mouse mrna & lncrna epitranscriptomic microarray (8 × 60k)  (Arraystar inc)

Journal: Clinical and Translational Medicine

Article Title: The antiprotease Spink7 promotes inflammation resolution by modulating multiple proteases activities during wound healing

doi: 10.1002/ctm2.70291

Figure Lengend Snippet: Spink7 is significantly upregulated during skin wound repair and knockdown Spink7 by siRNAs results in delayed healing. (A) Expression of Spink7 mRNA levels in skin wound healing examined by real‐time RT‐PCR ( n = 5 per time point). (B) Double staining for Spink7 mRNA (FISH in red) and Ly6G or E‐cadherin (IF in green) to identify Spink7 mRNA transcript in wound tissues. Left panel for Day 3 wound tissue and right panel for Day 7 wound tissue. The white dashed line separates the epidermis from the dermis. (C) Representative images of IHC staining for Spink7 in Day 3 wound tissue (left panel) and Day 7 wound tissue (right panel) to show positive signals in neutrophils and keratinocytes. The local magnification image of the left panel shows neutrophils with segmented nuclei that exhibit positive signals. (D) Macroscopic appearance of wound closure in si‐NC and si‐Spink7 treated wounds in mice at Day 0, 1, 3, and 7 after wounding. (E) Quantification of residual wound area in si‐NC and si‐Spink7 treated wounds in mice. Data are expressed as the percentage of the remaining area to the initial wound area. n = 5 samples/group. (F) Representative H&E sections from si‐NC and si‐Spink7 treated wounds on Day 7 after wounding. Black arrows delineate wound edges. Red arrows highlight epithelial migrating tongues. (G) Quantification and calculation percentages of wound re‐epithelialization. n = 5 samples/group. (H) High power fields of H&E sections from si‐NC and si‐Spink7 treated day 7 wounds (up panel) and representative images of IHC staining for MPO (down panel). (I) Quantification of MPO positive neutrophils of day 7 wounds of si‐NC and si‐Spink7 treated mice ( n = 5). Data are represented as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Data are representative of three independent experiments.

Article Snippet: Probe against mouse Spink7 (cat. no. 1191121‐C1, ACDBio) mRNA molecule was used.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Double Staining, Immunohistochemistry

Journal: Clinical and Translational Medicine

Article Title: The antiprotease Spink7 promotes inflammation resolution by modulating multiple proteases activities during wound healing

doi: 10.1002/ctm2.70291

Figure Lengend Snippet: Spink7 KO mice show impaired wound closure. (A) Expression of Spink7 in both KO and WT mice during wound healing examined by real‐time RT‐PCR (n = 5 per time point). (B) Macroscopic appearance of wound closure in wounds of KO and WT mice at different time points. (C) Quantification of wound area at different time points in wounds of KO and WT mice. n = 5 samples/group. (D) Representative H&E sections in 7‐day wounds of KO and WT mice. Black arrows delineate wound edges. Red arrows highlight epithelial migrating tongues. (E) Quantification of the epithelial tongue lengths of 7‐day wounds. n = 6–8 samples/group. (F) Quantification and calculation percentages of wound re‐epithelialization. n = 6–8 samples/group. (G) Quantification of the wound areas of 7‐day wounds. n = 6–8 samples/group. (H) Quantification of the wound widths of 7‐day wounds. n = 6–8 samples/group. (I) Representative images of IHC staining for MPO in 7‐day wounds of KO and WT mice. (J) Quantification of MPO positive neutrophils. n = 6–8 samples/group. (K) Expression of MPO mRNA levels in 7‐day wounds of KO and WT mice examined by real‐time RT‐PCR. n = 6–8 samples/group. Data are represented as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus WT mice group at same time points. Data are representative of three independent experiments.

Article Snippet: Probe against mouse Spink7 (cat. no. 1191121‐C1, ACDBio) mRNA molecule was used.

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry

Journal: Clinical and Translational Medicine

Article Title: The antiprotease Spink7 promotes inflammation resolution by modulating multiple proteases activities during wound healing

doi: 10.1002/ctm2.70291

Figure Lengend Snippet: Loss of Spink7 augments inflammatory response and enhances production of chemokines/cytokines during wound healing. (A) Graph of top 10 biological process terms for GO analysis from up‐regulated genes of microarray experiments of 7‐day wound samples (KO vs WT). (B) Top 10 molecular function terms for GO analysis. (C) Top 10 pathway terms for KEGG analysis from up‐regulated genes of microarray experiments. (D) Examining some proinflammatory chemokines/cytokines in wound tissue by real‐time RT‐PCR for validation of partial results of microarray experiments. n = 5 samples/group. Representative data are shown from two independent experiments. (E) Protein levels of some proinflammatory chemokines/cytokines in wound tissue detected by Bio‐Plex Pro™ Mouse Chemokine Assays Kit. n = 4–6 samples/group. Representative data are presented as the concentrations of chemokines/cytokines in total protein from two independent experiments performed in duplicate. Data are represented as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus WT mice group at same time points.

Article Snippet: Probe against mouse Spink7 (cat. no. 1191121‐C1, ACDBio) mRNA molecule was used.

Techniques: Microarray, Quantitative RT-PCR, Biomarker Discovery

Journal: Clinical and Translational Medicine

Article Title: The antiprotease Spink7 promotes inflammation resolution by modulating multiple proteases activities during wound healing

doi: 10.1002/ctm2.70291

Figure Lengend Snippet: Loss of Spink7 results in impaired M2 polarization of macrophages in wounds. (A) Double staining for F4/80 and iNOS to identify M1 macrophages in 7‐day wound tissues. Left panel for representative images and right panel for quantification analysis. n = 6 samples/group. (B) Double staining for F4/80 and CD206 to identify M2 macrophages in 7‐day wound tissues. Left panel for representative images and right panel for quantification analysis. n = 6 samples/group. (C) Examining protein levels of Arg1, CD206 and iNOS in 7‐day wound tissues of KO and WT mice. The experiments were performed in triplicate. (D) Detecting mRNA levels of Arg1 and Mrc1 in 7‐day wound tissues of KO and WT mice. n = 5 samples/group. (E) Secreted Spink7 protein was examined in conditioned medium. Total proteins were extracted from conditioned media (CM) and cells (C) for WB using Myc‐tag antibody. #, the non‐specific bands in samples from CM. (F) Levels of IL‐1α, IL‐1β, IL‐6 and iNOS mRNA were evaluated by RT‐PCR in CM‐treated LPS‐induced RAW264.7 M1 polarization samples. Data are represented as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. Data are representative of three independent experiments.

Article Snippet: Probe against mouse Spink7 (cat. no. 1191121‐C1, ACDBio) mRNA molecule was used.

Techniques: Double Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Clinical and Translational Medicine

Article Title: The antiprotease Spink7 promotes inflammation resolution by modulating multiple proteases activities during wound healing

doi: 10.1002/ctm2.70291

Figure Lengend Snippet: Loss of Spink7 leads to increased uPA and MMP2/9 activities in wounds. (A) Representative images of IHC staining for uPA in 7‐day wounds of KO and WT mice. (B) Analysis of uPA activity of both 3‐day and 7‐day wounds. n = 5 samples/group. (C and D) IHC staining forMMP2 and MMP9 in 7‐day wounds of KO and WT mice. Left panel for representative images and right panel for quantification analysis. ( E ) Detection of MMP9 and MMP2 protein levels in 7‐day wounds between KO and WT mice. The images are representative of experimental triplicates. (F) Gelatin zymography for MMP2/9 activities of 7‐day wounds. The images are representative of experimental triplicates. (G–I) Quantitative analysis of grayscale scanning of bands in gel zymography for MMP9, latent MMP2 and active MMP2. Data are represented as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. Data are representative of three independent experiments.

Article Snippet: Probe against mouse Spink7 (cat. no. 1191121‐C1, ACDBio) mRNA molecule was used.

Techniques: Immunohistochemistry, Activity Assay, Zymography

Journal: Clinical and Translational Medicine

Article Title: The antiprotease Spink7 promotes inflammation resolution by modulating multiple proteases activities during wound healing

doi: 10.1002/ctm2.70291

Figure Lengend Snippet: Loss of Spink7 exhibits enhanced KLKs activities, but inhibiting PAR2 signalling exacerbates the phenotype of impaired wound closure. (A) Representative images of IHC staining for KLK5 in 7‐day wounds of KO and WT mice. Right panel for quantification analysis. n = 5 samples/group. (B) Representative images of IHC staining for KLK7 in 7‐day wounds of KO and WT mice. Right panel for quantification analysis. n = 5 samples/group. (C) Trypsin‐like proteolytic activity measurement of normal skin and 7‐day wound tissues from KO and WT mice. n = 4 samples/group. (D) Evaluation of ability to inhibit proteolytic activity of KO wound samples by addition of CM containing Spink7 protein. The experiments were performed in triplicate. (E) Detection of PAR2 and TSLP protein levels in wounds of WT mice at different time points by WB. (F) Examination of TSLP protein levels in wounds to determine the effect of selective inhibition of PAR2 by ENMD‐1068 during wound healing. (G) Macroscopic appearance and quantification analysis of wound closure in both WT and KO mice treated with ENMD‐1068. Right panel for quantification analysis. n = 5 samples/group. (H) Examining mRNA levels of IL‐6, and IL‐1β in wounds treated with ENMD‐1068 by real‐time RT‐PCR. n = 5 samples/group. * p < 0.05, ** p < 0.01 and *** p < 0.001. Data are representative of two to three independent experiments.

Article Snippet: Probe against mouse Spink7 (cat. no. 1191121‐C1, ACDBio) mRNA molecule was used.

Techniques: Immunohistochemistry, Activity Assay, Inhibition, Quantitative RT-PCR

Journal: Clinical and Translational Medicine

Article Title: The antiprotease Spink7 promotes inflammation resolution by modulating multiple proteases activities during wound healing

doi: 10.1002/ctm2.70291

Figure Lengend Snippet: Knockdown of Spink7 promotes wound healing in R‐W‐CI model by augmenting inflammation. (A) Expression of Spink7 mRNA levels in wounds of R‐W‐CI and the in vivo knockdown effects of siRNAs gel against Spink7 examined by real‐time RT‐PCR (n = 5 per time point). (B) Macroscopic appearance of wound closure in si‐NC and si‐Spink7 treated wounds in R‐W‐CI mice at day 0, 7, and 12 after wounding. ( C ) Quantification of wound area in si‐NC and si‐Spink7 treated wounds in R‐W‐CI mice. n = 5 samples/group. (D) Representative H&E sections from si‐NC and si‐Spink7 treated wounds in R‐W‐CI mice on day 7 and 12 after wounding. Black arrows delineate wound edges. Red arrows highlight epithelial migrating tongues. (E) Quantification of wound areas, wound widths, percentages of re‐epithelialization and epithelial tongue lengths from si‐NC and si‐Spink7 treated wounds in R‐W‐CI mice on Day 7 after wounding. n = 5 samples/group. (F) Examining mRNA levels of CXCL1/2, CCL3/4, IL‐6, and IL‐1β in si‐NC and si‐Spink7 treated 7‐day R‐W‐CI wounds by real‐time RT‐PCR. n = 5 samples/group. * p < 0.05, ** p < 0.01 and *** p < 0.001. Data are representative of three independent experiments.

Article Snippet: Probe against mouse Spink7 (cat. no. 1191121‐C1, ACDBio) mRNA molecule was used.

Techniques: Knockdown, Expressing, In Vivo, Quantitative RT-PCR

Journal: Clinical and Translational Medicine

Article Title: The antiprotease Spink7 promotes inflammation resolution by modulating multiple proteases activities during wound healing

doi: 10.1002/ctm2.70291

Figure Lengend Snippet: Schematic diagram showing the mechanism of promoting inflammation resolution by Spink7 during skin wound healing.

Article Snippet: Probe against mouse Spink7 (cat. no. 1191121‐C1, ACDBio) mRNA molecule was used.

Techniques: