Journal: Cell death discovery

Article Title: Inhibition of EZH2 alleviates SAHA-induced senescence-associated secretion phenotype in small cell lung cancer cells.

doi: 10.1038/s41420-023-01591-y

Figure Lengend Snippet: Fig. 2 CCF associated with SAHA-induced SASP in SCLC cell lines. A Immunofluorescent images stained with γΗ2ΑΧ and 53BP1 were used to evaluate the DDR in H446 cells after treatment of DMSO or 3 μM SAHA for 6 days. Results were evaluated by the average values of five different fields with over 100 cells. Scale bars = 10 µm. n = 3 independent experiments. ***P < 0.001 compared with the corresponding control. B Immunofluorescent images stained with γΗ2ΑΧ and H3K27me3 were used to calculate the proportion of CCF in H446 cells after treatment of DMSO or 3 μM SAHA for 6 days. CCF was indicated by arrows. Scale bars = 10 µm. C The proportion of CCF was assessed as the average of five different fields with over 100 cells in H446 cells. Error bars indicate Mean ± SD, Student’s t-test. n = 3 independent experiments. **P < 0.01 compared with the corresponding control. D The protein levels of STING and p-STING were presented in H446 cells treated with DMSO or 3 μM SAHA by western blotting 6 days later. E H1688 cells determined the protein levels of STING and p-STING by western blotting. H1688 cells were treated with DMSO or 3 μM SAHA for 6 days and used for western blotting at SAHA withdrawal two days later. F Western blotting indicated the expression levels of cGAS-STING pathway-related proteins in H446 cells after co-treatment of 3 μM SAHA and 10 μM PF06928215. SAHA was treated for 6 days, and PF06928215 treatment time was 24 h. For positive control of the cGAS-STING activation, H446 cells were treated with 5 µM cGAMP for 4 h. G DMSO or 3 μM SAHA-treated H446 cells for 6 days were treated with 1 μM STING inhibitor C176 for 48 h and measured by western blotting.

Article Snippet: For treatment of STING inhibitor, 3 μM SAHA-treated H446 cells for 6 days were treated with 1 μM C176(Selleck, Catalog No.S6575) for 48 h [54] and measured, 3 μM SAHA-treated H1688 cells for 6 days were treated with 1 μM C176 for 48 h after SAHA withdrawal 2 days later and used for analysis.

Techniques: Staining, Control, Western Blot, Expressing, Positive Control, Activation Assay

Journal: Cell death discovery

Article Title: Inhibition of EZH2 alleviates SAHA-induced senescence-associated secretion phenotype in small cell lung cancer cells.

doi: 10.1038/s41420-023-01591-y

Figure Lengend Snippet: Fig. 3 The nuclear pore density increases and is related to CCF after treatment of SAHA in SCLC cells. A The relative mRNA level of Tpr in H446 cells after treatment of DMSO or 3 μM SAHA for 6 days was evaluated using RT-PCR. Error bars represent Mean ± SD, and the results are the average of three different experiments. mRNA levels were quantified relative to β-actin (control) mRNA. B Western blotting showed the expression levels of Tpr in H446 cells treated with DMSO or 3 μM SAHA for 6 days. C H446 cells were immunofluorescent stained with Tpr after treatment of DMSO or 3 μM SAHA for 6 days, evaluating the change of nucleoporin Tpr area. Scale bars = 10 µm. n = 3 independent experiments. D H446 cells transfected with a shTpr plasmid were treated with DMSO or 3 μM SAHA for 6 days and measured by western blotting. E Immunofluorescent images showing the incidence of CCF in H446 cells inhibited Tpr protein using interfering mRNA. Scale bars = 5 µm. F The proportion of CCF was assessed as the average of five different fields over 100 cells after treatment of DMSO or 3 μM SAHA for 6 days in H446 cells transfected with a shTpr plasmid. Error bars indicate Mean ± SD. n = 3 independent experiments. G The protein levels of the cGAS-STING pathway and senescence-associated proteins in H446 cells transfected with a shTpr plasmid were assessed by western blotting after treatment of DMSO or 3 μM SAHA for 6 days. ns, no significant; *P < 0.05; **P < 0.01; ***P < 0.001 compared with the corresponding control.

Article Snippet: For treatment of STING inhibitor, 3 μM SAHA-treated H446 cells for 6 days were treated with 1 μM C176(Selleck, Catalog No.S6575) for 48 h [54] and measured, 3 μM SAHA-treated H1688 cells for 6 days were treated with 1 μM C176 for 48 h after SAHA withdrawal 2 days later and used for analysis.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Expressing, Staining, Transfection, Plasmid Preparation

Journal: Cell death discovery

Article Title: Inhibition of EZH2 alleviates SAHA-induced senescence-associated secretion phenotype in small cell lung cancer cells.

doi: 10.1038/s41420-023-01591-y

Figure Lengend Snippet: Fig. 5 EZH2 inhibitor EPZ-6438 suppresses SAHA-induced SASP by decreasing CCF in SCLC cells. A, B H446 cells (A) and H1688 cells (B) were immunofluorescent stained with γΗ2ΑΧ and H3K27me3 to evaluate the incidence of CCF after treatment of DMSO or 3 μM SAHA and 0.5 μM EZP-6438, co-treatment of 3 μM SAHA and 0.5 μM EZP-6438, for 6 days, respectively. In H1688 cells, 3 μM SAHA was treated in a separate SAHA treatment area for 6 days and immunofluorescence was performed after 2 days. The incidence of CCF was presented as Mean ± SD of five different fields over 100 cells. n = 3 independent experiments. C, D Western blotting was used to assess the expression levels of cGAS- STING pathway and senescence-associated proteins in H446 (C) and H1688 (D) cells treated for 6 days with DMSO or 3 μM SAHA and 0.5 μM EZP-6438, co-treatment of 3 μM SAHA and 0.5 μM EZP-6438, respectively. According to the above method, SAHA alone treatment region was performed in H1688 cells. E, F The incidence of CCF in H446 (E) and H1688 (F) cells transfected with a shEZH2 plasmid were evaluated using immunofluorescence after treatment of 3 μM SAHA for 6 days. The values represent the Mean ± SD of five different fields over 100 cells. n = 3 independent experiments. G, H The expression levels of the cGAS-STING pathway and senescence-associated proteins in H446 (G) and H1688 (H) cells transfected with a shEZH2 plasmid were evaluated using western blotting after treatment of 3 μM SAHA for six days. ns no significant; *P < 0.05; **P < 0.01; ***P < 0.001 compared with the corresponding control.

Article Snippet: For treatment of STING inhibitor, 3 μM SAHA-treated H446 cells for 6 days were treated with 1 μM C176(Selleck, Catalog No.S6575) for 48 h [54] and measured, 3 μM SAHA-treated H1688 cells for 6 days were treated with 1 μM C176 for 48 h after SAHA withdrawal 2 days later and used for analysis.

Techniques: Staining, Western Blot, Expressing, Transfection, Plasmid Preparation, Control

Journal: Inflammation

Article Title: Aryl-functionalised α,α′-Trehalose 6,6′-Glycolipid Induces Mincle-independent Pyroptotic Cell Death

doi: 10.1007/s10753-023-01814-5

Figure Lengend Snippet: Aryl-trehalose glycolipid AF-2 induces Mincle-independent IL-1β production. a WT and Mincle −/− GM-CSF BMDMs and were incubated with plate-bound AF-2 or TDB (1 nmol/well; solubilised LPS (100 ng/mL) as a control) and IL-1β, IL-6, MIP-2, TNF-α, IL-12p40, and IL-10 production measured at 24 h by ELISA. b WT and Mincle −/− GM-CSF BMDMs were incubated with plate-bound AF-2 or TDB (1 or 8 nmol/well; solubilised LPS (100 ng/mL) or nigericin (10 μM) as a control) and IL-1β production measured at 24 h by ELISA. c WT and Mincle −/− GM-CSF BMDMs were stimulated with AF-2 or TDB (40 μM; solubilized in 2% DMSO in H 2 O) or LPS (100 ng/mL) and IL-1β was measured at 24 h by ELISA. d Negatively selected human monocytes were incubated with plate-bound AF-2 or TDB (1 or 8 nmol/well) with solubilised LPS (100 ng/mL) or nigericin (10 μM) as a control and IL-1β was measured. Data represent the Mean ± SEM of three to six independent experiments (murine BMDMs) or three (human monocyte) experiments performed in triplicate. Statistical significance was calculated in comparison to iPrOH using 2-way ANOVA (Dunnett’s multiple comparison test), **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05.

Article Snippet: 100 ng/mL LPS (Sigma) or nigericin (10 μM, ChemImpex) were used as controls [ ].

Techniques: Incubation, Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Inflammation

Article Title: Aryl-functionalised α,α′-Trehalose 6,6′-Glycolipid Induces Mincle-independent Pyroptotic Cell Death

doi: 10.1007/s10753-023-01814-5

Figure Lengend Snippet: Plate-coated aryl-trehalose glycolipid, AF-2, induces murine BMDM and human monocyte death. a and b WT and Mincle −/− GM-CSF BMDMs or c and d negatively enriched human monocytes were incubated in plates coated with AF-2 or TDB (8 nmol/well concentration in Sytox Green assay, 1 or 8 nmol/well concentration in LDH assay); solubilised LPS (100 ng/mL), nigericin (10 μM), or Triton X (1%). a and c Fluorescence of Sytox Green (1 μM) was measured over time; results are calculated relative to iPrOH control fluorescence. b and d LDH release was measured at 24 h from the supernatant. Data represent the Mean ± SEM of a a representative experiment repeated three times in triplicate, b - d three independent experiments performed in triplicate. GM-CSF BMDMs from WT mice were seeded on e cover slips alone or on coverslips coated overnight with AF-2 (8 nmol/cover slip) and imaged by scanning electron microscope (SEM) at ×150, ×500, and ×2,700 magnification. Scale bar: 100 μm for ×150 magnification and 10 µm for ×500 and ×2700 magnifications.

Article Snippet: 100 ng/mL LPS (Sigma) or nigericin (10 μM, ChemImpex) were used as controls [ ].

Techniques: Incubation, Concentration Assay, Lactate Dehydrogenase Assay, Fluorescence, Control, Microscopy

Journal: Inflammation

Article Title: Aryl-functionalised α,α′-Trehalose 6,6′-Glycolipid Induces Mincle-independent Pyroptotic Cell Death

doi: 10.1007/s10753-023-01814-5

Figure Lengend Snippet: Aryl-trehalose glycolipid AF-2 induces Caspase-1 mediated induced pyroptotic cell death. WT and Mincle −/− GM-CSF BMDMs were left untreated or pre-treated with Caspase-1 inhibitor Ac-YVAD-cmk (40 μM) before adding to plates coated with AF-2 or TDB (8 nmol/well concentration for Western Blot; 1 or 8 nmol/well concentration for IL-1β ELISA and LDL assay), or stimulation with solubilised LPS (100 ng/mL), nigericin (10 μM). a After 4 h the cells were lysed and whole cell lysates analysed for GSDMD cleavage by Western Blot. Data are representative of an experiment repeated three times. b IL-1β was measured in the supernatant at 24 h by ELISA. c and d LDH release was measured after 4 h and 24 h from the supernatant. Results are calculated relative to iPrOH control fluorescence. b , c and d Data represent the Mean ± SEM of three experiments performed in triplicate. Statistical significance was calculated in comparison to control using Multiple t-test (Mann-Whitney), **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05.

Article Snippet: 100 ng/mL LPS (Sigma) or nigericin (10 μM, ChemImpex) were used as controls [ ].

Techniques: Concentration Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Fluorescence, Comparison, MANN-WHITNEY

Journal: Inflammation

Article Title: Aryl-functionalised α,α′-Trehalose 6,6′-Glycolipid Induces Mincle-independent Pyroptotic Cell Death

doi: 10.1007/s10753-023-01814-5

Figure Lengend Snippet: K + efflux and NLRP3 inflammasomes are involved in AF-2 induces pyroptotic cell death. WT and Mincle −/− GM-CSF BMDMs were left untreated or pre-treated with KCl (50 nm) or NLRP3 inflammasome inhibitor CY-09 (20 or 40 µM) before being added to plates coated with AF-2 or TDB (8 nmol/well concentration for Sytox Green assay; 1 or 8 nmol/well concentration for IL-1β ELISA), or stimulated with solubilised nigericin (10 μM), or Triton X (1%). a Sytox Green fluorescence intensity (1 μM) was measured over time; results are calculated relative to iPrOH control fluorescence. b IL-1β was measured in the supernatant at 24 h by ELISA or c at 4 and 24 h by ELISA. a Data represent the Mean ± SEM of a representative experiment repeated three times. b , c Data represent the Mean ± SEM of three independent experiments performed in triplicate. Statistical significance was calculated in comparison to untreated control using Multiple t-test (Mann–Whitney), **** P ≤ 0.0001, *** P ≤ 0.001, * P ≤ 0.05.

Article Snippet: 100 ng/mL LPS (Sigma) or nigericin (10 μM, ChemImpex) were used as controls [ ].

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, Control, Comparison, MANN-WHITNEY

Journal: Inflammation

Article Title: Aryl-functionalised α,α′-Trehalose 6,6′-Glycolipid Induces Mincle-independent Pyroptotic Cell Death

doi: 10.1007/s10753-023-01814-5

Figure Lengend Snippet: Morphological changes induced by AF-2 are indicative of lytic cell death. a GM-CSF BMDMs from WT mice were seeded on cover slips coated with AF-2 or TDB (8 nmol/cover slip) or stimulated with nigericin (10 μM) and imaged by confocal microscopy after 4 h. Merged images of MPO (green), Histone 4 (red), and DAPI (blue). Scale bar: 50 μm. Scanning electron microscope (SEM) images of GM-CSF BMDMs and at b low (× 500) and c high (× 2,200–3,300) magnification following treatment with AF-2 (24 h, nmol/slide), staurosporine (5 h, 1 μm) as a control for apoptosis, or nigericin (1 h, 10 μM) as a control for pyroptosis. Scale bar: 10 μM). RAW264.7 cells were primed with LPS (1 μg/mL) and seeded on conductive silicon wafers coated with AF-2 (24 h, 8 nmol/slide) or stimulated with staurosporine (5 h, 1 μM) or nigericin (10 μM). Scanning electron cyro microscope (CyroSEM) images at d low (× 500) and e high (× 2,200–3,300) magnification. Scale bar: 10 μm.

Article Snippet: 100 ng/mL LPS (Sigma) or nigericin (10 μM, ChemImpex) were used as controls [ ].

Techniques: Confocal Microscopy, Microscopy, Control

Journal: Nucleic Acids Research

Article Title: Loss of the DNA repair protein, polynucleotide kinase/phosphatase, activates the type 1 interferon response independent of ionizing radiation

doi: 10.1093/nar/gkae654

Figure Lengend Snippet: Involvement of the cGAS-STING signalling pathway in the T1IFN response following PNKP depletion. ( A ) Effect of RNAi-mediated double downregulation of PNKP and cGAS or ZBP1 on the T1IFN response in MCF7 cells. ( B ) Effect of cGAS inhibition by G140 on the T1IFN response in PNKP-depleted MCF cells. For the western blots shown in (A) and (B), whole cell extracts were used to determine protein expression. ( C ) Densitometric analysis of the of the western blot signal for pSTAT1/STAT1 ratio in (B) above. ( D ) Measurement of cGAS activity in PNKP-depleted MCF7 cells relative to the control cells. 2′3′-cGAMP was determined from cell lysates using an ELISA. All data are presented as standard error of the mean of n = 4 experiments (** P < 0.01, *** P < 0.001). Statistical significance was determined using unpaired Student's t -test.

Article Snippet: Inhibitors of cGAS (G140, Inh-g140), cyclosporin A (CSA, 12088), JAK1/2 inhibitor (Ruxolitinib, 11609), STING inhibitor (C-178, 25860) were purchased from Cayman Chemical (Ann Arbor, Michigan, USA).

Techniques: Inhibition, Western Blot, Expressing, Activity Assay, Control, Enzyme-linked Immunosorbent Assay

Journal: Nucleic Acids Research

Article Title: Loss of the DNA repair protein, polynucleotide kinase/phosphatase, activates the type 1 interferon response independent of ionizing radiation

doi: 10.1093/nar/gkae654

Figure Lengend Snippet: Schematic diagram illustrating the underlying mechanism of how loss of PNKP activates the type 1 interferon response. Left panel: in the presence of PNKP, ROS-induced DNA damage fails to activate the T1IFN response as the strand break termini are constantly repaired by PNKP. However, in the absence of PNKP, the strand breaks induced by ROS are persistent, leading to the generation of smaller DNA fragments that are bound by ZBP1 and/or cGAS. Middle panel: activated cGAS synthesizes the second messenger molecule 2′3′-cGAMP, which binds to and activates STING leading to the downstream phosphorylation and activation of TBK1, IRF3 and subsequent synthesis and secretion of type 1 interferons such as IFNβ. Likewise, ZBP1, bound by oxidized DNA, is activated, and in complex with cGAS augments the activity of cGAS and STING. Right panel: the secreted IFNβ binds to its cognate receptor thereby promoting downstream phosphorylation events involving sequential JAK/TYK1 activation, STAT1/STAT2 activation and complex formation with IRF9. This ISGF3 complex translocates to the nucleus where it turns on interferon-stimulated genes. (Figure created with BioRender.com).

Article Snippet: Inhibitors of cGAS (G140, Inh-g140), cyclosporin A (CSA, 12088), JAK1/2 inhibitor (Ruxolitinib, 11609), STING inhibitor (C-178, 25860) were purchased from Cayman Chemical (Ann Arbor, Michigan, USA).

Techniques: Phospho-proteomics, Activation Assay, Activity Assay