Fig 1: RNA Pol II recruitment at the Atp1b3 promoter is regulated by FACT/TRIM33. a Atp1b3 and Rnf7 mRNA levels expressed as fold change over the WT BMDM. Mean ± SEM, n = 4. b RNA Pol II occupancy at the Atp1b3 gene. c (Left) Nascent Atp1b3 pre-mRNA levels from WT and Trim33-/- BMDM treated with DMSO or Flavopiridol (FLAVO) for 4 h, expressed as fold change over the DMSO-treated WT BMDM. RT-qPCR were performed using total RNA and intronic positions of primers are relative to TSS. (Right) Fold decrease, expressed as the ratio between DMSO and FLAVO-treated BMDM. Mean ± SEM, n = 5. d Antisense promoter transcripts levels (450nt upstream the TSS) in WT and Trim33-/- BMDM, relative to expression of WT BMDM. Mean ± SEM, n = 5. e ChIP-seq profiles of indicated chromatin modifications along with ChromHMM analysis at the Atp1b3 gene. f SPT16 ChIP-seq profiles at the Atp1b3 locus. g (Up) Schematic for EcoRI fragments and PCR primers positions used in Chromosome Conformation Capture (3C) at the Atp1b3/Rnf7 locus. (Down) 3C analysis of interaction between the - 35 kb region and the Atp1b3 promoter (- 35 kb/ Atp1b3 prom) in WT and Trim33-/- BMDM (two independent replicates). Negative controls (- 35 kb/- 92 kb and Atp1b3 prom/- 92 kb) included a region located 92 kb upstream the Atp1b3 TSS and not bound by TRIM33. Positive controls (Control) included digested and ligated genomic PCR fragments containing all ligation junctions. Genomic DNA after ligation (Input) is shown as a loading control. h (Left) RNA Pol II ChIP-qPCR at the Atp1b3 promoter 4 h after treatment of WT and Trim33-/- BMDM with the FACT inhibitor CBL0137 (CBL). (Right) Fold decrease, expressed as the ratio between DMSO and CBL-treated BMDM. Mean ± SEM, n = 3. i (Left) Atp1b3 pre-mRNA levels in WT and Trim33-/- BMDM 4 h after CBL treatment, relative to expression of DMSO-treated WT BMDM. RT-qPCR was performed using total RNA. (Right) Fold decrease, expressed as the ratio between DMSO and CBL-treated BMDM. Mean ± SEM, n = 3
Fig 2: The effect of ATP1B3 knockdown on cell cycle and apoptosis detected by flow cytometry(A) After 48h of transfection with ATP1B3-siRNA, four groups of cells were collected to detect the cell cycle distribution. It was found that the percentage of ATP1B3-siRNA1 and ATP1B3-siRNA3 cells in G2/M phase was increased while the percentage of cells in G0/G1 and S phase was decreased compared with the Mock and Control groups of SGC-7901 and MKN-45 cells. Data represent the mean±SD of three independent experiments. *P<0.05 vs. Control, **P<0.01 vs. Control. Thus, the knockdown of ATP1B3 could arrest cell cycle progression of gastric cancer cells. (B) Down-regulation of ATP1B3 induced apoptosis of gastric cancer SGC-7901 and MKN-45 cells, as shown by flow cytometry. The number of apoptotic cells in the ATP1B3-siRNA1 and ATP1B3-siRNA3 group was significantly increased compared with that of the Mock and Control groups of SG -7901 and MKN-45 cells. *P<0.05 vs. Control.
Fig 3: ATP1B3 knockdown suppressed activation of the PI3K/AKT signalling pathwayThe levels of PI3K, AKT, p-AKT and Caspase-3 protein in SGC-7901 and MKN-45 cells with altered expression of ATP1B3 were determined by Western blot.1 is the Mock group, 2 is the Control group, and 3 is the ATP1B3-siRNA group. Western blotting showed that knockdown of ATP1B3 resulted in the down-regulation of PI3K and AKT, attenuation of AKT phosphorylation, and activation of the apoptosis-related protein Caspase-3. GAPDH was used as an internal loading control. Each experiment was repeated three times, and similar results were obtained.
Fig 4: Nucleosome dynamics and non-coding RNA production at the - 35 kb regulatory region. a TRIM33, SPT16 and RNA Pol II occupancy along with ChromHMM analysis over the Atp1b3 - 35 kb site. b Nascent bidirectional transcripts at the - 35 kb site in WT and Trim33-/- BMDM, measured after 4sU-tagging and purification of newly transcribed RNA. Data are relative to WT BMDM. Mean ± SEM, n = 3. c Nucleosome positioning at the - 35 kb region in MNase-digested chromatin from WT and Trim33-/- BMDM. Data are relative to the undigested chromatin. Mean ± SEM, n = 3. d ChIP-qPCR of SPT16, PU.1 and TRIM33 occupancy at the - 35 kb site in WT BMDM treated with DMSO or CBL for 4 h. Mean ± SEM, n = 3. e Kinetics of non-coding transcripts at the - 35 kb region in WT and Trim33-/- BMDM after CBL treatment. RT-qPCR was performed using total RNA, and data are presented relative to expression of DMSO-treated WT BMDM. Mean ± SEM, n = 3. f SPT16 ChIP-qPCR (left) and nucleosome positioning (right) at the - 35 kb region in Trim33-/- IM (Trim33-/-) and in Trim33-/- IM that contained a lentivirus expressing TRIM33 (Trim33-/- + WT TRIM33). Mean ± SEM, n = 3. g Non-coding transcripts at the - 35 kb region (left), Atp1b3 mRNA (middle) and protein expression (right) in Trim33-/- and Trim33-/- + WT TRIM33 IM. Data are presented relative to expression of Trim33-/- IM. Mean ± SEM, n = 4. h Model for FACT/TRIM33-mediated repression at the Atp1b3 locus
Fig 5: ATP1B3 expression was knocked down by transfection with ATP1B3-siRNA in SGC-7901 cells(A) Representative photomicrographs of bright fluorescence in SGC-7901 cells were obtained at a magnification of 100x. At 48 h after infection, approximately 50% of the cells expressed FAM at the time points as determined by fluorescence microscopy. (B) Western Blot measured ATP1B3 protein expression in SGC-7901 cells and in cells transfected with siRNA1, siRNA2, siRNA3, or si-NC. It showed that ATP1B3 siRNA1 was the most efficient knockdown sequence compared with the other siRNAs. *P<0.05 compared with SGC-7901 and Control cells. (C) qRT-PCR assay analysis of ATP1B3 mRNA knockdown efficiency using siRNA1, siRNA2, siRNA3 and si-NC in SGC-7901 cells. *P<0.0.1 vs. Control. All of three siRNA fragments demonstrated good knockdown efficiency with respect to ATP1B3 mRNA, and siRNA1 had the highest interference efficiency from the average value.
Supplier Page from Abcam for Anti-ATP1B3 antibody [EPR8981]