Journal: Molecular Therapy

Article Title: Characterisation of a Novel Fc Conjugate of Macrophage Colony-stimulating Factor

doi: 10.1038/mt.2014.112

Figure Lengend Snippet: Pig CSF1-Fc produces viable CSF1-dependent proliferation in vitro and has extended plasma half-life in vivo . ( a ) CSF1-Fc molecule was produced by CSF1 joined to the hinge-CH3 region of pig IgG1a. ( b ) CSF1-dependent Ba/F3pCSF1R cells were cultured in rh-CSF1, harvested, washed twice in PBS, and plated for the optimized cell viability assay with either pig CSF1 or pig CSF1-Fc for 48 hours. Pig BM cells were flushed from an adult pig rib and placed in culture with either pig CSF1 or pig CSF1-Fc for 48 hours. Following addition of MTT solution and solubilization, optical density was read at 570nm using a plate reader. Results are the average of triplicate determinations ± SEM from three experiments. ( c ) Three weaner pigs were injected with either 0.5 mg/kg or 1.2 mg/kg pig CSF1 or Fc CSF1-Fc respectively and blood collected at time points above for CSF1 and Fc CSF1-Fc levels to be determined by ELISA. The mean ± SEM is graphed.

Article Snippet: A fusion protein comprising pig CSF1 joined to the hinge-CH3 region of pig IgG1a ( ) was expressed in HEK293F cells and purified using Protein A affinity chromatography under contract from Genscript.

Techniques: In Vitro, Clinical Proteomics, In Vivo, Produced, Cell Culture, Viability Assay, Injection, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Immunology Author Choice

Article Title: RNA helicase DDX3X promotes NK cell survival by supporting MCL1 expression

doi: 10.1093/jimmun/vkaf281

Figure Lengend Snippet: RNA helicase DDX3 supports the survival of mouse NK cells. Male mouse NK cells were isolated from the spleens of (A) C57BL/6 or (B) Cas9 transgenic mice using negative selection columns and incubated in culture medium supplemented with IL-2 and IL-15. (A) The DDX3 inhibitor RK33 (DDX3-i, red) or vehicle (DMSO, black) were added to cultures for 20 hours. Representative flow plots reveal uptake of NIR Zombie live/dead dye and loss of viability, with median and individual results for 5 mice/group. Statistical significance determined by Mann–Whitney test. Results are representative of >3 independent experiments. (B) Control or Ddx3x sgRNA (20 µM) was transfected into separate pools of Cas9 transgenic NK cells. After 48 hours, the viability of NK cells was assessed by flow cytometry, with representative and median outcomes displayed. Statistical significance determined by unpaired 2-tailed Student t -test. Data are representative of 2 independent experiments. (C) Immunoblot showing β-actin and DDX3X expression in isolated mouse NK cells from Cas9 transgenic mice 24 hours after transfection with scrambled or Ddx3x -specific sgRNA.

Article Snippet: Small guide RNAs (sgRNA) were obtained from Synthego (Redwood City, CA, USA) to target exon 4 of Ddx3x using a CRISPR evolution EZ sgRNA kit.

Techniques: Isolation, Transgenic Assay, Selection, Incubation, MANN-WHITNEY, Control, Transfection, Flow Cytometry, Western Blot, Expressing

Journal: The Journal of Immunology Author Choice

Article Title: RNA helicase DDX3X promotes NK cell survival by supporting MCL1 expression

doi: 10.1093/jimmun/vkaf281

Figure Lengend Snippet: DDX3X is required to sustain expression of the prosurvival protein MCL1. Mouse NK cells were isolated from the spleens of male (A–D) C57BL/6 or (E) Cas9 mice and cultured with IL-2/IL-15. (A) Cultures were treated with RK-33 (DDX3-i, red) or DMSO (black) for 24 hours prior to flow cytometry–based assessment of expression of prosurvival proteins BCL2, BCL-xL, and MCL1. Histograms depict representative staining and dot plots show individual mouse mean fluorescent intensity (MFI) and group mean. (B–D) Cultured C57BL/6 NK cells were transfected with scrambled (control) or (B–C) Ddx3x -specific siRNA or (C–D) Ddx3y-specific siRNA for 24 hours before flow cytometry analysis of cell viability. (B, D) Histograms depict representative intensity of prosurvival protein expression while dot plots show individual mouse MFI and group mean. (C) Real-time quantification of Ddx3x and Ddx3y transcripts in siRNA-treated cells normalized to expression in controls. (D) Cultured NK cells from Cas9 transgenic mice were transfected with 20 µM Ddx3x or control sgRNA. After 48 hours, MCL1 expression levels were determined and are presented as described for (A). Statistical significance determined by Mann–Whitney test. Results are representative of at least 2 independent experiments.

Article Snippet: Small guide RNAs (sgRNA) were obtained from Synthego (Redwood City, CA, USA) to target exon 4 of Ddx3x using a CRISPR evolution EZ sgRNA kit.

Techniques: Expressing, Isolation, Cell Culture, Flow Cytometry, Staining, Transfection, Control, Transgenic Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: Endothelial RAB5IF is required for pathological and developmental retinal angiogenesis

doi: 10.1038/s41467-025-66212-x

Figure Lengend Snippet: A The TIE1-Cre C57 mice (six-seven weeks old) were intravitreously injected with AAV5-FLEX-GFP viral particles (7 × 10 10 viral particles/0.5 µL/mouse), followed by frozen sections and flat mount staining 21 days later to observe GFP-positive infection area. Scale bar = 100 μm / 50 μm. B – H The TIE1-Cre C57 mice (six-seven weeks old) were intravitreously injected with AAV5-FLEX-sgRAB5IF (RAB5IF-eCKO#1 and RAB5IF-eCKO#2, encoding non-overlapping sgRNAs against murine RAB5IF ) or AAV5-FLEX-sgRNA control viral particles (“sgC), both at 7 × 10 10 viral particles/0.5 µL/mouse; After 21 days RAB5IF protein expression in the primary mRMECs extracted from adult mice was shown (pooled from 4 mice/sample, n = 5 samples/group, 15 μg protein/lane for WB) ( B ); IB4 staining of retinal flat mounts, showing representative images, and quantification of vascular branch numbers. Scale bar = 100 μm ( C ). PAS staining of retinal tissue, showing representative images, and quantification of acellular capillary numbers. Scale bar = 60 μm ( D ). Evans blue (EB) leakage assay assessing retinal vascular leakage, showing representative images, and quantification of EB leakage. Scale bar = 1 mm ( E ). Tubb3/NeuN double staining of retinal flat mounts to label RGCs, showing representative images, and quantification of Tubb3 + /NeuN + cell numbers. Scale bar = 60 μm ( F ). RBPMS staining of retinal sections to label RGCs, showing representative images, and quantification of RBPMS + cell numbers. GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar = 200 μm ( G ). Expression of listed neuronal marker proteins in the retinal tissues was also tested (individual samples, n = 5 mice/group, 15 μg protein/lane) ( H ). I At P1, endothelial-targeted AAV (1 × 10 11 viral particles/7.5 µL/mouse) was injected retro-orbitally for RAB5IF knockdown (RAB5IF-eKD). Subsequently, listed neuronal markers were detected by Western blot at P6. (pooled from 8 mice/sample, n = 8 samples/group, 15 μg protein/lane for WB). Data are presented as means ± S.D; One-way ANOVA with Bonferroni’s post hoc test. n = 5 mice per group. Source data are provided as a Source Data file.

Article Snippet: For in vitro studies, lentiviral short hairpin RNAs (shRNAs) and single guide RNAs (sgRNAs) obtained from Shanghai Genechem Co. (Shanghai, China) were utilized for gene knockdown and knockout (KO) in hRMECs.

Techniques: Injection, Staining, Infection, Control, Expressing, Double Staining, Marker, Knockdown, Western Blot

Journal: Nature Communications

Article Title: Endothelial RAB5IF is required for pathological and developmental retinal angiogenesis

doi: 10.1038/s41467-025-66212-x

Figure Lengend Snippet: A – E The human retinal microvascular endothelial cells (hRMECs) expressing the applied RAB5IF shRNAs (shRAB5IF#1, shRAB5IF#2 and shRAB5IF#3, with different shRNA sequences against RAB5IF ) or the scramble control shRNA (shC) were established, expression of RAB5IF mRNA and protein was shown ( n = 5 biological repeats, 15 μg protein/lane for WB) ( A ); Cells were further cultured for the indicated time periods, sprouting ( B ), cell proliferation (by testing EdU-positive nuclei ratio, C ), migration (Transwell assays, D ) and the capillary tube formation ( E ) were tested by the listed assays. F – J Stable hRMECs expressing the listed lentiviral CRISPR/Cas9-RAB5IF-KO construct (koRAB5IF#1/koRAB5IF#2, with different sgRNA sequences against RAB5IF ) or the control construct with non-sense sgRNA (koC) were established, and RAB5IF protein expression was tested ( n = 5 biological repeats, 15 μg protein/lane for WB) ( F ); Cells were further cultured for the indicated time periods, sprouting ( G ), cell proliferation (by testing EdU-positive nuclei ratio, H ), migration (Transwell assays, I ) and the capillary tube formation ( J ) and were tested. K , L Stable hRMECs with the designated genetic modifications on RAB5IF were cultured and subjected to Seahorse analyses to evaluate oxidative phosphorylation ( K ) and glycolysis ( L ). M – R Stable hRMECs with the lentiviral RAB5IF-expressing construct (oeRAB5IF-Sl1/oeRAB5IF-Sl2, two stable cell selections) or the empty vector (Vec) were established, expression of RAB5IF mRNA and protein was shown ( n = 5 biological repeats, 15 μg protein/lane for WB) ( M ); Cells were further cultured for the indicated time periods, sprouting ( N ), cell proliferation (by testing EdU-positive nuclei ratio, O ) and migration (Transwell assays, P ) were tested, with results quantified. Seahorse assays were also carried out to evaluate ATP contents ( Q ) and basal glycolysis levels ( R ). Data are presented as means ± S.D., n = 5 (biological repeats). “N.S.” indicates no statistical difference ( P > 0.05); One-way ANOVA with Bonferroni’s post hoc test. Scale bars = 100/200 μm. Source data are provided as a Source Data file.

Article Snippet: For in vitro studies, lentiviral short hairpin RNAs (shRNAs) and single guide RNAs (sgRNAs) obtained from Shanghai Genechem Co. (Shanghai, China) were utilized for gene knockdown and knockout (KO) in hRMECs.

Techniques: Expressing, shRNA, Control, Cell Culture, Migration, CRISPR, Construct, Phospho-proteomics, Stable Transfection, Plasmid Preparation