Journal: The EMBO Journal

Article Title: Termination of STING responses is mediated via ESCRT ‐dependent degradation

doi: 10.15252/embj.2022112712

Figure Lengend Snippet: A dCas9‐KRAB expressing iBMDMs, without (i.e. dCas9 alone) or with HRS‐targeting sgRNAs (i.e., sg1, sg2 or sg3) were treated for 48 h with 1 μg/ml doxycycline (Dox) to induce sgRNA expression. Cells were then left untreated (UT), or treated with 20 μg/ml DMXAA or 10 μg/ml 2′3′‐cGAM(PS)2 for 6 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown are representative of three independent experiments. B, C dCas9‐KRAB expressing iBMDMs, without (i.e. dCas9 alone) or with HRS‐targeting sgRNAs (i.e. sg1, sg2 or sg3) were treated for 48 h with 1 μg/ml Dox to induce sgRNA expression. Cells were then left UT, or treated with 20 μg/ml DMXAA or 5 μg/ml 2′3′‐cGAM(PS)2 for 6 h. Cell supernatant was collected and assayed for secreted IFNβ (B) and IL6 (C) by ELISA. Data are shown as mean ± SEM combined from N = 3 independent experiments. Statistical analysis was performed using two‐way ANOVA using Bonferroni's multiple comparisons test, where * P < 0.05, *** P < 0.001, **** P < 0.0001. n.d., not detected. D–F dCas9‐KRAB expressing iBMDMs, without (i.e. dCas9 alone) or with HRS‐targeting sgRNAs (i.e. sg1, sg2 or sg3) were treated for 24 h with 1 μg/ml Dox to induce sgRNA expression. iBMDMs were further treated with 1 μg/ml (i.e. 1,000 ng/ml) or 100 ng/ml H‐151 as indicated and incubated for a further 24 h. Cells were lysed for RNA purification and the expression of Ifnb1 (D), Isg15 (E) and Irf7 (F) was analysed by qPCR. Data are shown as mean ± SEM combined from N = 3 independent experiments. Statistical analysis was performed using two‐way ANOVA using Tukey's multiple comparisons test, where * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Bars display significant differences between dCas9 and sgRNA knockdowns in the absence of H‐151. Additional asterisks represent significant differences between the absence or presence of H‐151 across the same cell line. Source data are available online for this figure.

Article Snippet: Third‐generation lentiviral transduction (see below) was used to generate iBMDMs expressing blasticidin‐selectable dCas9‐KRAB, which were selected with 5 μg/ml blasticidin for ~ 72 h. CRISPRi sgRNA guides were designed using the Broad Institute CRISPick online tool ( https://portals.broadinstitute.org/gppx/crispick/public ) and cloned into the doxycycline‐inducible sgRNA plasmid pFgH1tUT, expressing GFP (see below).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation, Purification

Journal: The EMBO Journal

Article Title: Termination of STING responses is mediated via ESCRT ‐dependent degradation

doi: 10.15252/embj.2022112712

Figure Lengend Snippet: A–C dCas9‐KRAB expressing iBMDMs, without (i.e. dCas9 alone) or with HRS‐targeting sgRNAs (i.e. sg1, sg2 or sg3) were treated for 48 h with 1 μg/ml doxycycline (Dox) to induce sgRNA expression. Cells were then left untreated (UT), or treated with 500 ng/ml LPS or 200 ng/ml Pam3CSK4 (P3C) for 6 h. A Cells were lysed for immunoblot with the indicated antibodies. Data shown are representative of three independent experiments. (B, C) Cell supernatant was collected and assayed for secreted IL‐6 (B) or TNF (C) by ELISA. Data are shown as mean ± SEM combined from N = 3 independent experiments. Statistical analysis was performed using two‐way ANOVA using Bonferroni's multiple comparisons test, where no statically significant differences were observed across cell lines for the same treatment. D dCas9‐KRAB expressing iBMDMs, without (i.e. dCas9 alone) or with HRS‐targeting sgRNAs (i.e. sg1, sg2 or sg3) were treated for 24 h with 1 μg/ml Dox to induce sgRNA expression. iBMDMs were further treated with 1 μg/ml (i.e., 1,000 ng/ml) or 100 ng/ml H‐151 as indicated and incubated for a further 24 h. Cells were lysed for RNA purification and the expression of Il6 was analysed by qPCR. Data are shown as mean ± SEM combined from N = 3 independent experiments. Statistical analysis was performed using two‐way ANOVA using Bonferroni's multiple comparisons test, where no statistically significant changes were observed. E VPS4a WT or VPS4a E228Q dominant negative BMDMs were left UT or treated with 1 μg/ml Dox for 4 h. iBMDMs were then further left UT or treated with 10 μg/ml 2′3′‐cGAM(PS)2 for 4 h. Cell supernatant was collected and secreted IL‐6 was measured by ELISA. Data are shown as mean ± SEM combined from N = 3 independent experiments. Statistical analysis was performed using one‐way ANOVA using Bonferroni's multiple comparisons test, where ** P < 0.01, *** P < 0.001. n.d., not detected. Source data are available online for this figure.

Article Snippet: Third‐generation lentiviral transduction (see below) was used to generate iBMDMs expressing blasticidin‐selectable dCas9‐KRAB, which were selected with 5 μg/ml blasticidin for ~ 72 h. CRISPRi sgRNA guides were designed using the Broad Institute CRISPick online tool ( https://portals.broadinstitute.org/gppx/crispick/public ) and cloned into the doxycycline‐inducible sgRNA plasmid pFgH1tUT, expressing GFP (see below).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation, Purification, Dominant Negative Mutation

Journal: Cancer discovery

Article Title: The AMPK-related kinases SIK1 and SIK3 mediate key tumor suppressive effects of LKB1 in NSCLC.

doi: 10.1158/2159-8290.CD-18-1261

Figure Lengend Snippet: (A) Schematic of the experimental design using pSECC lentivirus to deliver Cre-recombinase, Cas9 and a Sik1 or Sik3 sgRNA as a single payload to the lungs of KrasG12D-mice (K) and Kras-Sik1 floxed mice (KSik1) after intratracheal virus delivery.

Article Snippet: As a cohort, KPSik1 mice showed an increase from 10% to 28% in tumor burden over KP mice, (a 2.8-fold increase) compared to a tumor burden of 52% in the KPL mice (5.25-fold increase over KP) , and this was consistent with the results observed in KP mice treated with SIK1 sgRNA guides ( Figure S3B , 2.5 fold tumor burden increase of KP+sgSik1 over KP+sgControls).

Techniques: Virus

Journal: Cancer discovery

Article Title: The AMPK-related kinases SIK1 and SIK3 mediate key tumor suppressive effects of LKB1 in NSCLC.

doi: 10.1158/2159-8290.CD-18-1261

Figure Lengend Snippet: (A) Schematic of the experimental design using pSECC lentivirus to deliver Cre-recombinase, Cas9 and a sgRNA of choice as a single payload to the lungs of KrasG12D-mice (K) and Kras-p53 floxed mice (KP) after intratracheal virus delivery.

Article Snippet: As a cohort, KPSik1 mice showed an increase from 10% to 28% in tumor burden over KP mice, (a 2.8-fold increase) compared to a tumor burden of 52% in the KPL mice (5.25-fold increase over KP) , and this was consistent with the results observed in KP mice treated with SIK1 sgRNA guides ( Figure S3B , 2.5 fold tumor burden increase of KP+sgSik1 over KP+sgControls).

Techniques: Virus

Journal: Cancer discovery

Article Title: The AMPK-related kinases SIK1 and SIK3 mediate key tumor suppressive effects of LKB1 in NSCLC.

doi: 10.1158/2159-8290.CD-18-1261

Figure Lengend Snippet: (A) ELISA analysis of IL-6 secretion in mouse KP lines. KP cells lines were transduced with a sgRNA guide targeting Lkb1 or with two independent sets of guides to target Sik1 and Sik3 simultaneously. 500,000 control or KO cells were seeded in 6 well plates and supernatant was collected at 48hrs to quantitate the amount of IL6 released by the cells into the media.

Article Snippet: As a cohort, KPSik1 mice showed an increase from 10% to 28% in tumor burden over KP mice, (a 2.8-fold increase) compared to a tumor burden of 52% in the KPL mice (5.25-fold increase over KP) , and this was consistent with the results observed in KP mice treated with SIK1 sgRNA guides ( Figure S3B , 2.5 fold tumor burden increase of KP+sgSik1 over KP+sgControls).

Techniques: Enzyme-linked Immunosorbent Assay, Transduction, Control