Journal: bioRxiv

Article Title: Lipid nanoparticle delivered intrabodies for inhibiting necroptosis and pyroptosis

doi: 10.1101/2025.05.06.652432

Figure Lengend Snippet: (A) Percentage of GFP-positive cells over time following LNP delivery of FLAG-MLKL-GFP intrabody mRNA (0.5 µg/mL) in Colo205, SW620, and HT-29 cells, measured using IncuCyte live cell imaging system. Error bars represent the SD of three technical replicates. See Figure S3 for representative images. (B) Immunoblot of total cell lysates 24 hours post-LNP transfection using the indicated mRNA concentrations. (C) Immunoblot of total lysates from cells treated with LNPs (1 µg/mL mRNA equivalent) for 24 hours, followed by cycloheximide (CHX, 40 µg/mL) and Q-VD-OPh (QVD, 20 µM) treatment as indicated. (D) Analysis of MLKL oligomerization and membrane translocation via Blue Native PAGE after LNP delivery of the indicated MLKL intrabody mRNA (0.5 µg/mL mRNA equivalent) overnight followed by TSI (TNF (50□ng/mL), Smac mimetic Compound A (1□µM), pan-caspase inhibitor IDN-6556 (10□µM)) treatment for 6 hours. GAPDH and VDAC were used as cytoplasmic and membrane markers, respectively. (E) Cell death kinetics measured by IncuCyte imaging following TSI (TNF (50□ng/mL), Smac mimetic Compound A (1□µM), pan-caspase inhibitor IDN-6556 (10□µM)) treatment in cells that were pretreated overnight with LNPs (0.5 µg/mL mRNA equivalent). Error bars represent SD from three technical replicates. All IncuCyte data (A, E) and immunoblots (B, C, D) are representative of two independent experiments.

Article Snippet: Uncapped RNA was synthesized using the HiScribe T7 High Yield RNA Synthesis Kit (NEB, Cat# E2040), while capped RNA was synthesized using the HiScribe T7 mRNA Kit with CleanCap® Reagent AG (NEB, Cat# E2080).

Techniques: Live Cell Imaging, Western Blot, Transfection, Membrane, Translocation Assay, Blue Native PAGE, Imaging

Journal: bioRxiv

Article Title: Lipid nanoparticle delivered intrabodies for inhibiting necroptosis and pyroptosis

doi: 10.1101/2025.05.06.652432

Figure Lengend Snippet: (A) A schematic of mRNA constructs. (B) Immunoblot analysis of whole-cell lysates harvested after LNP-mediated treatment overnight at the specified mRNA concentrations followed by nigericin (5 µM) stimulation for 1 hour. ( C and D ) iBMDMs were treated overnight with LNPs (0.5 µg/mL mRNA equivalent) and then stimulated with (C) nigericin (5 µM) with our without MCC950 (10 µM), or transfected with (D) poly(dA:dT) (1 µg/mL). Cell death was measured by IncuCyte imaging. Error bars represent the SD of three technical replicates. ( E and F ) Immunoblot analysis of total lysates from iBMDMs pre-treated with increasing concentrations of LNP-mRNA overnight followed by (E) LPS (50 ng/mL) priming for 3 hrs and then nigericin (5 µM) treatment for 1 hr with or without MCC950 (10 µM), or (F) poly(dA:dT) (1 µg/mL) transfection for 6 hrs. All data (B to F) are representative of two independent experiments.

Article Snippet: Uncapped RNA was synthesized using the HiScribe T7 High Yield RNA Synthesis Kit (NEB, Cat# E2040), while capped RNA was synthesized using the HiScribe T7 mRNA Kit with CleanCap® Reagent AG (NEB, Cat# E2080).

Techniques: Construct, Western Blot, Transfection, Imaging

Journal: Nature Communications

Article Title: 8q24 derived ZNF252P promotes tumorigenesis by driving phase separation to activate c-Myc mediated feedback loop

doi: 10.1038/s41467-025-56879-7

Figure Lengend Snippet: A Schematic diagram of ZNF252P promoter with the putative c-Myc binding sites and the information for mutations or deletions. B The expression of ZNF252P was positively correlated c-Myc expression by TCGA data in diverse cancers (two-tailed Spearman correlation analysis). C The immunoblotting validated the efficacy of c-Myc knockdown by siRNA and c-Myc overexpression by plasmid. Data are representatives of 3 independent experiments. D qPCR analysis tested ZNF252P expression after alteration of c-Myc. Data are mean ± s.d.; n = 3 independent experiments, two-tailed Student’s t -test. E The dual-luciferase reporter assay determined the transcription activity of ZNF252P with full-length or c-Myc motif deletion promoter (left). After c-Myc overexpression, the dual-luciferase reporter assay detected the transcription capacity of ZNF252P with wild-type or mutated c-Myc motif in the promoter (right). Data are mean ± s.d.; n = 3 independent experiments, two-tailed Student’s t -test. F The ChIP assay detected the enrichment of c-Myc at the ZNF252P promoter after alteration of c-Myc expression. Data are mean ± s.d.; n = 3 independent experiments, two-tailed Student’s t -test. G The MTT assay demonstrated the effect of indicated treatments on cell proliferation. Data are mean ± s.d.; n = 3 independent experiments, one-way ANOVA with Dunnett’s multiple comparisons test. Source data are provided as a Source data file.

Article Snippet: For qRT-PCR assays, we used the Reverse Transcription Kit (Vazyme) to reverse transcribe RNA to cDNA.

Techniques: Binding Assay, Expressing, Two Tailed Test, Western Blot, Knockdown, Over Expression, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay, MTT Assay

Journal: Nature Communications

Article Title: 8q24 derived ZNF252P promotes tumorigenesis by driving phase separation to activate c-Myc mediated feedback loop

doi: 10.1038/s41467-025-56879-7

Figure Lengend Snippet: A Transcription activity of c-My c with corresponding treatments. Data are mean ± s.d.; n = 3 independent experiments, two-tailed Student’s t -test. B The enrichment of HNRNPK at c-Myc promoter after ZNF252P knockdown. Data are mean ± s.d.; n = 3 independent experiments, two-tailed Student’s t -test. C Cells with positive localization of HNRNPK droplets at the sfGFP sites (c-Myc promoter). Scale bar, 5 μm (main images), 1 μm (insets). Data are representatives of 3 independent experiments. D The droplets formation with indicated treatments. Scale bar 10μm. Data are representatives or mean ± s.d.; n = 3 independent experiments, one-way ANOVA with Dunnett’s multiple comparisons test. E c-Myc promoter activity with corresponding treatments. Data are mean ± s.d.; n = 3 independent experiments, one-way ANOVA with Dunnett’s multiple comparisons test. F c-Myc expression with corresponding treatment. Data are mean ± s.d.; n = 3 independent experiments, one-way ANOVA with Dunnett’s multiple comparisons test. G Colocalization assay of ZNF252P /ILF3/c-Myc. Scale bar 10μm. Data are representatives of 3 independent experiments. H The enrichment of c-Myc immunoprecipitated with anti-ILF3. Data are mean ± s.d.; n = 3 independent experiments, two-tailed Student’s t -test. I The binding sites of ILF3 were mapped to 3’UTR of c-Myc . Data are representatives of 3 independent experiments. J Transcription activity of c-Myc after knockdown ILF3. Data are mean ± s.d.; n = 3 independent experiments, two-tailed Student’s t -test. K Remaining c-Myc mRNA after actinomycin D treatment. Data are mean ± s.d.; n = 3 independent experiments, one-way ANOVA with Dunnett’s multiple comparisons test. L mCherry-ILF3 was incubated with corresponding RNA. Scale bar 10 μm. Data are representatives of 3 independent experiments. M Remaining c-Myc mRNA after actinomycin D treatment. Data are mean ± s.d.; n = 3 independent experiments, one-way ANOVA with Dunnett’s multiple comparisons test. N c-Myc expression with ILF3 and its mutants. Data are mean ± s.d.; n = 3 independent experiments, one-way ANOVA with Dunnett’s multiple comparisons test. O Transcription activity of ZNF252P with corresponding treatments. Data are mean ± s.d.; n = 3 independent experiments, one-way ANOVA with Dunnett’s multiple comparisons test. Source data are provided as a Source data file.

Article Snippet: For qRT-PCR assays, we used the Reverse Transcription Kit (Vazyme) to reverse transcribe RNA to cDNA.

Techniques: Activity Assay, Two Tailed Test, Knockdown, Expressing, Immunoprecipitation, Binding Assay, Incubation