Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Pseudorabies Virus DNA Polymerase Processivity Factor UL42 Inhibits Type I IFN Response by Preventing ISGF3-ISRE Interaction.

doi: 10.4049/jimmunol.2001306

Figure Lengend Snippet: FIGURE 1. UL42 blocks IFN-a signaling. (A) UL42 suppressed the IFN-ainduced activation of ISRE. HeLa cells were cotransfected with 0.5 mg of plasmids expressing EV or PRV-UL42 or HSV1-UL42 or PRV-UL12, together with 100 ng of a firefly luciferase reporter driven by ISRE (pGL3-ISRE-Luc) or IFN-b (pGL3-IFNb-Luc) or MBT1 (pGL3-MBT1-Luc) and 10 ng of plasmid constitutively expressing Renilla luciferase (pRL-TK). pRL-TK was used as an internal con- trol of transfection efficiency. Twelve hours after transfection, cells were incubated in the media containing human IFN-a (1000 U/ml) or were transfected with 1 mg of poly(dA:dT) or plasmid expressing HIF1a for 24 h, and then were harvested and analyzed for luciferase activities (upper panels). The Flag-tagged UL42 and UL12 proteins were verified by Western blots (lower panels). The raw firefly and Renilla luciferase data are provided in Supplemental Table II. (B) UL42 of PRV and HSV1 suppressed the IFN-ainduced activation of ISRE in a dose-dependent manner. Porcine CRL cells (left panel) or human HeLa cells (right panel) were cotransfected with EV or 0.1, 0.3, 0.5, or 1 mg of plasmids expressing either PRV-UL42 or HSV1-UL42, together with 100 ng of a firefly luciferase reporter driven by ISRE (pGL3-ISRE-Luc) and 10 ng of plasmid constitutively expressing Renilla luciferase (pRL-TK). pRL-TK was used as an internal control of transfection effi- ciency. Twelve hours after transfection, cells were incubated in the media containing porcine IFN-a (1000 U/ml) or human IFN-a (1000 U/ml) for 24 h and were harvested and analyzed for luciferase activities (upper panels). The Flag-tagged UL42 proteins were verified by Western blot (lower panels). (C) UL42 of PRV and HSV1 inhibited IFN-amediated induction of ISGs at the mRNA level. HeLa cells were transfected with 1 mg of either the EV or the plasmid expressing PRV-UL42 or HSV1-UL42. Twenty-four hours after transfection, the cells were incubated in media containing human IFN-a (1000 U/ml) for an additional 4 h. Then, the mRNA levels of ISG56 (upper panel) and OAS1 (lower panel) were detected by qRT-PCR. For MBT1, HeLa cells were transfected with 1 mg of either EV or PRV-UL42 or HSV1-UL42, together with 1 mg of plasmid expressing HIF1a. Twenty-four hours after transfection, the mRNA levels of MBT1 were detected by qRT-PCR. (D) UL42 inhibited IFN-ainduced expression of ISG15 and PKR at the protein level. HeLa cells were transfected with 1 mg of either EV or Flag-tagged PRV-UL42 or HSV1-UL42 . Twenty-four hours after transfection, the cells were treated with human IFN-a (1000 U/ml) for 24 h. Then, the expression of ISG15 (upper panels), PKR (lower panels), and Flag were determined by Western blots. Tubulin was used as a reference control. The protein levels of ISG15 and PKR were quantified by densitometry and normalized to the levels of tubulin. The results in above experiments were obtained from three independent experiments (mean ± SD). Statistical analyses were performed using GraphPad Prism software with ANOVA or a t test. ***p < 0.001.

Article Snippet: Anti-HA (SC-805) and anti-ISG15 (sc-166755) Abs were purchased from Santa Cruz Biotechnology (CA).

Techniques: Activation Assay, Expressing, Luciferase, Plasmid Preparation, Transfection, Incubation, Western Blot, Control, Quantitative RT-PCR, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Pseudorabies Virus DNA Polymerase Processivity Factor UL42 Inhibits Type I IFN Response by Preventing ISGF3-ISRE Interaction.

doi: 10.4049/jimmunol.2001306

Figure Lengend Snippet: FIGURE 3. UL42 binds to ISRE and prevents ISGF3-ISRE interaction in a mutually exclusive manner. (A) UL42 binds to the promoters of ISGs. HEK293T cells were transfected with plasmids expressing either EV or Flag-tagged PRV-UL42 or HSV1-UL42 or PRV-UL12. Twenty-four hours after transfection, cells were harvested for ChIP analysis of promoters of ISG15, ISG56, or ISG54 using anti-Flag Ab. ISG54-exon was used as a negative control. (B) UL42 interacts with ISRE in vitro. Purified Flag-PRV-UL42 or Flag-HSV1-UL42 and Bio-ISRE or Bio-GFP were incubated in a DNA pulldown buffer with streptavidin beads. The incubated proteins were then immunoblotted using anti-Flag Ab. (C) The ISRE DNA oligonucleotides used for DNA pull-down and the core ISRE sequen- ces of ISGs amplified in the ChIP assay were aligned. The putative binding sites for ISRE are underlined. (D) UL42 prevents the association of ISGF3 and pro- moters of ISGs. HEK293T cells were transfected with plasmids expressing either EV or Flag-tagged PRV-UL42 or HSV1-UL42. Twenty-four hours after transfection, cells were treated with PBS (left panels) or IFN-a (500 U/ml) (right panels) for 1 h and harvested for ChIP analysis of promoters of ISG15, ISG56, or ISG54 binding to Flag-tagged UL42, STAT1, STAT2, or IRF9. (E) UL42 prevents the interactions of STAT1/STAT2/IRF9 with ISRE in vitro. Purified Flag- PRV-UL42 or Flag-HSV1-UL42, and HA-STAT1, HA-STAT2, HA-IRF9, and Bio-ISRE were incubated in DNA pull-down buffer with streptavidin beads. The incubated proteins were then immunoblotted using anti-Flag and anti-HA Abs. Data above in (A) and (D) are mean ± SD from three independent experi- ments. Statistical analyses were performed using GraphPad Prism software with ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Anti-HA (SC-805) and anti-ISG15 (sc-166755) Abs were purchased from Santa Cruz Biotechnology (CA).

Techniques: Transfection, Expressing, Negative Control, In Vitro, Purification, Incubation, Amplification, Binding Assay, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Pseudorabies Virus DNA Polymerase Processivity Factor UL42 Inhibits Type I IFN Response by Preventing ISGF3-ISRE Interaction.

doi: 10.4049/jimmunol.2001306

Figure Lengend Snippet: FIGURE 4. The DNA-binding sites of PRV-UL42 are necessary for UL42-mediated inhibition of IFN signaling. (A) Schematic diagram illustrating the truncated mutants and the DNA-binding site mutants of PRV-UL42. The four DNA-binding sites of PRV-UL42 are marked by arrows. NLS, nuclear localiza- tion signal. The ability of each truncated mutant in inhibiting IFN signaling is indicated. (B and C) CRL cells were cotransfected with 500 ng of plasmids expressing EV, or PRV WT UL42, or its deletion mutants (1371, 1353, 1255, 1116, 116255, 255371 and 116371 aa) (B), or its DNA-binding site mutants (C), along with 100 ng of pGL3-ISRE-Luc and 10 ng of pRL-TK. pRL-TK was used as an internal control of transfection efficiency. Twelve hours after transfection, cells were incubated in the media containing porcine IFN-a (1000 U/ml) for an additional 24 h, and they were harvested and analyzed for luciferase activities. Statistical analyses were performed using GraphPad Prism software with ANOVA. Different treatments marked by different letters repre- sent statistical significance between treatments (p < 0.05). (D) HeLa cells were transfected with 1 mg of either EV or the plasmid expressing Flag-tagged PRV WT UL42, or its DNA-binding site mutant (4M:R113A/R182A/R279A/R280A, 2M:R2791/R280A). Twenty-four hours after transfection, the cells were incubated in media containing human IFN-a (1000 U/ml) for an additional 4 h. Then, the mRNA levels of ISG56 and ISG15 were detected by qRT- PCR. For MBT1, HeLa cells were transfected with 1 mg of either EV or WT, or 4M, or 2M, together with 1 mg of plasmid expressing HIF1a. Twenty-four hours after transfection, the mRNA levels of MBT1 were detected by qRT-PCR. (E) HeLa cells were transfected with 1 mg of plasmids expressing either EV, or Flag-tagged PRV WT UL42, or its DNA-binding site mutants (4M:K124A/R196A/Q279A/R280A, 2M:Q2791/R280A). Twenty-four hours later, cells were harvested and the protein levels of ISG15, PKR, Flag, and tubulin were detected by Western blots. (F) The DNA-binding sites are necessary for PRV- UL42 to associate with the promoters of ISGs. HEK293T cells were transfected with plasmids expressing either EV or Flag-tagged PRV WT UL42 or its DNA-binding site mutant (4M). Twenty-four hours after transfection, cells were harvested for ChIP analysis of promoters of ISG15, ISG56, or ISG54 binding to Flag-tagged UL42. ISG54-exon was used as a negative control. Data in (D) and (F) are mean ± SD from three independent experiments. Statistical analyses were performed using GraphPad Prism software with ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Anti-HA (SC-805) and anti-ISG15 (sc-166755) Abs were purchased from Santa Cruz Biotechnology (CA).

Techniques: Binding Assay, Inhibition, Mutagenesis, Expressing, Control, Transfection, Incubation, Luciferase, Software, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Pseudorabies Virus DNA Polymerase Processivity Factor UL42 Inhibits Type I IFN Response by Preventing ISGF3-ISRE Interaction.

doi: 10.4049/jimmunol.2001306

Figure Lengend Snippet: FIGURE 5. The DNA-binding sites of HSV1-UL42 are necessary for UL42-mediated inhibition of IFN signaling. (A) Schematic diagram illustrating the truncated mutants and the DNA-binding sites mutants of HSV1-UL42. The four DNA-binding sites of HSV1-UL42 are marked by arrows. The ability of each truncated mutant in inhibiting IFN signaling is indicated. (B and C) HeLa cells were cotransfected with 500 ng of plasmids expressing either EV, HSV1 WT UL42 (WT), or its deletion mutants (1104, 104244, 244413, 104413, 1413 aa) (B), or its DNA-binding site mutants (C), along with 100 ng of pGL3-ISRE-Luc and 10 ng of pRL-TK. pRL-TK was used as an internal control of transfection efficiency. Twelve hours after transfection, cells were incu- bated in the media containing human IFN-a (1000 U/ml) for an additional 24 h, and were harvested and analyzed for luciferase activities. Statistical analyses were performed using GraphPad Prism software with ANOVA. Different treatments marked by different letters represent statistical significance between treatments (p < 0.05). (D) HeLa cells were transfected with 1 mg of either the EV or the plasmid expressing Flag-tagged HSV1 WT UL42, or its DNA-bind- ing site mutant (4M:R113A/R182A/R279A/R280A, 2M:R2791/R280A). Twenty-four hours after transfection, the cells were incubated in media containing human IFN-a (1000 U/ml) for an additional 4 h. Then, the mRNA levels of ISG56 and ISG15 were detected by qRT-PCR. For MBT1, HeLa cells were transfected with 1 mg of EV or WT, or 4M, or 2M, together with 1 mg of plasmid expressing HIF1a. Twenty-four hours after transfection, the mRNA levels of MBT1 were detected by qRT-PCR. (E) HeLa cells were transfected with 1 mg of plasmids expressing either EV, or Flag-tagged HSV1 WT UL42, or its DNA-binding site mutant (4M:R113A/R182A/R279A/R280A, 2M:R2791/R280A). Twenty-four hours later, cells were harvested and the protein level of ISG15, Flag, and tubulin were detected by Western blots. (F) The DNA-binding sites are necessary for HSV1-UL42 to associate with the promoters of ISGs. HEK293T cells were transfected with plasmids expressing either EV or Flag-tagged HSV1 WT UL42 or its DNA-binding site mutant (4M). Twenty-four hours after transfection, cells were harvested for ChIP analysis of promoters of ISG15, ISG56, or ISG54 binding to Flag-tagged UL42. ISG54-exon was used as a negative control. Data above in (D) and (F) are mean ± SD from three independent experiments. Statistical analyses were performed using GraphPad Prism software with ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Anti-HA (SC-805) and anti-ISG15 (sc-166755) Abs were purchased from Santa Cruz Biotechnology (CA).

Techniques: Binding Assay, Inhibition, Mutagenesis, Expressing, Control, Transfection, Luciferase, Software, Plasmid Preparation, Incubation, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Oncogene

Article Title: Tumor-associated mutant p53 promotes cancer cell survival upon glutamine deprivation through p21 induction.

doi: 10.1038/onc.2016.360

Figure Lengend Snippet: Figure 1. Cancer cells with mutp53 are more resistant to glutamine deprivation than cells with wtp53. (a) Lymphoma cell lines with known p53 status were cultured in complete or glutamine (Gln)-free medium for 2 days. Viability was determined by propidium iodide (PI) exclusion and normalized to cells cultured in complete medium. Mutp53 cells: CA46, SupT1 and DB. Wtp53 cells: EB3, DOHH2 and OCI-LY3 (LY3). (b) EB3 and CA46 cells were cultured in Gln-free medium for 24 h. Western blot was performed using antibodies as indicated. (c, d) EB3 and CA46 cells were cultured in complete or Gln-free medium for 48 h. Apoptosis was assessed by staining cells with annexin-V and PI. Positive staining was measured by flow cytometry and analyzed with FlowJo (Ashland, OR, USA). Representative graphs and percentage of late apoptotic cells (PI- and annexin-V-positive cells) are shown. Data represent mean ± s.d. of three independent experiments (***P ⩽0.001, Student’s t-test). (e) EB3 and CA46 cells were treated with L-DON for 3 days, compound 968 and BPTES for 4 days. Cell viability was assessed by PI exclusion and normalized to control (Cont) treated cells. Data represent mean ± s.d. of three independent experiments (**Po0.01, ***P ⩽0.001, Student’s t-test).

Article Snippet: Western blotting Immunoblotting was carried out as described previously.43 Briefly, cells were washed three times with PBS and lysed in ice-cold RIPA lysis buffer supplemented with protease inhibitor (Roche, Indianapolis, IN, USA) for 5 min. Immunoblotting was performed with the following antibodies: p53 (Santa Cruz Biotechnology, Dallas, TX, USA; DO-1, SC126), p-p53 Ser-15 (Cell Signaling Technology, Danvers, MA, USA; 9284), total PARP and cleaved-PARP (Cell Signaling Technology, 9542 and 5625), caspase 3 and cleaved-caspase 3 (Cell Signaling Technology, 9664 and 9665), beta actin (Sigma), and p21 (Cell Signaling Technology, 2947). shRNA knockdown p53 shRNA constructs (TRCN0000003753) were purchased from GE Dharmacon (Lafayette, CO, USA). shRNA lentiviral particles were generated in 293T cells as described previously.43 Briefly, 293T cells were co-transfected with pLKO.1 empty vector or p53 shRNA vector, pMDL, pCMV-VSV-G and pRSV-Rev at a ratio of 4:2:1:1.

Techniques: Cell Culture, Western Blot, Staining, Cytometry, Control

Journal: Oncogene

Article Title: Tumor-associated mutant p53 promotes cancer cell survival upon glutamine deprivation through p21 induction.

doi: 10.1038/onc.2016.360

Figure Lengend Snippet: Figure 2. Loss of mutp53 sensitizes cancer cells to glutamine deprivation and glutaminase inhibitor treatment. (a, b) p53 mRNA levels (a) relative to actin and protein levels (b) in control (cont) vector and p53 shRNA-transduced CA46 cells were assessed using qRT–PCR and western blot. (c) The indicated control vector or shRNA p53-transduced cells were cultured in complete or Gln-free medium for the indicated time points. Viability was assessed by PI exclusion and normalized to cells cultured in complete medium. Data represent mean ± s.d. of three independent experiments (***P ⩽0.001, Student’s t-test). (d, e) Annexin-V and PI staining of stably transfected CA46 cells cultured in Gln-free medium or complete medium for 24 h. Representative graphs of cells in late apoptosis (annexin-V- and PI positive) are shown. Data represent mean ± s.d. of three independent experiments (***P ⩽0.001, Student’s t-test). (f) The control vector-transduced cells and p53 shRNA- transduced cells were treated with 12.5 μM L-DON for 2 days, 40 μM compound 968 and 50 μM BPTES for 4 days. Cell viability was assessed by PI exclusion and normalized to control treated cells. Data represent mean ± s.d. of three independent experiments (*Po0.05, **Po0.01, ***P ⩽0.001, Student’s t-test). mRNA, messenger RNA; qRT–PCR, quantitative real-time reverse transcription PCR.

Article Snippet: Western blotting Immunoblotting was carried out as described previously.43 Briefly, cells were washed three times with PBS and lysed in ice-cold RIPA lysis buffer supplemented with protease inhibitor (Roche, Indianapolis, IN, USA) for 5 min. Immunoblotting was performed with the following antibodies: p53 (Santa Cruz Biotechnology, Dallas, TX, USA; DO-1, SC126), p-p53 Ser-15 (Cell Signaling Technology, Danvers, MA, USA; 9284), total PARP and cleaved-PARP (Cell Signaling Technology, 9542 and 5625), caspase 3 and cleaved-caspase 3 (Cell Signaling Technology, 9664 and 9665), beta actin (Sigma), and p21 (Cell Signaling Technology, 2947). shRNA knockdown p53 shRNA constructs (TRCN0000003753) were purchased from GE Dharmacon (Lafayette, CO, USA). shRNA lentiviral particles were generated in 293T cells as described previously.43 Briefly, 293T cells were co-transfected with pLKO.1 empty vector or p53 shRNA vector, pMDL, pCMV-VSV-G and pRSV-Rev at a ratio of 4:2:1:1.

Techniques: Control, Plasmid Preparation, shRNA, Quantitative RT-PCR, Western Blot, Cell Culture, Staining, Stable Transfection, Transfection, Reverse Transcription

Journal: Oncogene

Article Title: Tumor-associated mutant p53 promotes cancer cell survival upon glutamine deprivation through p21 induction.

doi: 10.1038/onc.2016.360

Figure Lengend Snippet: Figure 3. Expression of mutp53 promotes cell survival upon glutamine deprivation and glutaminase inhibitor treatment. (a) Mutp53 R248Q or R273H were stably expressed in p53−/ −HCT116 cells. Protein levels were assessed by western blot using antibodies against p53 (DO-1) and actin. (b) HCT116 p53−/ −cells expressing an empty vector or mutp53 protein were cultured in either Gln-free or complete medium for the indicated time points. Cell viability was determined by trypan blue exclusion and normalized to cells cultured in complete medium. Data represent mean ± s.e.m. of three independent experiments. (c) HCT116 p53−/ −cells expressing an empty vector or mutp53 protein were cultured in Gln-free medium or complete medium for 4 days. Lysate was extracted to perform western blot analysis using antibodies as indicated. (d) HCT116 p53−/ −cells expressing R248Q, R273H or empty vector, and HCT116 p53+/+ cells were cultured in Gln-free medium or complete medium for 3 days. (e) HCT116 p53−/ −cells expressing R248Q, R273H or empty vector were treated with 50 μM L-DON for 4 days, or 40 μM compound 968 for 5 days. (f) HCT116 p53−/ −cells expressing R248Q, R273H or empty vector were treated with 5 μM camptothecin (CPT) for 3 days, 10 μM doxorubicin (DOXO) for 4 days or 10 nM docetaxel (DTX) for 2 days. (d–f) Cell viability was determined by trypan blue exclusion and normalized to cells cultured in complete medium. Data represent mean ± s.e.m. of three independent experiments (**Po0.01, ***P ⩽0.001, Student’s t-test).

Article Snippet: Western blotting Immunoblotting was carried out as described previously.43 Briefly, cells were washed three times with PBS and lysed in ice-cold RIPA lysis buffer supplemented with protease inhibitor (Roche, Indianapolis, IN, USA) for 5 min. Immunoblotting was performed with the following antibodies: p53 (Santa Cruz Biotechnology, Dallas, TX, USA; DO-1, SC126), p-p53 Ser-15 (Cell Signaling Technology, Danvers, MA, USA; 9284), total PARP and cleaved-PARP (Cell Signaling Technology, 9542 and 5625), caspase 3 and cleaved-caspase 3 (Cell Signaling Technology, 9664 and 9665), beta actin (Sigma), and p21 (Cell Signaling Technology, 2947). shRNA knockdown p53 shRNA constructs (TRCN0000003753) were purchased from GE Dharmacon (Lafayette, CO, USA). shRNA lentiviral particles were generated in 293T cells as described previously.43 Briefly, 293T cells were co-transfected with pLKO.1 empty vector or p53 shRNA vector, pMDL, pCMV-VSV-G and pRSV-Rev at a ratio of 4:2:1:1.

Techniques: Expressing, Stable Transfection, Western Blot, Plasmid Preparation, Cell Culture

Journal: Oncogene

Article Title: Tumor-associated mutant p53 promotes cancer cell survival upon glutamine deprivation through p21 induction.

doi: 10.1038/onc.2016.360

Figure Lengend Snippet: Figure 4. Mutp53 induces expression of p53-target genes upon glutamine deprivation. (a) EB3 and CA46 cells were cultured in complete or Gln-free medium for 24 h. Cells were lysed for western blot using antibodies as indicated. (b) EB3 and CA46 cells were cultured in complete or Gln-free medium overnight. mRNA expression of p53-target genes relative to 18S was determined using qRT–PCR and normalized to the complete medium. (c) Cells were transduced with lentiviral particles followed by puromycin selection to generate stable knockdown of wtp53 in EB3 cells and mutp53 in CA46. p53 protein levels were determined by western blot. (d) EB3 and CA46 cells infected with virus containing control vector or shRNA against p53 were cultured in Gln-free medium overnight. mRNA expression of p53-target genes relative to actin was determined using qRT–PCR and normalized to the complete control medium. (e) HCT116 p53−/ −cells expressing R248Q, R273H or empty vector were cultured in Gln-free medium for 3 days. p53 activation and total p53 expression were determined by the western blot analysis using anti-phospho-p53 (Ser-15) and anti-p53 antibody. (f) HCT116 p53+/+ cells and HCT116 p53−/ −cells expressing R248Q, R273H or empty vector were cultured in complete or Gln-free medium overnight. mRNA expression of p53-target genes relative to actin was determined using qRT–PCR and normalized to the complete medium. Data represent mean ± s.d. of duplicates from two independent experiments (*Po0.05, **Po0.01, ***P ⩽0.001, Student’s t-test).

Article Snippet: Western blotting Immunoblotting was carried out as described previously.43 Briefly, cells were washed three times with PBS and lysed in ice-cold RIPA lysis buffer supplemented with protease inhibitor (Roche, Indianapolis, IN, USA) for 5 min. Immunoblotting was performed with the following antibodies: p53 (Santa Cruz Biotechnology, Dallas, TX, USA; DO-1, SC126), p-p53 Ser-15 (Cell Signaling Technology, Danvers, MA, USA; 9284), total PARP and cleaved-PARP (Cell Signaling Technology, 9542 and 5625), caspase 3 and cleaved-caspase 3 (Cell Signaling Technology, 9664 and 9665), beta actin (Sigma), and p21 (Cell Signaling Technology, 2947). shRNA knockdown p53 shRNA constructs (TRCN0000003753) were purchased from GE Dharmacon (Lafayette, CO, USA). shRNA lentiviral particles were generated in 293T cells as described previously.43 Briefly, 293T cells were co-transfected with pLKO.1 empty vector or p53 shRNA vector, pMDL, pCMV-VSV-G and pRSV-Rev at a ratio of 4:2:1:1.

Techniques: Expressing, Cell Culture, Western Blot, Quantitative RT-PCR, Transduction, Selection, Knockdown, Infection, Virus, Control, Plasmid Preparation, shRNA, Activation Assay

Journal: Oncogene

Article Title: Tumor-associated mutant p53 promotes cancer cell survival upon glutamine deprivation through p21 induction.

doi: 10.1038/onc.2016.360

Figure Lengend Snippet: Figure 5. Mutp53 directly binds to the promoter of p53-target genes upon glutamine deprivation. (a) CA46 cells (R248Q) were cultured in complete or Gln-free medium for 16 h. ChIP analysis was performed to determine p53 binding to the promoter of p53-target genes in response to Gln deprivation. (b) HCT116 p53+/+ cells or HCT116 p53−/ −cells expressing R248Q, R273H or vector control were cultured in complete or Gln-free medium for 16 h. ChIP analysis was performed to determine p53 binding to the promoter of p53-target genes. DNA-protein complexes were pulled down using total p53 antibody (DO-1) or isotype-matched IgG. All p53 binding sites were assessed by PCR. PCR products were separated using agarose electrophoresis.

Article Snippet: Western blotting Immunoblotting was carried out as described previously.43 Briefly, cells were washed three times with PBS and lysed in ice-cold RIPA lysis buffer supplemented with protease inhibitor (Roche, Indianapolis, IN, USA) for 5 min. Immunoblotting was performed with the following antibodies: p53 (Santa Cruz Biotechnology, Dallas, TX, USA; DO-1, SC126), p-p53 Ser-15 (Cell Signaling Technology, Danvers, MA, USA; 9284), total PARP and cleaved-PARP (Cell Signaling Technology, 9542 and 5625), caspase 3 and cleaved-caspase 3 (Cell Signaling Technology, 9664 and 9665), beta actin (Sigma), and p21 (Cell Signaling Technology, 2947). shRNA knockdown p53 shRNA constructs (TRCN0000003753) were purchased from GE Dharmacon (Lafayette, CO, USA). shRNA lentiviral particles were generated in 293T cells as described previously.43 Briefly, 293T cells were co-transfected with pLKO.1 empty vector or p53 shRNA vector, pMDL, pCMV-VSV-G and pRSV-Rev at a ratio of 4:2:1:1.

Techniques: Cell Culture, Binding Assay, Expressing, Plasmid Preparation, Control, Electrophoresis

Journal: Oncogene

Article Title: Tumor-associated mutant p53 promotes cancer cell survival upon glutamine deprivation through p21 induction.

doi: 10.1038/onc.2016.360

Figure Lengend Snippet: Figure 6. Mutp53 promotes cell survival upon glutamine deprivation through p21 induction. (a, b) HCT116 p53−/ −cells expressing mutp53 (R248Q) or empty vector were cultured in complete or Gln-free medium for 24 h. PI staining followed by flow cytometry was performed to assess cell cycle profile. Representative graphs of three independent experiments are shown. Data represent the mean±s.d. (c) HCT116 p53−/−cells expressing mutp53 R248Q or empty vector were cultured in complete or Gln-free medium for 24 h. Western blots were performed using antibodies as indicated. (d) p21 was transiently knocked down in HCT116 p53−/−cells expressing mutp53 R248Q or empty vector using siRNA (20 nM). Forty- eight hours after the siRNA transfection, cells were cultured in complete or Gln-free medium for 4 days. Cell viability was determined by trypan blue exclusion. Data represent the mean ±s.d. of four independent experiments, (***P⩽0.001, Student’s t-test). (e) Western blot was performed using antibodies as indicated.

Article Snippet: Western blotting Immunoblotting was carried out as described previously.43 Briefly, cells were washed three times with PBS and lysed in ice-cold RIPA lysis buffer supplemented with protease inhibitor (Roche, Indianapolis, IN, USA) for 5 min. Immunoblotting was performed with the following antibodies: p53 (Santa Cruz Biotechnology, Dallas, TX, USA; DO-1, SC126), p-p53 Ser-15 (Cell Signaling Technology, Danvers, MA, USA; 9284), total PARP and cleaved-PARP (Cell Signaling Technology, 9542 and 5625), caspase 3 and cleaved-caspase 3 (Cell Signaling Technology, 9664 and 9665), beta actin (Sigma), and p21 (Cell Signaling Technology, 2947). shRNA knockdown p53 shRNA constructs (TRCN0000003753) were purchased from GE Dharmacon (Lafayette, CO, USA). shRNA lentiviral particles were generated in 293T cells as described previously.43 Briefly, 293T cells were co-transfected with pLKO.1 empty vector or p53 shRNA vector, pMDL, pCMV-VSV-G and pRSV-Rev at a ratio of 4:2:1:1.

Techniques: Expressing, Plasmid Preparation, Cell Culture, Staining, Cytometry, Western Blot, Transfection

Journal: Oncogene

Article Title: Tumor-associated mutant p53 promotes cancer cell survival upon glutamine deprivation through p21 induction.

doi: 10.1038/onc.2016.360

Figure Lengend Snippet: Figure 7. Tumors expressing mutant p53 are more resistant to glutaminase inhibitor treatment in vivo. (a, b) Athymic nude mice at 7 weeks old were injected with HCT116 p53−/ −cells on the left flank. HCT116 p53−/ −cells expressing mutp53 R248Q were injected on the right flank. Once the tumor size reached an average of 60 mm3, the mice were treated with 15 mg/kg of L-DON every other day by intraperitonial injection. Tumor size was measured over time. Data represent the mean ± s.d. (n = 5 or 6 tumors as indicated), ***P ⩽0.001, Student’s t-test. (c) Tumors with L-DON or vehicle treatment were harvested at day 11. Western blot was performed using the indicated antibodies.

Article Snippet: Western blotting Immunoblotting was carried out as described previously.43 Briefly, cells were washed three times with PBS and lysed in ice-cold RIPA lysis buffer supplemented with protease inhibitor (Roche, Indianapolis, IN, USA) for 5 min. Immunoblotting was performed with the following antibodies: p53 (Santa Cruz Biotechnology, Dallas, TX, USA; DO-1, SC126), p-p53 Ser-15 (Cell Signaling Technology, Danvers, MA, USA; 9284), total PARP and cleaved-PARP (Cell Signaling Technology, 9542 and 5625), caspase 3 and cleaved-caspase 3 (Cell Signaling Technology, 9664 and 9665), beta actin (Sigma), and p21 (Cell Signaling Technology, 2947). shRNA knockdown p53 shRNA constructs (TRCN0000003753) were purchased from GE Dharmacon (Lafayette, CO, USA). shRNA lentiviral particles were generated in 293T cells as described previously.43 Briefly, 293T cells were co-transfected with pLKO.1 empty vector or p53 shRNA vector, pMDL, pCMV-VSV-G and pRSV-Rev at a ratio of 4:2:1:1.

Techniques: Expressing, Mutagenesis, In Vivo, Injection, Western Blot

Journal: Cell reports

Article Title: CRTC1/MAML2 directs a PGC-1α-IGF-1 circuit that confers vulnerability to PPARγ inhibition

doi: 10.1016/j.celrep.2021.108768

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ImmPRESS HRP Horse Anti-Rabbit IgG Polymer Detection Kit , Vector Labs , MP-7401.

Techniques: Western Blot, Immunohistochemistry, Virus, Recombinant, Protease Inhibitor, Reverse Transcription, SYBR Green Assay, Flow Cytometry, Luminescence Assay, Luciferase, Polymer, Plasmid Preparation, Expressing, Chromatin Immunoprecipitation, shRNA, Software

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Expression tests for ejection proteins gp14, gp15, and gp16 (A) Diagram of plasmids for gp14, gp15, and gp16. (B) Diagram of transformed colonies in different expression strains growing on agar plates treated with ampicillin (100 μg/mL). (C) Diagram of 100 mL starter cultures treated with ampicillin for different expression strains in 250 mL Erlenmeyer flasks. (D) Diagram of 1 L LB cultures treated with ampicillin and inoculated 1:50 with starter culture for different expression strains. (E–G) SDS-PAGE analysis for gp14, gp15, and gp16 before/after (−/+) induction with IPTG at OD600 = 0.6 and expression. Expected molecular weights are gp14, 21 kDa, gp15, 87 kDa and gp16, 146 kDa as incdicated on the SDS-PAGE gels in cyan, magenta, and green, respectively.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Expressing, Transformation Assay, SDS Page

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Workflow for the reconstitution of gp15:gp16 periplasmic tunnel of the T7 DNA-ejectosome.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques:

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Purification of T7 ejection proteins gp15 and gp16 (A) SDS-PAGE results for sonicated samples for gp15 and gp16. Abbreviations: M = Markers for molecular weight, T = Total sample post-sonication, S = Supernatant sample, and P = Pellet sample after centrifugation. (B) SDS-PAGE results for Ni-NTA purification for gp15 and gp16. Abbreviations: Ft = Flow-through sample that passed through the gravity-flow column without binding, W 1 and W 2 = Wash 1 and 2 eluent samples, E n = Elution samples, and B = Beads sample representing protein still bound to the Ni beads. (C) Chromatograms and SDS-PAGE results for gel filtration purified gp15 and gp16 on a calibrated analytical Superdex200 10/300 column. Only relevant lanes from the same gel are shown. Parts of this figure have been reprinted from Molecular Cell, Vol 81, Issue 15, Swanson, et al., Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution, Pages 3145-3159.e7, Copyright (2021), with permission from Elsevier.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Purification, SDS Page, Sonication, Molecular Weight, Centrifugation, Binding Assay, Filtration, Cryo-EM Sample Prep

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Purification of T7 ejection protein gp14 (A) SDS-PAGE results for sonicated, NLS detergent extracted, and Ni-NTA purified samples of gp14. Abbreviations: M = Markers for molecular weight, T = Total sample post-sonication, S = Supernatant sample, and P = Pellet sample after centrifugation, US = Ultracentrifuged Supernatant sample, UP = Ultracentrifuged Pellet sample, Ft = Flow-through sample that passed through the gravity-flow column without binding, W 1 and W 2 = Wash 1 and 2 eluent samples, E n = Elution samples, and B = Beads sample representing protein still bound to the Ni beads. (B) Chromatogram and SDS-PAGE results for gel filtration purified gp14 solubilized in NLS (extraction) detergent. (C) Chromatogram and SDS-PAGE results for gp14 detergent exchange from NLS to DDM detergent using an anion exchange column. (D) Chromatogram and SDS-PAGE results for gel filtration purified gp15 solubilized in DDM (mild) detergent. Only relevant lanes from the same gel are shown in (B–D). Parts of this figure have been reprinted from Molecular Cell, Vol 81, Issue 15, Swanson, et al., Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution, Pages 3145-3159.e7, Copyright (2021), with permission from Elsevier.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Purification, SDS Page, Sonication, Molecular Weight, Centrifugation, Binding Assay, Filtration, Extraction, Cryo-EM Sample Prep

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: In vitro assembly of the T7 periplasmic tunnel from purified gp15 and gp16 (A) Diagram of complex components incubated in a 3:1 molar ratio on ice for 60 min (B) Chromatogram and SDS-PAGE analysis of gel filtrated gp15:gp16 sample. Magenta arrow represents gp15 expected molecular weight at 87 kDa. Green arrow represents gp16 expected molecular weight at 146 kDa. Only relevant lanes from the same gel are shown. Parts of this figure have been reprinted from Molecular Cell, Vol 81, Issue 15, Swanson, et al., Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution, Pages 3145-3159.e7, Copyright (2021), with permission from Elsevier.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: In Vitro, Purification, Incubation, SDS Page, Molecular Weight, Cryo-EM Sample Prep

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Expected results for negative stain TEM and cryo-EM screening (A and B) Representative micrographs of (A) negatively stained and (B) vitrified gp15:gp16 complex. On the right-hand side are 2D-class averages. Scale bars, 100 nm. Reprinted from Molecular Cell, Vol 81, Issue 15, Swanson, et al., Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution, Pages 3145-3159.e7, Copyright (2021), with permission from Elsevier.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Staining, Cryo-EM Sample Prep

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet:

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Virus, Recombinant, Electron Microscopy, Filtration, Plasmid Preparation, Software, Chromatography, Electrophoresis, Microscopy

Journal: Cell reports

Article Title: The Cullin3-Rbx1-KLHL9 E3 ubiquitin ligase complex ubiquitinates Rheb and supports amino acid-induced mTORC1 activation

doi: 10.1016/j.celrep.2024.115101

Figure Lengend Snippet:

Article Snippet: The mouse mesenchymal stem cells (EMSC) were cultured in DMEM/F12 (#11330–032, Invitrogen) containing 15% FBS (Corning, #35–015-CV), 100 μL Primocin (#ant-pm-1, Invivogen) per 50 mL media, and 10 ng/mL recombinant murine FGF-basic (#450–33, Peprotech).

Techniques: Virus, Recombinant, Magnetic Beads, Control, Negative Control, shRNA, Plasmid Preparation, Immunofluorescence, Software

Journal: bioRxiv

Article Title: A mRNA-LNP vaccine against Dengue Virus elicits robust, serotype-specific immunity

doi: 10.1101/2021.01.05.425517

Figure Lengend Snippet: A) Schematic of the DENV genome and engineered mRNA construct. An mRNA encoding for the prM and ENV viral proteins was engineered with N-terminal signal peptide sequence, 5’ and 3’ untranslated regions (UTR) flanking the coding sequence, a 3’ poly-A tail, and a 5’ cap-1 structure. In vitro synthesized mRNA is encapsulated in a lipid nanoparticle for use in in vitro and in vivo experiments. B) 293T cells were transfected with the in vitro transcribed mRNA encoding for the wild-type sequence (WT), or a mutant version with amino acid substitutions in the fusion-loop epitope (ΔFL). Lysate was analyzed by western blot with the domain III specific 1A1D-2 monoclonal antibody and the fusion-loop specific 4G2 monoclonal antibody. C) Supernatant from transfected cells was purified and concentrated through ultracentrifugation and analyzed for VLPs by western blots with the 1A1D-2 monoclonal antibody or anti-GAPDH. Unpurified cell lysate from WT mRNA transfected cells is included as a control. Shown are representative blots. D) Electron microscopy image of VLPs from purified supernatant of transfected 293T cells showing homogenous shape and size of approximately 30nm.

Article Snippet: Membranes were blotted with envelope domain III specific 1A1D-2 (1:600) monoclonal antibody (CDC Arbovirus Reference Collection) or envelope fusion-loop specific 4G2 (3.33 mg/ml) (BEI Cat# NR-50327, Novus Biologicals Cat# NBP2-52709FR).

Techniques: Construct, Sequencing, In Vitro, Synthesized, In Vivo, Transfection, Mutagenesis, Western Blot, Purification, Control, Electron Microscopy

Journal: Cell reports

Article Title: Genetic and Functional Dissection of the Role of Individual 5-HT 2 Receptors as Entry Receptors for JC Polyomavirus

doi: 10.1016/j.celrep.2019.04.067

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-5-HT 2B R was obtained from Novus Biologicals cat# NLS1111 and recognizes the second extracellular loop.

Techniques: Generated, Virus, Recombinant, Mutagenesis, Luciferase, Control, Plasmid Preparation, Software, CRISPR