Journal: eLife

Article Title: Epidermal RAF prevents allergic skin disease

doi: 10.7554/eLife.14012

Figure Lengend Snippet: ( A ) Reduced ERK phosphorylation and increased JNK/p38 activation in primary Δ/Δep2 keratinocytes stimulated with EGF and/or TNFα and IL1β for 15 min. ( B ) Increased cytokine and chemokine production in primary Δ/Δep2 keratinocytes treated with EGF, TNFα and IL1β for 24 hr. Cytokine and chemokine production was determined by multiplex analysis, except for TSLP which was quantified by ELISA. Data represent mean ± SEM of 3–5 biological replicates. ( C–D ) Cells were pretreated with D-JNKI1 inhibitors prior to stimulation with EGF, TNFα and IL1β for 15 min ( C ) or 24 hr ( D ). Data represent the mean ± SEM of technical replicates (n = 3). ( E–F ) Effect of shRNA-mediated Mlk3 silencing on ERK and JNK phosphorylation and ICAM1 expression ( E ; stimulation with EGF, TNFα and IL1β for 15 min) and on the expression of Ccl2 and Tslp mRNA ( F ; stimulation with EGF, TNFα and IL1β for 24 hr) by F/F2 and Δ/Δep2 keratinocytes. shRen, shRNA targeting Renilla, used as a control; sh1 and sh2, targeting Mlk3 , binding sites nucleotide 2266–2285 and 2383–2402, respectively. The shRNAs were encoded by lentiviral vectors coexpressing GFP. GFP immunoblots are shown to confirm similar levels of infection in all samples. Data represent mean ± SEM of 4 biological replicates. Each keratinocyte culture represents a pool of three mice. Immunoblots are representative of three independent experiments. p1 = 0.041, p2 = 0.040, p3 = 1.89E-4, p4 = 0.018, p5 = 0.046, p6 = 0.020, p7 = 0.008, p8 = 0.016, p9 = 0.001, p10 = 0.018, p11 = 3.23E-4, p12 = 1.47E-4, p13 = 0.007, p14 = 0.03, p15 = 0.035, p16 = 0.023 and p17 = 0.046. DOI: http://dx.doi.org/10.7554/eLife.14012.018

Article Snippet: Cells were treated with EGF (2 ng/ml, R&D Systems, 2028-EG) and/or with TNFα (2.5 ng/ml, Millipore, Billerica, MA, 654245) and IL1β (2.5 ng/ml, R&D Systems, 401-ML/CF) as indicated.

Techniques: Phospho-proteomics, Activation Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, shRNA, Expressing, Control, Binding Assay, Western Blot, Infection

Journal: eLife

Article Title: Epidermal RAF prevents allergic skin disease

doi: 10.7554/eLife.14012

Figure Lengend Snippet: ( A ) Reduced ERK and increased JNK/p38 activation in BRAF and RAF1 knockdown (KD2) HaCat cells stimulated with EGF, TNFα and IL1β for 15 min. ( B ) D-JNKI1 reduces ICAM1 and CCL2 (n = 4) expression in KD2 cells treated with TNFα. ( C ) MEKi induces ICAM1 and CCL2 (n = 3) expression in RAF1KD cells treated with TNFα. In ( B–C ), ICAM1 expression was measured after a 3 hr, CCL2 expression after a 24 hr treatment with TNFα. ( D ) Effect of MLK3 silencing on ERK and JNK phosphorylation in WT and KD2 cells stimulated as in ( A ). MLK3 was silenced using a pool of oligonucleotides targeting the following regions: 686–704; 1489–1507; 2122–2138; and 2348–2366. MLK3 KD cells stimulated as in ( B–C ) show a decrease in JNK activation, ICAM1 and CCL2 (n = 7) expression. Immunoblots are representative of three independent experiments. qPCR data represent mean ± SEM of three independent experiments run in duplicates (p1 = 4.62E-4, p2 = 0.013, p3 = 0.050, p4 = 8.60E-8, p5 = 0.050, p6 = 0.001, p7 = 0.001 and p8 = 0.012). DOI: http://dx.doi.org/10.7554/eLife.14012.019

Article Snippet: Cells were treated with EGF (2 ng/ml, R&D Systems, 2028-EG) and/or with TNFα (2.5 ng/ml, Millipore, Billerica, MA, 654245) and IL1β (2.5 ng/ml, R&D Systems, 401-ML/CF) as indicated.

Techniques: Activation Assay, Knockdown, Expressing, Phospho-proteomics, Western Blot

Journal: Frontiers in Endocrinology

Article Title: Methanol extract of Inonotus obliquus improves type 2 diabetes mellitus through modifying intestinal flora

doi: 10.3389/fendo.2022.1103972

Figure Lengend Snippet: Effects of lipid metabolism and inflammatory cytokines. HDL-C (A) , TC (B) , TG (C) and LDL-C (D) . TNF-α (E) , IL-6 (F) , IL-10 (G) and IL-1β (H) inflammatory cytokines in the plasma of db/db mice. Data were expressed as the mean ± SD. ### p < 0.001 vs. K, * p < 0.05 vs. DB, ** p < 0.01vs. DB, *** p < 0.001 vs. DB.

Article Snippet: ELISA kit was used to test inflammatory factors purchased from Boster Biological Technology Co., LTD. Serum tumor necrosis factor-α (TNF-α, EK0527), interleukin-1β (IL-1β, PROTP10749), interleukin-6 (IL-6, EK0411), and interleukin-10 (IL-10, EK0417) assays were performed according to Boster manufacturer’s instructions.

Techniques: