Total RNA extracted from cancer cell lines and breast cancer and normal tissue samples were subjected to qRT-PCR and Western blot analysis. Cancer cell lines were transfected with scrambled-siRNA or METTL14-siRNA/ALKBH5-siRNA (Sigma-Aldrich) for 48 or 72 hours before they were subjected to qRT-PCR or Western blot analysis, as described previously (42 (link)). Primers used in this study are mentioned in table S1. Antibodies against ALKBH5 (#HPA 007196), β-actin (#A3854), GAPDH (#G9295), and METTL14 (#HPA038002) were purchased from Sigma-Aldrich. Antibodies against TGFβ1 (#ab155264), m6A (#ab151230), and YTHDF3 (#ab103328) were purchased from Abcam Inc. Antibodies against cyclin D1 (#2978), CDK4 (#12790), PARP (#5625S), SMAD3 (#9513), P27/KIP1 (#2552s), and pSMAD3 (#9520) were purchased from Cell Signaling Technology. The antibodies against cyclin E1 (#SC-4976), HuR (3A2) (#SC-5261), and pSMAD3 (#SC-11769) were purchased from Santa Cruz Biotechnology. The antibody against HMGA2 (GTX629478) was purchased from GeneTex. Actinomycin D (catalog no. A1410) was purchased from Sigma-Aldrich.
Protocol detail
Investigating m6A Regulators in Cancer Cells
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Actin
Actinomycin d
Antibodies
Antibody
Breast cancer
Cancer
Cell lines
Cyclin d1
Cyclin e1
Gapdh
Hmga2
Mettl14
Normal tissue
P27 kip1
Primers
Sirna
Smad3
Tgfβ1
Western blot analysis
Corresponding Organization : The University of Texas at San Antonio
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Protocol cited in 2 other protocols
Variable analysis
independent variables
- Transfection of cancer cell lines with scrambled-siRNA or METTL14-siRNA/ALKBH5-siRNA (Sigma-Aldrich) for 48 or 72 hours
- Expression levels measured by qRT-PCR and Western blot analysis
- Levels of TGFβ1, m6A, YTHDF3, cyclin D1, CDK4, PARP, SMAD3, P27/KIP1, pSMAD3, cyclin E1, HuR, and HMGA2 proteins
- Total RNA extracted from cancer cell lines and breast cancer and normal tissue samples
- Primers used for qRT-PCR (mentioned in table S1)
- Antibodies used for Western blot analysis (ALKBH5, β-actin, GAPDH, METTL14, TGFβ1, m6A, YTHDF3, cyclin D1, CDK4, PARP, SMAD3, P27/KIP1, pSMAD3, cyclin E1, HuR, and HMGA2)
- Actinomycin D (catalog no. A1410) purchased from Sigma-Aldrich
- Positive control: Not explicitly mentioned
- Negative control: Transfection with scrambled-siRNA
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