dy406 05 duo set  (R&D Systems)

Journal: eLife

Article Title: Mir221/222 drive synovial hyperplasia and arthritis by targeting cell cycle inhibitors and chromatin remodeling components

doi: 10.7554/eLife.84698

Figure Lengend Snippet: ( A ) qRT-PCR analysis of Mmp3 from huTNFtg (n = 5) and huTNFtg;TgCol6a1-Mir221/222 (n = 6) SFs. huTNFtg levels were used for normalization. B2m was used as a housekeeping gene. Student’s t -test statistical analysis was used, unpaired. ( B ) qRT-PCR analysis of Rankl from huTNFtg (n = 5) and huTNFtg;TgCol6a1-Mir221/222 (n = 6) SFs. huTNFtg levels were used for normalization. B2m was used as a housekeeping gene. Student’s t -test statistical analysis was used, unpaired. ( C ) IL-6 protein levels measured by ELISA in supernatants from WT (n = 2), TgCol6a1-Mir221/222 (n = 2), huTNFtg (n = 5), and huTNFtg;TgCol6a1-Mir221/222 mice (n = 5) SFs unstimulated or TNF stimulated for 24 or 48 hr. Student’s t -test statistical analysis was used, unpaired. ( D ) qRT-PCR analysis of Il6 from WT (n = 4), TgCol6a1-Mir221/222 (n = 5), huTNFtg (n = 8), and huTNFtg;TgCol6a1-Mir221/222 mice (n = 6) SFs. WT levels were used for normalization. B2m was used as a housekeeping gene. Student’s t -test statistical analysis was used, unpaired. ( E ) qRT-PCR analysis of TNF in SFs from huTNFtg (n = 3) and huTNFtg;TgCol6a1-Mir221/222 mice (n = 3). huTNFtg samples were used for normalization. B2m was used as a housekeeping gene. Student’s t -test statistical analysis was used, unpaired. ( F, G ) Human TNF protein levels measured by ELISA in SF supernatants and sera from WT (n = 3–7), TgCol6a1-Mir221/222 (n = 3–4), huTNFtg (n = 5), and huTNFtg;TgCol6a1-Mir221/222 mice (n = 4–5). Student’s t -test statistical analysis was used, unpaired. ( H, I ) Flow cytometry analysis of ICAM-1 and VCAM-1 expression as MFI in SFs from WT (n = 4), TgCol6a1-Mir221/222 (n = 5), huTNFtg (n = 8–9), and huTNFtg;TgCol6a1-Mir221/222 mice (n = 9). Student’s t -test statistical analysis was used, unpaired. Data represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0,0001, ns = not significant.

Article Snippet: Supernatants were collected at 0, 24, and 48 hr, and IL-6 quantification was performed using the mouse IL-6 Duo-Set ELISA kit, according to the manufacturer’s instructions (R&D Systems).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing

Journal: Proceedings of the National Academy of Sciences

Article Title: Oxysterol binding protein regulates the resolution of TLR-induced cytokine production in macrophages

doi: 10.1073/pnas.2406492121

Figure Lengend Snippet: Fig. 2. Osbp mediates macrophage inflammatory responses to 25HC. (A) Secretion of Il6 measured by ELISA in BMDMs left untreated or treated with the indicated Osbp ligands and then stimulated with LPS or PAM3 for 18 h. (B) BMDMs were left untreated or treated with 25HC, itraconazole, or OSW-1 then were left unstimulated or stimulated with LPS and their transcriptomes analyzed by RNA-seq. Top panels: Comparison of the effect of the indicated Osbp ligands on the expression of genes whose expression is altered by at least two-fold (FDR < 0.01; avg. log2(counts-per-million reads) > 2 across all conditions) by treatment with at least one of the indicated Osbp ligands. Bottom panels: The core LPS response was defined as the 637 genes whose expression at 6 h was altered by at least 10-fold (FDR < 0.01; avg. log2(CPM reads) > 2 across all conditions). The plots compare the effect of itraconazole or OSW-1 to that of 25HC on the expression at 18 h following stimulation of core response genes whose expression at 18 h was altered by at least two-fold by either of the indicated treatments on each plot. (C) Percentage of unedited sequences remaining in the indicated cell pools as measured by TIDE sequencing (29). (D) Western blot for Osbp in macrophages differentiated from Osbp g1, g5, and g6 pools. (E) Il6 protein level 18 h following LPS stimulation of macrophages derived from the indicated CIM lines and treated with the indicated compounds. Bars/points indicate the mean of three biological replicates; error bars indicate SEM (A and E). The blue line indicates best fit and r = Pearson correlation (B). Similar results were obtained in 3 (A) or 2 (E) independent experiments.

Article Snippet: All ELISA Duo Set kits were purchased from R&D Systems [Il6 (DY406), Cxcl1 (DY453), Cxcl2 (DY452), Il1b (DY401), and Csf2 (DY415)] or Cayman Chemical [PGE2 (514010)].

Techniques: Enzyme-linked Immunosorbent Assay, RNA Sequencing, Comparison, Expressing, Sequencing, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: Pharmacological CDK4/6 inhibition unravels a p53-induced secretory phenotype in senescent cells

doi: 10.1101/2020.06.05.135715

Figure Lengend Snippet: p16-3MR mice were treated with vehicle (PBS, 7 consecutive days), doxorubicin (5 mg/kg, 3 consecutive days) or abemaciclib (50 mg/kg, 7 consecutive days). N=6 mice/group. 14 dpt, bioluminescence was visualized and quantified by the IVIS spectrum in vivo imaging system, as shown by representative bioluminescence images ( A ) and quantification ( B ). ( C ) RNA isolated from treated kidneys and mRNA encoding p16 quantified by qRT-PCR. Representative images ( D ) to visualize SA-β-gal activities in vehicle-, doxorubicin-or abemaciclib-treated mouse kidney sections at 15 dpt (arrows indicated positive area; scale bar, 1 mm; N=3) and quantified ( E ). ( F ) 15 dpt, plasma was collected and expression levels of CXCL1 measured by ELISA. ( G ) Protein lysate obtained from drug treated kidneys to quantify IL6 by ELISA. ( H and I ) RNA isolated from kidneys and mRNA encoding indicated genes quantified by qRT-PCR. ( J ) Relative weight changes were calculated at 7 dpt and 14 dpt. Red blood cells ( K ) and white blood cells ( L ) were counted at 15 dpt. Percentage of T cells, B cells, granulocytes and macrophages were determined by flow cytometry analysis ( M ). Physical performance was measured by rotarods assay at 15 dpt ( N ), grip strength meter at 7 dpt and 14 dpt ( O ), and hanging tests were performed at 7 dpt and 14 dpt and normalized to weights ( P ). One-way ANOVA, data are means ±SD ( B, C, E, F, G, K, L and N ). Two-way ANOVA, data are means ±SD ( H, I, J, M, O and P ). *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant. dpt, days post treatment.

Article Snippet: 20 μg total protein was collected from each treated kidney and IL6 protein level was determined by the mouse IL6 duo-set (R&D Systems) ELISA.

Techniques: In Vivo Imaging, Isolation, Quantitative RT-PCR, Clinical Proteomics, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry