Fig 1: Fancd2-/- embryos overexpress the TGFβ pathway and error-prone DNA repair genes.(A) Fancd2/Smad3 heterozygous littermates were crossed to breed. Pregnant females were sacrificed at day E12.5 of gestation, uterine horns were dissected, and embryos genotyped. RNA from embryos was analyzed for expression of genes in the TGFβ pathway and DNA repair pathways. (B) Smad3 Fancd2-129B6F2 breeding has a Mendelian distribution at day E12.5, showing that Smad3 Fancd2-129B6F2 embryos can survive until day E12.5 of embryonic development (n = 141 embryos). (C) The frequency of Smad3 Fancd2-129B6F2 embryos at day E12.5 yields the expected Mendelian ratio, however the frequency of weaned Smad3 Fancd2-129B6F2 pups is halved, suggesting later lethality during embryonic development. (D) Gene expression analysis of E12.5 embryos. Left. Compared to WT embryos, Fancd2-/- embryos overexpress TGFβ pathway-related genes and overexpress the ligands for activation of the TGFβ pathway, Tgfb1 and Tgfb3, as well as their receptors Tgfbr2 and Tgfbr3. The oncogene Myc was also found to be overexpressed in Fancd2-/- embryos. Right. Compared to WT embryos, Fancd2-/- embryos overexpress genes related to alternative DNA repair pathways, including Prkdc, Mad2l2 and Parp1, as well as negative regulators of cell cycle progression, including Trp53 (p53) and Cdkn1a (p21). Red dots indicate differentially upregulated genes with respect to wild type embryos. Blue dots indicate differentially downregulated genes with respect to wild type embryos. Data in (B) and (C) are represented as bar plots. Data in (D) are represented as volcano plots. p values of 0.01 to 0.05 were considered significant (*), p values of 0.001 to 0.01 were considered very significant (**) and p values of < 0.001 were considered extremely significant (***, ****). See also S3 Fig.
Fig 2: MMP‐2 activity, Tgfb1, and androgen receptor gene expression in Fbn1C1039G/+ and WT aortic root/ascending and descending aortas from each sex.MMP zymography in (A) aortic root/ascending (ASC) and (B) descending (DES) aortic specimens from 16 week‐old Fbn1C1039G/+ mice (Fbn1) and littermate wild type control mice (WT) (n=10 ASC specimens per group, n=7 male and n=6 female DES specimens). Values expressed as fold difference compared with sex‐matched WT. C, RT‐PCR results for TGF‐β1 ligand (Tgfb1). D, ELISA results for active Tgfb1 ligand in 16‐week old ASC tissue. E, RT‐PCR for androgen receptor (AR) in ASC from Fbn1 at age 16 weeks (WT male=8; WT female=8; Fbn1 male=9; Fbn1 female=9). mRNA expression values are normalized to 18S as a loading control housekeeping gene and reported as fold change compared with WT using the 2−ΔΔCt method. Results presented as median±interquartile. Mann–Whitney U test used for the comparison between sex within each genotype. *P≤0.05, **P<0.01, *** P<0.001. MMP indicates matrix metalloproteinase; RT‐PCR, reverse transcription polymerase chain reaction; and TGF‐β1, transforming growth factor beta 1.
Fig 3: miR-21 silencing induces an antiinflammatory phenotype and upregulates TGF-β–related pathway in macrophages.Macrophages transfected with an miR-21 antagomir (antagomir-21) or scramble control followed by RT-qPCR and flow cytometry. (A) miR-21-5p fold change in BMDMs after 48-hour transfection with antagomir-21 (n = 8). (B) Spry2 mRNA fold change in BMDMs after 48-hour transfection with antagomir-21 (n = 8). (C) Tgfb1 fold change in BMDMs after transfection with antagomir-21, n = 8 per group pooled from 2 independent experiments. (D) Spearman’s correlation between miR-21-5p expression and Tgfb1 mRNA expression (n = 11). (E) RT-qPCR of Tgfbr2, Tgfbr1, Tgfbr3, and Smad7 in BMDMs after 48-hour transfection with antagomir-21 (n = 8). (F) RT-qPCR of proinflammatory genes Tnfa and Il6 in BMDMs transfected with antagomir-21 (n = 8). (G) RT-qPCR of antiinflammatory genes Mrc1 and Il10, n = 8 per group, pooled from 2 independent experiments. (H) Flow cytometry analysis of TGF-βR2 expression in BMDMs after silencing miR-21-5p, n = 4 per group. The bar graphs represent the percentage of F4/80+TGF-βR2+ cells. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 by unpaired, 2-tailed Student’s t test (A–C and E–G).
Fig 4: miR-21 induces a proinflammatory phenotype and downregulates TGF-β–related pathway in macrophages.Peritoneal macrophage (PM) transfection with miR-21 mimic (mimic-21) or scramble control (N5) followed by RT-qPCR and flow cytometry. (A) miR-21-5p fold change after 48 hours of transfection with mimic-21 (n = 6). (B) Spry2 (known target of miR-21-5p) mRNA fold change after 48-hour PM transfection (n = 6). (C) Tgfb1 fold change in PMs overexpressing miR-21-5p, n = 6 per group. (D) Spearman’s correlation between miR-21-5p expression and Tgfb1 mRNA expression (n = 11). (E) RT-qPCR of Tgfbr1, Tgfbr2, Tgfbr3, and (F) Smad7 fold change in PMs overexpressing miR-21-5p, n = 6 per group. (G) Histograms of TGF-βR2 expression in BMDMs transfected with mimic-21 or scramble N5 by quantitative flow cytometry using fluorescence minus one (FMO) controls. The bar graphs represent the MFI (left) and percentage of cells (right), n = 4 per group. (H) RT-qPCR of proinflammatory genes Tnfa and Il6 in PMs transfected with mimic-21 (n = 6). (I) RT-qPCR of polarization markers Arg1 and Nos2 in PMs transfected with mimic-21, n = 5–6 per group. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 by unpaired, 2-tailed Student’s t test (A–C and E–I).
Fig 5: miR-21 silencing in sensory neurons prevents neuropathic hypersensitivity and regulates TGF-βR2 expression.(A) RT-qPCR of miR-21-5p in DRG cultures of WT and miR-21–cKO mice, n = 6–7 cultures per group. (B) RT-qPCR of miR-21-5p in DRGs of WT and miR-21–cKO mice on day 7 after SNI (n = 6–9). (C) ImageStream analyses of exosomes (extracellular vesicles, EVs) isolated from culture media of WT and miR-21–cKO DRG neurons incubated with vehicle or capsaicin (CAPS, 1 μM) for 3 hours (n = 4). (D) Attenuated allodynia in miR-21–cKO mice up to day 7 after SNI in males and females. Data are presented as 50% paw withdrawal thresholds (PWT). +P < 0.05, +++P < 0.001 compared with miR-21–cKO contralateral thresholds; ***P < 0.001 compared with WT contralateral thresholds; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with WT ipsilateral thresholds; by 2-way ANOVA followed by Tukey’s multiple-comparison test (n = 10–14 per group). (E) Intrathecal injection of TGF-βR1 inhibitor SB431542 (100 pmol/mouse) abolished the anti-allodynic effect in miR-21 cKO. Arrow indicates the time of injection given on day 7 after SNI, n = 6. (F) Representative scatterplots of DRG CD11b+F4/80+ cells stained for TGF-βR2 on day 7 after SNI. Bar graphs represent TGF-βR2+ cell number (n = 4). (G) Representative histograms of TGF-βR2 expression in DRG CD11b+F4/80+ cells on day 7 after SNI (MFI), n = 4. (H) RT-qPCR of Tgfbr1, Tgfb1, Tgfbr2, and Tgfbr3 in F4/80+ cells isolated from DRGs of WT and miR-21–cKO mice on day 7 after SNI, n = 5 independent experiments from 4–6 pooled animals in each. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by unpaired, 2-tailed Student’s t test (A) or 1-way ANOVA followed by Tukey’s multiple-comparison test (B, C, and E–H).
Supplier Page from Abcam for Mouse TGF beta 1 ELISA Kit