Journal: European journal of cell biology

Article Title: Septin-dependent defense mechanisms against Pseudomonas aeruginosa are stalled in cystic fibrosis bronchial epithelial cells.

doi: 10.1016/j.ejcb.2024.151416

Figure Lengend Snippet: Fig. 1. (A) Septin (SEPT) transcript expressions using RNA sequencing ("RNAseq") in the lungs (public data extracted from GTEXportal.org, TPM: transcripts per million). (B) SEPT mRNA expression in BEAS-2B cells (n=6) using quantitative polymerase chain reaction (qPCR) (reference: SEPT1, Cycle threshold means given between brackets). (C) SEPT7 immunostaining (red) in BEAS-2B cells infected with P. aeruginosa for 3 h [multiplicity of infection (MOI) =20] (green). Nuclei are labelled with DAPI (cyan). (D) Western blot of SEPT7 and reference protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in BEAS-2B cells after infecting with PAK (MOI = 5) for 3 h or not infected (NI) (representative of n= 3 experiments).

Article Snippet: The following SEPT antibodies have already been validated (Pfanzelter et al., 2018): SEPT2 (60075–1, 1/20 000 Proteintech, Manchester, UK), SEPT6 (HPA005665, 1/1000, Atlas Antibody, Bromma, Sweden), SEPT7 (18991; 1/1000, IBL, Minneapolis, MN, USA), SEPT9 (A302–353A-M, 1/1000 Bethyl Laboratories, Montgomery, Texas), and SEPT11 (A304–1761-M, 1/1000, Bethyl laboratories).

Techniques: RNA Sequencing, Expressing, Real-time Polymerase Chain Reaction, Immunostaining, Infection, Western Blot

Journal: European journal of cell biology

Article Title: Septin-dependent defense mechanisms against Pseudomonas aeruginosa are stalled in cystic fibrosis bronchial epithelial cells.

doi: 10.1016/j.ejcb.2024.151416

Figure Lengend Snippet: Fig. 2. SEPT7 mRNA expression analysis using qPCR (A) and SEPT7 (B, C), SEPT2 (B, D), SEPT6 (B, D), SEPT9 (B, D), SEPT11 (B, D) protein expression using western blot in BEAS-2B after 72 h transfection of 5 nM control siRNA (siCTL, white circles) or SEPT7 siRNA (blue circles) (n=4, *p<0.05, Mann–Whitney test). (E) Amount of intracellular P. aeruginosa (Pa) (CFU/mL) in BEAS-2B cells transfected for 72 h with 5 nM of siCTL or SEPT7 siRNA and then infected for 30 min (orange circles, n=6), 1 h (purple circles, n=5) and 3 h (red circles, n=10) with Pa (MOI=5) [(*p<0.05, analysis of variance (ANOVA), Bonferroni correction)]. (F) IL-6 production by BEAS-2B cells transfected for 72 h with 5 nM of siCTL or SEPT7 siRNA in the non-infected (-, white circles) condition and after 3 h of Pa (red cir cles) infection (MOI=5) [(n= 9; *p<0.05, **p<0.01, ****p<0.0001 analysis of variance (ANOVA), Bonferroni correction)].

Article Snippet: The following SEPT antibodies have already been validated (Pfanzelter et al., 2018): SEPT2 (60075–1, 1/20 000 Proteintech, Manchester, UK), SEPT6 (HPA005665, 1/1000, Atlas Antibody, Bromma, Sweden), SEPT7 (18991; 1/1000, IBL, Minneapolis, MN, USA), SEPT9 (A302–353A-M, 1/1000 Bethyl Laboratories, Montgomery, Texas), and SEPT11 (A304–1761-M, 1/1000, Bethyl laboratories).

Techniques: Expressing, Western Blot, Transfection, Control, MANN-WHITNEY, Infection

Journal: European journal of cell biology

Article Title: Septin-dependent defense mechanisms against Pseudomonas aeruginosa are stalled in cystic fibrosis bronchial epithelial cells.

doi: 10.1016/j.ejcb.2024.151416

Figure Lengend Snippet: Fig. 4. (A) Kinetics of SEPT mRNA expression measured by RNAseq in primary bronchial epithelial cells from patients without CF (Non-CF, N = 4) or with CF (N = 4) and infected with P. aeruginosa (PAK strain, MOI 0.25) for 0, 2, 4, or 6 h (*p<0.05: CF vs. non-CF; #p<0.05: CF 4 h vs. CF 2 h, Benjamini and Hochberg corrected p- value) [data were extracted from (Balloy et al., 2015)]. (B) Western blot and quantification of SEPT7 and reference protein GAPDH expression in primary bronchial epithelial cells from non-CF donors (non-CF1, non-CF2) and CF patients (CF1, CF2) at baseline and (C) from non-CF3 and CF2 after 3 h of Pa infection (red circled) (MOI = 10) or non-infected (-; white circle) (n=4; ns: non-significant Mann–Whitney test). (D) Immunostaining of SEPT7 (red) in primary bronchial epithelial cells from non-CF1 and CF1 infected for 3 h with Pa (MOI=20) (green). Nuclei are labelled with DAPI (cyan). (E) Western blot of SEPT7 and reference protein GAPDH in Non-CF2 and CF2 cells after 96 h transfection with 10 nM siCTL or SEPT7 siRNA. Amount of intracellular Pa (CFU/mL) in primary non-CF2 and CF2 cells (n=6) transfected for 72 h with 10 nM CTL (white circle) or SEPT7 (red circles) siRNA and then infected for 4 h with Pa (MOI=10) (n=4; ***p<0.001, ANOVA, Bonferroni correction). (F) Interleukin-6 (IL-6) production by non-CF2 and CF2 cells transfected for 72 h with 10 nM CTL (white circle) or SEPT7 (red circle) siRNA in non-infected and after 4 h of Pa infection (MOI=10) (n= 4; **p<0.01, ***p<0.001, ****p<0.0001, ANOVA, Bonferroni correction).

Article Snippet: The following SEPT antibodies have already been validated (Pfanzelter et al., 2018): SEPT2 (60075–1, 1/20 000 Proteintech, Manchester, UK), SEPT6 (HPA005665, 1/1000, Atlas Antibody, Bromma, Sweden), SEPT7 (18991; 1/1000, IBL, Minneapolis, MN, USA), SEPT9 (A302–353A-M, 1/1000 Bethyl Laboratories, Montgomery, Texas), and SEPT11 (A304–1761-M, 1/1000, Bethyl laboratories).

Techniques: Expressing, Infection, Western Blot, MANN-WHITNEY, Immunostaining, Transfection