Journal: Journal of Crohn's & Colitis

Article Title: Therapeutic siRNAs Targeting the JAK/STAT Signalling Pathway in Inflammatory Bowel Diseases

doi: 10.1093/ecco-jcc/jjab129

Figure Lengend Snippet: Selectivity and efficiency of JAK -targeting siRNAs. Selectivity of the sorted siRNAs was assessed on PC3-JAK3-GFP-engineered cells transfected using lipofectamine RNAiMAX and different siRNAs at 10 nM. Representative western blot analysis is shown on the top panel, with the corresponding quantification at the bottom [ N = 4 independent experiments] for the expression of JAK1 [A], JAK3 [B], JAK2 [C] or TYK2 [D] proteins. For A, * represents respectively p -values = 0.0307 and 0.0324; for B, * represents respectively p -values = 0.0477 and 0.0456; for D, * represents respectively p -values = 0.0212 and 0.0359. [E] Representative western blot of PC3 cells transfected by JAK1 -targeting siRNAs at different concentrations ranging from 0.5 to 12.5 pM. [F] Quantification of JAK1 protein expression [ N = 3 independent experiments] from the experiment shown in E. Quantification by RTqPCR of JAK1 mRNA levels in PC3 cells [G, H] or JAK3 mRNA levels in PC3-JAK3-GFP-engineered cells [I, J] transfected using lipofectamine RNAiMAX and siRNAs at concentration ranging from 0.5 to 12.5 pM [ N = 3 independent experiments]. GAPDH was used as a housekeeping gene.

Article Snippet: Protein lysates of CACO-2 cells transfected for 3 days with 10 nM siRNA using lipofectamine RNAiMAX were analysed by the SomaLogic company.

Techniques: Transfection, Western Blot, Expressing, Concentration Assay

Journal: Journal of Crohn's & Colitis

Article Title: Therapeutic siRNAs Targeting the JAK/STAT Signalling Pathway in Inflammatory Bowel Diseases

doi: 10.1093/ecco-jcc/jjab129

Figure Lengend Snippet: Analysis of sequence-based transcriptomic effects and phenotypic side effects in response to JAK modulation. Quantification by RTqPCR of target genes at mRNA levels in T47D cells transfected using lipofectamine RNAiMAX at 10 nM with siHJ1D8 [A], siHJ1D2 [B], siHMJ3D1 [C] or siHJ3D41 [D], dot plot representing N = 4 independent experiments. p -values: for A: ** = 0.0014; * = 0.0264; for B: ** = 0.0079; for D: ***** = 0.0001, ** = 0.0086. [E] THP1-XBlue-MD2-CD14 cells were treated for 24 h with Pam3CSK4 [1 ng/mL], HKLM [10 7 cells/mL], Poly[I:C] [low/high molecular weight] [10 µg/mL], LPS [10 ng/mL], FLA-ST [10 ng/mL], CpG [10 µg/mL] or siRNAs at final concentration 10 nM. Alkaline phosphatase activity was quantified using a QUANTI Blue Assay, and absorbance was measured at 655 nm and presented as mean ± SD [ N = 6 independent experiments]. [F] Proliferation analysis in CACO-2 cells, following transfection using lipofectamine RNAiMAX at 10 nM of siRNA or treatment by different concentrations of tofacitinib or filgotinib for 72 h. Cells were incubated with 5 µM EdU for 5 h, before harvesting and fixating the cells for downstream FACS analysis. Dot plot showing mean percentage ± SD [ N = 4 independent experiments]; p -values: * = 0.0157, *** = 0.0010. [G] Analysis of apoptotic cell death in CACO-2 cells occurring after transfection by lipofectamine RNAiMAX at 10 nM of siRNAs or treated by different concentrations of tofacitinib or filgotinib, in the presence of 2 µM of CellEvent reagent for 72 h. Cells were harvested and fixated before downstream FACS analysis. Dot plot showing mean percentage ± SD [ N = 4 independent experiments]; p -values: * = 0.0137 and 0.0204. [H] Mitochondrial metabolism and ATP levels were assessed in CACO-2 cells, following transfection by lipofectamine RNAiMAX at 10 nM of siRNAs or treated by different concentrations of tofacitinib or filgotinib for 72 h. Cells were then analysed using a ViaLight Plus Cell Proliferation and Cytotoxicity Bioassay kit. Dot plot showing mean percentage ± SD [ N = 4 independent experiments]; p -values: * = 0.0143, ** = 0.0051.

Article Snippet: Protein lysates of CACO-2 cells transfected for 3 days with 10 nM siRNA using lipofectamine RNAiMAX were analysed by the SomaLogic company.

Techniques: Sequencing, Transfection, High Molecular Weight, Concentration Assay, Activity Assay, Incubation, Bioassay

Journal: Journal of Crohn's & Colitis

Article Title: Therapeutic siRNAs Targeting the JAK/STAT Signalling Pathway in Inflammatory Bowel Diseases

doi: 10.1093/ecco-jcc/jjab129

Figure Lengend Snippet: Comparative analysis of JAK -targeting siRNA and Jakinibs on JAK/STAT signalling in CACO-2 cells. Western blot analysis of JAK/STAT signalling in CACO-2 cells transfected at 10 nM siRNA using lipofectamine RNAiMAX for 48 h. Untransfected cells were then exposed to tofacitinib at 1 µM or filgotinib at 2 or 5 µM for 1 h. To trigger the activation of the JAK/STAT signalling pathway, cells were then exposed for 30 min to 10 ng/mL IL22 [A], IFNγ [B], IFNβ [C] or IL4 [D]. Quantification of at least N = 4 independent experiments, with each dot representing one ‘ N ’, with means in black. p -values: * = <0.05, ** = 0.0079. Exact p -values are given in .

Article Snippet: Protein lysates of CACO-2 cells transfected for 3 days with 10 nM siRNA using lipofectamine RNAiMAX were analysed by the SomaLogic company.

Techniques: Western Blot, Transfection, Activation Assay

Journal: Journal of Crohn's & Colitis

Article Title: Therapeutic siRNAs Targeting the JAK/STAT Signalling Pathway in Inflammatory Bowel Diseases

doi: 10.1093/ecco-jcc/jjab129

Figure Lengend Snippet: Efficiency of JAK -targeting siRNAs over time. RT-qPCR analysis of CACO-2 cells transfected by 10 nM siHJ1D8/2 with lipofectamine RNAiMAX for 1–7 days [ N = 4 independent experiments, mean and SD represented] for the expression of JAK1 [A] or JAK3 [B]. Western-blot analysis of CACO-2 cells transfected by 10 nM siHJ1D8/2 with lipofectamine RNAiMAX for 1–7 days with a representative western blot analysis [C], and the corresponding quantification at the bottom [ N = 6 independent experiments, mean and SD represented] for the expression of JAK1 [D]. Western blot analysis of CACO-2 cells pulse transfected for 6 h by 10 nM siHJ1D8/2 with lipofectamine RNAiMAX and analysed 1–14 days after transfection with a representative western blot analysis [E] and the corresponding quantification at the bottom [ N = 3 independent experiments, mean and SD represented] for the expression of JAK1 [F].

Article Snippet: Protein lysates of CACO-2 cells transfected for 3 days with 10 nM siRNA using lipofectamine RNAiMAX were analysed by the SomaLogic company.

Techniques: Quantitative RT-PCR, Transfection, Expressing, Western Blot