Journal: Frontiers in Ophthalmology

Article Title: Brief research report: ETS-1 blockade increases ICAM-1 expression in activated human retinal endothelial cells

doi: 10.3389/fopht.2024.1384428

Figure Lengend Snippet: Impact of ETS-1 silencing on the levels of human retinal endothelial cell ICAM1 transcript and membrane-bound ICAM-1 protein. Human retinal endothelial cells were transfected with ETS-1 siRNA s4847, s4848, s4847 plus s4848, or non-targeted control siRNA for 24 hours, and subsequently stimulated for another 24 hours with TNF-α (10 ng/mL), IL-1β (10 ng/mL) or fresh medium alone. (A-C) Graphs show expression of ICAM1 transcript after transfection with (A) ETS-1 siRNA s4847, (B) ETS-1 siRNA s4848, and (C) ETS-1 siRNA s4847 plus s4848, versus the common non-targeted control siRNA. Bars represent mean expression normalized to the expression of reference genes, RPLP0 and GAPDH (n = 3-4 monolayers/condition). Error bars show standard deviation. (D, E) Graph shows membrane-bound ICAM-1 protein after treatment with siRNA, followed by (D) TNF-α or fresh medium, and (E) IL-1β or fresh medium. Bars represent mean relative fluorescence of immunolabelled cell monolayers after corrections for background fluorescence and cell number (n = 4 cell monolayers/condition). Error bars show standard deviation. x = below detectable level. Data were analyzed by (A-C) two-tailed unpaired t-test and (D, E) two-way ANOVA, with Sídák post-hoc testing: *p < 0.05; **p<0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Mouse monoclonal IgG2b K anti-human ICAM-1 (clone LB-2) and isotype-matched negative control anti-dansyl (clone 27-35) antibodies were bought from BD Pharmingen (San Jose, CA), and used at a working concentration of 1 μg/mL.

Techniques: Membrane, Transfection, Control, Expressing, Standard Deviation, Fluorescence, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Antibody Conjugates Bispecific for Pollen Allergens and ICAM-1 with Potential to Prevent Epithelial Allergen Transmigration and Rhinovirus Infection

doi: 10.3390/ijms24032725

Figure Lengend Snippet: Antibody conjugates (αPhl p 5/αICAM-1) bind to Phl p 5 and human ICAM-1. Conjugates formed with αPhl p 5-IgG*Strep and αICAM-1-IgG*Bio ( x -axes: αPhl p 5/αICAM-1; 5 µg/mL, 1 µg/mL and 0.2 µg/mL), αPhl p 5-IgG*Strep or αICAM-1-IgG*Bio alone were tested for reactivity to Phl p 5 ( A ) and ICAM-1 ( B ) and detected by anti-human F(ab´) 2 (green bars) or anti-mouse IgG (blue bars). Optical density (OD) values ( y -axes) corresponding to bound αPhl p 5/αICAM-1 conjugates, αPhl p 5-IgG*Strep or αICAM-1-IgG*Bio are shown as means of 10-fold analysis (deriving from duplicate determination of five independent experiments) ± standard deviation (SD)s. ( C , D ) are schematic representations of the experiments in ( A , B ).

Article Snippet: Human ICAM-1-specific mouse IgG 1 antibody labeled with biotin (clone 15.2) was purchased from LifeSpan BioSciences (Seattle, WA, USA) and termed αICAM-1-IgG*Bio.

Techniques: Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Antibody Conjugates Bispecific for Pollen Allergens and ICAM-1 with Potential to Prevent Epithelial Allergen Transmigration and Rhinovirus Infection

doi: 10.3390/ijms24032725

Figure Lengend Snippet: Detection of ICAM-1 or Phl p 5 captured by αPhl p 5/αICAM-1 conjugates on 16HBE14o- cells through flow cytometric analysis. ( A ) Expression of human ICAM-1 on the surface of 16HBE14o- cells was confirmed by using αICAM-1-IgG*Bio ( upper panel: white) or an isotype control ( upper panel: gray) and is shown as the percentage of ICAM-1-positive cells out of the alive cells in the scatter plot ( lower panel). ( B ) Cells were incubated with αPhl p 5/αICAM-1 conjugates and different amounts of Phl p 5 (1 µg/50 µL, 2 µg/50 µL and 5 µg/50 µL) and were subsequently probed for αPhl p 5/αICAM-1-bound Phl p 5 with specific rabbit serum or pre-immune serum as an isotype control. Results are displayed as overlayed histograms of Phl p 5-specific rabbit serum (white) and the isotype control (gray) ( upper panels), or as percentages of Phl p 5-positive cells out of the alive cells in the scatter plots ( lower panels). Scatter plots depict the forward scatter (FSC-A) ( x -axes) against ( y -axes) ( A ) Steptavidin Dylight 488 and ( B ) anti-rabbit Alexa Fluor 405. Experiments were performed in triplicates, and displayed data are representatives of two independent experiments.

Article Snippet: Human ICAM-1-specific mouse IgG 1 antibody labeled with biotin (clone 15.2) was purchased from LifeSpan BioSciences (Seattle, WA, USA) and termed αICAM-1-IgG*Bio.

Techniques: Expressing, Incubation

Journal: BMC Biotechnology

Article Title: Interfering ribonucleic acids that suppress expression of multiple unrelated genes

doi: 10.1186/1472-6750-9-57

Figure Lengend Snippet: Design of CODEMIRs targeting VEGF-A and ICAM-1 . (a) Example of seed site alignment and consensus target sequence design for VEGF-A and ICAM-1 using a 12 nucleotide seed. (b) and (c) Schematic illustration of CODEMIR-1 and -2 (respectively) and guide strand binding to the VEGF-A and ICAM-1 mRNAs. Top strand represents the target mRNA (5' to 3'), bottom strand indicates the guide strand (3' to 5').

Article Snippet: ICAM-1 expression was assayed 48 hours after transfection and 24 hours after stimulation with 1 ng/mL recombinant human Interleukin-1β (R&D systems): cells were trypsinized, stained with 0.5 μg anti-human ICAM-1 mouse IgG 1 antibody (Becton Dickinson) at 4°C for 20 minutes, washed with PBS, stained with 0.2 μg Phycoerythrin-labelled anti-mouse IgG 1 antibody (Becton Dickinson) at 4°C for 20 minutes, washed with PBS and analysed using a FACScalibur flow cytometer (Becton Dickinson).

Techniques: Sequencing, Binding Assay

Journal: BMC Biotechnology

Article Title: Interfering ribonucleic acids that suppress expression of multiple unrelated genes

doi: 10.1186/1472-6750-9-57

Figure Lengend Snippet: Suppression of VEGF-A and ICAM-1 expression by CODEMIR-1 and -2 . Unless otherwise indicated, VEGF-A (ELISA) or ICAM-1 (FACS) were assayed in ARPE-19 cells 48 hours post-transfection and 24 hours post-stimulation with 130 μM Deferoxamine or 1 ng/mL IL-1β respectively. All data points indicate the mean of triplicate samples. Error bars show standard deviation. Statistical significance was determined by two-way ANOVA using a Bonferroni post-test (* p < 0.001 as compared to either untransfected and/or irrelevant siRNA; ** p < 0.001 as compared to untransfected and p < 0.01 as compared to irrelevant siRNA; † p < 0.001 compared to CODEMIR-1). (a) Gene suppression by CODEMIR-1 and -2. (b) Dose responsiveness of gene suppression by CODEMIR-1. Cells were transfected at the indicated concentrations. (ND = not determined). (c) Effect of mismatches on gene suppression by CODEMIR-1. (d) Suppression of VEGF-A and ICAM-1 reporter constructs by CODEMIR-1ARPE-19 cells were co-transfected with the AmCyan reporter and AsRed control plasmids and 40 nM indicated RNA duplexes. Fluorescence was assessed by FACS 48 hours post-transfection. (e) Suppression of VEGF-A and ICAM-1 mRNA expression by selected CODEMIRs. For VEGF-A , stimulation was performed 24 hours post-transfection using 65 μM Deferoxamine and the QuantiGene ® assay was performed on cell lysates prepared 24 hours post-stimulation. For ICAM-1 the QuantiGene ® assay was performed on cell lysates prepared 24 hours post-transfection.

Article Snippet: ICAM-1 expression was assayed 48 hours after transfection and 24 hours after stimulation with 1 ng/mL recombinant human Interleukin-1β (R&D systems): cells were trypsinized, stained with 0.5 μg anti-human ICAM-1 mouse IgG 1 antibody (Becton Dickinson) at 4°C for 20 minutes, washed with PBS, stained with 0.2 μg Phycoerythrin-labelled anti-mouse IgG 1 antibody (Becton Dickinson) at 4°C for 20 minutes, washed with PBS and analysed using a FACScalibur flow cytometer (Becton Dickinson).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Standard Deviation, Construct, Fluorescence

Journal: BMC Biotechnology

Article Title: Interfering ribonucleic acids that suppress expression of multiple unrelated genes

doi: 10.1186/1472-6750-9-57

Figure Lengend Snippet: VEGF-A and ICAM-1 suppressive activity of 32 variants of CODEMIR-1 . ARPE-19 cells were transfected with 40 nM of the indicated RNA duplex, and VEGF-A (ELISA) and ICAM-1 (FACS) were assayed 48 hours post-transfection and 24 hours after stimulation with 130 μM Deferoxamine or 1 ng/mL IL-1β respectively. The guide strand of each CODEMIR is shown in the 5' to 3' direction, with blue bases indicating mismatches to the VEGF-A target sequence and red bases indicating mismatches to the ICAM-1 target sequence. Data points represent the mean of triplicate samples. Error bars indicate standard deviation.

Article Snippet: ICAM-1 expression was assayed 48 hours after transfection and 24 hours after stimulation with 1 ng/mL recombinant human Interleukin-1β (R&D systems): cells were trypsinized, stained with 0.5 μg anti-human ICAM-1 mouse IgG 1 antibody (Becton Dickinson) at 4°C for 20 minutes, washed with PBS, stained with 0.2 μg Phycoerythrin-labelled anti-mouse IgG 1 antibody (Becton Dickinson) at 4°C for 20 minutes, washed with PBS and analysed using a FACScalibur flow cytometer (Becton Dickinson).

Techniques: Activity Assay, Transfection, Enzyme-linked Immunosorbent Assay, Sequencing, Standard Deviation

Journal: BMC Biotechnology

Article Title: Interfering ribonucleic acids that suppress expression of multiple unrelated genes

doi: 10.1186/1472-6750-9-57

Figure Lengend Snippet: (a) VEGF and ICAM expression in ARPE-19 cells after transfection with inosine containing CODEMIRs (#100–102) . ARPE-19 cells were transfected with 40 nM duplex RNA and VEGF-A (ELISA) or ICAM-1 (FACS) were assayed 48 hours post-transfection. (b) Comparison of VEGF-A suppressive activity of CODEMIRs containing inosine bases or mismatches at positions 13 and/or 15 of the guide strand. ARPE-19 cells were transfected with 10 nM duplex RNA and VEGF-A (ELISA) was assayed 48 hours post-transfection. Each bar represents the mean of triplicate samples. Error bars indicate standard deviation.

Article Snippet: ICAM-1 expression was assayed 48 hours after transfection and 24 hours after stimulation with 1 ng/mL recombinant human Interleukin-1β (R&D systems): cells were trypsinized, stained with 0.5 μg anti-human ICAM-1 mouse IgG 1 antibody (Becton Dickinson) at 4°C for 20 minutes, washed with PBS, stained with 0.2 μg Phycoerythrin-labelled anti-mouse IgG 1 antibody (Becton Dickinson) at 4°C for 20 minutes, washed with PBS and analysed using a FACScalibur flow cytometer (Becton Dickinson).

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Activity Assay, Standard Deviation

Journal: BMC Biotechnology

Article Title: Interfering ribonucleic acids that suppress expression of multiple unrelated genes

doi: 10.1186/1472-6750-9-57

Figure Lengend Snippet: Suppression of VEGF-A and ICAM-1 expression by variants (shifted 5' of the guide strand) of CODEMIR-1 . ARPE-19 cells were transfected with 40 nM of the indicated duplex RNA. 24 hours after transfection cells were stimulated with 130 μM Deferoxamine ( VEGF-A ) or 1 ng/ml IL-1β ( ICAM-1 ). Gene expression was assayed 48 hours after transfection by ELISA on cell supernatant (VEGF-A) or FACS (ICAM). All data points indicate the mean of triplicate samples. Error bars indicate standard deviation.

Article Snippet: ICAM-1 expression was assayed 48 hours after transfection and 24 hours after stimulation with 1 ng/mL recombinant human Interleukin-1β (R&D systems): cells were trypsinized, stained with 0.5 μg anti-human ICAM-1 mouse IgG 1 antibody (Becton Dickinson) at 4°C for 20 minutes, washed with PBS, stained with 0.2 μg Phycoerythrin-labelled anti-mouse IgG 1 antibody (Becton Dickinson) at 4°C for 20 minutes, washed with PBS and analysed using a FACScalibur flow cytometer (Becton Dickinson).

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Standard Deviation