Journal: APL Bioengineering

Article Title: Pulsatile low shear stress increases susceptibility to endothelial inflammation via upregulation of IFT and activation of YAP

doi: 10.1063/5.0263936

Figure Lengend Snippet: Endothelial inflammation caused by TNF-α is blocked by siRNAs to YAP and IFT88. Cells were treated with either YAP siRNA, IFT88 siRNA, or Ctrl siRNA for 5 h, followed by a further 4 h of treatment with ± TNF-α (20 ng/ml) prior to fixation and analysis. (a) Representative confocal images of P65 (red) and (b) corresponding nuclear to cytoplasmic (N/C) ratio of P65 staining intensity (n = 100 cells) and (c) percentage of nuclear positive P65 cells in which the N/C ratio was greater than 1.1 [dashed line in (b)] (n = 9 fields). (d) Confocal images of ICAM-1 (green) counterstained for nuclei (blue) and (e) corresponding ICAM-1 integrated intensity normalized to cell number (n = 9 fields). Bars represent mean ± SD with data from three independent experiments. Scale bars = 50 μ m. Statistical analysis was based on a two-way ANOVA with Bonferroni post hoc indicated ± TNF-α and between YAP siRNA, IFT88 siRNA relative to Ctrl siRNA. Statistical analysis was based on a two-way ANOVA with Bonferroni post hoc indicated ± TNF-α (*) and between YAP siRNA, IFT88 siRNA relative to Ctrl siRNA ( # ).

Article Snippet: Gene knockdown was performed using siRNA and compared to scramble control siRNA (sc-37007, Santa Cruz Biotechnology) according to the manufacturer's protocols.

Techniques: Staining

Journal: APL Bioengineering

Article Title: Pulsatile low shear stress increases susceptibility to endothelial inflammation via upregulation of IFT and activation of YAP

doi: 10.1063/5.0263936

Figure Lengend Snippet: Endothelial inflammation caused by LSS is blocked by siRNAs to YAP, MYH10, and IFT88. Cells were treated with siRNAs to either YAP, IFT88, MYH10, or scrambled control for 5 h, followed by a further 4 h pulsatile flow at either HSS or LSS in the presence of TNF-α (20 ng/ml) prior to fixation and analysis. (a) Confocal images of ICAM-1 (green) counterstained for nuclei (blue) and (b) corresponding ICAM-1 integrated intensity normalized to cell number (n = 9 fields). Bars represent mean ± SD with data from three independent experiments. Scale bars = 50 μ m. Statistical analysis was based on a two-way ANOVA with Bonferroni post hoc indicated between LSS and HSS ( * ) and between YAP, IFT88, and MYH10 siRNAs relative to Ctrl siRNA ( # ).

Article Snippet: Gene knockdown was performed using siRNA and compared to scramble control siRNA (sc-37007, Santa Cruz Biotechnology) according to the manufacturer's protocols.

Techniques: Control

Journal: APL Bioengineering

Article Title: Pulsatile low shear stress increases susceptibility to endothelial inflammation via upregulation of IFT and activation of YAP

doi: 10.1063/5.0263936

Figure Lengend Snippet: Inter-dependency between YAP and IFT88/primary cilia expression. Cells were treated with either YAP siRNA, IFT88 siRNA, or Ctrl siRNA for 5 hrs prior to fixation. (a) Representative confocal images of primary cilia (arrows) showing axoneme and basal body labeled using anti-acetylated α-tubulin (green) and anti-pericentrin (red), nucleus (blue) and (b) corresponding cilia length (n = 100 cilia) and (c) the percentage of ciliated cells (n > 600 cells). (d) Representative confocal images of YAP (green) and (e) corresponding nuclear/cytoplasmic (N/C) YAP intensity ratios (n = 200 cells) and (f) the percentage of nuclear YAP positive cells in which the N/C ratio was greater than 1.1 (dashed line in E). Scale bar = 20 μ m. Data from three independent experiments. Bars represent mean ± SD. Statistical analysis was based on one-way ANOVA test with differences shown relative to control group.

Article Snippet: Gene knockdown was performed using siRNA and compared to scramble control siRNA (sc-37007, Santa Cruz Biotechnology) according to the manufacturer's protocols.

Techniques: Expressing, Labeling, Control

Journal: The EMBO Journal

Article Title: A bipartite bacterial virulence factor targets the complement system and neutrophil activation

doi: 10.1038/s44318-024-00342-8

Figure Lengend Snippet: ( A , B ) co-IP assays. For this experiment, dHL-60 cell lysates were incubated with or without His-tagged N0362 protein (10 µg) for 3 h at 4 °C. Then, 5 µl Ni-NTA resin slurry was added to the mixture and further incubated at 4 °C for overnight. Following the incubation, the mixture was washed five times with PBS containing 0.05% Tween 20. The final pellets were suspended in Laemmli sample buffer, boiled for 5 min, and then briefly centrifuged to collect the supernatants, which were then subjected to immunoblotting analysis probed against different antibodies, including mAb-N7, a monoclonal antibody that recognizes N0362, anti-FPR1 (αFPR1), anti-CXCR1 (αCXCR1), anti-CXCR2 (αCXCR2), anti-CD11b (αCD11b), and anti-C5αR (αC5αR, Fig. ). ( C , D ) Immunofluorescence analysis of dHL-60 cells. The cells were incubated with Alexa Fluor 488-labeled N0362 proteins for 2 h, stained with αFPR1 or αCXCR1, followed by staining with Alexa Fluor 594. The micrographs were captured under fluorescence microscopy using FITC, TRITC, and DAPI emission filters, and the images were merged. The scale bars represent 20 µm. ( E , F ) Downregulation of FPR1 or CXCR1 expression using siRNA impairs the binding of N0362 to dHL-60 cells. For this study, dHL-60 cells were treated using siRNA specific to either ( E ) FPR1 or ( F ) CXCR1 for 96 h, followed by immunofluorescence analysis as above described. The scale bars represent 20 µm. .

Article Snippet: dHL-60 cells (1 × 10 6 ) were seeded into 24-well cell culture plates and transfected with 0.5 μg of either CXCR1 siRNA (sc-40026), FPR1 siRNA (sc-40121) or scramble siRNA (sc-37007, Santa Cruz Biotechnology) using Lipofectamine LTX with PLUS reagent (Invitrogen) for 96 h at 37 °C and 5% CO 2 .

Techniques: Co-Immunoprecipitation Assay, Incubation, Western Blot, Immunofluorescence, Labeling, Staining, Fluorescence, Microscopy, Expressing, Binding Assay

Journal: The EMBO Journal

Article Title: A bipartite bacterial virulence factor targets the complement system and neutrophil activation

doi: 10.1038/s44318-024-00342-8

Figure Lengend Snippet: ( A – C ) For this experiment, DMSO-differentiated HL-60 cell (dHL-60) lysates were incubated with or without His-tagged N0362 protein (10 µg) for 3 h at 4 °C, followed by precipitation using Ni-NTA resin. The resulted co-IP samples were subjected to immunoblotting analysis probed against mAb-N7, a monoclonal antibody that recognizes N0362. ( A ) anti-CXCR2 (αCXCR2), ( B ) anti-CD11b (αCD11b), and ( C ) anti-C5aR (αC5aR). ( D ) siRNA knockdown of FPR1 or CXCR1 reduces the binding of N0362 to HL-60 cells. For this experiment, activated HL-60 cells were transfected with scramble siRNA (control), FPR1 siRNA or CXCR1 siRNA for 3 days and then the cells were co-incubated with His-tagged N0362 for 2 h. Cells were then fixed and stained anti-His antibody. Fluorescent cells were counted and recorded as percentage of positive cells in relative to total cells. * P value < 0.05. ( E , F ) Detection of FPR1 and CXCR1 by immunoblotting analysis. Lane 1: scramble siRNA; Lane siRNA-FPR1 or CXCR1 72 h after the knockdown; and Lane 3: siRNA-FPR1 or CXCR1 96 h. β-actin was used as a loading control.

Article Snippet: dHL-60 cells (1 × 10 6 ) were seeded into 24-well cell culture plates and transfected with 0.5 μg of either CXCR1 siRNA (sc-40026), FPR1 siRNA (sc-40121) or scramble siRNA (sc-37007, Santa Cruz Biotechnology) using Lipofectamine LTX with PLUS reagent (Invitrogen) for 96 h at 37 °C and 5% CO 2 .

Techniques: Incubation, Co-Immunoprecipitation Assay, Western Blot, Knockdown, Binding Assay, Transfection, Control, Staining

Journal: The EMBO Journal

Article Title: A bipartite bacterial virulence factor targets the complement system and neutrophil activation

doi: 10.1038/s44318-024-00342-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: dHL-60 cells (1 × 10 6 ) were seeded into 24-well cell culture plates and transfected with 0.5 μg of either CXCR1 siRNA (sc-40026), FPR1 siRNA (sc-40121) or scramble siRNA (sc-37007, Santa Cruz Biotechnology) using Lipofectamine LTX with PLUS reagent (Invitrogen) for 96 h at 37 °C and 5% CO 2 .

Techniques: Isolation, Recombinant, Plasmid Preparation, Sequencing, Labeling, Staining, Transfection, Software, Binding Assay, Microscopy, Electron Microscopy

Journal: Cancer Cell International

Article Title: Mithramycin targets head and neck cancer stem cells by inhibiting Sp1 and UFMylation

doi: 10.1186/s12935-024-03609-6

Figure Lengend Snippet: The expression of the UFMylation system proteins. We have compared the untreated tumor spheres or tumor spheres treated with poly (I: C) or poly (A: U). UFSP2 expression in Detroit 562 spheres and adherent cells after the treatment with poly (I: C) or poly (A: U) (Fig. 3a). UFSP2 expression in FaDu cells transfected with inducible shRNA for TLR3 treated with poly (I: C) or poly (A: U), and in Detroit 562 cells after transient transfection with TLR3 siRNA and treatment with poly (I: C) or poly (A: U) (Fig. 3b). UFSP2 expression by immunocytochemistry in Detroit 562 adherent cells and tumor spheres (Fig. 3c). DDRGK1 and UFL1 expression in Detroit 562 control adherent cells or tumor spheres after the treatment with poly (I: C) or poly (A: U) (Fig. 3d).* p > 0.05 (compared to adherent cells). UFSP2 expression by immunocytochemistry in Detroit 562 untreated tumor spheres, and spheres treated with poly (I: C), and poly (A: U) (Fig. 3e)

Article Snippet: Double-stranded small interfering RNA (siRNA) for knocking down the endogenous TLR3 (sc-36685), and UFM1 (sc-76804) including scrambled-sequence (control) siRNA (control siRNA-A, sc-37007), were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, shRNA, Immunocytochemistry, Control

Journal: Cancer Cell International

Article Title: Mithramycin targets head and neck cancer stem cells by inhibiting Sp1 and UFMylation

doi: 10.1186/s12935-024-03609-6

Figure Lengend Snippet: The UFM1 expression is increased in tumor spheres and associated with their size and stemness. The expression and colocalization of UFM1 in adherent cells and tumor spheres treated with poly (I: C) or poly (A: U); * p > 0.05, ** p > 0.03, ** p > 0.01 (compared to adherent cells) (Fig. 5a). Tumor spheres size in cells transfected with control siRNA and those transfected with UFM1 siRNA; * p > 0,05, ** p > 0,01, *** p > 0,005 (compared to control siRNA) (Fig. 5b). The expression of UFM1 after the siRNA silencing shown by western blot (Fig. 5c). The survival of UFM1-silenced tumor spheres (Fig. 5d). The expression of UFM1, OCT4, CD133, ABCG2, fibronectin, and vimentin after UFM1 silencing shown by qPCR; * p > 0.05, ** p > 0.03, *** p > 0.01, **** p > 0,001 (compared to control siRNA) (Fig. 5e). Immunocytochemistry shows reduced expression of CD133 and ALDH1 after UFM1 silencing; * p > 0.05, ** p > 0.03, *** p > 0.005 (compared to control siRNA) (Fig. 5f). Co-localization of CD133 and ALDH1 with UFM1 in untreated tumor spheres (Fig. 5g)

Article Snippet: Double-stranded small interfering RNA (siRNA) for knocking down the endogenous TLR3 (sc-36685), and UFM1 (sc-76804) including scrambled-sequence (control) siRNA (control siRNA-A, sc-37007), were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, Control, Western Blot, Immunocytochemistry