strll  (ATCC)

Journal: Cell death & disease

Article Title: The eEF2 kinase coordinates the DNA damage response to cisplatin by supporting p53 activation.

doi: 10.1038/s41419-024-06891-4

Figure Lengend Snippet: Fig. 1 eEF2K mediates cellular susceptibility towards cisplatin. A Viability of Eef2k+/+ and Eef2k−/−MEFs treated with the indicated concentrations of cisplatin (CisPt) or vehicle control for 48 h as measured by an MTT assay (n = 3). Untreated condition was set to 100% for each cell line. B Viability of HEK293 cells stably expressing individual shRNAs targeting eEF2K (sh-eEF2K1 and sh-eEF2K2) or scrambled control (sh-scr) treated with the indicated concentrations of CisPt or vehicle control for 72 h as measured by an MTT assay (n = 3). Untreated condition was set to 100% for each cell line. C Cell death rates of Eef2k+/+ and Eef2k−/−MEFs treated with the indicated concentrations of CisPt or vehicle control for 48 h as indicated, as measured using trypan blue staining (n = 3). D Level of apoptosis markers in Eef2k+/+ and Eef2k−/−

Article Snippet: Pooled mouse eEF2K siRNA (sc-39011, Santa Cruz, Dallas, Texas, USA) was used to knockdown eEF2K expression. siRNAs-mediated knockdown P53 expression was performed using mouse p53 siRNAs with the following sequences si-p53-1 GUA AAC GCU UCG AGA UGU U and si-p53-2 AAA UUU GUA UCC CGA GUA U (Dharmacon, Lafayette, Colorado, USA).

Techniques: Control, MTT Assay, Stable Transfection, Expressing, Staining

Journal: Cell death & disease

Article Title: The eEF2 kinase coordinates the DNA damage response to cisplatin by supporting p53 activation.

doi: 10.1038/s41419-024-06891-4

Figure Lengend Snippet: Fig. 2 eEF2K supports the induction of the DDR pathways in response to cisplatin. A Scheme illustrating ATM/ATR signalling in response to DNA damage. B Level of activity of the ATM/ATR-dependent DDR pathways in Eef2k+/+ and Eef2k−/−MEFs treated with CisPt (5 μM) for the indicated times, as measured with immunoblot analysis using the indicated antibodies. The immunoblots are representative of three independent experiments. C Level of activity of the ATM/ATR-dependent DDR pathways in MEFs transfected with siRNA targeting eEF2K (si-Eef2k) or scrambled control (scr) treated with CisPt (5 μM) for the indicated times, as measured with immunoblot analysis using the indicated antibodies. The immunoblots are representative of three independent experiments. * indicates unspecific bands (as previously reported [32]). D Level of γH2AX foci formation in Eef2k+/+ and Eef2k−/−MEFs treated with CisPt (50 μM) or vehicle control for 2 h, as determined by immunofluorescence using a γH2AX antibody (n = 3). E Level of 53BP1 foci formation in Eef2k+/+ and Eef2k−/−MEFs treated with CisPt (50 μM) or vehicle control for 2 h, as determined by immunofluorescence using a 53BP1 antibody (n = 3). Data are expressed as mean ± SD; ***P < 0.005.

Article Snippet: Pooled mouse eEF2K siRNA (sc-39011, Santa Cruz, Dallas, Texas, USA) was used to knockdown eEF2K expression. siRNAs-mediated knockdown P53 expression was performed using mouse p53 siRNAs with the following sequences si-p53-1 GUA AAC GCU UCG AGA UGU U and si-p53-2 AAA UUU GUA UCC CGA GUA U (Dharmacon, Lafayette, Colorado, USA).

Techniques: Activity Assay, Western Blot, Transfection, Control

Journal: Cell death & disease

Article Title: The eEF2 kinase coordinates the DNA damage response to cisplatin by supporting p53 activation.

doi: 10.1038/s41419-024-06891-4

Figure Lengend Snippet: Fig. 3 eEF2K promotes enhanced DNA damage repair in response to cisplatin. A Level of DNA damage in Eef2k+/+ and Eef2k−/−MEFs treated with vehicle (-) or with CisPt (5 μM) for 2 h followed by 2 or 24 h period of recovery, as measured by modified alkali comet assay (n = 3). B Level of DNA damage in HEK293 cells stably expressing sh-eEF2K1 or sh-scr treated with vehicle (-) or with CisPt (5 μM) for 2 h followed by 2 or 24 h period of recovery, as measured by modified alkali comet assay (n = 3). C Level of γH2AX foci formation and resolution in Eef2k+/+ and Eef2k−/−MEFs treated with vehicle (-) or with CisPt (5 μM) for 2 h followed by 0 to 48 h period of recovery, as determined by immunofluorescence using a γH2AX antibody (n = 3). D Micronuclei formation in Eef2k+/+ and Eef2k−/−MEFs treated with vehicle (-) or with CisPt (5 μM) for 2 h followed by 16 or 24 h period of recovery, as measured using DAPI staining. Red arrows indicating micronuclei positive cells and grey arrows indicating micronuclei negative cells (n = 3). E Level of ERCC1 and XPF expression in Eef2k+/+ and Eef2k−/−MEFs treated with CisPt (5 μM) for the indicated times, as measured with immunoblot analysis using the indicated antibodies. Data are expressed as mean ± SD; *P < 0.05, ***P < 0.005.

Article Snippet: Pooled mouse eEF2K siRNA (sc-39011, Santa Cruz, Dallas, Texas, USA) was used to knockdown eEF2K expression. siRNAs-mediated knockdown P53 expression was performed using mouse p53 siRNAs with the following sequences si-p53-1 GUA AAC GCU UCG AGA UGU U and si-p53-2 AAA UUU GUA UCC CGA GUA U (Dharmacon, Lafayette, Colorado, USA).

Techniques: Single Cell Gel Electrophoresis, Stable Transfection, Expressing, Staining, Western Blot

Journal: Cell death & disease

Article Title: The eEF2 kinase coordinates the DNA damage response to cisplatin by supporting p53 activation.

doi: 10.1038/s41419-024-06891-4

Figure Lengend Snippet: Fig. 4 eEF2K-mediated cellular susceptibility towards cisplatin is dependent on p53 activation and expression. A Level of p53 activation in Eef2k+/+ and Eef2k−/−MEFs treated with CisPt (50 μM) for the indicated times, as measured with immunoblot analysis using the indicated antibodies. B Level of p53 activation in HEK293 cells stably expressing sh-eEF2K1, sh-eEF2K2, or sh-scr treated with CisPt (50 μM) for the indicated times, as measured with immunoblot using the indicated antibodies. C, D Viability of Eef2k+/+ MEFs (C) or Eef2k−/−MEFs (D) transfected with individual siRNA targeting p53 (si-p53-1 and si-p53-2) or scrambled control (si-scr) treated with the indicated concentrations of cisplatin (CisPt) or vehicle control for 48 h, using an MTT assay (n = 3). Data are expressed as mean ± SD; *P < 0.05, ***P < 0.005.

Article Snippet: Pooled mouse eEF2K siRNA (sc-39011, Santa Cruz, Dallas, Texas, USA) was used to knockdown eEF2K expression. siRNAs-mediated knockdown P53 expression was performed using mouse p53 siRNAs with the following sequences si-p53-1 GUA AAC GCU UCG AGA UGU U and si-p53-2 AAA UUU GUA UCC CGA GUA U (Dharmacon, Lafayette, Colorado, USA).

Techniques: Activation Assay, Expressing, Western Blot, Stable Transfection, Transfection, Control, MTT Assay

Journal: Cell death & disease

Article Title: The eEF2 kinase coordinates the DNA damage response to cisplatin by supporting p53 activation.

doi: 10.1038/s41419-024-06891-4

Figure Lengend Snippet: Fig. 5 The eEF2K ortholog, efk-1, mediates germ cell death in Caenorhabditis elegans in response to cisplatin. A Level of germ cell death in wild type (wt) and efk-1(ok3609) (efk-1) adult C. elegans treated with the indicated concentrations of CisPt for 48 h, as measured by number of germ cell corpses per gonad arm. B Egg production in wild type (wt) and efk-1 knockout (efk-1) adult C. elegans treated with the indicated concentrations of CisPt for 48 h, as measured by number of eggs produced per worm per hour. C Percentage of viable eggs produced by wt and efk-1 knockout (efk-1) adult C. elegans treated as in (B). D Proposed model for the role of eEF2K in the DDR triggered by cisplatin. Data are expressed as mean ± SD; ***P < 0.001.

Article Snippet: Pooled mouse eEF2K siRNA (sc-39011, Santa Cruz, Dallas, Texas, USA) was used to knockdown eEF2K expression. siRNAs-mediated knockdown P53 expression was performed using mouse p53 siRNAs with the following sequences si-p53-1 GUA AAC GCU UCG AGA UGU U and si-p53-2 AAA UUU GUA UCC CGA GUA U (Dharmacon, Lafayette, Colorado, USA).

Techniques: Knock-Out, Produced

Journal: EMBO reports

Article Title: BRD9 is a druggable component of interferon-stimulated gene expression and antiviral activity.

doi: 10.15252/embr.202152823

Figure Lengend Snippet: Figure 7. Hypothetical model of BRD9 and ncBAF function at selected interferon-stimulated gene promoters.

Article Snippet: cDNAs encoding BRD7, BRD9, BRD9-dBD, and BRD9-N216A were PCR-amplified from existing vectors (Addgene plasmids #65379 (Gong et al, 2015) and #75114-6 (Hohmann et al, 2016); gifts from Kyle Miller and Christopher Vakoc) and cloned into pLVX-IRESPuro (Takara) via XhoI/NotI or EcoRI/NotI sites.

Techniques: