Prolidase-specific siRNAs and non-specific scrambled controls were purchased from Santa Cruz Biotechnology. NIH3T3 (2 × 105 cells/well) grown in 6-well culture plates were transfected with 100 to 300 PM of Prolidase-specific siRNAs or scrambled controls using INTERFERIN (Polypus) as per the manufacturer’s protocol. Post transfection, cells were incubated for 36 to 48 h at 37 °C/5% CO2, washed with PBS (1×) and harvested by gentle scraping for protein isolation. Knock-down of prolidase was confirmed by immunoblot analysis.
For KLF6 knock-down studies, AUMsilenceTM FANA Antisense Oligo (FANA ASOs) targeting the mouse KLF6 (5′-AATGAATTTGGTCCACAGGTC-3′) was designed and synthesized by AUM LifeTech, LLC. The day before ASO addition, NIH-3T3 cells were seeded at 50 to 60% cell density in 12-well plate. The KLF6 ASO or scrambled ASO were gymnotically delivered to the cells twice at 24-h interval and at a final concentration of 5 μM. 24 h post second round ASO delivery, cells were collected for further analysis. For TGF-β1 treatment, cells were serum starved for 1 h before treatment with 5 ng/ml of TGF-β1 for 6 h. Kncok-down of KLF6 was analyzed by immunoblot analysis.
For KLF6 knock-down studies, AUMsilenceTM FANA Antisense Oligo (FANA ASOs) targeting the mouse KLF6 (5′-AATGAATTTGGTCCACAGGTC-3′) was designed and synthesized by AUM LifeTech, LLC. The day before ASO addition, NIH-3T3 cells were seeded at 50 to 60% cell density in 12-well plate. The KLF6 ASO or scrambled ASO were gymnotically delivered to the cells twice at 24-h interval and at a final concentration of 5 μM. 24 h post second round ASO delivery, cells were collected for further analysis. For TGF-β1 treatment, cells were serum starved for 1 h before treatment with 5 ng/ml of TGF-β1 for 6 h. Kncok-down of KLF6 was analyzed by immunoblot analysis.