Journal: Oncotarget

Article Title: Curcumin modulates chronic myelogenous leukemia exosomes composition and affects angiogenic phenotype via exosomal miR-21

doi: 10.18632/oncotarget.8483

Figure Lengend Snippet: ( A ) miR-21 expression levels in HUVECs treated with 20 μg/ml of K562 Curcu-exosomes and control exosomes were determined by quantitative Real time PCR analysis. We also analyzed miR-21 expression in HUVECs treated with K562 Curcu-exosomes and control exosomes and/or transfected with miR-21 inhibitor or miR-21 mimic. Values (FOI: fold of induction) are the mean ± SD of 3 independent experiments * p ≤ 0.05, ** p ≤ 0.01. ( B ) Pre-miR-21 expression in HUVECs treated with different amounts of K562 exosomes. Pre-miR-126 expression levels in HUVECs treated with 20 and 50 μg/ml of K562 exosomes were determined by quantitative Real time PCR analysis. We also analyzed pre-miR-21 expression in HUVECs treated with K562 Curcu-exosomes and control exosomes and/or transfected with miR-21 inhibitor or miR-21 mimic. ( C ) Luciferase activity of HUVECs transfected with reporter plasmid (RhoB-pEZX), treated with K562 Curcu-exosomes and control exosomes and/or cotransfected with miR-21 inhibitor or miR-21 mimic.

Article Snippet: Soon after, a RhoB unconjugated primary antibody (Novus Biologicals) was added for 30 min at room temperature.

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Activity Assay, Plasmid Preparation

Journal: Oncotarget

Article Title: Curcumin modulates chronic myelogenous leukemia exosomes composition and affects angiogenic phenotype via exosomal miR-21

doi: 10.18632/oncotarget.8483

Figure Lengend Snippet: ( A ) Real time PCR analysis showed that RhoB mRNA expression decreased in HUVECs treated with Curcu-exosomes compared to control exosomes. Expression of RhoB was evaluated in HUVECs transfected with 2-Ome-miR-21 (miR-21 inhibitor) treated or not with 20 μg/ml of control exosomes (miR-21 inhibitor + Exo 20 μg/ml) and Curcu-exosomes (miR-21 inhibitor + Curcu-Exo 20 μg/ml). Expression of RhoB was also evaluated in HUVECs transfected with miR-21 mimic (miR-21 mimic) treated or not with 20 μg/ml of control exosomes (miR-21 mimic + Exo 20 μg/ml) and Curcu-exosomes (miR-21 mimic + Curcu-Exo 20 μg/ml). Values (FOI: fold of induction) are the mean ± SD of 3 independent experiments * p ≤ 0.05, ** p ≤ 0.01. ( B ) Histogram shows the MFI (Mean Fluorescence Intensity) relative to the expression of RhoB in HUVECs after treatment with low serum medium (Control), 20 μg/ml of exosomes (Exo 20 μg/ml) and 20 μg/ml of Curcu-exosomes (Curcu-exo 20 μg/ml). Expression of RhoB was evaluated, with FACS analysis, in HUVECs transfected with 2-Ome-miR-21(miR-21 inhibitor) and treated with 20 μg/ml of control exosomes (miR-21 inhibitor + Exo 20 μg/ml) and Curcu-exosomes (miR-21 inhibitor + Curcu-Exo 20 μg/ml). Surface expression of VCAM1 was evaluated in HUVECs transfected with miR-21 mimic (miR-21 mimic) and treated with 20 μg/ml of control exosomes (miR-21 mimic + Exo 20 μg/ml) and 20 μg/ml of Curcu-exosomes (miR-21 mimic + Curcu-Exo 20 μg/ml). Values are the mean ± SD of 3 independent experiments * p ≤ 0.05 ** p ≤ 0.01. ( C ) Curcu-exosomes inhibit HUVECs migration. Addition of control exosomes (20, 50 μg/ml) to the upper wells of the chamber induces dose-dependent increase of HUVEC migration, the addition of Curcu-exosomes reverts this effects. Values are the mean ± SD of 3 fields in three independent experiments * p ≤ 0.05, ** p ≤ 0.01. The ability of migrating of HUVECs transfected with 2-Ome-miR-21 (miR-21 inhibitor) treated or not with 20 μg/ml of control exosomes (miR-21 inhibitor + Exo 20 μg/ml) and with 20 μg/ml of Curcu-exosomes (miR-21 inhibitor + Curcu-Exo 20 μg/ml), was evaluated. The ability of migration of HUVECs transfected with miR-21 mimic (miR-21 mimic) treated or not with 20 μg/ml of control exosomes (miR-21 mimic + Exo 20 μg/ml) and with 20 μg/ml of Curcu-exosomes (miR-21 mimic + Curcu-Exo 20 μg/ml) was also measured.

Article Snippet: Soon after, a RhoB unconjugated primary antibody (Novus Biologicals) was added for 30 min at room temperature.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Transfection, Fluorescence, Migration

Journal: Oncotarget

Article Title: Curcumin modulates chronic myelogenous leukemia exosomes composition and affects angiogenic phenotype via exosomal miR-21

doi: 10.18632/oncotarget.8483

Figure Lengend Snippet: ( A ) The permeability of HUVEC monolayer increased after treatment, for 3 and 6 hours, with K562 control exosomes (Exo K562) compared to untreated HUVEC monolayer (Ctrl), the treatment with Curcu-exosomes (Curcu-Exo K562), protected the endothelial monolayer. ( B ) Upper panel: Analysis at confocal microscopy of endothelial monolayer. The integrity of the monolayer was altered, after treatment with K562 control exosomes (Exo K562 20 μg/ml); the treatment of Curcu-exosomes alleviated the alteration of the EC monolayer (Exo K562 20 μg/ml). Lower panel: Analysis at confocal microscopy of RhoB expression in HUVECs treated with K562 Curcu-exosomes and control exosomes. K562 control exosomes (Exo K562 20 μg/ml) induce an increase of immunostaining for RhoB compared to untreated cells (Ctrl). The treatment with K562 Curcu-exosomes (Curcu-Exo K562, 20 μg/ml) reverted this effect.

Article Snippet: Soon after, a RhoB unconjugated primary antibody (Novus Biologicals) was added for 30 min at room temperature.

Techniques: Permeability, Control, Confocal Microscopy, Expressing, Immunostaining

Journal: Disease Models & Mechanisms

Article Title: RhoB blockade selectively inhibits autoantibody production in autoimmune models of rheumatoid arthritis and lupus

doi: 10.1242/dmm.029835

Figure Lengend Snippet: Anti-RhoB Ig inhibits autoantibody production and attenuates arthritis. K/BxN mice were treated with 500 µg anti-RhoB Ig at (A-D,F) 21 days or (E) 28 days of age. (A) The number of anti-GPI ASCs was measured by ELISpot at the termination of the experiment (6 weeks of age). Data show means±s.e.m. for n =19 control-Ig- and n =16 anti-RhoB-Ig-treated mice. (B) Titers of anti-GPI Ig in the serum were also measured at 6 weeks of age by ELISA. Data show means±s.e.m. for n =8 mice of each treatment group. (C,E) Joint inflammation was measured by the change in ankle thickness. Data show means±s.e.m. for (C) n =12 control Ig (▲) and n =14 anti-RhoB Ig (◊), and (E) n =9 control Ig (▲) and n =10 anti-RhoB Ig (◊)-treated mice. (D) Metatarsal joint harvested at 6 weeks of age stained with H&E. Representative sections from a total of n =5 mice for each treatment group are shown. Scale bar: 100 µm. (F) K/BxN mice were treated with 500 µg anti-RhoB Ig at 21 days of age. Three weeks later, the LNs draining the arthritic joints were harvested, and the isolated cells were stimulated with PMA+ionomycin overnight. Inflammatory cytokines were measured in culture supernatants using the cytometric bead array and flow cytometry. Each symbol depicts an individual mouse (control Ig, n =18; anti-RhoB Ig, n =15) with the mean indicated by a solid bar. *** P <0.001. ** P <0.01.

Article Snippet: Recombinant proteins RhoB (OPPA00123), RhoA (OPPA00043), Rac1 (OPPA01858) and Cdc42 (OPPA00141) were purchased from Aviva Systems Biology (San Diego, CA, USA).

Techniques: Enzyme-linked Immunospot, Control, Enzyme-linked Immunosorbent Assay, Staining, Isolation, Flow Cytometry

Journal: Disease Models & Mechanisms

Article Title: RhoB blockade selectively inhibits autoantibody production in autoimmune models of rheumatoid arthritis and lupus

doi: 10.1242/dmm.029835

Figure Lengend Snippet: Arthritis, but not autoantibody level, is attenuated in RhoB ko mice. The RhoB ko allele was crossed into the KRN model on the C57BL/6 background. (A) Joint inflammation was determined by measuring ankle thickness. Data show means±s.e.m. for n =25 KRN.g7 and n =14 RhoB ko KRN.g7 mice. (B) Metatarsal joint harvested at 6 weeks of age stained with H&E. Representative sections from a total of n =5 mice for each treatment group are shown. Scale bar: 100 µm. At 6 weeks of age, (C) the number of anti-GPI ASCs was measured by ELISpot; data show means±s.e.m. for n =15 KRN.g7 and n =17 RhoB ko KRN.g7 mice; n.s., not significant; and (D) titers of anti-GPI Ig in the serum were measured by ELISA. Data show means±s.e.m. for n =23 KRN.g7 and n =11 RhoB ko KRN.g7 mice. (E) At 6 weeks of age, LNs draining the arthritic joints were harvested and the isolated cells were stimulated with PMA+ionomycin overnight. Inflammatory cytokines were measured in culture supernatants using the cytometric bead array and flow cytometry. Each symbol depicts an individual mouse with the mean indicated by a solid bar (KRN B6.g7, n =25; RhoB ko KRN B6.g7, n =12).

Article Snippet: Recombinant proteins RhoB (OPPA00123), RhoA (OPPA00043), Rac1 (OPPA01858) and Cdc42 (OPPA00141) were purchased from Aviva Systems Biology (San Diego, CA, USA).

Techniques: Staining, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay, Isolation, Flow Cytometry

Journal: Disease Models & Mechanisms

Article Title: RhoB blockade selectively inhibits autoantibody production in autoimmune models of rheumatoid arthritis and lupus

doi: 10.1242/dmm.029835

Figure Lengend Snippet: Anti-RhoB Ig has no effect in RhoB ko arthritic mice. RhoB ko KRN.g7 mice were treated with 500 µg anti-RhoB Ig at 21 days of age. Data show means±s.e.m. for n =5 control-Ig- and n =5 anti-RhoB-Ig-treated mice. n.s., not significant. (A) Joint inflammation was measured by the change in ankle thickness. At 6 weeks of age, (B) titers of anti-GPI Ig in the serum were measured by ELISA and (C) the number of anti-GPI ASCs was measured by ELISpot.

Article Snippet: Recombinant proteins RhoB (OPPA00123), RhoA (OPPA00043), Rac1 (OPPA01858) and Cdc42 (OPPA00141) were purchased from Aviva Systems Biology (San Diego, CA, USA).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Disease Models & Mechanisms

Article Title: RhoB blockade selectively inhibits autoantibody production in autoimmune models of rheumatoid arthritis and lupus

doi: 10.1242/dmm.029835

Figure Lengend Snippet: Serum from RhoB ko KRN.g7 mice does not induce arthritis in a serum-transfer model. (A) Pooled serum from K/BxN mice was injected into wt or RhoB ko C57BL/6 mice. Joint inflammation was measured by the change in ankle thickness. Data show means±s.e.m. from a representative experiment of two, with n =5 mice for each group. (B) Pooled serum from 6-week-old KRN.g7 or RhoB ko KRN.g7 mice was equalized for anti-GPI titer and injected into C57BL/6 recipient mice. Data show mean ankle thickness±s.e.m. from n =8 mice for each group. * P <0.05.

Article Snippet: Recombinant proteins RhoB (OPPA00123), RhoA (OPPA00043), Rac1 (OPPA01858) and Cdc42 (OPPA00141) were purchased from Aviva Systems Biology (San Diego, CA, USA).

Techniques: Injection

Journal: Disease Models & Mechanisms

Article Title: RhoB blockade selectively inhibits autoantibody production in autoimmune models of rheumatoid arthritis and lupus

doi: 10.1242/dmm.029835

Figure Lengend Snippet: Targeting RhoB does not affect immune-cell repertoire or function in either RhoB ko or anti-RhoB-Ig-treated C57BL/6 mice. (A) The frequency of individual lymphoid populations in wt or RhoB ko C57BL/6 mice was measured by flow cytometry. Data show means±s.e.m., n =6 mice of each genotype. Gating strategies are shown in Fig. S6 B . (B) The amount of serum Ig in wt or RhoB ko C57BL/6 mice was measured by ELISA. n =6 mice of each genotype. (C) wt and RhoB ko C57BL/6 mice or (D) control-Ig- and anti-RhoB-Ig-treated C57BL/6 mice were immunized with 100 µg NP-KLH. Serum was harvested 10 days later and serum anti-NP titers were measured by ELISA. Data show means±s.e.m. for n =5 mice of each group. All analyses were performed three times.

Article Snippet: Recombinant proteins RhoB (OPPA00123), RhoA (OPPA00043), Rac1 (OPPA01858) and Cdc42 (OPPA00141) were purchased from Aviva Systems Biology (San Diego, CA, USA).

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control

Journal: Disease Models & Mechanisms

Article Title: RhoB blockade selectively inhibits autoantibody production in autoimmune models of rheumatoid arthritis and lupus

doi: 10.1242/dmm.029835

Figure Lengend Snippet: Anti-RhoB Ig lowers autoantibody levels in the MRL/ lpr mouse model of SLE. Weekly doses (500 µg) of anti-RhoB IgG or control mouse IgG were administered to MRL/ lpr mice starting at 4 weeks of age. At 16 weeks of age, serum dsDNA-autoantibody titers were determined by ELISA. Data show means±s.e.m. from two separate experiments combined for n =14 mice per group. Comparisons were made between control and anti-RhoB IgG groups at the specified weeks of age. * P <0.05.

Article Snippet: Recombinant proteins RhoB (OPPA00123), RhoA (OPPA00043), Rac1 (OPPA01858) and Cdc42 (OPPA00141) were purchased from Aviva Systems Biology (San Diego, CA, USA).

Techniques: Control, Enzyme-linked Immunosorbent Assay