Journal: Cell Communication and Signaling : CCS

Article Title: Inhibition of bromodomain and extra-terminal proteins targets constitutively active NFκB and STAT signaling in lymphoma and influences the expression of the antiapoptotic proteins BCL2A1 and c-MYC

doi: 10.1186/s12964-024-01782-9

Figure Lengend Snippet: BET inhibitors/-degraders change the protein expression and induce cell death in DLBCL. a ) Induction of cell death [%] after 24 h treatment with the indicated BET inhibitors/-degraders at 1µM measured by AnnexinV-FITC/PI staining and flow cytometry [data are shown as mean + standard deviation (SD) with n = 3]. b - d ) Western Blot of BRD4, c-MYC and BCL2-familiy proteins after 24 h treatment with the indicated BET inhibitors/-degraders at 1µM in b ) SUDHL2, c ) TMD8 and d ) U2932 cells with GAPDH serving as housekeeping control [one representative blot out of three independent experiments is shown]

Article Snippet: SUDHL2 (RRID: CVCL_8795) and Pfeiffer (RRID: CVCL_3326) cells were obtained from American Type Culture Collection, SUDHL6 (RRID: CVCL_2206) and U2932 (RRID: CVCL_1896) were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and TMD8 cells (RRID: CVCL_A442) were kindly provided by Martin Dyer (Leicester, UK).

Techniques: Expressing, Staining, Flow Cytometry, Standard Deviation, Western Blot, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Inhibition of bromodomain and extra-terminal proteins targets constitutively active NFκB and STAT signaling in lymphoma and influences the expression of the antiapoptotic proteins BCL2A1 and c-MYC

doi: 10.1186/s12964-024-01782-9

Figure Lengend Snippet: Viability and protein levels are influenced by selected BET inhibitors/-degraders. a ) Cell viability determined by CTG assay after 48 h treatment with the indicated BET inhibitors/ degraders in ABC and CGB cell lines [data are shown as mean +/- standard deviation (SD) with n = 3]. b ) Western Blot of BRD4 and BCL2 familiy proteins in SUDHL2, TMD8 and Pfeiffer cells treated with JQ1 [1µM], ABBV-075 [SUDHL2 and Pfeiffer 100nM/TMD8 300nM], ARV-825 [SUDHL2 100nM/TMD8 30nM/Pfeiffer 10 nM] and dBET6 [SUDHL2 100nM/TMD8 30nM/Pfeiffer 10 nM]. Cell viability determined by AnnexinV-FITC/PI staining and flow cytometry after 24 h incubation is indicated below the Western blot [one representative blot out of three independent experiments is shown]

Article Snippet: SUDHL2 (RRID: CVCL_8795) and Pfeiffer (RRID: CVCL_3326) cells were obtained from American Type Culture Collection, SUDHL6 (RRID: CVCL_2206) and U2932 (RRID: CVCL_1896) were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and TMD8 cells (RRID: CVCL_A442) were kindly provided by Martin Dyer (Leicester, UK).

Techniques: CTG Assay, Standard Deviation, Western Blot, Staining, Flow Cytometry, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: Inhibition of bromodomain and extra-terminal proteins targets constitutively active NFκB and STAT signaling in lymphoma and influences the expression of the antiapoptotic proteins BCL2A1 and c-MYC

doi: 10.1186/s12964-024-01782-9

Figure Lengend Snippet: c-Myc and BCL2A1 synergize to prevent cell death. a ) Protein expression of c-MYC and BCL2A1 in SUDHL2 and TMD8 cells upon treatment with JQ1 [1µM], ABBV-075 [SUDHL2 100nM/TMD8 300nM], ARV-825 [SUDHL2 100nM/TMD8 30nM] or dBET6 [SUDHL2 100nM/TMD8 30nM] for up to 24 h. GAPDH serves as loading control and one representative blot out of three independent experiments is shown. b ) mRNA expression of c-Myc and Bcl2a1 was assessed by qRT-PCR in the same experiments. Data are shown as mean + standard deviation (SD) with n = 3. c ) Silencing of c-MYC was performed using two individual siRNAs (#1 and #2) followed by analysis of c-Myc and Bcl2a1 mRNA expression at 6 and 24 h after 2nd electroporation. d - e ) siRNA-mediated knockdown of Bcl2a1 and c-Myc alone and combined in SUDHL2 and TMD8 cells was performed with two pooled siRNA sequences for each target. d ) Knockdown efficiency was confirmed by Western Blot 24 h after the 2nd electroporation. e ) Viability of electroporated cells 6 and 24 h after the 2nd electroporation measured by AnnexinV-FITC/PI and flow cytometry [data are shown as mean + standard deviation (SD) with n = 3]. For individual and combined knockdown, the concentration of non-targeting siCtrl was adapted to match the concentration of targeting siRNA

Article Snippet: SUDHL2 (RRID: CVCL_8795) and Pfeiffer (RRID: CVCL_3326) cells were obtained from American Type Culture Collection, SUDHL6 (RRID: CVCL_2206) and U2932 (RRID: CVCL_1896) were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and TMD8 cells (RRID: CVCL_A442) were kindly provided by Martin Dyer (Leicester, UK).

Techniques: Expressing, Control, Quantitative RT-PCR, Standard Deviation, Electroporation, Knockdown, Western Blot, Flow Cytometry, Concentration Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Inhibition of bromodomain and extra-terminal proteins targets constitutively active NFκB and STAT signaling in lymphoma and influences the expression of the antiapoptotic proteins BCL2A1 and c-MYC

doi: 10.1186/s12964-024-01782-9

Figure Lengend Snippet: BET inhibitors/-degraders downregulate STAT3 and canonical NFκB signaling. a ) Western Blot of SUDHL2, TMD8 and Pfeiffer cells treated with JQ1 [1µM] or dBET6 [SUDHL2 100nM/TMD8 and Pfeiffer 30nM] for 8 h and 24 h [one representative blot out of three independent experiments is shown]. b ) Ratio of phosphorylated to total protein, determined by quantification of Western Blot bands. c ) mRNA levels of IκBα, p65 and TNFα in SUDHL2, TMD8 and Pfeiffer cells treated with JQ1 [1µM] or dBET6 [SUDHL2 100nM/TMD8 and Pfeiffer 30nM] for 1 h and 8 h. d ) mRNA levels of Stat3, Stat5 and Pim-1 in SUDHL2, TMD8 and Pfeiffer cells treated with JQ1 [1µM] or dBET6 [SUDHL2 100nM/TMD8 and Pfeiffer 30nM] for 1 h and 8 h [data are shown as mean + standard deviation (SD) with n = 3]

Article Snippet: SUDHL2 (RRID: CVCL_8795) and Pfeiffer (RRID: CVCL_3326) cells were obtained from American Type Culture Collection, SUDHL6 (RRID: CVCL_2206) and U2932 (RRID: CVCL_1896) were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and TMD8 cells (RRID: CVCL_A442) were kindly provided by Martin Dyer (Leicester, UK).

Techniques: Western Blot, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: Inhibition of bromodomain and extra-terminal proteins targets constitutively active NFκB and STAT signaling in lymphoma and influences the expression of the antiapoptotic proteins BCL2A1 and c-MYC

doi: 10.1186/s12964-024-01782-9

Figure Lengend Snippet: BCL2A1 protein levels and STAT3 phosphorylation are downregulated upon TPCA-1 treatment in SUDHL2, TMD8 and Pfeiffer cells. a ) Western Blot of SUDHL2, TMD8 and Pfeiffer cells after 1, 8 and 24 h of TPCA-1 treatment with increasing concentrations [one representative blot out of three independent experiments is shown]. b ) mRNA expression of IκBα, p65 and TNFα and c ) mRNA expression of Stat3, Stat5 and Pim-1 after 1 and 8 h of TPCA-1 [1µM] treatment [data are shown as mean + standard deviation (SD) with n = 3]. d ) Cell death induction upon TPCA-1 treatment for 1, 8 and 24 h determined by FACS FSC/SSC [data are shown as mean + standard deviation (SD) with n = 3]

Article Snippet: SUDHL2 (RRID: CVCL_8795) and Pfeiffer (RRID: CVCL_3326) cells were obtained from American Type Culture Collection, SUDHL6 (RRID: CVCL_2206) and U2932 (RRID: CVCL_1896) were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and TMD8 cells (RRID: CVCL_A442) were kindly provided by Martin Dyer (Leicester, UK).

Techniques: Phospho-proteomics, Western Blot, Expressing, Standard Deviation