 h post-transfection by western blotting.
Further experiments, to determine the effects of CAV1 depletion or amplification on HER2 stability at the cell membrane, were performed at 48 h post-transfection.

Galectin-1 or GLUT1 was depleted in human bladder cancer cells using a pool of three target-specific 20–25 nt siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). UM-UC-3 and HT-1376 bladder cancer cells were transfected in 6- or 96-well culture plates, at 60–80% confluence, with galectin-1 and GLUT1, respectively. Cells were also transfected with a scrambled siRNA in parallel as controls.
For each transfection, cells were treated for 5 h with 2.4 µM of siRNA in transfection medium (Santa Cruz) containing 0.5 µL/cm² of transfection reagent (Santa Cruz). After incubation, complete media was added and the cells were incubated for 24 or 48 h. Galectin-1 or GLUT1 downregulation was evaluated 24 h or 48 h post-transfection by Western blotting. The uptake and PDT experiments were performed 24 h or 48 h post-transfection with GLUT1 hsiRNA or galectin-1 hsiRNA, respectively.

Panc-1 cells plated at 40–50% density were grown to ~75% confluency overnight as described above. The next day, cells were treated with 50-60 pmols of three CPE siRNAs custom synthesized by Invitrogen, (Carlsbad, CA) which target both CPE-WT and CPE-ΔN mRNAs:
siCPE Seq1#: GAU UUG UCC GAG ACC UUC AAG GUA A;
siCPE Seq2#: UUA CCU UGA AGG UCU CGG ACA AAU C;
siCPE Seq3#: GAU CCU GAG AGU UCC GAA CGU UUA A,
or scrambled siRNA, which is a non-targeting 20-25nt siRNA (Santa Cruz Biotechnology, Dallas, TX), using Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA) according to manufacturers’ protocol. After 24h incubation, the cells were dissociated by trypsinization, cell count were obtained, and seeded in various plates for cell invasion, cell migration, colony formation assay, and cell proliferation assays.