pdonr223 smarcb1 wt donor plasmid  (Addgene inc)

Journal: Scientific Reports

Article Title: SMARCB1 deletion in atypical teratoid rhabdoid tumors results in human endogenous retrovirus K (HML-2) expression

doi: 10.1038/s41598-021-92223-x

Figure Lengend Snippet: SMARCB1 regulates HERV-K (HML-2) env expression. ( a ) Restored SMARCB1 expression in CHLA 02 and CHLA 04 AT/RT cell lines results in downregulation of HML-2 transcription measured at 48 h by qRT-PCR. ( b , c ) SMARCB1 binds the HML-2 LTR significantly more than the promoter of control gene, HPRT . ( b ) SMARCB1 transfected 293 T cells show a significantly greater proportion of HML-2 LTR bound to SMARCB1 following immunoprecipitation compared to control, non-targeting IgG. ( c ) SMARCB1 transfected 293 T cells show no significant difference between non-targeting IgG bound to genomic HPRT and SMARCB1 bound to genomic HPRT . Percent input is a normalized value with input set to 100% (e.g. % input = 100*2^(input Ct − immunoprecipitated chromatin Ct). Ct is cycle threshold. ( d , e ) SMARCB1 knockdown results in increased transcription of HML-2 transcripts as measured with qRT-PCR. ( d ) HML-2 transcripts in 293 T cells transfected with scrambled shRNA control compared to shRNA targeting SMARCB1 at 24 h as measured by qRT-PCR. ( e ) HML-2 transcripts in neural stem cells transfected with scrambled shRNA control are significantly higher compared to transcripts in cells transfected with shRNA targeting SMARCB1 at 48 h. (qRT-PCR) Data was entered into Prism v9 for graph creation. [Error bars represent SEM. Statistics in Supplemental Table ].

Article Snippet: The CAT construct was used to control for effects of transfection as a plasmid of similar size to the SMARCB1 plasmid . pDONR223_SMARCB1_WT was a gift from Jesse Boehm & William Hahn & David Root (Addgene plasmid # 81791; http://n2t.net/addgene:81791 ; RRID:Addgene_81791) . pHAGE-NRAS was a gift from Gordon Mills & Kenneth Scott (Addgene plasmid # 116767; http://n2t.net/addgene:116767 ; RRID:Addgene_116767).

Techniques: Expressing, Quantitative RT-PCR, Control, Transfection, Immunoprecipitation, Knockdown, shRNA

Journal: Scientific Reports

Article Title: SMARCB1 deletion in atypical teratoid rhabdoid tumors results in human endogenous retrovirus K (HML-2) expression

doi: 10.1038/s41598-021-92223-x

Figure Lengend Snippet: C-MYC binding to the HERV-K (HML-2) LTR. ( a – d ) C-MYC binds HML-2 LTR significantly less when SMARCB1 is expressed. Panel a: Relative quantification of HML-2 LTR bound to C-MYC transcription factor binding protein immunoprecipitated from either 293 T cells transfected with a SMARCB1 plasmid or a control ( CAT ) plasmid. ( b ) Relative quantification of housekeeping gene HPRT bound to C-MYC post-immunoprecipitation from 293 T cells transfected with either SMARCB1 or a control ( CAT ) plasmid. ( c ) Relative quantification of C-MYC bound to the HML-2 LTR post immunoprecipitation in CHLA 02 cells transfected with either SMARCB1 or with CAT plasmid. ( d ) Relative quantification of CMYC bound to housekeeping gene HPRT post immunoprecipitation in CHLA 02 cells transfected with either SMARCB1 or with CAT plasmid. Percent input is a normalized value with input set to 100% (e.g. % input = 100*2^(input Ct − immunoprecipitated chromatin Ct). Ct is cycle threshold. ( e ) C-MYC binds the HML-2 LTR in vitro. Biotinylated nucleotides with a non-targeting sequence, a scrambled canonical C-MYC binding sequence, or varying sequences from the Chr7p22.1a or Chr7p22.1b HML-2 LTR were incubated with recombinant C-MYC protein, washed, and run on an immunoblot to detect specific binding between C-MYC and different sequences. There is a white line between the the final LTR_939_944 and another ladder depicting that a portion of the gel was deleted to simplify the figure. The full gel image can be found in the supplemental information. ( f ) Contains a diagram of the mechanism by which HML-2 expression is regulated in the absence or presence of SMARCB1 protein. ( f1 ) shows how HML-2 is actively expressed when C-MYC is bound to the LTR (promoter) when SMARCB1 protein is not expressed, while ( f2 a,b) depict the SMARCB1 mediated inhibition of C-MYC activation of HML-2 transcription. Graphs generated with Prism v9. ( f ) Generated in Microsoft PowerPoint. [Error bars represent SEM. Statistics in Supplemental Table ].

Article Snippet: The CAT construct was used to control for effects of transfection as a plasmid of similar size to the SMARCB1 plasmid . pDONR223_SMARCB1_WT was a gift from Jesse Boehm & William Hahn & David Root (Addgene plasmid # 81791; http://n2t.net/addgene:81791 ; RRID:Addgene_81791) . pHAGE-NRAS was a gift from Gordon Mills & Kenneth Scott (Addgene plasmid # 116767; http://n2t.net/addgene:116767 ; RRID:Addgene_116767).

Techniques: Binding Assay, Quantitative Proteomics, Immunoprecipitation, Transfection, Plasmid Preparation, Control, In Vitro, Sequencing, Incubation, Recombinant, Western Blot, Expressing, Inhibition, Activation Assay, Generated

Journal: Journal of Virology

Article Title: Concurrent Control of the Kaposi's Sarcoma-Associated Herpesvirus Life Cycle through Chromatin Modulation and Host Hedgehog Signaling: a New Prospect for the Therapeutic Potential of Lipoxin A4

doi: 10.1128/JVI.02177-19

Figure Lengend Snippet: Effect of SMARCB1 on KSHV lytic cycle induction and ATPase activity of the SWI/SNF complex. (A) Effect of SMARCB1 on the KSHV ORF50 promoter. HEK293T cells were transfected, using the Lipofectamine 2000 reagent, with an increasing quantity of SMARCB1 expression plasmids (0, 0.5, 1, or 2 μg), 1 μg of the ORF50 promoter-luciferase constructs, and 0.2 μg of the Renilla reporter construct (as a transfection control). Cells were harvested at 36 h after transfection. Alteration in ORF50 promoter activity was determined after normalization to the Renilla activity as a transfection efficiency control. The data represent the mean fold induction from at least three independent experiments ± SD. *, P < 0.05; ***, P < 0.001. (B) SMARCB1 silencing. TREx-BCBL-1-RTA cells were transduced using 5 μg/ml Polybrene at an MOI of 0.1 with the SMART vector-inducible lentiviral shRNAs directed against control shRNA (sh-Control) or SMARCB1 (sh-SMARCB1). Cells were selected with 3 μg/ml puromycin for 72 h. shRNA expression was then induced with 1.0 μg/ml doxycycline. (i) Total protein was extracted 72 h after doxycycline induction and subjected to Western blotting using antibody to cellular proteins (SMARCB1, Brg1, GAPDH) and the viral protein RTA (K8). Membranes exposed to LI-COR secondary antibodies were scanned using a LI-COR Odyssey infrared scanner. Representative data from three experiments are shown here. (ii) cDNA was isolated, and real-time qPCR for SMARCB1 was done. Expression of the gene for SMARCB1 was normalized to that of the gene for GAPDH. Results are presented as the mean ± SD from three independent experiments. Statistical analysis was done by using Student's t test. ***, P < 0.001. (C) HMVEC-d cells were transduced with either control shRNA lentiviral particles (sh-Control; catalog no. sc-108080; Santa Cruz Biotechnology, Inc.) or an shRNA lentiviral particle against SMARCB1 (sh-SMARCB1; catalog no. sc-35668-V; Santa Cruz Biotechnology, Inc.) and selected with 3 μg/ml puromycin for 5 to 7 days. These transduced cells were either left uninfected or infected with KSHV (30 DNA copies/cell). Total proteins were extracted in RIPA lysis buffer and subjected to Western blotting using antibody for viral protein (K8, ORF50) and cellular proteins Brg1, SMARCB1, and GAPDH. Membranes exposed to LI-COR secondary antibodies were scanned using a LI-COR Odyssey infrared scanner. A representative blot for the experiments is presented. GAPDH was used as the loading control. (D) ATPase activity. The ATPase activity of isolated chromatin-remodeling complex from TREx-BCBL-1-RTA cells (untreated), TREx-BCBL-1-RTA SMARCB1 KD cells, and TREx-BCBL-1-RTA cells with an expression plasmid for SMARCB1 overexpression was measured in terms of ATP hydrolysis on immunoprecipitation Dynabeads. This ATPase activity was further quantified by a luciferase assay, performed using the ADP-Glo Max assay (catalog number V7001; Promega), as discussed in Materials and Methods. Results are presented as the mean ± SD from three independent experiments, and statistical analysis was done by using Student's t test. *, P <0.05; ***, P < 0.001.

Article Snippet: The KSHV ORF50 promoter construct (p2500Luc) was obtained from George Miller (Yale University School of Medicine, New Haven, CT) ( 68 ). pDONR223_SMARCB1_WT was a gift from Jesse Boehm, William Hahn, and David Root (Addgene overexpression plasmid no. 81791).

Techniques: Activity Assay, Transfection, Expressing, Luciferase, Construct, Control, Plasmid Preparation, shRNA, Western Blot, Isolation, Transduction, Infection, Lysis, Over Expression, Immunoprecipitation

Journal: Journal of Virology

Article Title: Concurrent Control of the Kaposi's Sarcoma-Associated Herpesvirus Life Cycle through Chromatin Modulation and Host Hedgehog Signaling: a New Prospect for the Therapeutic Potential of Lipoxin A4

doi: 10.1128/JVI.02177-19

Figure Lengend Snippet: Expression of Gli-1. (A) Gli-1 gene expression relative to GAPDH gene expression for TREx-BCBL-1-RTA cells treated with sh-Control and sh-SMARCB1 was determined using real-time PCR. Results are presented as the mean ± SD from three independent experiments, and statistical analysis was done by using Student's t test. ***, P < 0.001. (B) The amount of Gli-1 in lysates obtained from TREx-BCBL-1-RTA cells (untreated), TREx-BCBL-1-RTA SMARCB1 KD cells, and TREx-BCBL-RTA cells with an expression plasmid for SMARCB1 overexpression was estimated using ELISA. Results are presented as the mean ± SD from three independent experiments, and statistical analysis was done by using Student's t test. *, P < 0.05; ***, P < 0.001. (C) Healthy skin and KS skin tissue specimens were deparaffinized, hydrated, permeabilized, blocked with signal enhancer, reacted with anti-SMARCB1 and anti-Gli-1 antibodies, washed, and incubated with Alexa Fluor 594 (red) and Alexa Fluor 488 (green) secondary antibodies, respectively. The images were merged with those for DAPI-stained nuclei. (D) BCBL-1 cells and LXA4-treated BCBL-1 cells were fixed, permeabilized, washed, incubated with anti-Gli-1, washed, and incubated with Alexa Fluor 594 (red). Nuclei were stained with DAPI. Immunofluorescence assay staining for Gli-1 in LXA4-treated BCBL-1 cells showed the nuclear translocation of Gli-1. (E) Gli-1 expression in lysates obtained from healthy B cells, KSHV-negative cells (BJAB), and BJAB-KSHV cells (i) and from KSHV-positive cells (BCBL-1 cells and LXA4-treated BCBL-1 cells) (ii) was estimated using the ELISA method. The experiments were repeated three times. The results are presented as the mean ± SD, and statistical analysis was done by using Student's t test. **, P < 0.01; ***, P < 0.001.

Article Snippet: The KSHV ORF50 promoter construct (p2500Luc) was obtained from George Miller (Yale University School of Medicine, New Haven, CT) ( 68 ). pDONR223_SMARCB1_WT was a gift from Jesse Boehm, William Hahn, and David Root (Addgene overexpression plasmid no. 81791).

Techniques: Expressing, Gene Expression, Control, Real-time Polymerase Chain Reaction, Plasmid Preparation, Over Expression, Enzyme-linked Immunosorbent Assay, Incubation, Staining, Immunofluorescence, Translocation Assay