Journal: Nature protocols

Article Title: Nonviral ultrasound-mediated gene delivery in small and large animal models

doi: 10.1038/s41596-019-0125-y

Figure Lengend Snippet: a, Representative image obtained using bioluminescence imaging (IVIS Spectrum) to visualize the measured luciferase signal 4 d after sonoporation of pUb-Luc2 into the thigh muscle in mice. The injected thigh muscle is indicated by a red oval. b, Luciferase expression profile in mice injected with plasmid DNA premixed with microbubbles and treated with sonoporation (DNA + MBs + US group; n = 6) compared to control group injected with plasmid DNA premixed with microbubbles (DNA + MBs group; n = 6). The treatment effect was monitored using bioluminescent imaging for 21 d after treatment (***P = 0.0003); P values were determined by two-way ANOVA with multiple comparisons. c, BMP-6 expression and secretion monitored using RT-qPCR (gene expression measured as relative quantification (RQ), left-side y axis label) and ELISA (protein secretion measured in picograms (pg), right-side y axis label), respectively, at days 2, 5 and 10 following sonoporation into a tibia bone fracture model in pigs. Data are means ± s.e.m. BMP, bone morphogenetic protein. Institutional regulatory board permission was obtained from the Institutional Animal Care and Use Committee of the Cedars-Sinai Medical Center for all procedures performed within this protocol. a,b, adapted with permission from Shapiro et al.26, Elsevier. c adapted with permission from Bez et al.27, American Association for the Advancement of Science.

Article Snippet: We analyzed its expression using an ABI 7500 Prism system with Hs00233470_m1 primer.

Techniques: Imaging, Luciferase, Injection, Expressing, Plasmid Preparation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Data in Brief

Article Title: Liver metal levels and expression of genes related to iron homeostasis in rhesus monkeys after inhalational manganese exposure

doi: 10.1016/j.dib.2016.01.055

Figure Lengend Snippet: Expression levels of factors in the BMP6-HAMP-SLC40A1 iron trafficking axis in livers from rhesus monkeys after manganese inhalation. (A–D) Liver RNA levels of bone morphogenetic protein 6 ( BMP6), hepcidin antimicrobial peptide ( HAMP ), inhibitor of differentiation 1 ( ID1) and mothers against decapentaplegic homology 7 ( SMAD7) were measured by QPCR and plotted relative to liver GAPDH RNA levels vs. manganese exposure level. ⁎ Indicates statistical significance ( p <0.05) compared to control group (0 mg/m 3 ) as calculated by one-way ANOVA with Dunnett׳s method. (E) Liver BMP6:GAPDH ratios were plotted vs. liver manganese (Mn), iron (Fe) and ID1:GAPDH levels along with rho ( ρ ) and p values as calculated by Spearman׳s rank correlation test. (F) Levels of SLC40A1 (ferroportin) were measured by immunoblot in protein lysates extracted from livers of monkeys after manganese inhalation.

Article Snippet: RNA was extracted from thawed liver tissue and analyzed by quantitative polymerase chain reaction (QPCR) using Taqman assays (Life Technologies) Rh02839540_m1 ( BMP6 ), Rh02819165_m1 ( HAMP ), Rh02913303_m1 ( ID1 ), Rh00998191_m1 ( SMAD7 ), Rh02621719_u1 ( IL6 ), Rh02621758_m1 ( TFRC ) and Rh02621745_g1 ( GAPDH ) as previously described .

Techniques: Expressing, Control, Western Blot

Journal: Data in Brief

Article Title: Liver metal levels and expression of genes related to iron homeostasis in rhesus monkeys after inhalational manganese exposure

doi: 10.1016/j.dib.2016.01.055

Figure Lengend Snippet: Liver transferrin receptor levels in rhesus monkeys after manganese inhalation . (A) Levels of transferrin receptor (TFRC) and GAPDH were measured by immunoblot in protein lysates extracted from livers of monkeys after manganese inhalation. (B) Liver TFRC:GAPDH ratios were plotted relative to manganese (Mn) exposure level. (C) Liver TFRC:GAPDH ratios were plotted vs. BMP6:GAPDH RNA levels along with rho ( ρ ) and p values as calculated by Spearman׳s rank correlation test. (D) Liver TFRC and GAPDH RNA levels were measured by QPCR and plotted as a ratio vs. manganese exposure level. (E) Liver TFRC:GAPDH ratios were plotted relative to liver manganese (Mn) levels along with rho ( ρ ) and p values as calculated by Spearman׳s rank correlation test.

Article Snippet: RNA was extracted from thawed liver tissue and analyzed by quantitative polymerase chain reaction (QPCR) using Taqman assays (Life Technologies) Rh02839540_m1 ( BMP6 ), Rh02819165_m1 ( HAMP ), Rh02913303_m1 ( ID1 ), Rh00998191_m1 ( SMAD7 ), Rh02621719_u1 ( IL6 ), Rh02621758_m1 ( TFRC ) and Rh02621745_g1 ( GAPDH ) as previously described .

Techniques: Western Blot

Journal: Stem Cells International

Article Title: Bone Morphogenetic Protein 6 Inhibits the Immunomodulatory Property of BMMSCs via Id1 in Sjögren's Syndrome

doi: 10.1155/2018/9837035

Figure Lengend Snippet: BMP6 was overexpressed in BMMSCs of SS patients and NOD mice and regulated BMMSCs differentiation. Real-time RT-PCR results indicated that the mRNA level of BMP6 is about five times higher in SS patient BMMSCs (a) and eight times higher in NOD BMMSCs (b) than in normal BMMSCs ( ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001). (c) ELISA showed that the BMP6 protein level is higher in the supernatant of NOD BMMSCs culture medium compared with BALB/c ( ∗∗∗∗ P < 0.0001). (d, e) BMP6-treated BMMSCs showed a significant increase in ALP activity compared to untreated controls ( ∗∗∗ P < 0.001). Real-time RT-PCR analysis demonstrated elevated expression of BGLAP mRNA (h) and ALP mRNA (i) in BMP6-treated cells ( ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001). (f, g) Oil Red O staining showed that BMP6 has no effect on cellular lipid accumulation in BMMSCs. Real-time RT-PCR results revealed that BMP6 do not increase the expression of PPAR γ mRNA (j) and FABP4 mRNA (k). N.S.: no significant difference.

Article Snippet: The following primers were used: BMP6 (Hs01099594_m1, Invitrogen), GAPDH (Hs02786624_g1, Invitrogen), Bmp6 (Mm01332882_m1, Invitrogen), Gapdh (Mm99999915_g1, Invitrogen), Bglap (Mm03413826_mH, Invitrogen), Alp (Mm01187117_m1, Invitrogen), Pparg (Mm00440940_m1, Invitrogen), Fabp4 (Mm00445878_m1, Invitrogen), Ido1 (Mm00492590_m1, Invitrogen), Nos2 (Mm00440502_m1, Invitrogen), TGFb1 (Mm00441727_g1, Invitrogen), Id1 (Mm00775963_g1, Invitrogen), Id2 (Mm00711781_m1, Invitrogen), Id3 (Mm00492575_m1, Invitrogen), Id4 (Mm00499701_m1, Invitrogen), and Cox2 (Mm03294838_g1, Invitrogen).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Activity Assay, Expressing, Staining

Journal: Stem Cells International

Article Title: Bone Morphogenetic Protein 6 Inhibits the Immunomodulatory Property of BMMSCs via Id1 in Sjögren's Syndrome

doi: 10.1155/2018/9837035

Figure Lengend Snippet: BMP6 impaired the immunomodulatory properties of BMMSCs by downregulating PGE2 and upregulating Th1 and Th17 cells. (a, b) CFSE assays showed T cells proliferation was inhibited by BMMSCs, and this effect could be attenuated by BMP6 ( ∗∗ P < 0.01). (c, d, e) Flow cytometric analysis indicated that BMP6 significantly inhibited BMMSC-mediated downregulation of Th1 and Th17 cells ( ∗∗∗ P < 0.001). ELISA assays revealed that BMP6-treated BMMSCs showed a decreased concentration of PGE2 (f) and increased concentration of IFN-gamma (g) in the supernatants of the BMMSCs/T cell coculture system ( ∗ P < 0.05). (h) Although no significant difference was detected, there was an increasing trend in IL-17 concentration in the supernatants of the BMMSCs/T cell coculture system (i, j). CFSE assays showed that T cells proliferated less when cocultured with anti-BMP6-pretreated NOD BMMSCs compared with the isotype antibody group ( ∗∗ P < 0.01). (k, l, m) Flow cytometric analysis showed that anti-BMP6-treated NOD BMMSCs exert an effect on downregulating Th1 and Th17 cells ( ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001). ELISA assays revealed that anti-BMP6-pretreated NOD BMMSCs significantly increased the levels of PGE2 (n) and decreased the levels of IFN-gamma (o) but had no effect on IL-17 (p) in the supernatants of the BMMSCs/T cell coculture system ( ∗ P < 0.05, ∗∗ P < 0.01).

Article Snippet: The following primers were used: BMP6 (Hs01099594_m1, Invitrogen), GAPDH (Hs02786624_g1, Invitrogen), Bmp6 (Mm01332882_m1, Invitrogen), Gapdh (Mm99999915_g1, Invitrogen), Bglap (Mm03413826_mH, Invitrogen), Alp (Mm01187117_m1, Invitrogen), Pparg (Mm00440940_m1, Invitrogen), Fabp4 (Mm00445878_m1, Invitrogen), Ido1 (Mm00492590_m1, Invitrogen), Nos2 (Mm00440502_m1, Invitrogen), TGFb1 (Mm00441727_g1, Invitrogen), Id1 (Mm00775963_g1, Invitrogen), Id2 (Mm00711781_m1, Invitrogen), Id3 (Mm00492575_m1, Invitrogen), Id4 (Mm00499701_m1, Invitrogen), and Cox2 (Mm03294838_g1, Invitrogen).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Stem Cells International

Article Title: Bone Morphogenetic Protein 6 Inhibits the Immunomodulatory Property of BMMSCs via Id1 in Sjögren's Syndrome

doi: 10.1155/2018/9837035

Figure Lengend Snippet: BMP6 impaired immunomodulatory properties of BMMSCs and downregulated PGE2 via Id1. (a, c, d) Real-time RT-PCR results demonstrated a higher level of Id1 and Id4 mRNA expression in BMMSCs derived from NOD mice compared with BALB/c, while Id2 and Id3 transcripts showed no significant difference between two groups (a) ( ∗∗∗∗ P < 0.0001). (b, e, f) BMP6-treated BALB/c BMMSCs showed an increase in Id1, rather than in Id2, Id3, or Id4 mRNA expression ( ∗∗∗∗ P < 0.0001) Knockdown of Id1 increased BMP6-induced Cox2 (g) and PGE2 (h) downregulation, as evidenced by real-time RT-PCR and ELISA, respectively. ( ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001). (i) CFSE assays indicated that BMP6 inhibited downregulation of T cells proliferation mediated by BMMSCs, while Id1 knockdown attenuated this effect ( ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001). N.S.: no significant difference.

Article Snippet: The following primers were used: BMP6 (Hs01099594_m1, Invitrogen), GAPDH (Hs02786624_g1, Invitrogen), Bmp6 (Mm01332882_m1, Invitrogen), Gapdh (Mm99999915_g1, Invitrogen), Bglap (Mm03413826_mH, Invitrogen), Alp (Mm01187117_m1, Invitrogen), Pparg (Mm00440940_m1, Invitrogen), Fabp4 (Mm00445878_m1, Invitrogen), Ido1 (Mm00492590_m1, Invitrogen), Nos2 (Mm00440502_m1, Invitrogen), TGFb1 (Mm00441727_g1, Invitrogen), Id1 (Mm00775963_g1, Invitrogen), Id2 (Mm00711781_m1, Invitrogen), Id3 (Mm00492575_m1, Invitrogen), Id4 (Mm00499701_m1, Invitrogen), and Cox2 (Mm03294838_g1, Invitrogen).

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: Stem Cells International

Article Title: Bone Morphogenetic Protein 6 Inhibits the Immunomodulatory Property of BMMSCs via Id1 in Sjögren's Syndrome

doi: 10.1155/2018/9837035

Figure Lengend Snippet: Knockdown of Id1 rescued impaired immunosuppressive capacity of BMMSCs induced by BMP6 in vivo . (a, b) BMMSCs treatment significantly reduced the number of CD4 + T cells, while BMP6-treated BMMSCs showed no significant reduction. When Id1 was blocked by siRNA in BMP6-treated BMMSCs, a greater reduction in CD4 + T cells number was observed ( ∗ P < 0.05, ∗∗∗∗ P < 0.0001). (c, d) A greater ratio of Th1 cells were detected in the presence of BMP6 compared with BMMSCs alone. Knockdown of Id1 significantly decreased the proportion of Th1 cells. ( ∗∗∗∗ P < 0.0001).

Article Snippet: The following primers were used: BMP6 (Hs01099594_m1, Invitrogen), GAPDH (Hs02786624_g1, Invitrogen), Bmp6 (Mm01332882_m1, Invitrogen), Gapdh (Mm99999915_g1, Invitrogen), Bglap (Mm03413826_mH, Invitrogen), Alp (Mm01187117_m1, Invitrogen), Pparg (Mm00440940_m1, Invitrogen), Fabp4 (Mm00445878_m1, Invitrogen), Ido1 (Mm00492590_m1, Invitrogen), Nos2 (Mm00440502_m1, Invitrogen), TGFb1 (Mm00441727_g1, Invitrogen), Id1 (Mm00775963_g1, Invitrogen), Id2 (Mm00711781_m1, Invitrogen), Id3 (Mm00492575_m1, Invitrogen), Id4 (Mm00499701_m1, Invitrogen), and Cox2 (Mm03294838_g1, Invitrogen).

Techniques: Knockdown, In Vivo