Journal: The EMBO Journal

Article Title: A shorter splicing isoform antagonizes ZBP1 to modulate cell death and inflammatory responses

doi: 10.1038/s44318-024-00238-7

Figure Lengend Snippet: ( A ) Schematic depicting the alternatively-spliced transcript variants of murine Zbp1 , which encode the full-length protein (ZBP1-Long) and a short isoform that contains the two Zα domains but lacks the RHIMs (R). ( B ) UCSC Genome browser snapshot of the Zbp1 gene (173,206,612–173,218,923) on chromosome 2. Single-cell RNA sequencing data displayed as genome coverage from the Tabula Muris project (Tabula Muris et al, ). ( C – E ) Immunoblot of ZBP1 in total lysates of thymus and spleen ( C ), IFNα (1,000 U/ml)-treated lung fibroblasts ( D ), and BMDMs ( E ) from WT and Zbp1 −/− mice. TBP, GAPDH, and VINCULIN serve as loading controls. Data information: Data in ( D ) are representative of two independent experiments. .

Article Snippet: Previously generated vectors containing a single flag-tagged mouse ZBP1 coding sequence based on pCW-Cas9 (#50661, Addgene) were used to generate the required vectors for the expression of fluorescent fusion proteins (Jiao et al, ).

Techniques: RNA Sequencing Assay, Western Blot

Journal: The EMBO Journal

Article Title: A shorter splicing isoform antagonizes ZBP1 to modulate cell death and inflammatory responses

doi: 10.1038/s44318-024-00238-7

Figure Lengend Snippet: ( A ) Schematic depicting the generation of novel ZBP1-deficient mice ( Zbp1 −/− ) using CRISPR-Cas9-mediated gene targeting in C57BL/6N zygotes, as indicated. To generate Zbp1 −/− mice, most of the Zbp1 gene was removed by cutting once upstream of exon 2 and once within the 3′UTR in exon 8, leading to loss of the intervening region via nonhomologous end joining. The deletion event was confirmed by Sanger sequencing as indicated. ( B ) qPCR on RNA extracted from primary lung fibroblasts from three WT and three ZBP1 −/− mice. Cells were either untreated, treated for 20 h with IFNα, or pulsed with IFNα (20 h IFNα treatment followed by washing and 24 h without IFNα), and primers were used to amplify only the short (ZBP1 Short), only the long (ZBP1 Long) or both (ZBP1 L + S) Zbp1 isoforms. Data were presented as fold change relative to untreated WT samples. Dots represent individual mice. Mean ± SD is shown. ( C , D ). Immunoblot analysis with total lysates of lung fibroblasts from three WT mice and one Zbp1 −/− mouse treated with IFNα (18 h pretreatment) and indicated chemicals (CHX, 10 µg/ml ( C ) or MG132, 5 µM ( D )) for 6 or 12 h. Data information: Data were representative of 2 ( B ) independent experiments. .

Article Snippet: Previously generated vectors containing a single flag-tagged mouse ZBP1 coding sequence based on pCW-Cas9 (#50661, Addgene) were used to generate the required vectors for the expression of fluorescent fusion proteins (Jiao et al, ).

Techniques: CRISPR, Sequencing, Western Blot

Journal: The EMBO Journal

Article Title: A shorter splicing isoform antagonizes ZBP1 to modulate cell death and inflammatory responses

doi: 10.1038/s44318-024-00238-7

Figure Lengend Snippet: ( A , B ) Schematic depicting the generation of mice expressing only the long ( ZBP1 L ) ( A ) or short isoform of ZBP1 ( ZBP1 S ) ( B ) using CRISPR-Cas9-mediated gene targeting in C57BL/6 N zygotes, as indicated. To generate Zbp1 L/L mice, alternative exon 4 (alt. Ex4) was removed. To generate Zbp1 S/S mice, the region including exons 4 to 8 was removed. The resulting deletion (1218 bp for Zbp1 L and 6900 bp for Zbp1 S ) was confirmed by sequencing the new fusion site, and for Zbp1 S an additional 4 bp insertion was detected. ( C ) Immunoblot analysis of total lysates of lung fibroblasts from WT, Zbp1 L/L , Zbp1 S/S , and Zbp1 −/− mice treated with IFNα (1000 U/ml) for 24 h. ( D , E ). Cell death measured by DRAQ7 uptake ( D ) and immunoblot analysis of total lysates ( E ) of lung fibroblasts from WT, Zbp1 L/L , Zbp1 S/S and Zbp1 −/− mice stimulated with IFNα (1000 U/ml) (24-h pretreatment) alone or in combination with Emricasan (em) (2.5 µM) and etanercept (et) (50 µg/ml). ( F , G ) Cell death measured by DRAQ7 uptake ( F ) and immunoblot analysis of total lysates ( G ) of lung fibroblasts from WT, Zbp1 L/L , Zbp1 S/S , and Zbp1 −/− mice stimulated with IFNα (1000 U/ml) (24-h pretreatment) alone or in combination with KPT-330 (10 µM). Graphs show the percentage of cell death normalized to the total cell number obtained by 0.1% Triton X-100-induced cell lysis. All values are mean ± SEM from triplicate wells for each genotype ( n = 3). In immunoblot analyses, GAPDH was used as a loading control. Data information: Data were representative of 3 ( C – G ) independent experiments. .

Article Snippet: Previously generated vectors containing a single flag-tagged mouse ZBP1 coding sequence based on pCW-Cas9 (#50661, Addgene) were used to generate the required vectors for the expression of fluorescent fusion proteins (Jiao et al, ).

Techniques: Expressing, CRISPR, Sequencing, Western Blot, Lysis, Control

Journal: The EMBO Journal

Article Title: A shorter splicing isoform antagonizes ZBP1 to modulate cell death and inflammatory responses

doi: 10.1038/s44318-024-00238-7

Figure Lengend Snippet: ( A ) Representative macroscopic mouse pictures from mice with the indicated age and genotypes. ( B ) Kaplan–Meier plot depicting the time of sacrifice due to lesion severity of mice with the indicated genotypes. ( C ) Macroscopic skin score of mice with the indicated age and genotypes. ( D ) Representative images of skin sections from 6–10-week-old mice with the indicated genotypes, stained with haematoxylin and eosin (H&E), Trichrome, anti-keratin 6 (K6) antibodies, and Hoechst (DNA stain) or anti-keratin 10 (K10), anti-keratin 14 (K14) antibodies and Hoechst. Histological pictures shown are representative of control ( n = 7), RIPK1 E-KO Zbp1 WT/WT ( n = 16 for H&E and Trichrome), ( n = 10 for K6 and K10/K14); RIPK1 EKO Zbp1 WT/S ( n = 4), RIPK1 EKO Zbp1 S/S ( n = 3). Scale bars, 100 µm. ( E ) Microscopic quantification of epidermal thickness on the skin sections from 6–10- or 14–19 (old)-week-old mice with the indicated genotypes. ( F ) Flow cytometric quantification of CD45 + cells, CD4 + T cells (CD45 + CD3 + CD4 + ), neutrophils (CD45 + CD3 - NK1.1 - CD19 - CD11b + Ly6G + ), and inflammatory monocytes (CD45 + CD3 - NK1.1 - CD19 - Ly6G - CD11b + Ly6C + ) in skin from 6–10 or 14–19 (old) weeks old mice with indicated genotypes. Data information: Control mice include Ripk1 fl/fl that do not express K14-Cre or Ripk1 fl/wt K14-Cre mice, with wild type or Zbp1 S alleles. Dots represent individual mice. Mean ± SEM is shown. P values were calculated by one-way ANOVA with Tukey´s multiple comparisons test. .

Article Snippet: Previously generated vectors containing a single flag-tagged mouse ZBP1 coding sequence based on pCW-Cas9 (#50661, Addgene) were used to generate the required vectors for the expression of fluorescent fusion proteins (Jiao et al, ).

Techniques: Staining, Control

Journal: The EMBO Journal

Article Title: A shorter splicing isoform antagonizes ZBP1 to modulate cell death and inflammatory responses

doi: 10.1038/s44318-024-00238-7

Figure Lengend Snippet: Gating strategy for analysing immune cells in digested skin with flow cytometry. The dot plots from RIPK1 E-KO Zbp1 L/L mouse is used as representative. The viability of cells is determined by the staining with LIVE/DEAD TM Fixable Near-IR Dead Cell Stain Kit (Live/Dead). Dead cells are presented as Live/Dead + , and live cells are presented as Live/Dead ─ . FSC forward scatter, SSC side scatter.

Article Snippet: Previously generated vectors containing a single flag-tagged mouse ZBP1 coding sequence based on pCW-Cas9 (#50661, Addgene) were used to generate the required vectors for the expression of fluorescent fusion proteins (Jiao et al, ).

Techniques: Flow Cytometry, Staining

Journal: The EMBO Journal

Article Title: A shorter splicing isoform antagonizes ZBP1 to modulate cell death and inflammatory responses

doi: 10.1038/s44318-024-00238-7

Figure Lengend Snippet: ( A ) Representative macroscopic mouse pictures from mice with the indicated age and genotype. The images of the control and RIPK1 E-KO mice at P45 are the same as in Fig. and are included here for comparison. ( B ) Kaplan–Meier plot depicting the time of sacrifice due to lesion severity of mice with the indicated genotype. The same group of RIPK1 E-KO mice as in Fig. 3B is included here for comparison. ( C ) Macroscopic skin scores of 1–2-week-old mice with the indicated genotypes. ( D ) Representative images of skin sections from 1–2-week-old mice with the indicated genotypes, stained with haematoxylin and eosin (H&E), Trichrome, anti-keratin 6 (K6) antibodies and Hoechst or anti-keratin 10 (K10), anti-keratin 14 (K14) antibodies and Hoechst. Histological pictures shown are representative of control ( n = 24), RIPK1 E-KO Zbp1 WT/WT ( n = 7), RIPK1 E-KO Zbp1 WT/L ( n = 13), RIPK1 E-KO Zbp1 L/L ( n = 6). Scale bars, 100 µm. ( E ) Microscopic quantification of epidermal thickness on the skin sections from 1–2-week-old mice with the indicated genotypes. ( F ) Flow cytometric quantification of CD45 + cells, CD4 + T cells (CD45 + CD3 + CD4 + ), neutrophils (CD45 + CD3 - NK1.1 - CD19 - CD11b + Ly6G + ), and inflammatory monocytes (CD45 + CD3 - NK1.1 - CD19 - Ly6G - CD11b + Ly6C + ) in the skin from 1–2-week-old mice with indicated genotypes. Data information: Control mice include Ripk1 fl/fl that do not express K14-Cre, or Ripk1 fl/wt K14-Cre mice, with wild type or Zbp1 L alleles. Dots represent individual mice. Mean ± SEM is shown. P values were calculated by one-way ANOVA with Tukey´s multiple comparisons test. .

Article Snippet: Previously generated vectors containing a single flag-tagged mouse ZBP1 coding sequence based on pCW-Cas9 (#50661, Addgene) were used to generate the required vectors for the expression of fluorescent fusion proteins (Jiao et al, ).

Techniques: Control, Comparison, Staining

Journal: The EMBO Journal

Article Title: A shorter splicing isoform antagonizes ZBP1 to modulate cell death and inflammatory responses

doi: 10.1038/s44318-024-00238-7

Figure Lengend Snippet: ( A ) Schematic depicting the fusion proteins of ZBP1-L-NG, ZBP1mZα-L-NG, or ZBP1-S-Sc and the Dox-inducible systems. ( B ) Immunoblot analysis of total lysates from immortalized MEFs expressing doxycycline (dox)-inducible ZBP1-L-NG, ZBP1-S-Sc, ZBP1-L-NG with moderate or high expression levels of ZBP1-S-Sc, ZBP1mZα-L-NG, and empty vector (EV) stimulated with doxycycline for 24 h. ( C , D ) Cell death measured by DRAQ7 uptake ( C ) and immunoblot analysis of total lysates ( D ) in iMEFs expressing the indicated fusion proteins stimulated with doxycycline (1 µg/ml) (24 h pretreatment) and Emricasan (5 µM). ( E , F ) Cell death measured by DRAQ7 uptake ( E ) and immunoblot analysis of total lysates ( F ) in iMEFs expressing the indicated fusion proteins stimulated with doxycycline (1 µg/ml) (24 h pretreatment) and KPT-330 (10 µM). Cell death graphs show the percentage of cell death normalized to the total cell number obtained by 0.1% Triton X-100-induced cell lysis. Each value is presented as mean ± SEM from triplicate wells for each cell population ( n = 3). For immunoblot analyses, α-TUBULIN ( D ) and GAPDH ( F ) were used as loading controls. Data information: Data were representative of 1 ( B ), 3 ( C – E ), 4 ( F ) independent experiments. .

Article Snippet: Previously generated vectors containing a single flag-tagged mouse ZBP1 coding sequence based on pCW-Cas9 (#50661, Addgene) were used to generate the required vectors for the expression of fluorescent fusion proteins (Jiao et al, ).

Techniques: Western Blot, Expressing, Plasmid Preparation, Lysis

Journal: The EMBO Journal

Article Title: A shorter splicing isoform antagonizes ZBP1 to modulate cell death and inflammatory responses

doi: 10.1038/s44318-024-00238-7

Figure Lengend Snippet: ( A ) Schematic depicting the fusion proteins of ZBP1-L-NG, ZBP1-S-Sc, or ZBP1mZα-S-Sc and the combination of the Dox and cumate-inducible systems. ( B ) Immunoblot analysis of total lysates from iMEFs expressing doxycycline (dox)-induced empty vector (EV), the combination of dox-induced ZBP1-L-NG with ZBP1-S-Sc or ZBP1mZα-S-Sc stimulated with a combination of doxycycline (1 µg/ml) and cumate (100 µg/ml). ( C , D ) Cell death measured by DRAQ7 uptake ( C ) and immunoblot analysis of total lysates ( D ) in iMEFs expressing the indicated fusion proteins stimulated with combinations of doxycycline (1 µg/ml), cumate (50 µg/ml) (24 h pretreatment), and Emricasan (5 µM). ( E , F ). Cell death measured by DRAQ7 uptake ( E ) and immunoblot analysis of total lysates ( F ) in iMEFs expressing the indicated fusion proteins stimulated with combinations of doxycycline (1 µg/ml), cumate (50 µg/ml) (24 h pretreatment) and KPT-330 (10 µM). Cell death graphs show the percentage of cell death normalized to the total cell number obtained by 0.1% Triton X-100-induced cell lysis. Each value is presented as mean ± SEM from triplicate wells for each cell population ( n = 3). For immunoblot analyses, GAPDH ( B ) and α-TUBULIN ( D , F ) were used as loading controls. Data information: Data were representative of 2 ( B ), 3 ( C – F) independent experiments. .

Article Snippet: Previously generated vectors containing a single flag-tagged mouse ZBP1 coding sequence based on pCW-Cas9 (#50661, Addgene) were used to generate the required vectors for the expression of fluorescent fusion proteins (Jiao et al, ).

Techniques: Western Blot, Expressing, Plasmid Preparation, Lysis

Journal: The EMBO Journal

Article Title: A shorter splicing isoform antagonizes ZBP1 to modulate cell death and inflammatory responses

doi: 10.1038/s44318-024-00238-7

Figure Lengend Snippet: ( A ) Immunoblot analysis of total lysates from iMEFs expressing doxycycline (dox)-induced ZBP1-L-NG, dox-induced ZBP1-S-Sc, the combination of dox-induced ZBP1-L-NG with ZBP1-S-Sc, dox-induced ZBP1mZα-L-NG,dox-induced empty vector (EV) stimulated with doxycycline (1 µg/ml) or cumate-induced ZBP1mZα-S-Sc stimulated with cumate (10 µg/ml) for 24 h. ( B ) Cell death measured by DRAQ7 uptake in iMEFs expressing the indicated fusion proteins stimulated with a combination of doxycycline (1 µg/ml) or cumate (10 µg/ml) (24 h pretreatment) and Emricasan (2.5 µM, left) or KPT-330 (10 µM, right). Graphs show the percentage of cell death normalized to the total cell number obtained by 0.1% Triton X-100-induced cell lysis. Each value is presented as mean ± SEM from triplicate wells for each cell population ( n = 3). ( C ) Immunoblot analysis of anti-NeonGreen (left) and anti-mScarlet (right) immunoprecipitates and total lysates (input) from iMEFs expressing the indicated fusion proteins stimulated with doxycycline (1 µg/ml) alone for 24 h ( D ) or the combination of doxycycline (24 h pre-stimulation) with Emricasan (2.5 µM) or KPT-330 (10 µM) for 6 h. ( D ) Immunoblot analysis of J2 anti-dsRNA monoclonal antibody immunoprecipitates and total lysates (input) from iMEFs expressing indicated fusion proteins stimulated with doxycycline (1 µg/ml) for 24 h. GAPDH ( C ) and VINCULIN ( A , D ) serve as loading control. Data information: Data were representative of 2 ( A ), 3 ( B – D ) independent experiments. .

Article Snippet: Previously generated vectors containing a single flag-tagged mouse ZBP1 coding sequence based on pCW-Cas9 (#50661, Addgene) were used to generate the required vectors for the expression of fluorescent fusion proteins (Jiao et al, ).

Techniques: Western Blot, Expressing, Plasmid Preparation, Lysis, Control