The human KRT18-YFP expression vector28 (link) was a generous gift from Dr. Rudolf Leube and Dr. Nicole Schwarz. E0771 cells were transiently transfected with the plasmid or a YFP-only transfection control, and the resulting cellular fluorescence was monitored using a 20x objective lens on an IncuCyte Zoom Imaging System (Essen Bioscience, Ann Arbor, MI, USA). A processing definition was created in the IncuCyte Zoom software to quantitate the number of fluorescent cells in a given microscopy field. Data were graphed as the number of fluorescent cells per microscopy field after transfection with the KRT18-YFP vector minus the number of fluorescent cells visible after transfection with the YFP-only control.
The Krtap5-5 shRNA-resistant vector (Genscript, Piscataway, NJ, USA) used in the shRNA targeting experiment was designed to have the putative P3 shRNA binding site mutated and was Myc-tagged, where the sequence 153-CTCCAGCTGCTGTTGC-168 in wild-type Krtap5-5 was altered to 153-AAGCTCCTGTTGCTGT-168 in the shRNA resistant vector. After transfecting this construct into E0771 cells, stable transfectants were generated after 7-day selection in G418. Transduction with the P3 shRNA proceeded as described above. For experiments involving the shRNA-resistant line, Krtap5-5 gene expression was assessed using a primer pair designed against the gene’s open reading frame.
The Krtap5-5 shRNA-resistant vector (Genscript, Piscataway, NJ, USA) used in the shRNA targeting experiment was designed to have the putative P3 shRNA binding site mutated and was Myc-tagged, where the sequence 153-CTCCAGCTGCTGTTGC-168 in wild-type Krtap5-5 was altered to 153-AAGCTCCTGTTGCTGT-168 in the shRNA resistant vector. After transfecting this construct into E0771 cells, stable transfectants were generated after 7-day selection in G418. Transduction with the P3 shRNA proceeded as described above. For experiments involving the shRNA-resistant line, Krtap5-5 gene expression was assessed using a primer pair designed against the gene’s open reading frame.