pcdna3 1 matf4  (Addgene inc)

Journal: bioRxiv

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1101/2025.03.12.642754

Figure Lengend Snippet: (A) Gene set enrichment analysis of RNA-seq data of RPE1 TP53 KO control and EIF2A KO cells (more detailed analysis in Suppl. Fig. 3A). Circle size represents gene set size. (B, C) SILAC-based proteomics analysis of RPE1 TP53 KO control and EIF2A KO cells, showing differential protein expression at baseline (panel B) and upon AZD1775 treatment for 6, 24 and 48 h (panel C). (D) RPE1 TP53 KO control and EIF2A KO cells were treated with AZD1775 or Debio 0123 (1 µM) for 24 h, followed by a 10’ puromycin pulse. Puromycin incorporation was visualized by immunoblotting (left panel). Quantification of immunoblots is indicated (right panel). Data represent mean ± SD; RPE1 TP53 KO EIF2A KO treated with Debio 0123 (n=4); control RPE1 TP53 KO treated with DMSO or AZD1775 (n=7); Other conditions (n=5). (E) RPE1 TP53 KO PAC KO cells were treated with AZD1775 (250 nM) and ISRIB for 24 h and subsequently labeled with L-azidohomoalanine (AHA). Immunoblot of AHA-labeled proteins and quantification is shown. Data represent mean ± SD (n=2). (F) Schematic overview of the CRISPR/Cas9 genome-wide AZD1775 resistance screen. (G) MAGeCK scores for individual genes in the RPE1 TP53 KO AZD1775 positive selection screen (day 12 vs day 0). (H) Cell confluency analysis of parental RPE1 TP53 KO PAC KO or GCN2 KO cells. Mean ± SD, n=3 for control and GCN2 KO #1, n=2 for GCN2 KO #2. (I) Quantification of clonogenic survival assays of parental RPE1 TP53 KO PAC KO and ATF4 KO #1 and #2 cells (corresponding images provided in Suppl. Fig. 3H). Mean ± SD, n=3 for all conditions. (J, K) Representative histograms (panel J) and quantification (panel K) of ATF4-mScarlet flow cytometry measurements in RPE1 TP53 KO ATF4-mScarlet reporter cells treated with thapsigargin (1 µM), AZD1775 (1 µM), Debio 0123 (1 µM) and/or A92 (1 µM) for 24 h. Data represent mean ± SD (n=3). (L) Parental RPE1 TP53 KO PAC KO , ATF4 KO #1 and GCN2 KO #2 cells were treated with AZD1775 (0, 250 or 500 nM) for 24 h, and indicated proteins were immunoblotted. Statistical analysis of panels D, K: unpaired t-test with p ≤ 0.05 considered significant.

Article Snippet: Proteins were separated by SDS-PAGE and the gel was stained with Ponceau S. Afterwards, proteins were transferred onto nitrocellulose membranes and subsequently blocked with 5% milk or in 0.1% TBST and blotted with the following primary antibodies for 1 h at room temperature (RT) or overnight at 4°C: 1:1000 rabbit anti-phospho-eIF2a Ser51 (Cell Signaling, #3398), 1:1000 rabbit anti-phospho-eIF2a Ser51 (Cell Signaling, #9721), 1:1000 rabbit anti-eIF2a (Cell Signaling, #9722), 1:1000 rabbit anti-phospho-GCN2 Thr899 (Abcam, ab75836), 1:1000 rabbit anti-GCN2 (Abcam, ab134053), 1:1000 rabbit anti-ZNF598 (Sigma, HPA041760), 1:1000 rabbit anti-PERK (Cell Signaling, #5683), 1:1000 rabbit anti-phospho-cdc2 (CDK1) Tyr15 (Cell Signaling, #4539), 1:500 rabbit anti-ATF4 (Cell Signaling, #11815), 1:500 mouse anti-ATF4 (Cell Signaling, #97038), 1:500 rabbit anti-WEE1 (Cell Signaling, #13084).

Techniques: RNA Sequencing, Control, Expressing, Western Blot, Labeling, CRISPR, Genome Wide, Selection, Flow Cytometry

Journal: bioRxiv

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1101/2025.03.12.642754

Figure Lengend Snippet: (A) RPE1 TP53 KO PAC KO cells were treated with AZD1775 (250 nM) for 6 h in the presence or absence of ISRIB (1 μM), pulse labeled with EdU, and processed for quantitative image-based cytometry. Cell cycle stage was defined by DNA content and EdU positivity. Median ATF4 intensity from n=2 experiments is shown. (B, C) Experimental set-up (panel B). RPE1 TP53 KO ATF4-mScarlet-NLS reporter cells were treated overnight with nocodazole. Mitotic cells were isolated and replated in the presence or absence of palbociclib. After 2 h AZD1775 (1 µM) and/or A92 (1 µM) was added for 20 h (Panel B). mScarlet levels in RPE1 TP53 KO ATF4-mScarlet reporter cells were measured by flow cytometry. Data represent mean ± SD (n=3)(panel C). (D, E) Flow cytometry gating strategy (panel D) and analysis of γH2AX in RPE1 TP53 KO cells after treatment with AZD1775 (1 µM) and/or A92 (1 µM) in the absence (panel E, left) or presence (panel E, right) of nocodazole. Data represent mean ± SD (n=4). (F) Flow cytometry analysis of MPM2-positivity in RPE1 TP53 KO cells after treatment with nocodazole, doxorubicin, AZD1775 (1 µM) and/or A92 (1 µM). Data represent mean ± SD (n=3). (G) Flow cytometry analysis of MPM2-positivity in control RPE1 TP53 KO PAC KO and ATF4 KO #2 or GCN2 KO #2 cells after treatment with nocodazole, doxorubicin and AZD1775 (1 µM). Data represent mean ± SD (n=3). (H) Immunoblot of resting PBMCs or PBMCs stimulated with anti-CD3/CD28 beads. PBMCs were treated with DMSO, AZD1775 (500 nM) or thapsigargin (500 nM) for 24 h. (I) RPE1 TP53 KO PAC KO and ATF4 KO #2 or GCN2 KO #2 cells were treated with AZD1775 and/or cisplatin for 5 days. Cell survival was analyzed by MTT conversion. Data represent mean ± SD (n=4). Statistical analysis was performed using unpaired t-tests, with p ≤ 0.05 considered significant.

Article Snippet: Proteins were separated by SDS-PAGE and the gel was stained with Ponceau S. Afterwards, proteins were transferred onto nitrocellulose membranes and subsequently blocked with 5% milk or in 0.1% TBST and blotted with the following primary antibodies for 1 h at room temperature (RT) or overnight at 4°C: 1:1000 rabbit anti-phospho-eIF2a Ser51 (Cell Signaling, #3398), 1:1000 rabbit anti-phospho-eIF2a Ser51 (Cell Signaling, #9721), 1:1000 rabbit anti-eIF2a (Cell Signaling, #9722), 1:1000 rabbit anti-phospho-GCN2 Thr899 (Abcam, ab75836), 1:1000 rabbit anti-GCN2 (Abcam, ab134053), 1:1000 rabbit anti-ZNF598 (Sigma, HPA041760), 1:1000 rabbit anti-PERK (Cell Signaling, #5683), 1:1000 rabbit anti-phospho-cdc2 (CDK1) Tyr15 (Cell Signaling, #4539), 1:500 rabbit anti-ATF4 (Cell Signaling, #11815), 1:500 mouse anti-ATF4 (Cell Signaling, #97038), 1:500 rabbit anti-WEE1 (Cell Signaling, #13084).

Techniques: Labeling, Cytometry, Isolation, Flow Cytometry, Control, Western Blot

Journal: bioRxiv

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1101/2025.03.12.642754

Figure Lengend Snippet: (A) Relative gene and protein expression upon treatment with AZD1775 (24 h, 1 µM) measured by Ribo-seq and RNA-seq. (B) Differential protein expression upon treatment with AZD1775 (24 h, 1 µM) measured by Ribo-seq. (C) Pathway enrichment analysis of Ribo-seq data shown in (M). (D) Gene expression of ATF4-target genes DDIT4, DDIT3, ATF3 and PPP1R15A upon treatment with AZD1775 (24 h, 1 µM). (E) Codon usage in RPE1 TP53 KO cells upon treatment with DMSO or AZD1775 (24 h, 1 µM).

Article Snippet: Proteins were separated by SDS-PAGE and the gel was stained with Ponceau S. Afterwards, proteins were transferred onto nitrocellulose membranes and subsequently blocked with 5% milk or in 0.1% TBST and blotted with the following primary antibodies for 1 h at room temperature (RT) or overnight at 4°C: 1:1000 rabbit anti-phospho-eIF2a Ser51 (Cell Signaling, #3398), 1:1000 rabbit anti-phospho-eIF2a Ser51 (Cell Signaling, #9721), 1:1000 rabbit anti-eIF2a (Cell Signaling, #9722), 1:1000 rabbit anti-phospho-GCN2 Thr899 (Abcam, ab75836), 1:1000 rabbit anti-GCN2 (Abcam, ab134053), 1:1000 rabbit anti-ZNF598 (Sigma, HPA041760), 1:1000 rabbit anti-PERK (Cell Signaling, #5683), 1:1000 rabbit anti-phospho-cdc2 (CDK1) Tyr15 (Cell Signaling, #4539), 1:500 rabbit anti-ATF4 (Cell Signaling, #11815), 1:500 mouse anti-ATF4 (Cell Signaling, #97038), 1:500 rabbit anti-WEE1 (Cell Signaling, #13084).

Techniques: Expressing, RNA Sequencing, Gene Expression

Journal: bioRxiv

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1101/2025.03.12.642754

Figure Lengend Snippet: (A) Relative ATF4-mScarlet MFI in RPE1 ATF4-mScarlet reporter cells after treatment with 125, 250, 500, or 1000 nM of AZD1775, RP-6306, VE-822 or AZD7762. Data represent mean ± SD (DMSO: n=4; other conditions: n=3). Statistical analysis was performed using a one-way ANOVA multiple comparisons with p≤0.05 considered significant. (B) Immunoblot of RPE1 TP53 KO cells treated with siRNA targeting WEE1 for 72 h and AZD1775 (250 nM) for 2.5 or 5 h. (C, D) NanoBRET kinase target engagement assay performed with HEK293T cells transfected with WEE1-NanoLuc (panel C) and NanoLuc-GCN2 (panel D) and incubated with K-10 tracer (0.5 μM) and indicated doses of CC1, AZD1775, Debio 0123 and ZNL-02-096 for 2 h prior to substrate addition and BRET signal detection.

Article Snippet: Proteins were separated by SDS-PAGE and the gel was stained with Ponceau S. Afterwards, proteins were transferred onto nitrocellulose membranes and subsequently blocked with 5% milk or in 0.1% TBST and blotted with the following primary antibodies for 1 h at room temperature (RT) or overnight at 4°C: 1:1000 rabbit anti-phospho-eIF2a Ser51 (Cell Signaling, #3398), 1:1000 rabbit anti-phospho-eIF2a Ser51 (Cell Signaling, #9721), 1:1000 rabbit anti-eIF2a (Cell Signaling, #9722), 1:1000 rabbit anti-phospho-GCN2 Thr899 (Abcam, ab75836), 1:1000 rabbit anti-GCN2 (Abcam, ab134053), 1:1000 rabbit anti-ZNF598 (Sigma, HPA041760), 1:1000 rabbit anti-PERK (Cell Signaling, #5683), 1:1000 rabbit anti-phospho-cdc2 (CDK1) Tyr15 (Cell Signaling, #4539), 1:500 rabbit anti-ATF4 (Cell Signaling, #11815), 1:500 mouse anti-ATF4 (Cell Signaling, #97038), 1:500 rabbit anti-WEE1 (Cell Signaling, #13084).

Techniques: Western Blot, Transfection, Incubation

Journal: bioRxiv

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1101/2025.03.12.642754

Figure Lengend Snippet: (A) Relative ATF4-mScarlet MFI in RPE1 TP53 KO ATF4-mScarlet reporter cells after treatment with the indicated doses of Debio 0123. Data represent mean ± SD (n=3). (B) Cell viability in parental RPE1 TP53 KO PAC KO , GCN2 KO #1 and GCN2 KO #2 cells after treatment with Debio 0123 and RP-6306 at the indicated doses (n=5). (C) ZIP synergy scores from cell survival matrices shown in and . Data represent mean ± SD (n=5). (D) Synergy plots with ZIP synergy scores of heatmaps shown in . (E) Relative ATF4-mScarlet MFI in RPE1 TP53 KO ATF4-mScarlet reporter cells treated with the indicated doses of AZD1775, Debio 0123 and RP-6306. Data represent mean ± SD (n=4).

Article Snippet: Proteins were separated by SDS-PAGE and the gel was stained with Ponceau S. Afterwards, proteins were transferred onto nitrocellulose membranes and subsequently blocked with 5% milk or in 0.1% TBST and blotted with the following primary antibodies for 1 h at room temperature (RT) or overnight at 4°C: 1:1000 rabbit anti-phospho-eIF2a Ser51 (Cell Signaling, #3398), 1:1000 rabbit anti-phospho-eIF2a Ser51 (Cell Signaling, #9721), 1:1000 rabbit anti-eIF2a (Cell Signaling, #9722), 1:1000 rabbit anti-phospho-GCN2 Thr899 (Abcam, ab75836), 1:1000 rabbit anti-GCN2 (Abcam, ab134053), 1:1000 rabbit anti-ZNF598 (Sigma, HPA041760), 1:1000 rabbit anti-PERK (Cell Signaling, #5683), 1:1000 rabbit anti-phospho-cdc2 (CDK1) Tyr15 (Cell Signaling, #4539), 1:500 rabbit anti-ATF4 (Cell Signaling, #11815), 1:500 mouse anti-ATF4 (Cell Signaling, #97038), 1:500 rabbit anti-WEE1 (Cell Signaling, #13084).

Techniques:

Journal: Shock (Augusta, Ga.)

Article Title: TARGETING S100A9-TLR2 AXIS CONTROLS MACROPHAGE NLRP3 INFLAMMASOME ACTIVATION IN FATTY LIVER ISCHEMIA REPERFUSION INJURY

doi: 10.1097/SHK.0000000000002470

Figure Lengend Snippet: IR-induced injury leads to increased S100A9 and related pathway activation in fatty livers HFD fed C57 mice were used to establish liver IR models. Samples were harvested after 90-min ischemia and 6 h of reperfusion. (A) Serum ALT levels. (B) Immunofluorescence staining and quantification of CD11b + macrophages in ischemia fatty livers, scale bars: 50 μm, 20 μm. (C) Volcano Plot displaying 748 genes to be upregulated and 625 genes downregulated. (D) KEGG pathway enrichment analysis of major biological pathways contributing to fatty liver IR stress. (E) Heatmap showing the expression of different genes. (F) Western-assisted analysis and relative density ratio of S100A9, TLR2, and ATF4 in liver tissues. (G) Immunochemistry staining and quantification of S100A9 positive cells in ischemia fatty livers, scale bars: 50 μm, 20 μm. (H) Detection of cytokines IL-1β, CXCL1, CXCL-2, and IL-6 by qRT-PCR in the livers. N = 3–6/group. All data represent the mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001. ALT, alanine aminotransferase; HFD, high-fat diet; KEGG, Kyoto Encyclopedia of Genes and Genomes; IR, ischemia reperfusion.

Article Snippet: Macophage ATF4 was detected using primary ATF4 mouse mAb (60035-1-Ig, Proteintech).

Techniques: Activation Assay, Immunofluorescence, Staining, Expressing, Western Blot, Quantitative RT-PCR

Journal: Shock (Augusta, Ga.)

Article Title: TARGETING S100A9-TLR2 AXIS CONTROLS MACROPHAGE NLRP3 INFLAMMASOME ACTIVATION IN FATTY LIVER ISCHEMIA REPERFUSION INJURY

doi: 10.1097/SHK.0000000000002470

Figure Lengend Snippet: S100A9 induces inflammatory injury via macrophage TLR2 activation BMMs were isolated from WT mice and incubated with rS100A9 or rS100A8 respectively for 24 h. (A) Western-assisted analysis of TLR2 and ATF4 in rS100A9-stressed macrophages. (B) Western-assisted analysis of TLR2 and ATF4 in rS100A8-stressed macrophages; BMMs were transfected with the TLR2-siRNA or control vector followed by incubation with rS100A9. (C) Detection of cytokines IL-1β, CXCL1, CXCL-2, and IL-6 by qRT-PCR in BMMs. (D) Western-assisted analysis of ATF4 in nuclear extracts. (E) Immunofluorescence staining for ATF4 distribution in macrophages. DAPI was used to visualize nuclei. Scale bars: 50 μm, 20 μm. N = 3–6/group. All data represent the mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001. BBM, bone-derived macrophage. WT, wild type; qRT-PCR, quantitative real-time PCR.

Article Snippet: Macophage ATF4 was detected using primary ATF4 mouse mAb (60035-1-Ig, Proteintech).

Techniques: Activation Assay, Isolation, Incubation, Western Blot, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Immunofluorescence, Staining, Derivative Assay, Real-time Polymerase Chain Reaction

Journal: Shock (Augusta, Ga.)

Article Title: TARGETING S100A9-TLR2 AXIS CONTROLS MACROPHAGE NLRP3 INFLAMMASOME ACTIVATION IN FATTY LIVER ISCHEMIA REPERFUSION INJURY

doi: 10.1097/SHK.0000000000002470

Figure Lengend Snippet: S100A9 knockdown attenuates liver dysfunction and inflammatory damage following IR of fatty livers C57 mice were injected with AAV9-S100A9-shRNA 48 h before establishing liver IR models using HFD-fed mice. Samples were harvested after 90-min ischemia and 6 h of reperfusion. (A) Western-assisted analysis and relative density ratio of S100A9 in liver tissues. (B) H&E staining of ischemic fatty livers, scale bar: 200 μm, 100 μm. (C) Serum ALT levels. (D) Immunofluorescence staining and quantification of CD11b positive macrophages and immunochemistry staining and quantification of Ly6G positive cells in ischemia fatty livers, scale bar: 40 μm. (E) Western-assisted analysis of TLR2 and ATF4 in liver tissues. (F) Dual immunofluoresence staining for CD68 (red) and TLR2 (green) co-localization in injured fatty livers after IR stimulation, scale bars: 50 μm, 20 μm. (G) Detection of cytokines IL-1β, CXCL1, CXCL-2, and IL-6 by qRT-PCR in the livers. N = 3–6/group. All data represent the mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001. HFD, high-fat diet; IR, ischemia reperfusion. qRT-PCR, quantitative real-time PCR.

Article Snippet: Macophage ATF4 was detected using primary ATF4 mouse mAb (60035-1-Ig, Proteintech).

Techniques: Knockdown, Injection, shRNA, Western Blot, Staining, Immunofluorescence, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Shock (Augusta, Ga.)

Article Title: TARGETING S100A9-TLR2 AXIS CONTROLS MACROPHAGE NLRP3 INFLAMMASOME ACTIVATION IN FATTY LIVER ISCHEMIA REPERFUSION INJURY

doi: 10.1097/SHK.0000000000002470

Figure Lengend Snippet: ATF4 is responsible for regulating S100A9-triggered NEK7/NLRP3 inflammasome activation in macrophages BMMs were transfected with the ATF4-siRNA or control vector followed by incubation with rS100A9 . (A) Detection of cytokines IL-1β, CXCL1, CXCL-2, and IL-6 by qRT-PCR in BMMs. (B) IL-1β release from BMMs. (C) Western-assisted analysis of NEK7 and NLRP3 in BMMs. (D) Dual immunofluoresence staining for NEK7 (red) and NLRP3 (green) co-localization in BMMs, scale bars: 40 μm, 20 μm. N = 3–6/group. All data represent the mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001. BBM, bone-derived macrophage.

Article Snippet: Macophage ATF4 was detected using primary ATF4 mouse mAb (60035-1-Ig, Proteintech).

Techniques: Activation Assay, Transfection, Control, Plasmid Preparation, Incubation, Quantitative RT-PCR, Western Blot, Staining, Derivative Assay

Journal: Shock (Augusta, Ga.)

Article Title: TARGETING S100A9-TLR2 AXIS CONTROLS MACROPHAGE NLRP3 INFLAMMASOME ACTIVATION IN FATTY LIVER ISCHEMIA REPERFUSION INJURY

doi: 10.1097/SHK.0000000000002470

Figure Lengend Snippet: The schematic diagram of S100A9 regulating IR-induced macrophage NLRP3 inflammasome activation in fatty livers. During fatty liver IR stress, S100A9 is released and accumulated in injured livers. S100A9 interacts with macrophage TLR2, promoting ATF4 activation and the formation of NEK7/NLRP3 inflammasome. It leads to subsequent inflammatory response and liver damage. IR, ischemia reperfusion.

Article Snippet: Macophage ATF4 was detected using primary ATF4 mouse mAb (60035-1-Ig, Proteintech).

Techniques: Activation Assay