pgl3 zscan4 promoter plasmid  (Addgene inc)

Journal: bioRxiv

Article Title: Mitochondrial Respiratory Chain Function is crucial for Muscle Toxicity in Facioscapulohumeral Muscular Dystrophy

doi: 10.1101/2025.11.25.690559

Figure Lengend Snippet: (A) Left: longitudinal quantitation of DUX4 and ZSCAN4-tdTomato containing nuclei in iDUX4 ZSCAN4-tdT reporter myoblasts at 2h intervals after DOX (62.5 ng/mL). Right: immunofluorescence micrographs of iDUX4 ZSCAN4-tdT reporter myoblasts before (0h), and 4h and 16h after exposure to DOX (62.5 ng/mL; green=DUX4, red=ZSCAN4-tdTomato, blue=nuclei; scale bar=100μm). (B) Normalised oxygen consumption rate (OCR) curves of iDUX4 control (CTRL) and DOX-induced (62.5 ng/mL DOX for 4h and 16h) myoblasts. (C) Respirometric assessment of mitochondrial respiration reveals reduction of basal, maximal and ATP-linked respiration in iDUX4 myoblasts 4h after DOX (62.5 ng/mL). (D) Normalised extracellular acidification rate (ECAR) curves of iDUX4 control (CTRL) and DOX-induced (62.5 ng/mL DOX for 4h and 16h) myoblasts. (E) ATP production rates demonstrating reduction of ATP in iDUX4 myoblasts 4h after DOX (62.5 ng/mL), mainly arising from defective oxidative phosphorylation (OXPHOS; mitoATP), while glycoATP is unaffected before 16h. (F) Assaying of Casp9 (red; onset 4h after DOX) and Casp3/7 activation (grey; onset 8h after DOX), measured at 2h intervals over 16h. (G) Assaying Annexin V as a marker of apoptosis in iDUX4 myoblasts after DOX (62.5 ng/mL), measured at 2h intervals over 16h. (H) Casp3/7 activation in iDUX4 myoblasts 8h after DOX (62.5 ng/mL) is prevented by a Casp9 inhibitor (Z-LEHD-FMK TFA; 10 μM). (I) Casp3/7 and Casp9 activation in iDUX4 myoblasts 8h after DOX (62.5 ng/mL) with non-targeted (Q10; 20 μM) or mitochondria-targeted (mitoQ10; 20μM) Coenzyme Q10. [n=3-6, data is mean ± s.d., where * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001].

Article Snippet: The inducible iDUX4 ZSCAN4-tdT reporter line was generated by transduction of iDUX4 myoblasts with a Lentiviral vector encoding tdTomato with nuclear localisation signals on both ends under control of the minimal promoter of ZSCAN4 (Fig. S6). pGL3 Zscan4 promoter plasmid was a gift from Stephen Tapscott (Addgene plasmid #69249; http://n2t.net/addgene:69249 ; RRID: Addgene_69249) [ ].

Techniques: Quantitation Assay, Immunofluorescence, Control, Phospho-proteomics, Activation Assay, Marker

Journal: bioRxiv

Article Title: Mitochondrial Respiratory Chain Function is crucial for Muscle Toxicity in Facioscapulohumeral Muscular Dystrophy

doi: 10.1101/2025.11.25.690559

Figure Lengend Snippet: (A) Workflow for generation of ρ 0 -like iDUX4 myoblasts: cells were treated with ethidium bromide (50, 100 or 150 nM EtBr) for two weeks, yielding ρ 0 -like iDUX4 cells with varying degrees of OXPHOS impairment (iDUX4-ρ 0+ : mild OXPHOS reduction, iDUX4-ρ 0++ : severe OXPHOS reduction and iDUX4-ρ 0+++ : full OXPHOS inhibition). Scheme was created with BioRender.com . (B) Percentage of OXPHOS versus glycolytic ATP production rate in iDUX4-ρ 0 myoblasts, with mitoATP and glycoATP production rates indicated, as determined by Seahorse respirometry. (C) Immunofluorescence (green=DUX4, blue=nuclei; scale bar=100μm) with percentage of DUX4-positive nuclei of unmodified iDUX4 and iDUX4- ρ 0+++ myoblasts after DOX (62.5 ng/mL) for 16h. (D) Expression levels of DUX4 target genes ZSCAN4 , PRAMEF1 and TRIM43 , as assessed by RT-qPCR (relative to housekeeper RPLP0 ). (E) DUX4-induced increases in mitoROS and ΔΨm in unmodified iDUX4 cells are not observed in iDUX4- ρ 0+++ myoblasts after DOX (62.5 ng/mL) for 16h. (F) DUX4-induced Casp9 activation in unmodified iDUX4 myoblasts does not occur in iDUX4- ρ 0+++ cells after DOX (62.5 ng/mL) for 8h. (G) Annexin V and Casp3/7 activation in iDUX4, iDUX4-ρ 0+ , iDUX4-ρ 0++ and iDUX4-ρ 0+++ myoblasts after DOX (62.5 ng/mL) for 36h shows inverse correlation between DUX4-induced apoptosis and mitochondrial respiratory chain impairment. DUX4 is unable to induce apoptosis in fully OXPHOS-deficient iDUX4-ρ 0+++ myoblasts. (H) Both non-DUX4-induced iDUX4 and iDUX4-ρ 0+++ myoblasts undergo apoptosis (Annexin V) after exposure to the apoptosis inducer staurosporine (STSP; 1 μM) for 48h. [n=3-12, data is mean ± s.d., where * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001].

Article Snippet: The inducible iDUX4 ZSCAN4-tdT reporter line was generated by transduction of iDUX4 myoblasts with a Lentiviral vector encoding tdTomato with nuclear localisation signals on both ends under control of the minimal promoter of ZSCAN4 (Fig. S6). pGL3 Zscan4 promoter plasmid was a gift from Stephen Tapscott (Addgene plasmid #69249; http://n2t.net/addgene:69249 ; RRID: Addgene_69249) [ ].

Techniques: Inhibition, Immunofluorescence, Expressing, Quantitative RT-PCR, Activation Assay

Journal: Cell & bioscience

Article Title: ZSCAN4 interacts with PARP1 to promote DNA repair in mouse embryonic stem cells.

doi: 10.1186/s13578-023-01140-1

Figure Lengend Snippet: Fig. 1 ZSCAN4 expression is reversely correlated with the extent of DSBs in mESCs. A Immunofluorescence images of the pZscan4-GFP mESCs. Note only a small subpopulation of cells (boxed in top row) express ZSCAN4 at a given time. Scale bar: 10 µm. B Left: western blot of γH2AX, PARP1, and ZSCAN4 the GFP+ (indicative of ZSCAN4 expressing) and GFP− (indicative of ZSCAN4-negative) mESCs. Middle and right: quantitative levels of γH2AX and PARP1. Data are normalized to the GFP− group and are represented as mean ± SEM. C Left: western blot of γH2AX, PARP1, and ZSCAN4 in the wildtype mESCs transiently expressing FLAG-ZSCAN4. Middle and right: quantitative levels of γH2AX and PARP1. Data are normalized to the GFP− group and are represented as mean ± SEM. D Left: IF images of FLAG and γH2AX in wildtype mESCs transiently overexpressing FLAG-ZSCAN4. Scale bar: 20 µm. Right: quantitative percentage of cells with ≧10 γH2AX foci. Data are represented as mean ± SEM. E Left: IF images of FLAG and γH2AX in wildtype mESCs transiently overexpressing FLAG-ZSCAN4 with 0.01% H2O2 treatment. Scale bar: 20 µm. Middle: quantitative percentage of cells with ≧10 γH2AX foci. Data are represented as mean ± SEM. Right: fold change of the percentage of cells with ≧10 γH2AX foci after the H2O2 treatment. See also Additional file 1: Figs. S1, S2 and Table S2

Article Snippet: To generate the pZscan4-GFP mESCs, the Zscan4 promoter sequence cloned from 2570 bp upstream of Zscan4c start codon [2] and a 720 bp eGFP coding sequence were cloned into the pSin vector (16578, Addgene) and the plasmid was transfected into HEK293T cell along with pSPAX2 (12260, Addgene) and pMD2.G (12259, Addgene) to produce lentivirus.

Techniques: Expressing, Immunofluorescence, Western Blot

Journal: Cell & bioscience

Article Title: ZSCAN4 interacts with PARP1 to promote DNA repair in mouse embryonic stem cells.

doi: 10.1186/s13578-023-01140-1

Figure Lengend Snippet: Fig. 2 ZSCAN4-associated DSB reduction is dependent on PARP1. A Left: western blot of γH2AX and ZSCAN4 in wildtype mESCs transiently overexpressing FLAG-ZSCAN4 treated with or without 3-AB. 0.1% DMSO serves as the vehicle control (no 3-AB). Right: quantitative levels of γH2AX. Data are normalized to no 3-AB (control) group and are represented as mean ± SEM. B IF images of FLAG and γH2AX in wildtype mESCs transiently overexpressing FLAG-ZSCAN4 treated with or without 3-AB. Scale bar: 20 µm. Right: quantitative percentage of cells with ≧10 γH2AX foci. Data are represented as mean ± SEM. C Left: western blot of γH2AX, PARP1 and ZSCAN4 in wildtype (Parp1+/+) and PARP1 knockout (Parp1−/−) mESCs. For each genotype, two clones (#1 and #2) were used. Right: quantitative levels of γH2AX. Data are normalized to clone #1 of Parp1+/+ mESCs and are represented as mean ± SEM. (D) Left: IF images of FLAG and γH2AX in wildtype (Parp1+/+) and PARP1 knockout (Parp1−/−) mESCs. Right: quantitative percentage of cells with ≧10 γH2AX foci. Data are represented as mean ± SEM. See also Figure S2, S3 and Additional file 1: Table S2

Article Snippet: To generate the pZscan4-GFP mESCs, the Zscan4 promoter sequence cloned from 2570 bp upstream of Zscan4c start codon [2] and a 720 bp eGFP coding sequence were cloned into the pSin vector (16578, Addgene) and the plasmid was transfected into HEK293T cell along with pSPAX2 (12260, Addgene) and pMD2.G (12259, Addgene) to produce lentivirus.

Techniques: Western Blot, Control, Knock-Out, Clone Assay

Journal: Cell & bioscience

Article Title: ZSCAN4 interacts with PARP1 to promote DNA repair in mouse embryonic stem cells.

doi: 10.1186/s13578-023-01140-1

Figure Lengend Snippet: Fig. 3 PARP1 has a protein–protein interaction with ZSCAN4. A Co-IP analysis of FLAG-ZSCAN4 and HA-PARP1 expression plasmids in HEK293T cells. B IF images of FLAG-ZSCAN4 and HA-PARP1 expression plasmids in HEK293T cells. Scale bar: 15 µm. See also Additional file 1: Fig. S4

Article Snippet: To generate the pZscan4-GFP mESCs, the Zscan4 promoter sequence cloned from 2570 bp upstream of Zscan4c start codon [2] and a 720 bp eGFP coding sequence were cloned into the pSin vector (16578, Addgene) and the plasmid was transfected into HEK293T cell along with pSPAX2 (12260, Addgene) and pMD2.G (12259, Addgene) to produce lentivirus.

Techniques: Co-Immunoprecipitation Assay, Expressing

Journal: Cell & bioscience

Article Title: ZSCAN4 interacts with PARP1 to promote DNA repair in mouse embryonic stem cells.

doi: 10.1186/s13578-023-01140-1

Figure Lengend Snippet: Fig. 4 ZSCAN4 and PARP1 interacts with each other. A The illustration of ZSCAN4 protein domains. The numbers on the top indicate the residue positions. SCAN: the SCAN domain (amino acid residue 1–163 of ZSCAN4); LS: the linker sequence (amino acid residue 164–396 of ZSCAN4); ZF: the zinc finger domain (amino acid residue 397–506 of ZSCAN4). B The illustration of PARP1 protein domains. DB: the DNA binding domain (amino acid residue 1–382 of PARP1); AM: the auto-modification domain (amino acid residue 383–655 of PARP1); CAT: the catalytic domain (amino acid residue 656–1014 of PARP1). C Co-IP of individual FLAG-ZSCAN4 domains (SCAN, LS or ZF) and full-length HA-PARP1. D IF images of FLAG (indicative of ZSCAN4 domains) and HA (indicative of full length PARP1) in mouse BNL CL.2 transiently expressing full length HA-PARP1 and a FLAG tagged ZSCAN4 domain. Scale bar: 25 µm. E Co-IP of individual HA-PARP1 domains (DB, AM or CAT) and full-length FLAG-ZSCAN4. F IF images of FLAG (indicative of full length ZSCAN4) and HA (indicative of PARP1 domains) in mouse BNL CL.2 transiently expressing full length FLAG-ZSCAN4 and a HA tagged PARP1 domain. Scale bar: 25 µm. G Co-IP of the HA-DB and HA-AM (of PARP1) with FLAG-SCAN (of ZSCAN4). The top arrow indicates the full-length FLAG-ZSCAN4 bands. The lower arrow indicates the FLAG-SCAN domain (of ZSCAN4) bands. WT: wildtype. H Co-IP results of the HA-DB and HA-AM (of PARP1) with FLAG-ZF (of ZSCAN4). The top arrow indicates the full-length FLAG-ZSCAN4 bands. The lower arrow indicates the FLAG-ZF domain (of ZSCAN4) bands. The middle panel is an overexposure of the top panel to reveal the FLAG-ZF bands. WT: wildtype

Article Snippet: To generate the pZscan4-GFP mESCs, the Zscan4 promoter sequence cloned from 2570 bp upstream of Zscan4c start codon [2] and a 720 bp eGFP coding sequence were cloned into the pSin vector (16578, Addgene) and the plasmid was transfected into HEK293T cell along with pSPAX2 (12260, Addgene) and pMD2.G (12259, Addgene) to produce lentivirus.

Techniques: Residue, Sequencing, Binding Assay, Modification, Co-Immunoprecipitation Assay, Expressing

Journal: Cell & bioscience

Article Title: ZSCAN4 interacts with PARP1 to promote DNA repair in mouse embryonic stem cells.

doi: 10.1186/s13578-023-01140-1

Figure Lengend Snippet: Fig. 5 The ⍺2 motif on the SCAN domain and the ZF4 motif on the ZF domain are critical for the binding of ZSCAN4 with PARP1. A Illustration of the SCAN domain (top) and its truncated variants (△⍺1 to △⍺5). B Illustration of the ZF domain (top) and its truncated variants (△ZF1 to △ZF4). C Co-IP of the full and truncated FLAG-SCAN domain with the full-length HA-PARP1. D Co-IP of the full and truncated FLAG-ZF domain with the full-length HA-PARP1. E Illustration of the full-length FLAG-ZSCAN4 and two truncated variants (△⍺2ZF2 and △⍺2ZF4). F Co-IP of FLAG-ZSCAN4 (full length), △⍺2ZF2 and △⍺2ZF4 (truncated variants) with the full-length HA-PARP1. G Illustration of the identified interactions between different motifs of ZSCAN4 and PARP1. The arrows indicate inter-domain bindings. The ⍺2 motif on the SCAN domain and the ZF4 motif are labeled to highlight their importance. See also Additional file 1: Table S1

Article Snippet: To generate the pZscan4-GFP mESCs, the Zscan4 promoter sequence cloned from 2570 bp upstream of Zscan4c start codon [2] and a 720 bp eGFP coding sequence were cloned into the pSin vector (16578, Addgene) and the plasmid was transfected into HEK293T cell along with pSPAX2 (12260, Addgene) and pMD2.G (12259, Addgene) to produce lentivirus.

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Labeling

Journal: Cell & bioscience

Article Title: ZSCAN4 interacts with PARP1 to promote DNA repair in mouse embryonic stem cells.

doi: 10.1186/s13578-023-01140-1

Figure Lengend Snippet: Fig. 6 The ⍺2 and ZF4 motifs of ZSCAN4 are required for ZSCAN4-associated DSB reduction. A Left: western blot of γH2AX and FLAG (indicative of full length or truncated ZSCAN4) in wildtype mESCs transfected with plasmids expressing either the full length (WT) or truncated (△⍺2ZF4) ZSCAN4. EV: empty vector. Right: quantitative levels of γH2AX. Data are normalized to EV transfected mESCs and are represented as mean ± SEM. B Left: IF images of FLAG and γH2AX in wildtype mESCs transfected with plasmids expressing either the full length (WT) or truncated (△⍺2ZF4) ZSCAN4. Right: quantitative percentage of cells with ≧10 γH2AX foci. Data are represented as mean ± SEM. See also Additional file 1: Table S2

Article Snippet: To generate the pZscan4-GFP mESCs, the Zscan4 promoter sequence cloned from 2570 bp upstream of Zscan4c start codon [2] and a 720 bp eGFP coding sequence were cloned into the pSin vector (16578, Addgene) and the plasmid was transfected into HEK293T cell along with pSPAX2 (12260, Addgene) and pMD2.G (12259, Addgene) to produce lentivirus.

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation