Figure 2
Bortezomib and LBH589 up-regulate KLF9 in MM1.S cells. (A) KLF9/ACTB Q-RT-PCR signal ratios were obtained from total cellular RNAs corresponding to each group of MM1.S cells treated with indicated amounts of bortezomib (BTZ) for 24 hours. Signal ratios were normalized by the corresponding ratio in untreated cells (0nM). (B) MM1.S cells were treated with indicated amounts of bortezomib for 24 hours followed by Western blotting of total protein extracts with antibodies indicated on the left. Numbers below the panels show the fold increase of KLF9/tubulin and acetylated histone H3 (Ac-H3)/tubulin ratios normalized to that in the first lane (untreated cells). (C) MM1.S cells treated or not with 5nM of bortezomib for 24 hours were cross-linked, sonicated and subjected to chromatin immunoprecipitation with acetylated histone H3 (Ac-H3)–specific antibodies or nonspecific (IgG) antibodies followed by the reversal of the cross-linking and DNA isolation. Isolated DNA was used in quantitative PCR with GAPDH or KLF9 promoter-specific primers, positions of which are designated by the numbers. All PCR signals were normalized by GAPDH-specific PCR signals and by the corresponding PCR signals obtained in reactions on DNA precipitated with IgG antibodies. (D) KLF9/ACTB Q-RT-PCR signal ratios were obtained from total cellular RNAs corresponding to each group of MM1.S cells treated with indicated amounts of LBH589 (LBH) for 24 hours. Signal ratios were normalized by the corresponding ratio in untreated cells (0nM). (E) MM1.S cells were treated with the indicated amounts of LBH589 (LBH) for 24 hours followed by Western blotting of total protein extracts with antibodies indicated on the left. Numbers below the panels show the fold increase of KLF9/tubulin and acetylated histone H3 (Ac-H3)/tubulin ratios normalized to that in the first lane (untreated cells). (F) MM1.S cells were treated with the indicated amounts of tunicamycin (TM) or bortezomib (BTZ) for 24 hours followed by cell collection and Western blotting of cell total protein extracts with antibodies indicated on the left.

Bortezomib and LBH589 up-regulate KLF9 in MM1.S cells. (A) KLF9/ACTB Q-RT-PCR signal ratios were obtained from total cellular RNAs corresponding to each group of MM1.S cells treated with indicated amounts of bortezomib (BTZ) for 24 hours. Signal ratios were normalized by the corresponding ratio in untreated cells (0nM). (B) MM1.S cells were treated with indicated amounts of bortezomib for 24 hours followed by Western blotting of total protein extracts with antibodies indicated on the left. Numbers below the panels show the fold increase of KLF9/tubulin and acetylated histone H3 (Ac-H3)/tubulin ratios normalized to that in the first lane (untreated cells). (C) MM1.S cells treated or not with 5nM of bortezomib for 24 hours were cross-linked, sonicated and subjected to chromatin immunoprecipitation with acetylated histone H3 (Ac-H3)–specific antibodies or nonspecific (IgG) antibodies followed by the reversal of the cross-linking and DNA isolation. Isolated DNA was used in quantitative PCR with GAPDH or KLF9 promoter-specific primers, positions of which are designated by the numbers. All PCR signals were normalized by GAPDH-specific PCR signals and by the corresponding PCR signals obtained in reactions on DNA precipitated with IgG antibodies. (D) KLF9/ACTB Q-RT-PCR signal ratios were obtained from total cellular RNAs corresponding to each group of MM1.S cells treated with indicated amounts of LBH589 (LBH) for 24 hours. Signal ratios were normalized by the corresponding ratio in untreated cells (0nM). (E) MM1.S cells were treated with the indicated amounts of LBH589 (LBH) for 24 hours followed by Western blotting of total protein extracts with antibodies indicated on the left. Numbers below the panels show the fold increase of KLF9/tubulin and acetylated histone H3 (Ac-H3)/tubulin ratios normalized to that in the first lane (untreated cells). (F) MM1.S cells were treated with the indicated amounts of tunicamycin (TM) or bortezomib (BTZ) for 24 hours followed by cell collection and Western blotting of cell total protein extracts with antibodies indicated on the left.

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