Journal: Poultry Science

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells

doi: 10.1016/j.psj.2026.106722

Figure Lengend Snippet: PGCs are more sensitive to DNA double-strand breaks than somatic cells. (A) Cell viability curves after 24 h treatment with increasing concentrations of etoposide (ETP; x-axis shows log 10 [ETP]). PGC viability dropped sharply even at low ETP doses, whereas CEF cells were more tolerant (viability data are mean ± SD of triplicates). (B) Apoptosis detection in PGCs treated with low-dose ETP. The proportion of Annexin V + /PI + cells (late apoptosis) was significantly elevated even at 0.03 µM ETP. (C) Western blot analysis of γ-H 2 AX protein in PGCs treated with varying ETP concentrations (left panel; β-actin as loading control). The bar graph (right) shows the ratio of γ-H 2 AX to β-actin band intensity, with a marked increase at 3 µM ETP (p-values are indicated in the figure). (D) Quantification of γ-H 2 AX foci per cell in THP-1 cells, male PGCs, and female PGCs after exposure to X-ray doses of 0, 2, 4, 6, and 8 Gy. After 48 h recovery, γ-H 2 AX foci increased significantly with higher radiation in THP-1, male PGCs, and female PGCs. Female PGC data points are red squares; male PGCs are blue squares; THP-1 are black circles. (E) Cell-cycle distribution of male vs. female PGCs after DNA damage. PGCs were irradiated (2, 4, 6 Gy), cultured 48 h, and analyzed by flow cytometry for cell-cycle phase (propidium iodide staining). Stacked bars show the percentage of cells in G 0 G 1 , S, and G 2 /M phases in untreated vs. irradiated cells. After damage, female PGCs predominantly arrested in G 2 /M (increased G 2 fraction), whereas male PGCs accumulated in S phase. Statistical significance by one-way ANOVA (p-values are indicated in the figure).

Article Snippet: For chemical induction of DSBs, PGC cultures were exposed to etoposide (a topoisomerase II inhibitor; MCE 33419-42-0) at various concentrations (0.01, 0.1, 1, 10, 100 μM, and 1 mM) for 24 h. Control cells were treated with vehicle for the same durations.

Techniques: Western Blot, Control, Irradiation, Cell Culture, Flow Cytometry, Staining

Journal: ACS chemical neuroscience

Article Title: Passage Variation of PC12 Cells Results in Inconsistent Susceptibility to Externally Induced Apoptosis

doi: 10.1021/acschemneuro.6b00208

Figure Lengend Snippet: Optimization and validation of etoposide-induced cell death in PC12 cells (passage 9–13). 50 × 103 cells/well. (a) Approximately 50% cell death was observed after 48 h. Four replicates per experiment. Values are represented as mean ± SD. Multiple t test, Holm-Sidak method, alpha = 5.000%, ****p < 0.0001. (b) Fluorescence measured after 48 h. Flupirtine at 3 μM concentration rescued the cells from apoptosis. Experiment repeated thrice. Values are represented as mean ± SD. Unpaired two-tailed t test, 95% CI, ****p < 0.0001 in comparison to vehicle. ####p < 0.0001 in comparison to etoposide treatment.

Article Snippet: 5 Etoposide (Chem-Impex International, 28435) was stored as a working stock solution of 150 μ g/mL at −20 °C for up to 3 months.

Techniques: Fluorescence, Concentration Assay, Two Tailed Test, Comparison

Journal: ACS chemical neuroscience

Article Title: Passage Variation of PC12 Cells Results in Inconsistent Susceptibility to Externally Induced Apoptosis

doi: 10.1021/acschemneuro.6b00208

Figure Lengend Snippet: NGF differentiated PC12 cells show a statistically significant difference in sensitivity to apoptosis induced by etoposide between early and late passages at 72 h post insult. (a) Passage 10. Three replicates per experiment. Values are represented as mean ± SD. One-way ANOVA, Dunnett test, 95% CI. ****p < 0.0001. (b) Passage 17. Three replicates per experiment. Values are represented as mean ± SD. One-way ANOVA, Dunnett test, 95% CI. ****p < 0.0001. (c) Comparison of 72 h etoposide treatment in passage 10 and 17 PC12 cells differentiated using NGF. Unpaired two-tailed t test, 95% CI, ****p < 0.0001.

Article Snippet: 5 Etoposide (Chem-Impex International, 28435) was stored as a working stock solution of 150 μ g/mL at −20 °C for up to 3 months.

Techniques: Comparison, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Metabolic reprogramming tips vaccinia virus infection outcomes by stabilizing interferon-γ induced IRF1

doi: 10.1371/journal.ppat.1012673

Figure Lengend Snippet: (A) Western blot analysis of VACV proteins–early (SSB) and late (A27)–in A549 cells primed with either IFN-α or IFN-γ, grown in glucose or galactose. (B) qPCR of VACV transcripts–early ( I3L ) and late ( F17R ) genes (N = 3). (C) Top: Quantification of plaque assay for VACV-Luc-GFP infectious units from A549 cells (N = 3). Bottom: representative image of plaque assay with various dilutions of viral titers (10 −1 –10 −3 ). (D) Western blot analysis for HSV-1 viral immediate early (ICP27) and tegument proteins (VP16) in A549 cells. (E) Quantification of plaque forming units from HSV-1-GFP A549 infected cells primed with either glucose or galactose as well as IFN-α or IFN-γ (N = 3). (F) VSV (luciferase) replication assay from IFN-pretreated A549 cells grown in glucose or galactose. Bottom: images showing presence or absence of cytopathic effect (with interferon priming). Plaque forming unit: P.F.U. Glc: glucose (25 mM). Gal: galactose (10 mM). Loading control for western blot and qPCR: β-actin. For viral infection, MOI = 0.01 for VACV-Luc-GFP and VSV-Luc; MOI = 1 for HSV-1-GFP. Concentration for IFN-α and IFN-γ: 1000 units/mL. Statistical analysis was performed using an unpaired t-test in GraphPad Prism 9.5.1: n.s. not significant, * P ≤ 0.05, ** P < 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: The following primary antibodies used in this study were diluted in 1:1000 in 5% milk in TBST: STAT1 rabbit mAB (CST, cat. no. 14994T), phospho-STAT1 rabbit mAB (CST, cat. no. 9167S), IRF1 mAB (CST, cat. no. 8478T), GBP2 pAB (Proteintech, cat no. 11854-1-AP), ISG15 mAB (Santa Cruz Biotech, cat. no. sc-166755), IFITM3 pAB (Proteintech, cat. no. 11714-1-AP), IDO1 pAB (Novus Biologicals, cat. no. NBP1-87703), IFIT1 mAB (CST, cat. no. 14769S), HDAC1 pAB (Proteintech, cat. no. 10197-1-AP), SDHA mAB (CST, cat. no. 11998S), TOM70 pAB (ABclonal, cat. no. A4349), vaccinia virus A27L pAB (BEI resources, cat. no. NR-627), vaccinia virus I3L mAB (a generous gift from Dr. David Evans) [ ], HSV-1 VP16 mAB (Santa Cruz Biotech, cat. no. sc-7545), HSV-1 ICP27 mAB (Santa Cruz Biotech, cat. no. sc-69806), PARP mAB (CST, cat. no. 9532S), and β-actin mAB (Sigma, cat. no. A5316).

Techniques: Western Blot, Plaque Assay, Infection, Luciferase, Control, Concentration Assay

Journal: Nature Communications

Article Title: Single-component near-infrared optogenetic systems for gene transcription regulation

doi: 10.1038/s41467-021-24212-7

Figure Lengend Snippet: a Schematics of the mCherry gene transcription repression in the iLight bacterial system. Expression of mCherry from the constitutively active ColE promoter with LexA408 operator is controlled by the oligomeric state of iLight fused with DNA-binding domain of LexA408. b Schematics of the reporter gene transcription activation in the iLight mammalian system. To induce reporter expression from the plasmid with 12× (upstream activating sequence) UAS upstream of minimal promoter, a nucleus localization signal (NLS)-tagged iLight was fused with a Gal4 DNA-binding domain and a VP16 transcriptional activation domain. The 660 nm light-induced iLight tetramerization brings the Gal4 DNA-binding domains into proximity, enabling them to bind 12× UAS and allowing VP16 to recruit transcription initiation complexes.

Article Snippet: A transcription activating domain of transactivating tegument protein VP16 from Herpes simplex and Gal4(1–65) DBD were PCR amplified from a pGV-2ER plasmid (Systasy).

Techniques: Expressing, Binding Assay, Activation Assay, Plasmid Preparation, Sequencing

Journal: Nature Communications

Article Title: Single-component near-infrared optogenetic systems for gene transcription regulation

doi: 10.1038/s41467-021-24212-7

Figure Lengend Snippet: a Dependence of the SEAP reporter expression on concentration of the exogenous biliverdin in HeLa cells expressing the iLight optogenetic system. SEAP signal was detected after 48 h of 660 nm illumination. b The 72 h-long kinetics of the SEAP reporter expression in HeLa cells with the iLight optogenetic system. Cells were illuminated in one of the following regimes: cells were kept in darkness (black dots); illumination with 660 nm light for 72 h (red dots); illumination with 660 nm light for 24 h followed by 48 h of darkness (blue dots); or illumination with 660 nm light for 24 h followed by 4 h of 780 nm light and 44 h of darkness (green dots). The culture medium samples were collected every 12 h to measure the SEAP signal. c Dependence of the SEAP reporter expression on the power of 660 nm activation light. HeLa cells were kept in darkness or under 660 nm light of the respective light intensities. Numbers indicate fold increase in the SEAP signal over darkness. In b , c , cells were supplemented with 10 µM biliverdin. In a – c , HeLa cells bearing NLS-Gal4-DBD-iLight-VP16 were transfected with pG12-SEAP (12× UAS) reporter plasmid. Box plots show the median (center line), first and third quartiles (box edges), 1× the SD (whiskers), and individual data points. n = 4 independent experiments for all conditions. a.u., arbitrary units. Source Data are available as a Source Data file.

Article Snippet: A transcription activating domain of transactivating tegument protein VP16 from Herpes simplex and Gal4(1–65) DBD were PCR amplified from a pGV-2ER plasmid (Systasy).

Techniques: Expressing, Concentration Assay, Activation Assay, Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: Single-component near-infrared optogenetic systems for gene transcription regulation

doi: 10.1038/s41467-021-24212-7

Figure Lengend Snippet: a RLuc8 luciferase reporter signals detected in mice after the hydrodynamic co-transfection of the livers with the NLS-Gal4-DBD-iLight-VP16 and pG12-RLuc8 plasmids. Mice kept in darkness (top) or illuminated with 660 nm light of 3.2 mW cm −2 (bottom) for 48 h are shown. b Kinetics of the RLuc8 reporter expression in mice shown in a kept in darkness or illuminated for up to 96 h. Box plots show the median (center line), first and third quartiles (box edges), 1× the SD (whiskers) and individual data points. n = 3 individual animals. Source Data are available as a Source Data file.

Article Snippet: A transcription activating domain of transactivating tegument protein VP16 from Herpes simplex and Gal4(1–65) DBD were PCR amplified from a pGV-2ER plasmid (Systasy).

Techniques: Luciferase, Cotransfection, Expressing

Journal: Science advances

Article Title: egr3 is a mechanosensitive transcription factor gene required for cardiac valve morphogenesis.

doi: 10.1126/sciadv.adl0633

Figure Lengend Snippet: Fig. 2. Endothelial-specific deletion of egr3 recapitulates the global mutant phenotype. (A) Schematic of the knock-in reporter [Pt(egr3:Gal4-VP16)] generated by inserting Gal4-VP16 in exon 2 of egr3. (B) Pt(egr3:Gal4-VP16); Tg(UAS:eGFP) expression (gray) at 72 hpf; EdCs are marked by Tg(kdrl:Hsa.HRAS-mCherry) expression (cyan) and myocardial cells by Tg(myl7:BFP-CAAX) expression (magenta); yellow arrowheads point to AV canal egr3 reporter+ cardiomyocytes. (B′) Maximum projection of Pt(egr3:Gal4-VP16); Tg(UAS:eGFP) expression with image segmentation of egr3 reporter+ AV canal EdCs (cyan), egr3 reporter+ AV canal myocardial cells (magenta), and egr3 reporter+ OFT cells (yellow). (C) Schematic of egr3 floxed allele Pt(egr3:loxP-egr3-loxP). (D) Confocal images of representative hearts from 72 hpf egr3+/+, endothelial cell–specific egr3−/−, and myocardial cell–specific egr3−/− larvae using the egr3 floxed allele line. (E and F) Percentage of 72 hpf egr3+/?, endothelial cell–specific egr3−/− (E), and myocardial cell–specific egr3−/− (F) larvae with a superior valve leaflet and without a superior valve leaflet (i.e., endocardial monolayer); one and two independent experiments, respectively; Fisher’s exact test. Scale bars, 20 μm.

Article Snippet: To generate the egr3:Gal4 line, the pGTag- Gal4- VP16- beta- actin plasmid was a gift from J. Essner (Addgene # 117817) and homology arms were added by vector ligation (98).

Techniques: Mutagenesis, Knock-In, Generated, Expressing

Journal: Science advances

Article Title: egr3 is a mechanosensitive transcription factor gene required for cardiac valve morphogenesis.

doi: 10.1126/sciadv.adl0633

Figure Lengend Snippet: Fig. 3. egr3 is necessary for EdC migration, which is required for cardiac valve formation. (A) RNA-seq heatmap analysis of differentially expressed genes; fold change ± 1.5, Padj < 0.05, mean > 5. (B) scRNA-seq endocardial expression pattern of Egr3 targets nr4a2b and spp1. (C) In situ hybridization for Egr3 targets nr4a2b and spp1 in egr3+/+ and egr3−/− siblings at 48 hpf. (D) Confocal images of representative hearts from 48 hpf egr3+/? and egr3−/− sibling embryos displaying, respectively, AV EdC protru- sions toward the ECM and no protrusions; EdCs are marked by Tg(kdrl:eGFP) expression (cyan). (E) Percentage of hearts from 48 hpf egr3+/? and egr3−/− sibling embryos displaying AV EdC protrusions toward the ECM; three independent experiments. (F) Confocal images of representative hearts from 55 hpf egr3+/− and egr3−/− sibling embryos displaying, respectively, collective AV EdC migration and no migration; EdCs are marked by Tg(kdrl:NLS-mCherry) expression (gray) and AV EdCs by Pt(egr3:Gal4- VP16);Tg(UAS:eGFP) expression (green). (G) Percentage of hearts from 55 hpf egr3+/− and egr3−/− sibling embryos displaying AV EdC migration; three independent ex- periments. (H) Number of migrating AV EdCs per heart in 55 hpf egr3+/− and egr3−/− sibling embryos; n = 18 and 17 embryos; three independent experiments. Student’s t test, mean ± SEM. (E) and (G) Fisher’s exact test. Scale bars, 20 μm.

Article Snippet: To generate the egr3:Gal4 line, the pGTag- Gal4- VP16- beta- actin plasmid was a gift from J. Essner (Addgene # 117817) and homology arms were added by vector ligation (98).

Techniques: Migration, RNA Sequencing, Expressing, In Situ Hybridization

Journal: Science advances

Article Title: egr3 is a mechanosensitive transcription factor gene required for cardiac valve morphogenesis.

doi: 10.1126/sciadv.adl0633

Figure Lengend Snippet: Fig. 4. egr3 triggers EdC migration and Alcama expression. (A) 3D reconstruction of representative hearts from control [Tg(fli1a:Gal4);Tg(UAS:Kaede)] and endothelial- specific egr3-overexpressing [Tg(fli1a:Gal4);Tg(UAS:Kaede);Tg(UAS:egr3-p2a-dTomato)] 72 hpf larvae; magnified regions show single z planes of the atrium from control and endothelial-specific egr3-overexpressing 72 hpf larvae; yellow arrowheads point to Tg(UAS:egr3-p2a-dTomato)+ atrial EdCs in the adjacent ECM. (B) Confocal images of representative AV VIC quantification through the spot render function in Imaris; VICs (magenta spots). (C) Quantification of EdCs present in the ECM per cardiac region in 72 hpf endothelial-specific egr3-overexpressing larvae; n = 9 control and 22 egr3 OE larvae, n = 0 and 0.14 EdCs (ventricle), n = 5 and 13.36 EdCs (AV canal), n = 0 and 0.59 EdCs (atrium). (D) Confocal images of representative hearts from control [Tg(fli1a:Gal4);Tg(UAS:Kaede)] and endothelial-specific egr3-overexpressing [Tg(fli1a:Gal4);Tg (UAS:Kaede);Tg(UAS:egr3-p2a-dTomato)] 54 hpf embryos immunostained for Alcama; magnified regions show Alcama+ cells in the AV canal of the control heart and in the atrium of the Tg(UAS:egr3-p2a-dTomato) heart. (E) Quantification of Alcama+ EdCs per cardiac region of control and endothelial-specific egr3-overexpressing embryos at 54 hpf; n = 13 control and 14 egr3 OE embryos, n = 0.07 and 3.28 EdCs (ventricle), n = 31.77 and 42.86 EdCs (AV canal), n = 0.38 and 4.71 EdCs (Atrium); three independent experiments. (F) Confocal images of representative hearts from control Pt(egr3:Gal4-VP16) and egr3-driven nr4a2b-overexpressing [Pt(egr3:Gal4-VP16); Tg(UAS:nr4a2b- p2a-dTomato)] egr3−/− larvae at 72 hpf. (G) Quantification of EdCs present in the AV canal ECM of control and nr4a2b-overexpressing egr3−/− larvae at 72 hpf; n = 16 and 15 larvae; two independent experiments. (C), (E), and (G) Student’s t test, mean ± SEM. Scale bars, 20 μm.

Article Snippet: To generate the egr3:Gal4 line, the pGTag- Gal4- VP16- beta- actin plasmid was a gift from J. Essner (Addgene # 117817) and homology arms were added by vector ligation (98).

Techniques: Migration, Expressing, Control

Journal: Science advances

Article Title: egr3 is a mechanosensitive transcription factor gene required for cardiac valve morphogenesis.

doi: 10.1126/sciadv.adl0633

Figure Lengend Snippet: Fig. 5. egr3 expression in the heart is regulated by biomechanical forces. (A) In situ hybridization for egr3 expression in 48 hpf tnnt2a+/+ and tnnt2a−/− sibling embryos. (B) Illustration of bead insertion into a 48 hpf heart. (C) Pt(egr3:Gal4-VP16); Tg(UAS:eGFP) expression (gray) at 72 hpf in bead-inserted larva versus sham; myocar- dial cells are marked by Tg(myl7:BFP-CAAX) expression (magenta) and EdCs by Tg(kdrl:Hsa.HRAS-mCherry) expression (cyan). Red dotted circle highlights inserted bead; yellow arrowhead points to egr3+ cells near the bead. (D) Quantification of total volume of egr3 reporter+ cells in bead-inserted larvae and sham; n = 3 sham and 5 experimental larvae; one experiment. (E) Confocal images of representative hearts from control [Tg(fli1a:Gal4);Tg(UAS:Kaede)] and endothelial-specific egr3- overexpressing [Tg(fli1a:Gal4);Tg(UAS:Kaede);Tg(UAS:egr3-p2a-dTomato)] tnnt2a MO–injected embryos immunostained for Alcama at 54 hpf; magnified regions show the AV canal of tnnt2a MO–injected embryos with rescued Alcama expression in the Tg(UAS:egr3-p2a-dTomato) embryo. (F) Quantification of Alcama+ EdCs per cardiac region of tnnt2a MO–injected embryos at 54 hpf; n = 6 control and 6 egr3 OE embryos, n = 0.00 and 8.00 EdCs (ventricle), n = 0.00 and 13.00 EdCs (AV canal), n = 0.00 and 4.83 EdCs (Atrium); two independent experiments. (G) Oscillatory shear stress induces the expression of the mechanosensitive transcription factor gene egr3 in valve EdCs, where it orchestrates valvulogenesis by promoting Alcama expression and their migration via Nr4a2b. (H) Schematic of aortic valve collection from LVAD patients and control donors. (I) Relative mRNA levels of EGR3, KLF2, NR4A2, and SPP1 from aortic valves of LVAD patients and control donors; n = 4 and 4 to 6; one experiment. Ct values can be found in data S1. (D), (F), and (I) Student’s t test, mean ± SEM. (B) and (H) Illustrations were generated with Biorender.com. Scale bars, 20 μm.

Article Snippet: To generate the egr3:Gal4 line, the pGTag- Gal4- VP16- beta- actin plasmid was a gift from J. Essner (Addgene # 117817) and homology arms were added by vector ligation (98).

Techniques: Expressing, In Situ Hybridization, Control, Injection, Shear, Migration, Generated

Journal: Advanced Science

Article Title: A Novel Engineering Cell Therapy Platform Mimicking the Immune Thrombocytopenia‐Derived Platelets to Inhibit Cytokine Storm in Hemophagocytic Lymphohistiocytosis

doi: 10.1002/advs.202404571

Figure Lengend Snippet: Schematic illustration of the anti‐CD41‐PLT‐VP16 process and relevant therapeutic effect on HLH mice. Anti‐CD41‐PLT‐VP16 blocks phagocytosis reduces cytokine storms, and indirectly inhibits the function of other hyperinflammation‐associated immune cells in the form of “besieger”.

Article Snippet: Colocalization of the anti‐CD41‐PLT‐VP16 components was assessed under a CLSM, and the PLTs were stained with phalloidin (MedChemExpress, US) and imaged by CLSM with FITC‐conjugated anti‐CD41 (Thermo Fisher Scientific, US).

Techniques:

Journal: Advanced Science

Article Title: A Novel Engineering Cell Therapy Platform Mimicking the Immune Thrombocytopenia‐Derived Platelets to Inhibit Cytokine Storm in Hemophagocytic Lymphohistiocytosis

doi: 10.1002/advs.202404571

Figure Lengend Snippet: Characterization and properties of anti‐CD41‐PLT‐VP16. A) PLT morphology was assessed by electron microscopy. Bar = 2 µm. B) DLS was used to measure the size of the PLTs in each group. C) CLSM image showing the colocalization of anti‐CD41‐PLT‐VP16 components. (PLT: red, anti‐CD41: green, bar = 20 µm) D) FCM verified the labeling efficiency of anti‐CD41 on PLTs. E) HPLC was used to determine the encapsulation rate of anti‐CD41‐PLT‐VP16 at multiple concentrations. VP16 at 320 µg mL −1 resulted in the optimal encapsulation rate, so this concentration was used for constructing anti‐CD41‐PLT‐VP16 in subsequent experiments. F) HPLC was used to detect the loading rate of multiple concentrations of anti‐CD41‐PLT‐VP16. G) HPLC was used to detect the drug release rate from pharmacophores in different pH environments; pH 7.4 was used to simulate in vivo circulation, and pH 5.5 was used to simulate macrophage lysosomes. Anti‐CD41‐PLT‐VP16 is highly stable in a neutral environment, and the drug release rate can reach 85% at 35 h in an acidic environment. H) WB analysis was utilized to detect platelet function‐related proteins. I) WB detection of characteristic proteins on the surface of platelets from each group. J) The turbidimetric assay was utilized to determine platelet aggregation. The control group was untreated platelet‐rich plasma and PPP was platelet‐poor plasma, which had an absorbance value of 0 in the agglutination assay. The absorbance was measured after the addition of the procoagulant ADP to the remaining four groups, except for the control group, and the addition of the anti‐CD41‐PLT‐VP16 had no effect on platelet agglutination. K) Zeta potentials of the PLT, anti‐CD41‐PLT, and anti‐CD41‐PLT‐VP16 groups. *** p < 0.001, ns = not significant.

Article Snippet: Colocalization of the anti‐CD41‐PLT‐VP16 components was assessed under a CLSM, and the PLTs were stained with phalloidin (MedChemExpress, US) and imaged by CLSM with FITC‐conjugated anti‐CD41 (Thermo Fisher Scientific, US).

Techniques: Electron Microscopy, Labeling, Encapsulation, Concentration Assay, In Vivo, Turbidimetric Assay, Control, Clinical Proteomics, Agglutination

Journal: Advanced Science

Article Title: A Novel Engineering Cell Therapy Platform Mimicking the Immune Thrombocytopenia‐Derived Platelets to Inhibit Cytokine Storm in Hemophagocytic Lymphohistiocytosis

doi: 10.1002/advs.202404571

Figure Lengend Snippet: The effect of anti‐CD41‐PLT‐VP16 on macrophages. A) CLSM observation of phagocytosis of PLT‐VP16 and anti‐CD41‐PLT‐VP16 by macrophages. (PLT: red, DAPI: blue, bar = 10 µm) B) Phagocytosis of PLT‐VP16 and anti‐CD41‐PLT‐VP16 by macrophages was photographed by CLSM after 1, 2, and 4 h of administration. (DAPI: blue, PLT: red, anti‐CD41: green, bar = 10 µm) C) CCK‐8 measurement of macrophage viability in different treatment groups after 12 h of administration. D) WB analysis was used to detect changes in the levels of mitochondrial apoptosis pathway‐related proteins, including Bax, Caspase‐3, and Bcl‐2, in the different treatment groups after 12 h of administration. E) FCM with Annexin V‐FITC/PI was used to detect the apoptosis of macrophages in different treatment groups 12 h after drug administration. F) Cycle detection was performed on macrophages from different treatment groups after 12 h of drug administration, and an increase in (S+G2)/G1 could indicate cycle arrest due to blockage of DNA replication. G) ROS levels were detected in macrophages from each treatment group 12 h after administration and observed by fluorescence microscopy. The green fluorescence intensity indicates the level of ROS in the cells. Bar = 50 µm. ** p < 0.01.

Article Snippet: Colocalization of the anti‐CD41‐PLT‐VP16 components was assessed under a CLSM, and the PLTs were stained with phalloidin (MedChemExpress, US) and imaged by CLSM with FITC‐conjugated anti‐CD41 (Thermo Fisher Scientific, US).

Techniques: CCK-8 Assay, Fluorescence, Microscopy

Journal: Advanced Science

Article Title: A Novel Engineering Cell Therapy Platform Mimicking the Immune Thrombocytopenia‐Derived Platelets to Inhibit Cytokine Storm in Hemophagocytic Lymphohistiocytosis

doi: 10.1002/advs.202404571

Figure Lengend Snippet: The therapeutic effect of anti‐CD41‐PLT‐VP16 in humanized mice with HLH. A) Treatment schedules for the different groups of the humanized HLH model mice. B) MR images of liver and spleen tissues from mice in different treatment groups before and after treatment ( n = 6). The liver and spleen were photographed separately at the same level, and the regions circled by the red dotted line are the liver and spleen tissue. Bar = 5 mm. C) In vivo imaging of small animals showing the circulation and enrichment of anti‐CD41‐PLT‐VP16 in the tail vein over time after the injection of Cy5‐labeled anti‐CD41‐PLT‐VP16 ( n = 6). D) Measurement of IL‐1β, IFN‐γ, IL‐6, and TNF‐α levels in the PB after treatment of mice in different treatment groups ( n = 6). Dashed lines indicate normal values. E) Quantitative results of F4/80 in liver and spleen tissues determined by IHC after treatment of mice in different treatment groups ( n = 6). F) IVM real‐time in vivo imaging of liver, spleen, and BM macrophage activity in different treatment groups of mice 12 h after the corresponding treatments. The blue fluorescence intensity indicates macrophage activity. (Blood: red, cathepsin K probe: blue, n = 6). G) Wright‐Giemsa staining of PB and BM from mice in different treatment groups. Arrows show hemophagy ( n = 6). Bar = 50 µm. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Colocalization of the anti‐CD41‐PLT‐VP16 components was assessed under a CLSM, and the PLTs were stained with phalloidin (MedChemExpress, US) and imaged by CLSM with FITC‐conjugated anti‐CD41 (Thermo Fisher Scientific, US).

Techniques: In Vivo Imaging, Injection, Labeling, Activity Assay, Fluorescence, Staining

Journal: Advanced Science

Article Title: A Novel Engineering Cell Therapy Platform Mimicking the Immune Thrombocytopenia‐Derived Platelets to Inhibit Cytokine Storm in Hemophagocytic Lymphohistiocytosis

doi: 10.1002/advs.202404571

Figure Lengend Snippet: Detection of the biosafety of anti‐CD41‐PLT‐VP16. A) Changes in the body weight of mice in different treatment groups ( n = 6). The weight of the anti‐CD41‐PLT‐VP16 group after treatment converged on that of normal mice before modeling. B) Renal function measurements, including creatinine (CREA) and nitrogen (BUN), in mice in different treatment groups after treatment ( n = 6). Liver function measurements, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, in mice in different treatment groups after treatment ( n = 6). C) In vivo real‐time imaging of large blood vessels in mice at 0, 1, 2, and 4 h after tail vein injection of anti‐CD41‐PLT‐VP16 (blood: red, cathepsin K probe: blue, n = 3, bar = 100 µm). D) H&E staining of mouse lung, heart, and kidney tissues in different treatment groups ( n = 6). Bar = 50 µm. * p < 0.05, *** p < 0.001.

Article Snippet: Colocalization of the anti‐CD41‐PLT‐VP16 components was assessed under a CLSM, and the PLTs were stained with phalloidin (MedChemExpress, US) and imaged by CLSM with FITC‐conjugated anti‐CD41 (Thermo Fisher Scientific, US).

Techniques: In Vivo, Imaging, Injection, Staining

Journal: Advanced Science

Article Title: A Novel Engineering Cell Therapy Platform Mimicking the Immune Thrombocytopenia‐Derived Platelets to Inhibit Cytokine Storm in Hemophagocytic Lymphohistiocytosis

doi: 10.1002/advs.202404571

Figure Lengend Snippet: Mechanism of inhibition of inflammation‐associated cytokine storm by anti‐CD41‐PLT‐VP16. A) Schematic illustration of the in vivo process in which anti‐CD41‐PLT‐VP16 prevents antigen presentation by occupying the Fc receptor on the surface of macrophages in HLH through ADCP; after being phagocytosed by macrophages, anti‐CD41‐PLT‐VP16 can induce the apoptosis of macrophages and inhibit cytokine release. B) Volcano plots show the upregulation and decrease in gene expression. The expression of some of the genes associated with Ras/MAPK, inflammation, and FcγR‐mediated phagocytosis are labeled in the figures ( n = 3). C) Heatmap showing the expression of cytokine‐related genes in each group ( n = 3, p < 0.05). D) KEGG enrichment analysis comparing the anti‐CD41‐PLT‐VP16 group with the VP16 group. Bubble plots showing the pathways in which differentially expressed proteins were concentrated ( p < 0.05). E) Differentially expressed genes in the anti‐CD41‐PLT‐VP16 group versus the control group were analyzed by PPI ( p < 0.05). F) Quantitative real‐time PCR (qPCR) was performed on the control, VP16, and anti‐CD41‐PLT‐VP16 groups, and the graph shows the expression levels of genes involved in the Ras‐MAPK pathway in each group. G) qPCR results showing the expression levels of genes involved in the FcγR‐mediated phagocytosis pathway. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Colocalization of the anti‐CD41‐PLT‐VP16 components was assessed under a CLSM, and the PLTs were stained with phalloidin (MedChemExpress, US) and imaged by CLSM with FITC‐conjugated anti‐CD41 (Thermo Fisher Scientific, US).

Techniques: Inhibition, In Vivo, Immunopeptidomics, Gene Expression, Expressing, Labeling, Control, Real-time Polymerase Chain Reaction

Journal: Advanced Science

Article Title: A Novel Engineering Cell Therapy Platform Mimicking the Immune Thrombocytopenia‐Derived Platelets to Inhibit Cytokine Storm in Hemophagocytic Lymphohistiocytosis

doi: 10.1002/advs.202404571

Figure Lengend Snippet: Schematic diagram of the preliminary clinical trial. The control group was normally treated patients, and VP16 at the time of treatment was replaced with anti‐CD41‐PLT‐VP16 in equal final concentration in the trial group. A significantly higher disease remission rate was found in the anti‐CD41‐PLT‐VP16 group. “ORR” = Objective response rate; “NR” = No response.

Article Snippet: Colocalization of the anti‐CD41‐PLT‐VP16 components was assessed under a CLSM, and the PLTs were stained with phalloidin (MedChemExpress, US) and imaged by CLSM with FITC‐conjugated anti‐CD41 (Thermo Fisher Scientific, US).

Techniques: Control, Concentration Assay

Journal: PLOS Biology

Article Title: ERR2 and ERR3 promote the development of gamma motor neuron functional properties required for proprioceptive movement control

doi: 10.1371/journal.pbio.3001923

Figure Lengend Snippet: (A-D’) Overview of E12 chick spinal cord: stable transfection with expression vector driving eGFP expression (A) in ventral horn motor neurons. Higher magnification of eGFP expressing neurons further identified as motor neurons by retrograde cholera toxin B (CTxB) tracing upon in ovo injection into the hind limb (A’) (scale bar: 100 μm). Examples of expression and nuclear localization of mouse ERR2 (B) and ERR3 (C) in chick motor neurons (detected via N-terminal V5 epitope tags) (scale bar: 30 μm). eGFP + motor neuron recorded with patch pipette containing Alexa Fluor 568 dye (red) (D and D’) (scale bar: 50 μm). (E, F) Example traces of current clamp recordings of chick motor neurons (in acute spinal cord slice preparations) forcedly expressing eGFP only (control) (E) or ERR2:VP16 and eGFP (F) , upon 100, 200, and 400 pA, 1 s square current pulses: motor neurons expressing elevated ERR2 levels exhibit higher firing rates and gear up their firing rates more rapidly in response to current pulses (F) . (G) Forced ERR2 and ERR2:VP16 expression ( n = 21, n = 20, respectively) shifts chick motor neuron properties towards a gamma motor neuron-like biophysical signature (high firing rates, low rheobases) compared to control (white, n = 23). (H) Decrease in rheobase upon ERR2, ERR3 ( n = 21) transfection, yet not significant, but significant decrease in rheobase upon ERR2:VP16 ( n = 20) or ERR3:VP16 ( n = 10). No decrease in rheobase upon ERR2:EnR ( n = 17) transfection. (I) Significant increases in gain upon transfection of ERR2, ERR3, ERR2:VP16 or ERR3:VP16, but not upon ERR2:EnR transfection. (H, I) Similar decreases in rheobase (H) , increase in gain (I) upon ERR2+ERR3 ( n = 13) cotransfection, compared to ERR2 or ERR3 single transfection. No differences detected between control 1 and control 2 with parameters recorded during two different experiments at different time points upon expressing “eGFP only” control vectors, thus demonstrating robustness of the assay. Data are presented as mean ±SEM. n = # of neurons. Statistically significant differences are indicated as: * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. = not significant, Student t test). Data for Fig 6G-J can be found in .

Article Snippet: ERR2VP16 was generated by fusing the open reading frame for the herpes simplex virus-1 (HSV-1) VP16 (amplified from pActPL-VP16AD plasmid, Addgene plasmid #15305, Watertown, USA) activation domain to ERR2.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, In Ovo, Injection, Transferring, Control, Transfection, Cotransfection

Journal: PLOS Biology

Article Title: ERR2 and ERR3 promote the development of gamma motor neuron functional properties required for proprioceptive movement control

doi: 10.1371/journal.pbio.3001923

Figure Lengend Snippet: (A) Heatmap based on transcript reads (transcript per million (TPM)) detected by RNA sequencing: ERR2 ( n = 4) and Dlk1 ( n = 4) promote different gene expression signatures in chick motor neurons when compared to control ( n = 4), including Kcna10 (red) by ERR2 (only up-regulated genes are shown). (B) Kcna10 genomic locus: 3 clustered ERR2/3 binding sites within promoter region. (C) Reporter tdTomato fluorescence driven by wild-type (WT) Kcna10 promoter is boosted by ERR2 cotransfection (red, n = 102) in chick motor neurons when compared to control (gray, n = 100) and decrease in tdTomato reporter fluorescence upon mutating the ERR2/3 binding sites (black, n = 101). (D) Whole-cell patch-clamp recordings: forced ERR2:VP16 expression (red, n = 20) shifts chick motor neuron properties towards a gamma motor neuron-like biophysical signature (high firing rates, low rheobases) compared to control (white, n = 23). Forced Kcna10 (black, n = 24) expression partially recapitulates the promotion of a gamma motor neuron biophysical signature by ERR2 in chick motor neurons, when compared to control (white, n = 23) motor neurons. (E) Significant decreases in rheobase upon Kcna10 ( n = 24) transfection. (F) Significant increase in firing frequency upon Kcna10 ( n = 24) transfection. (G) No significant increase in gain upon Kcna10 ( n = 24) transfection. Data are presented as mean ± SEM, n = number of experiments or neurons. Statistically significant differences are indicated as: * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. = not significant, Student t test). Data for Fig 7A and 7C can be found in and data for can be found in . The complete set of values for the RNAseq experiments can be found in .

Article Snippet: ERR2VP16 was generated by fusing the open reading frame for the herpes simplex virus-1 (HSV-1) VP16 (amplified from pActPL-VP16AD plasmid, Addgene plasmid #15305, Watertown, USA) activation domain to ERR2.

Techniques: RNA Sequencing, Gene Expression, Control, Binding Assay, Fluorescence, Cotransfection, Patch Clamp, Expressing, Transfection