Journal: Nature Communications

Article Title: Cryptosporidium uses CSpV1 to activate host type I interferon and attenuate antiparasitic defenses

doi: 10.1038/s41467-023-37129-0

Figure Lengend Snippet: a HE morphological features of small intestine of Ifnar1 fl/fl and Villin.Ifnar1 −/− neonates following infection. Neonatal mice (5 days old) were orally inoculated with C. parvum oocysts (10 6 oocysts per animal) and ileal epithelium (4 cm of small intestine tissue from the ileocecal junction) was collected (72 h p.i.) Representative HE images from four independent experiments are shown. Insets are higher magnification of the boxed regions showing the parasites (arrows). Bars = 50 µm. b C. parvum intestinal infection burden in Ifnar1 fl/fl and Villin.Ifnar1 −/− mice. Neonatal mice were inoculated with C. parvum oocysts (10 6 oocysts per animal) for 48 h and 72 h. Infection burden was evaluated by RT-qPCR of cp18S gene in the isolated intestinal epithelium (fold changes to Ifnar1 fl/fl normalized to host Gapdh ) and parasite counting of intestine sections after immunofluorescent staining of C. parvum (average number/60X field). Data are presented as mean values ± SD, compiled from 4 independent experiments and the dots represent the mean value of each experiment with 6 mice in each group. Statistical significance was determined by two-tailed unpaired Student’s t -test. c Immunofluorescent staining of C. parvum , and PCNA and EpCAM staining of small intestine of Ifnar1 fl/fl and Villin.Ifnar1 −/− neonates with and without C. parvum infection (at 72 h p.i.). Representative images from 4 independent experiments are shown. Insets are higher magnification of the boxed regions showing the parasites revealed by immunofluorescent staining for parasite counting (arrowheads). Blue: DAPI (DNA), green: PCNA or EpCAM, red: C. parvum (arrows). Bars = 50 µm. d No significant difference in infection burden between IEC4.1 and IEC4.1- Ifnar1 −/− cells following C. parvum infection in vitro. IEC4.1 and IEC4.1- Ifnar1 −/− cells were infected with C. parvum for 2 h and 24 h. Infection burden was evaluated by RT-qPCR ( cp18S , fold changes to IEC4.1 normalized to Gapdh ). The dots represent data from three biological replicates. Data are presented as mean values ± SD. Blue: DAPI (DNA), red: C. parvum (arrows). Bars = 5 µm. Source data are provided as a Source Data file.

Article Snippet: Other antibodies used for immunofluorescent staining include anti-PCNA antibody (Cell Signaling, catalogue no. 13110, 1:1000), anti-EpCAM antibody (Abcam, catalogue no. ab71916, 1:200), anti-Lysozyme antibody (Abcam, catalogue no. ab108508, 1:500), anti-Villin antibody (Santa Cruz, catalogue no. sc-58897, 1:200), anti-mouse-Cruz fluor 488-conjugated (Santa Cruz, catalogue no. sc-516176, 1:200), anti-rabbit-Cruz fluor 488-conjugated (Santa Cruz, catalogue no. sc-516248, 1:200), anti-bovine Alexa fluor 594-conjugated (Jackson Immuno Research lab, catalogue no. 101-585-003, 1:250).

Techniques: Infection, Quantitative RT-PCR, Isolation, Staining, Two Tailed Test, In Vitro

Journal: Mediators of Inflammation

Article Title: IFN- α -2b Reduces Postoperative Arthrofibrosis in Rats by Inhibiting Fibroblast Proliferation and Migration through STAT1/p21 Signaling Pathway

doi: 10.1155/2023/1699946

Figure Lengend Snippet: The inhibitory effect of IFN- α -2b on the proliferation of fibroblasts by STAT1/P21 signaling pathway. (a) After treating fibroblasts with IFN- α -2b or combined with fludarabine, perform EdU staining to analyze the result image. The cells shown in red are marked as positive. (b) The results of the EdU incorporation test showed that as the concentration of IFN- α -2b increased, the percentage of positive cells decreased significantly. This trend was partially reversed by fludarabine. (c, e) Western blotting shows the increase of PCNA, cyclin A, and collagen I and predicts the expression levels of related pathway proteins. (d, f) The analysis results showed that IFN- α -2b significantly reduced the relative expression levels of PCNA, cyclin A, and collagen I in a concentration-dependent manner, while increasing the relative expression levels of P-STAT1 and P21. And after fludarabine application, the trend of action was partially reversed. The histogram shows the results of three repeated detections of the gray value of the Western blot band. ∗ Compared with the control group. ∗∗ Compared between the two groups, P < 0.05 ( n = 3).

Article Snippet: The mouse monoclonal antibodies PCNA (#2586), rabbit monoclonal antibodies cyclin A (#67955), horseradish peroxidase-conjugated goat anti-mouse (#7056), and goat anti-rabbit (#7074) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Staining, Concentration Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: Human Cytomegalovirus Induces Significant Structural and Functional Changes in Terminally Differentiated Human Cortical Neurons

doi: 10.1101/2023.03.03.531045

Figure Lengend Snippet: (A) Representative western blot showing banding patterns for cyclin B1 and cyclin-dependent kinase inhibitor 1 (p21), with included total protein stain. (B) Quantification of cyclin B1 western blots demonstrates a trend of HCMV downregulation across all lines, with significance in the PSEN2N141I forebrain neurons (t-tests. (C) Analysis of p21 blotting reveals an unclear trend, with two of the five tested lines demonstrating significant downregulation. (D) Representative immunofluorescent staining for proliferative marker Ki67. Key areas of overlap between Ki67 and viral eGFP are denoted with red arrows. (E) In both controls and the sAD line, HCMV induces an increase in Ki67+ cells. (F) Representative Western Blot demonstrating banding patter for proliferative cell nuclear antigen (PCNA), with included total protein stain. (G) Quantification of PCNA blotting demonstrates a trend of HCMV-mediated upregulation in forebrain neurons. Data are presented as mean ± SEM, with n = 2-4. Student’s t-tests were used to determine statistical differences in B, C and G, while two-way ANOVA was used in E. *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: Primary antibodies used for western blotting in this study include rabbit anti-cyclin B1 (1:1000; Cell Signaling Technology), mouse anti-p21 (1:1000; Millipore-Sigma), and mouse anti-PCNA (1:500-600; Cell Signaling Technology).

Techniques: Western Blot, Staining, Marker