paper pmt3 p38α human n ha fl kyriakis lab  (Addgene inc)

Journal: Cells

Article Title: p38 Mediates Resistance to FGFR Inhibition in Non-Small Cell Lung Cancer

doi: 10.3390/cells10123363

Figure Lengend Snippet: Involvement of p38 kinase in resistance to FGFR inhibition. ( A ) Protein expression/phosphorylation levels of FGFR and its direct downstream effectors were analysed with Western blot. Experiments were conducted in triplicates. Representative blots are shown. ( B ) A circos plot showing a copy number variation (relativeLog) in resistant variant of NCI-H1581 (outer circle) and NCI-H1703 (inner circle) cell lines in comparison to respective sensitive variant. Enlarged circos plot with a legible gene names is shown in . ( C ) A heat map showing genes with copy number variation (CNV) between sensitive versus resistant variant (data subjected to rlog transformation). ( D ) A heat map showing differentially expressed genes between sensitive versus resistant cell line variants (only genes with the mutual direction of change in both analysed cell lines are shown; data subjected to rlog transformation).

Article Snippet: To generate cells overexpressing p38 MAPK NCI-H1581 and NCI-H1703 cells were seeded onto 6 cm plates and, after 24 h, transfected with pMT3-p38-HA plasmid (Addgene, #12658) using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, MA, USA).

Techniques: Inhibition, Expressing, Phospho-proteomics, Western Blot, Variant Assay, Comparison, Transformation Assay

Journal: Cells

Article Title: p38 Mediates Resistance to FGFR Inhibition in Non-Small Cell Lung Cancer

doi: 10.3390/cells10123363

Figure Lengend Snippet: p38 activity mediates in CPL304110-induced cell growth inhibition. ( A ) Sensitive and resistant variants of NCI-H1581 and NCI-H1703 cells were grown with CPL304110 (0.1 µΜ for NCI-H1581 and NCI-H1581R; 1 µΜ for NCI-H1703 and NCI-H1703R) and/or SB202190 (2 µM) in 3D BD Matrigel ® . Cell growth was measured with ImageJ software after 14 days of culture. Representative pictures were taken. Scale bar represents 100 µm, n = 3. ( B ) Proliferation analysis was evaluated by MTT in sensitive and resistant cells exposed to CPL304110 (0.1 µΜ for NCI-H1581 and NCI-H1581R; 1 µΜ for NCI-H1703 and NCI-H1703R) and/or SB202190 (2 µM) for 96 h. Data are expressed as mean ± SD, ** p ≤ 0.005, n = 3.

Article Snippet: To generate cells overexpressing p38 MAPK NCI-H1581 and NCI-H1703 cells were seeded onto 6 cm plates and, after 24 h, transfected with pMT3-p38-HA plasmid (Addgene, #12658) using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, MA, USA).

Techniques: Activity Assay, Inhibition, Software

Journal: Cells

Article Title: p38 Mediates Resistance to FGFR Inhibition in Non-Small Cell Lung Cancer

doi: 10.3390/cells10123363

Figure Lengend Snippet: p38 MAPK overexpression induces resistance to FGFR inhibition. ( A ) p38 kinase overexpression was established in NCI-H1581 and NCI-H1703 cells and confirmed with Western blot. Experiments were conducted in triplicates. Representative blots are shown. ( B ) Cell growth in 3D BD Matrigel ® and ( C ) cell proliferation in the presence of CPL304110 (0.1 µΜ for NCI-H1581, NCI-H1581R, NCI-H1581/p38↑; 1 µΜ for NCI-H1703, NCI-H1703R, NCI-H1703/p38↑) was assessed. Cells were cultured in 3D BD Matrigel ® for 14 days. Representative pictures were taken. Scale bar represents 100 µm, n = 3. Cell proliferation was assessed using MTT viability assay after 96 h. Data are expressed as mean ± SD, *** p ≤ 0.001, n = 3.

Article Snippet: To generate cells overexpressing p38 MAPK NCI-H1581 and NCI-H1703 cells were seeded onto 6 cm plates and, after 24 h, transfected with pMT3-p38-HA plasmid (Addgene, #12658) using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, MA, USA).

Techniques: Over Expression, Inhibition, Western Blot, Cell Culture, MTT Viability Assay