cell extraction buffer  (Thermo Fisher)

Journal: bioRxiv

Article Title: DNA topological regulation by topoisomerase IIβ-DNA-PK interaction is important for controlled hypoxia-inducible gene expression

doi: 10.1101/2025.01.06.631366

Figure Lengend Snippet: (A) qRT-PCR results presenting the effect of hypoxic-stress caused by CoCl 2 treatment (+CoCl 2 , throughout the figures) on the transcription of representative HIGs, p21 , ALDOC , and IL1 β, in SH-SY5Y cells (n= 3 biologically independent samples). An equal amount of H 2 O, the solvent for CoCl 2 , was applied as the control (–CoCl 2 , throughout the figures). Data are presented as mean values and SD. P -values for the bar graphs were calculated with the unpaired, one sided Student’s t-test. (B) Representative ChIP-PCR results showing HIF1α and TOP2B occupancies on the representative HIGs, p21 , ALDOC , and IL1 β under normoxia and hypoxia in SH-SY5Y cells. (C) Representative ChIP-PCR results showing TOP2A occupancy on the representative HIGs, p21 , ALDOC , and IL1 β under normoxia and hypoxia in SH-SY5Y cells. (D) Representative ICE assay results showing the TOP2A and TOP2B proteins engaged with the chromosomal DNA under normoxia and hypoxia, with or without etoposide treatment (+Eto, 50 μM, 1 h; –Eto, DMSO). Three dots per –CoCl 2 or +CoCl 2 set are 22, 44, and 66 ng genomic DNA (chromosome) loaded onto nitrocellulose membrane, followed by immunoblotting using TOP2A or TOP2B antibody. (E) qRT-PCR results indicating that under hypoxic stresses, inhibiting TOP2B using etoposide and ICRF193 stimulates transcription at the representative HIGs, p21 , ALDOC , and IL1 β (n= 3 biologically independent samples). Data are presented as mean values and SEM. P -values for the bar graphs were obtained through one-way ANOVA with multiple comparisons.

Article Snippet: HCT116 or SH-SY5Y cell extracts were prepared using a RIPA buffer (Cell Signaling Technology) in the presence of freshly added protease inhibitors [1 mM benzamidine, 0.25 mM PMSF, 1 mM Na-metabisulfite, 1 mM dithiothreitol (DTT); chemicals purchased from Sigma].

Techniques: Quantitative RT-PCR, Solvent, Control, Membrane, Western Blot

Journal: bioRxiv

Article Title: DNA topological regulation by topoisomerase IIβ-DNA-PK interaction is important for controlled hypoxia-inducible gene expression

doi: 10.1101/2025.01.06.631366

Figure Lengend Snippet: (A) Immunoblots showing the protein levels of TOP2B and DNA-PK in WT and TOP2B KO SH-SY5Y cells. ACTIN, a reference. (B) ChIP-qPCR results showing DNA-PK occupancies on representative HIGs with or without TOP2B (n = 3 independent experiments). Data are presented as mean values and SD. P -values for the bar graphs were calculated with the unpaired, one sided Student’s t-test. (C) ChIP-qPCR results showing pDNA-PK occupancies on representative HIGs with or without TOP2B (n = 3 independent experiments). Data are presented as mean values and SD. P -values for the bar graphs were calculated with the unpaired, one sided Student’s t-test. (D) ChIP-qPCR results showing HIF1α occupancies on representative HIGs with or without TOP2B (n = 3 independent experiments). Data are presented as mean values and SD. P -values for the bar graphs were calculated with the unpaired, one sided Student’s t-test. (E) qRT-PCR results presenting the effects of TOP2B KO on representative HIG transcription (n = 3 biological samples). Data are presented as mean values and SD. P -values for the bar graphs were calculated with the unpaired, one sided Student’s t-test.

Article Snippet: HCT116 or SH-SY5Y cell extracts were prepared using a RIPA buffer (Cell Signaling Technology) in the presence of freshly added protease inhibitors [1 mM benzamidine, 0.25 mM PMSF, 1 mM Na-metabisulfite, 1 mM dithiothreitol (DTT); chemicals purchased from Sigma].

Techniques: Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: DNA topological regulation by topoisomerase IIβ-DNA-PK interaction is important for controlled hypoxia-inducible gene expression

doi: 10.1101/2025.01.06.631366

Figure Lengend Snippet: (A) Primary sequences of representable TOP2B fragments identified by phosphopeptide mass spectrometry. The sequences unique to TOP2B are underlined. The phosphorylatable residues within the unique sequences and mutations generated in this study are in red. (B) Immunoblots of TOP2B and γH2AX. Plasmids expressing WT and mutant TOP2B proteins were transfected into TOP2B KO SH-SY5Y cells. Vec, an empty plasmid as a control. ACTIN, a reference. (C) qRT-PCR results showing the effects of mutant TOP2B proteins on representative HIG transcription (n = 3). Data are presented as mean values and SD. P -values for the bar graphs were calculated with the unpaired, one sided Student’s t-test. (D) qRT-PCR results showing the effects of T1403A and S1526A TOP2B mutations on representative HIG transcription under hypoxic conditions (n = 3). Data are presented as mean values and SD. P -values for the bar graphs were calculated with the unpaired, one sided Student’s t-test.

Article Snippet: HCT116 or SH-SY5Y cell extracts were prepared using a RIPA buffer (Cell Signaling Technology) in the presence of freshly added protease inhibitors [1 mM benzamidine, 0.25 mM PMSF, 1 mM Na-metabisulfite, 1 mM dithiothreitol (DTT); chemicals purchased from Sigma].

Techniques: Mass Spectrometry, Generated, Western Blot, Expressing, Mutagenesis, Transfection, Plasmid Preparation, Control, Quantitative RT-PCR