Journal: bioRxiv

Article Title: Neural precursor cells rescue symptoms of Rett syndrome by activation of the Interferon γ pathway

doi: 10.1101/2024.01.07.574507

Figure Lengend Snippet: A) Principal component analysis (PCA) plot for the sequenced samples of WT, Mecp2 KO, WT+NPCs and Mecp2 KO+NPCs cerebella. Percentage of variance is reported for both PC1 (first component) and PC2 (second component). B) The number of deregulated genes (DEGs) for the different comparison is reported, considering a p.adj<0.05. The number of DEGs with a LogFoldChange lower than −1 (down-regulated genes) or LogFoldChange greater than 1 (up-regulated genes) is also indicated. C) Dot plot of Gene Ontology (GO) enriched pathway analysis in the cerebellum, indicating the top 30 most enriched pathways of the comparison between KO+NPCs versus KO samples. D) Gene set enrichment analysis (GSEA) of IFNγ response in cerebellar samples, indicating a significant enrichment of the gene set in KO+NPCs vs KO comparison, as well as in KO+NPCs vs WT comparison. E) The histogram reports the transcriptional levels of genes (Parp9, Iftm3, Irf8 and Bst2) associated to IFNγ pathway. Data, expressed as percentage of WT animals, are shown as mean ± SEM. *p<0.05, **p<0.01 by two-way ANOVA followed by Tukey post hoc test. F) Western blot analysis of phosphorylated STAT1 (P-STAT1) in WT or KO neurons cultured with NPCs (for 14 days) (n=7-9). Data are represented as mean ± SEM and expressed as percentage of WT neurons cultured alone. Representative bands of P-STAT1 and the corresponding lanes of TGX-stain free gel are reported.

Article Snippet: Then, membranes were incubated for 1 hour in blocking solution [5% BSA in 0.1% Tween-20 in Tris-buffered saline containing (TBST)], and incubated overnight at 4°C with the following primary antibodies: rabbit anti-phosphorylated Stat1 (clone 58D6, 1:1000; #9167, Cell Signalling) or rabbit anti-Bdnf (1:1000; ab108319, Abcam).

Techniques: Comparison, Western Blot, Cell Culture, Staining

Journal: PLoS Pathogens

Article Title: Human cytomegalovirus protein UL42 antagonizes cGAS/MITA-mediated innate antiviral response

doi: 10.1371/journal.ppat.1007691

Figure Lengend Snippet: (A) HCMV UL42 inhibits cGAS-MITA-induced IFNβ promoter and ISRE activation in a dose-dependent manner. HEK293T/MITA cells (1x10 5 ) were transfected with the IFNβ promoter (0.05 μg) or ISRE (0.03 μg) reporter plasmid, and expression plasmids for cGAS (0,01 μg) and increased amounts of UL42 (0, 0.025, 0.05, and 0.1 μg) for 20 hrs before luciferase assays. (B) Effects of UL42 on IFN-β-induced STAT1/2 activation. HEK293 cells (1x10 5 ) were transfected with STAT1/2 reporter (0.005 μg) and UL42 expression (0.05 μg) plasmids for 20 hrs. The cells were then untreated or treated with IFN-β for 12 hrs before luciferase assays. (C) HCMV UL42 inhibits HCMV-, HSV-1-, and VACV-induced transcription of antiviral genes in HFFs. UL42-stable HFFs (4x10 5 ) were un-infected or infected with HCMV (MOI = 1), HSV-1 (MOI = 1), or VACV (MOI = 1) for the indicated times (upper histographs) or 12 h (lower histographs) before qPCR analysis. The immunoblots show the expression levels of UL42 in the HFF-UL42 stable cell lines. (D) HCMV UL42 inhibits dsDNA-induced transcription of antiviral genes in HFFs. UL42 stable HFFs (4x10 5 ) were transfected with HSV120 (2 μg), DNA90 (2 μg), VACV70 (2 μg), or ISD (2 μg) for the indicated times before qPCR analysis. (E) UL42 impairs HCMV- and HSV120-induced phosphorylation of downstream components. UL42 stable HFFs (4x10 5 ) were infected with HCMV (MOI = 1) or transfected with HSV120 (2 μg/ml) for the indicated times before immunoblot analysis. The lower panels are results of qPCR analysis for HCMV UL123 mRNA or HSV120 DNA. (F) Effect of UL42 on IFN-β-induced phosphorylation of STAT1. UL42 stable HFFs (4x10 5 ) were untreated or treated with IFN-β (100 ng/ml) for the indicated times before immunoblot analysis. Graphs show mean ± SD, n = 3. *p<0.05, **p<0.01 (unpaired t test).

Article Snippet: 2’ 3’-cGAMP, and lipofectamine 2000 (Invitrogen); polybrene (Millipore); puromycin and RNase inhibitor (Thermo); dual-specific luciferase assay kit (Promega); SYBR (BIO-RAD); digitonin (Sigma); streptavidin agarose (Solulink); mouse antibodies against Flag, and β-actin (Sigma), and HA (Covance); rabbit monoclonal antibodies against cGAS (66546S/31659S), MITA (13647S), phosphor-MITA (85735S), phosphor-p65, and phosphor-IRF3 (4947S) (Cell Signaling Technology), phosphor-TBK1(ab109272) and TBK1(ab40676) (Abcam), IRF3 (sc-9082), phosphor-Tyrosine701-STAT1(9167S) and STAT1(sc-346) (Santa Cruz Biotechnology) were purchased from the indicated manufacturers.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Infection, Western Blot, Stable Transfection

Journal: Nature Communications

Article Title: Age-dependent Pdgfrβ signaling drives adipocyte progenitor dysfunction to alter the beige adipogenic niche in male mice

doi: 10.1038/s41467-023-37386-z

Figure Lengend Snippet: a Experimental schema: TMX-induced 12-month-old Sma-Control and Sma-Pdgfrβ-KO male mice were administered one dose of mouse IgG (1 µg/mouse) or α-IL-33 (1 µg/mouse) antibodies for 5 consecutive days and subsequently cold exposed for 2 or 7 days. b mRNA levels of IL-13 and IL-5 within iWAT depots from 2-day cold exposed mice described in ( f ) ( n = 4 mice/group). c Representative H&E staining of dorsolumbar iWAT sections from 7-day cold exposed mice described in ( a ) (×10 magnification, scale bars 100 µm). d Representative Plin1 (blue) and Ucp1 (green) immunostaining from iWAT sections from 7-day cold exposed mice described in ( a ) (×20 magnification, scale bars 100 µm) (Images representative of 2 independent experiments). e Experimental schema: 2-month-old TMX-induced Sma-Control and Sma-Pdgfrβ D849V were administered one dose of vehicle (0.1%BSA in 1xPBS) or recombinant murine IL-33 (12 µg/kg) for 5 consecutive days and subsequently cold exposed for 2 or 7 days. f mRNA levels of IL-13 and IL-5 within iWAT depots from 2-day cold exposed mice described in ( e ) ( n = 4 mice/group). g Representative H&E staining of dorsolumbar iWAT sections from vehicle or IL-33 treated Pdgfrβ D849V 7-day cold exposed mice as described in ( e ) (×10 magnification, scale bars 100 µm). h Representative of Plin1 (blue) and Ucp1 (green) immunostaining of dorsolumbar iWAT sections from Control and Pdgfrβ D849V 7-day cold exposed mice (×20 magnification, scale bars 100 µm) (Images representative of 2 independent experiments). Data are presented as mean values ± SEM. Data were analyzed by two-tailed Student’s t -test or two-way ANOVA for multiple comparisons. Source data are provided within the Source data file.

Article Snippet: Primary antibodies used were phosphorylated Stat1 (1:200; 9167S Cell Signaling) or total Pdgfrβ (1:200; 4564S Cell Signaling).

Techniques: Control, Staining, Immunostaining, Recombinant, Two Tailed Test

Journal: Nature Communications

Article Title: Age-dependent Pdgfrβ signaling drives adipocyte progenitor dysfunction to alter the beige adipogenic niche in male mice

doi: 10.1038/s41467-023-37386-z

Figure Lengend Snippet: a Sma- mGFP+ cells were FACS isolated from 2-month-old TMX-induced Sma-Control and Sma-Pdgfrβ D849V or from 12-month-old TMX-induced Sma-Control and Sma-Pdgfrβ-KO mice and examined for phosphorylated Stat1 by flow cytometry. b Experimental schema: 12-month-old TMX-induced Sma-Control mice were administered one dose of vehicle (5% DMSO) or fludarabine (3 mg/kg) for 5 consecutive days and subsequently cold challenged for 2 or 7 days. c Representative H&E staining of dorsolumbar iWAT sections from 7-day cold exposed mice described in ( b ) (×10 magnification, scale bars 100 µm). d Quantification of beige and white adipocyte area per section ( n = 3 images/mouse; 3 mice/group) from immunostained images (Supplementary Fig. ). e mRNA levels of denoted thermogenic genes within dorsolumbar iWAT depots from mice described in ( b ) after 7 days of cold exposure ( n = 4 mice/group). f Experimental schema: 2-month-old TMX-induced Sma-Control and Sma-Pdgfrβ D849V male mice were administered one dose of vehicle (5% DMSO) or fludarabine (3 mg/kg) for 5 consecutive days and subsequently cold exposed for 2 or 7 days. g mRNA levels of IL-33 and IL-13 markers within iWAT depots from 2-day cold exposed mice described in ( f ) ( n = 4 mice/group). h , i Representative images of H&E staining ( h ) and Plin1 (blue) and Ucp1 (green) immunostaining ( i ) of dorsolumbar iWAT sections from cold exposed mice described in ( f ) (×10 and ×20 magnification, scale bars 100 µm) (Images representative of 2 independent experiments). j Quantification of beige and white adipocyte area per section ( n = 3 images/mouse; 3 mice/group) from immunostained images in ( i ). k mRNA levels of denoted thermogenic gene expression within iWAT depots from mice described in ( f ) ( n = 4 mice/group). Data are presented as mean values ± SEM. Data were analyzed by two-tailed Student’s t -test or two-way ANOVA for multiple comparisons. Source data are provided within the Source data file.

Article Snippet: Primary antibodies used were phosphorylated Stat1 (1:200; 9167S Cell Signaling) or total Pdgfrβ (1:200; 4564S Cell Signaling).

Techniques: Isolation, Control, Flow Cytometry, Staining, Immunostaining, Gene Expression, Two Tailed Test