expression vectors n terminal myc epitope tagged tmprss2  (Addgene inc)

Journal: Clinical Cancer Research

Article Title: BRD9 Degradation Disrupts Ribosome Biogenesis in Multiple Myeloma

doi: 10.1158/1078-0432.CCR-22-3668

Figure Lengend Snippet: Inhibition or knockdown of BRD9 downregulates expression of ribosome biogenesis genes. A, Venn diagram overlaps of genes differentially expressed by dBRD9-A and shBRD9 (top). Bar graphs showing the top 10 gene ontology enrichment results (bottom). B and C, GSEA plots for ribosome biogenesis and rRNA metabolic process after treatment with dBRD9-A ( B ) and BRD9 knockdown ( C ). D and E, Expression levels of representative ribosome biogenesis genes were assessed by qRT-PCR after treatment with 100 nmol/L of dBRD9-A or DMSO control for 24 hours ( D ) or transduction of shBRD9 or shLuc ( E ) in OPM2 cells. Data are normalized against the housekeeping control gene GAPDH. The expression relative to DMSO or shLuc are shown as mean ± SD of triplicate samples. F and G, Immunoblot analysis for ribosome biogenesis proteins after treatment with or without dBRD9-A (100 nmol/L) for 24 hours ( F ), or transduction of shBRD9 or shLuc ( G ) in OPM2 cells. H and I, CD138 + primary MM cells separated from the BMMCs of 4 patients with MM were treated with or without dBRD9-A (100 nmol/L) for 24 hours, and the representative ribosome biogenesis mRNA level was measured by qRT-PCR ( H ) or BRD9 and MYC expression was assessed by immunoblotting ( I ). J, IHC analysis for BRD9, MYC, RRS1, PES1, and BOP1 in the subcutaneous tumor samples from the OPM2 TurboGFP-Luc–injected mice treated with dBRD9-A or vehicle control. Scale bars, 50 μmol/L. Data are representative of three independent tumors per treatment group. FDR Q-val, false discovery rate Q-value; NES, normalized enrichment score; NOM P -val, nominal P -value. ***, P < 0.001.

Article Snippet: The human MYC expression vector was constructed by amplifying MYC cDNA using PCR and inserting into the pLenti-DDK-P2A-Puro empty vector (OriGene Technologies, PS100092).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Transduction, Western Blot, Injection

Journal: Clinical Cancer Research

Article Title: BRD9 Degradation Disrupts Ribosome Biogenesis in Multiple Myeloma

doi: 10.1158/1078-0432.CCR-22-3668

Figure Lengend Snippet: BRD9 colocalized with BRD4 and MYC in ribosome biogenesis gene promoters. A, Tornado plots showing BRD9 enrichment at regions bound by MYC, BRD4, and RNAPII ChIP-signal ± 5 kb in OPM2 cells. B, Tornado plots showing BRD9, MYC, BRD4, and RNAPII ChIP-signal ± 5 kb of ribosome biogenesis gene promoters in OPM2 cells. Promoters are ranked by RNAPII ChIP-signal. C, Representative gene tracks showing BRD9, MYC, BRD4, RNAPII, and H3K27Ac ChIP-seq occupancy at the indicated genomic loci in dBRD9-A or DMSO-treated OPM2 cells. D, Violin plot representing changes in MYC, BRD4, and RNAPII occupancy at each peak region in ChIP-Rx experiments of dBRD9-A (100 nmol/L)-treated OPM2 cells following a 24-hour treatment. E, Venn diagram overlaps of all identified BRD9 and BRD4 ChIP-seq peaks in OPM2 cells. F, GSEA of BRD9, MYC, and BRD4 overlapped genes for Gene Ontology (Biologic Process, MSigDB) summarized by a dot plot is shown. G and H, FLAG-tagged MYC or empty vector was overexpressed in OPM2 cells. Expression level of MYC measured by immunoblotting after treatment with or without dBRD9-A (100 nmol/L) for 24 hours (top, G ). Expression level of indicated mRNAs was measured by qRT-PCR ( H ). I, FLAG-tagged MYC or empty vector overexpressed in OPM2 cells that were cultured with the indicated concentrations of dBRD9-A for 5 days. Viable cells were determined by MTT assay. Data represent mean ± SD of triplicate cultures. J, GSEA plots showed MYC target genes were downregulated by dBRD9-A (top) or shBRD9 (bottom). K, Proposed model of BRD9 (ncSWI/SNF) functions in MM cells. BRD9 binds to acetylated BRD4 and strengthens the interaction of ncBAF and BRD4, enhancing expression of ribosome biogenesis genes. Pharmacologic degradation or knockdown of BRD9 destabilizes the binding of ncSWI/SNF to BRD4 and reduces the transcription of the ribosome biogenesis genes.

Article Snippet: The human MYC expression vector was constructed by amplifying MYC cDNA using PCR and inserting into the pLenti-DDK-P2A-Puro empty vector (OriGene Technologies, PS100092).

Techniques: ChIP-sequencing, Plasmid Preparation, Expressing, Western Blot, Quantitative RT-PCR, Cell Culture, MTT Assay, Binding Assay