Journal: Nature Communications

Article Title: Exploring the role of stromal osmoregulation in cancer and disease using executable modelling

doi: 10.1038/s41467-018-05414-y

Figure Lengend Snippet: The qualitative network explains cellular phenotypes of Stromal cells in varying experimental conditions. Membrane proteins deregulated under varying stimuli, showing a Transport proteins deregulated in Early (ETDLN) and Late (LTDLN) stage after exposure to TFs, and after exposure to LPS. Proteins are upregulated (red arrows), downregulated (blue arrows), or unchanged (grey) in response to TFs or LPS. b Phenotype change for FRCs upon exposure to TFs for Early (ETDLN) and Late (LTDLN) stage. The Model output represents the physiological behaviour predicted by the model, and the experimental output represents the behaviour observed or implied at the cellular level from experiments or the gene array, and is independent of the model (see methods). Boxes containing two colours indicate phenotypes where there is contradictions/data is unclear. The model predicts an increase in viability, and a sustained increase in attachment and movement/membrane dynamics. Specific protein activity loss predictions are verified with experiments. c , d Knockdown of genes for ( c ) ATP2A3 , and its effect on cellular attachment. The cascade predicted by the model to be underpinning this behaviour change is seen in part ( d ). e , f Also shown are siRNA knockdowns for SLC9A1 and its effect on cell viability ( e ), and FXYD5 and its effect on viability and attachment ( f ). Predicted mechanisms for these knockdowns are included in Supplementary Fig. . * P < 0.05, ** P < 0.01, *** P < 0.001 using two-tailed unpaired t -test. Error bars represent standard deviation. Shown are 11 replicates ( c ), and 9 replicates ( e , f )

Article Snippet: One microgram of total RNA was reverse transcribed using the First Strand cDNA synthesis Kit (Thermo Scientific) with oligo(dT) primers. qRT-PCR was performed using TaqMan assays ( ATP2A3 (Mm00443911_m1), FXYD5 (Mm00435435_m1), SLC9A1 (Mm00444270_m1)) and a StepOne Real Time PCR System instrument (both Life Technologies).

Techniques: Membrane, Activity Assay, Knockdown, Cell Attachment Assay, Two Tailed Test, Standard Deviation

Journal: Science translational medicine

Article Title: Targeting the NF-κB signaling pathway in chronic tendon disease

doi: 10.1126/scitranslmed.aav4319

Figure Lengend Snippet: (A) Heat map of NF-κB profiling gene expression array from healthy human hamstrings tendons (control, n = 4) and early-stage diseased tendons (tendinopathy, n = 5). (B) Volcano plot of NF-κB profiling gene expression array. P < 10−6 are scaled for visualization purposes. (C) NF-κB complex protein coding genes NFKB1, REL, and RELB in control and tendinopathy tendon samples. (D) Regulatory NF-κB protein coding genes CHUK, IKBKB, IKBKE, and NFKBIE in control and tendinopathy tendon samples. Data are shown as means ± SD with individual points representing biologically independent samples. Statistically significant differences were calculated using multiple t tests with Holm-Šídák correction, **P < 0.01 and *P < 0.05.

Article Snippet: Gene expression changes were measured for Ikbkb (IKKβ, Mm01222247_m1), RelA (p65, Mm00501346_m1), RelB (p50, Mm01268877_m1), Chuk (IKKα, Mm00432529_m1), Ikbkg (NEMO, Mm00494927_m1), and Gapdh (Mm99999915_g1).

Techniques: Expressing

Journal: Science translational medicine

Article Title: Targeting the NF-κB signaling pathway in chronic tendon disease

doi: 10.1126/scitranslmed.aav4319

Figure Lengend Snippet: (A) Schematic of NF-κB signaling and gene transcription. NF-κB signaling was controlled by targeting inhibitor of NF-κB kinase subunit p (IKKβ), which acts upstream of the NF-κB complex. Tendon fibroblast IKKβ modulation was achieved by deletion of IKKβ (IKKβKOScx) and activation of IKKβ (IKKβCAScx) using Cre-loxP-mediated recombination under the Scx promoter. (B) Expression of IKKβ in tendon fibroblasts from WT, IKKβKOScx, and IKKβCAScx mice. Cultured mouse osteoclasts were used as a positive control (pos. CTL) (51). (C) Photograph of 16-week-old mice to demonstrate hair loss. (D) Secreted cytokines and growth factors in vehicle and IL-1β-treated tendon fibroblasts from WT, IKKβKOScx, and IKKβCAScx mice (n = 5 per group). (E) Immunolabeling for CD68 (brown) in the supraspinatus tendon from WT, IKKβKOScx,and IKKβCAScx mice. T, tendon; E, enthesis. (F) Microcomputed tomography (μCT) three-dimensional reconstruction of coronal section from proximal humerus. Arrows denote the supraspinatus tendon attachment site. (G) Quantification of bone morphometry: Bone volume normalized to total volume (BV/TV), trabecular thickness (Tb.Th), cortical thickness (Ct.Th), and total cortical area (Tt.Ar) (n = 8 to 9 per genotype). (H) Quantification of mechanical properties of the supraspinatus tendon-to-bone attachment (n = 8 to 9 per genotype). Data are shown as means ± SD with individual points representing biologically independent samples. Statistically significant differences were calculated using one-way analysis of variance (ANOVA) (genotype) with Fisher’s least significant difference (LSD) post hoc test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

Article Snippet: Gene expression changes were measured for Ikbkb (IKKβ, Mm01222247_m1), RelA (p65, Mm00501346_m1), RelB (p50, Mm01268877_m1), Chuk (IKKα, Mm00432529_m1), Ikbkg (NEMO, Mm00494927_m1), and Gapdh (Mm99999915_g1).

Techniques: Activation Assay, Expressing, Cell Culture, Positive Control, Immunolabeling, Tomography

Journal: Science translational medicine

Article Title: Targeting the NF-κB signaling pathway in chronic tendon disease

doi: 10.1126/scitranslmed.aav4319

Figure Lengend Snippet: (A) Ten-week-old mice were subjected to a chronic overuse protocol with 1 week of progressive training, followed by 4 weeks of downhill running. Control mice were permitted normal cage activity. (B) NF-κB pathway-related gene regulation due to overuse. (C) Hematoxylin and eosin (H&E) images of WT, IKKβKOScx, and IKKβCAScx mice. B, bone; black arrowhead, spindle-shaped tendon fibroblast; white arrowhead, enthesis chondrocyte. (D) mRNA expression of Ikbkb, IL-1β, Scx, Col1a1, Col3a1, and Bgn in tendon from control cage-active or treadmill overuse-subjected WT (n = 4 to 6 per group), IKKβKOScx (n = 3 to 4 per group), and IKKβCAScx (n = 3 per group) mice. (E) Failure load, ultimate stress, and Young’s modulus of the supraspinatus tendon-to-bone attachment in cage-active and treadmill overuse-subjected mice (n = 5 to 13 per group). Data are shown as means ± SD with individual points representing biologically independent samples. Statistically significant differences were calculated using two-way ANOVA (genotype, overuse) with Fisher’s LSD post hoc test. **P < 0.01 and *P < 0.05.

Article Snippet: Gene expression changes were measured for Ikbkb (IKKβ, Mm01222247_m1), RelA (p65, Mm00501346_m1), RelB (p50, Mm01268877_m1), Chuk (IKKα, Mm00432529_m1), Ikbkg (NEMO, Mm00494927_m1), and Gapdh (Mm99999915_g1).

Techniques: Activity Assay, Expressing

Journal: Science translational medicine

Article Title: Targeting the NF-κB signaling pathway in chronic tendon disease

doi: 10.1126/scitranslmed.aav4319

Figure Lengend Snippet: (A) Volcano plots of NF-κB signaling array in healthy human tendon fibroblasts treated with IL-1β with or without IKKβ inhibitor. (B) Proinflammatory cytokines IL-6 and CCL-2 produced by healthy human tendon fibroblasts treated with IL-1β with or without IKKβ inhibitor. Data are shown as means ± SD. Statistically significant differences were calculated using one-way ANOVA (treatment) with Fisher’s LSD post hoc test. *P < 0.05.

Article Snippet: Gene expression changes were measured for Ikbkb (IKKβ, Mm01222247_m1), RelA (p65, Mm00501346_m1), RelB (p50, Mm01268877_m1), Chuk (IKKα, Mm00432529_m1), Ikbkg (NEMO, Mm00494927_m1), and Gapdh (Mm99999915_g1).

Techniques: Produced