Mapk14 sirna

MDA-MB-231, T47D, U20S, MG63 and SAOS-2 cell lines were purchased from ATCC. BBL358 cells were generated as described [20 (link)]. All cells were maintained in DMEM high glucose supplemented with 10% FBS, 1% L-Glutamine and 1% penicillin-streptomycin at 37°C and 5% CO₂. Treatments were performed with PROTACs at the indicated concentrations or with 1 μM of PH-797804 (Selleckchem #S2726). To induce p38α pathway activation, cells were stimulated with UV (40 J/m²) and collected after 1 h. Cells were treated with 20 μM MG132 (Calbiochem #474790) or 1 μM NAE inhibitor (NEDD8-Activating Enzyme inhibitor, Calbiochem #505477) for 1 h before PROTAC addition. For siRNA transfection, cells were cultured in OPTIMEM, and MAPK14 siRNA (Ambion, #s3586) was used with Lipofectamine RNAiMAX following the manufacturer’s instructions.

Sub-confluent PC3 cells in six-well dishes were transfected with the following siRNAs using Dharmafect (Thermo Scientific, Waltham, MA, USA): DKK-1 siRNA ID#s: s22723 and s22721 (Ambion, Life Technologies, Carlsbad, CA, USA); MAPK11 siRNA ID#s: MAPK11HSS183382, MAPK11HSS183383 and MAPK11HSS183384 (Invitrogen, Life Technologies, Carlsbad, CA, USA); MAPK12 siRNA ID#s: MAPK12HSS109466, MAPK12HSS109467 and MAPK12HSS109405 (Invitrogen, Life Technologies, Carlsbad CA, USA); MAPK14 siRNA ID#s: s3585, s3586 and s3587 (Ambion, Life Technologies, Carlsbad, CA, USA). Per six-well transfection, 100 nM siRNA were diluted in 50 μl of OPTI-MEM and 2 μl (should be 100 nM amount as varied) of Dharmafect (Invitrogen) in 100 μl of OPTI-MEM. SiRNA and Dharmafect dilutions were incubated at room temperature for 5 min. The diluted siRNA was then combined with the diluted Dharmafect at a ratio of 1 : 2, and incubated at room temperature for 20 min. Cells were washed twice with HBSS and medium replaced with 850 μl of OPTI-MEM supplemented with 10% FCS. In all, 150 μl of the siRNA and Dharmafect mixture was then introduced drop-wise to the cells. After 5 h, the Dharmafect mixture was replaced with the normal culture medium containing both FCS and P/S. The cells were further cultured for 24 h before supernatant was collected and cells lysed for either protein or RNA analysis.