Journal: Cell reports

Article Title: Dectin-1 Binding to Annexins on Apoptotic Cells Induces Peripheral Immune Tolerance via NADPH Oxidase-2.

doi: 10.1016/j.celrep.2019.11.086

Figure Lengend Snippet: Figure 3. Annexin-Induced ROSs Are Derived from NADPH Oxidase-2 H2DCFDA ROS detection experiments were per- formed as described in Figure 2. (A–D) Dectin-1-expressing BMDCs were pre- incubated for 30–60 min with the indicated ROS scavenger before addition of the treatment. Results represent mean ± SD of at least two (B), three (D), or four (A and C) independent experiments. (E) BMDCs from NOX-2-deficient mice or WT littermates were treated with indicated ligands. Quantification of six independent experiments. The relative ROS-in- crease is normalized to untreated cells and the ROS-increase of WT control cells were set to 1 (WT BMDCs: black circles; NOX-2 KO BMDCs: orange squares). FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity. ***p < 0.001, **p < 0.01, *p < 0.05 (paired, two-tailed t test). Significant outliers (r values < 0.05) as determined by GraphPad outlier calculator were removed. See also Figure S5.

Article Snippet: Specific NADPH-oxidase 2 (NOX-2) inhibition experiments were performed using the peptide inhibitor gp91 ds-TAT aside with appropriate control scrambled gp91 ds-TAT (Anaspec), and the small molecule inhibitor GSK2795039 diluted in DMSO (MedChemExpress).

Techniques: Derivative Assay, Expressing, Incubation, Control, Two Tailed Test

Journal: Cell reports

Article Title: Dectin-1 Binding to Annexins on Apoptotic Cells Induces Peripheral Immune Tolerance via NADPH Oxidase-2.

doi: 10.1016/j.celrep.2019.11.086

Figure Lengend Snippet: Figure 6. Effective Immunosuppression of AC-Derived Annexins Requires ROSs (A–G) Quantification of suppression experiments performed as in Figure 5 for indicated cytokines. WT BMDCs were preincubated with NOX-2 in- hibitors GSK2795039 (A–C) or gp91-TAT (D–G) for 30 min. Subsequently, apoptotic Jurkat T cells (aJ; ratio of 0.5:1) (C, F, and G) or eukaryotically expressed mAnxA1DN cells (1,000 nM) (A, B, D, and E) were added, and BMDCs were further incubated for 8 h. (H–K) BMDCs generated from NOX-2-deficient mice and WT littermate controls were incubated with aJ cells (ratio of 2:1) (J and K) or mAnxA1DN (500 nM) (H and I). After preincubation, BMDCs were stimulated with the TLR agonist CpG. Indi- cated cytokines in the supernatants were analyzed by ELISA 20-24 h after stimulation. The rangeofcytokine secretion was 75–3,746 pg/mL (A), 270–10,798 pg/mL (B), 745–4,298 pg/mL (C), 1,017–3,111 pg/mL (D), 4,197–6,832 pg/mL (E), 384– 2,543 pg/mL (F), 2,153–8,465 pg/mL (G), 93–1,159 pg/mL (H), 438–3,540 pg/mL (I), 1,702–3,318 pg/mL (J),and 2,885–5,766pg/mL(K).Resultsrepresentthe means of three (F–K), four (D and E), or seven (A–C) independent experiments. ***p < 0.001; **p < 0.01; *p < 0.05 (paired, two-tailed t test). See also Figure S7.

Article Snippet: Specific NADPH-oxidase 2 (NOX-2) inhibition experiments were performed using the peptide inhibitor gp91 ds-TAT aside with appropriate control scrambled gp91 ds-TAT (Anaspec), and the small molecule inhibitor GSK2795039 diluted in DMSO (MedChemExpress).

Techniques: Derivative Assay, Incubation, Generated, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Biomedicines

Article Title: Secretome of hESC-Derived MSC-like Immune and Matrix Regulatory Cells Mitigate Pulmonary Fibrosis through Antioxidant and Anti-Inflammatory Effects.

doi: 10.3390/biomedicines11020463

Figure Lengend Snippet: Figure 1. Elevated Nox-mediated oxidative stress and Tlr4/MyD88-mediated inflammation in lungs of mice with BLM-induced pulmonary fibrosis (PF). (a) Schematic diagram for the creation of the BLM-induced PF mouse model. C57BL/6 mice that were 6–8 weeks old were injured by intratracheal instillation of 1.5 mg/kg BLM at day 0 (d0) and d7, and the lung tissues were harvested at d14 post-injury for evaluation (N = 8). (b) Representative histochemical images of H&E and Masson staining demonstrated alveolar structural damage (H&E staining) with ECM deposition (Masson staining). (c) Representative immunoblotting blots demonstrated an increased expression of proteins of Nox family numbers (Nox2, Nox3, and Nox4), oxidization stress markers (Nrf2 and Ho-1), and EMT markers (α-SMA and Vimentin) in the lungs of BLM-induced PF mice compared with the saline group. (d) Semi-quantitation of relative levels of proteins of interest as determined by the relative density over Gapdh in (c). (e) IF staining validated the increased abundance of α-SMA in the lungs of mice at d14 post-BLM challenge compared with saline controls. (f) Representative blots of immunoblotting assay demonstrated the enhanced activation of Tlr4/MyD88 signaling in the lung of BLM-induced PF mice model compared with the saline group. (g) Semi-quantitation of the relative levels of proteins of interest Tlr4, MyD88, and p-NF-κB as evaluated by the relative density over Gapdh in (f). Data in (d,g) represent mean ± SD from the indicated number of mice (N = 3), as analyzed by GraphPad prism one-way ANOVA Tukey’s multiple comparisons test.

Article Snippet: After blocking with 5% non-fat milk or 3% BSA in PBS for 1 h at RT, the membrane was incubated overnight at 4 ◦C with primary antibodies: rabbit monoclonal IgG HO-1 (27282-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NRF2 (Proteintech, 16396-1-AP), rabbit monoclonal IgG SMA (14395-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX4 (BA2813, Boster, Wuhan, China), mouse monoclonal IgG NOX2 (BA2811, Boster, Wuhan, China), mouse monoclonal IgG antiα-tubulin (T5168, SIGMA, St. Louis, MO, USA), rabbit monoclonal IgG ATF6 (24169- 1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG N-cadherin (22018-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX3 (20065-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG Toll-like receptor 4 (TLR4) (BS20594R, Bioss, Woburn, MA, USA), and rabbit monoclonal IgG Myeloid differentiation primary response 88 (MyD88) (NB100-56698, Novusbio, Centennial, CO, USA).

Techniques: Staining, Western Blot, Expressing, Saline, Quantitation Assay, Activation Assay

Journal: Biomedicines

Article Title: Secretome of hESC-Derived MSC-like Immune and Matrix Regulatory Cells Mitigate Pulmonary Fibrosis through Antioxidant and Anti-Inflammatory Effects

doi: 10.3390/biomedicines11020463

Figure Lengend Snippet: Elevated Nox-mediated oxidative stress and Tlr4/MyD88-mediated inflammation in lungs of mice with BLM-induced pulmonary fibrosis (PF). ( a ) Schematic diagram for the creation of the BLM-induced PF mouse model. C57BL/6 mice that were 6–8 weeks old were injured by intratracheal instillation of 1.5 mg/kg BLM at day 0 (d0) and d7, and the lung tissues were harvested at d14 post-injury for evaluation (N = 8). ( b ) Representative histochemical images of H&E and Masson staining demonstrated alveolar structural damage (H&E staining) with ECM deposition (Masson staining). ( c ) Representative immunoblotting blots demonstrated an increased expression of proteins of Nox family numbers (Nox2, Nox3, and Nox4), oxidization stress markers (Nrf2 and Ho-1), and EMT markers (α-SMA and Vimentin) in the lungs of BLM-induced PF mice compared with the saline group. ( d ) Semi-quantitation of relative levels of proteins of interest as determined by the relative density over Gapdh in ( c ). ( e ) IF staining validated the increased abundance of α-SMA in the lungs of mice at d14 post-BLM challenge compared with saline controls. ( f ) Representative blots of immunoblotting assay demonstrated the enhanced activation of Tlr4/MyD88 signaling in the lung of BLM-induced PF mice model compared with the saline group. ( g ) Semi-quantitation of the relative levels of proteins of interest Tlr4, MyD88, and p-NF-κB as evaluated by the relative density over Gapdh in ( f ). Data in ( d , g ) represent mean ± SD from the indicated number of mice (N = 3), as analyzed by GraphPad prism one-way ANOVA Tukey’s multiple comparisons test.

Article Snippet: After blocking with 5% non-fat milk or 3% BSA in PBS for 1 h at RT, the membrane was incubated overnight at 4 °C with primary antibodies: rabbit monoclonal IgG HO-1 (27282-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NRF2 (Proteintech, 16396-1-AP), rabbit monoclonal IgG SMA (14395-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX4 (BA2813, Boster, Wuhan, China), mouse monoclonal IgG NOX2 (BA2811, Boster, Wuhan, China), mouse monoclonal IgG anti-α-tubulin (T5168, SIGMA, St. Louis, MO, USA), rabbit monoclonal IgG ATF6 (24169-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG N-cadherin (22018-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX3 (20065-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG Toll-like receptor 4 (TLR4) (BS20594R, Bioss, Woburn, MA, USA), and rabbit monoclonal IgG Myeloid differentiation primary response 88 (MyD88) (NB100-56698, Novusbio, Centennial, CO, USA).

Techniques: Staining, Western Blot, Expressing, Saline, Quantitation Assay, Activation Assay

Journal: Biomedicines

Article Title: Secretome of hESC-Derived MSC-like Immune and Matrix Regulatory Cells Mitigate Pulmonary Fibrosis through Antioxidant and Anti-Inflammatory Effects

doi: 10.3390/biomedicines11020463

Figure Lengend Snippet: BLM induces NOX4-mediated oxidative stress in A549 lung epithelial cells. ( a ) Representative images of immunoblotting blots revealed an increased expression of α-SMA, NOX4 and NRF2 in A549 cells exposed to indicated concentrations of BLM (1.0, 2.5, 5.0, 10.0, and 20.0 μg/mL) for 48 h. ( b ) Semi-quantitation of the relative levels of α-SMA, NOX4, and NRF2 proteins as evaluated by the relative density over GAPDH in ( a ). ( c ) Representative images of A549 cells treated with indicated concentrations of BLM for 48 h and stained with CellROX ® Orange reagent. The BLM-induced A549 injury increased ROS production, especially at the concentration of 5 μg/mL, compared to untreated cells (NC). ( d ) Mean gray value of ROS production in ( c ). ( e ) Representative images of IF exhibited more abundant HO-1, NRF2, NOX4, ATF6, α-SMA, and N-Cad proteins in A549 cells exposed to 5 μg/mL of BLM for 48 h as compared with the NC control. ( f ) Representative images of the fluorescent intensity of the relevant proteins in ( e ). Data in ( b , d , f ) represent mean ± SD from three independent experiments (N = 3), as analyzed by GraphPad prism one-way ANOVA Tukey’s multiple comparisons test. Scale bars in ( c ), 100 µm; ( e ), 10 µm.

Article Snippet: After blocking with 5% non-fat milk or 3% BSA in PBS for 1 h at RT, the membrane was incubated overnight at 4 °C with primary antibodies: rabbit monoclonal IgG HO-1 (27282-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NRF2 (Proteintech, 16396-1-AP), rabbit monoclonal IgG SMA (14395-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX4 (BA2813, Boster, Wuhan, China), mouse monoclonal IgG NOX2 (BA2811, Boster, Wuhan, China), mouse monoclonal IgG anti-α-tubulin (T5168, SIGMA, St. Louis, MO, USA), rabbit monoclonal IgG ATF6 (24169-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG N-cadherin (22018-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX3 (20065-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG Toll-like receptor 4 (TLR4) (BS20594R, Bioss, Woburn, MA, USA), and rabbit monoclonal IgG Myeloid differentiation primary response 88 (MyD88) (NB100-56698, Novusbio, Centennial, CO, USA).

Techniques: Western Blot, Expressing, Quantitation Assay, Staining, Concentration Assay, Control

Journal: Biomedicines

Article Title: Secretome of hESC-Derived MSC-like Immune and Matrix Regulatory Cells Mitigate Pulmonary Fibrosis through Antioxidant and Anti-Inflammatory Effects

doi: 10.3390/biomedicines11020463

Figure Lengend Snippet: hESC-MSC-IMRC-CM reduces the BLM-induced oxidative stress and EMT in A549 lung epithelial cells. ( a ) Representative immunoblots showed that hESC-MSC-IMRC-CM could reduce BLM-induced A549 cells oxidative injury by decreasing the expression of NRF2, HO-1, NOX4, and EMT markers’ N-Cad. ( b ) The relative levels of NRF2, HO-1, NOX4, and N-Cad proteins as evaluated by a densitometric analysis in ( a ). ( c ) Representative images of A549 cells treated with indicated conditions for 48 h and stained with CellROX ® Orange reagent. hESC-MSC-IMRC-CM significantly reduced the ROS production compared to the BLM group. ( d ) Mean gray value of ROS production in ( c ). ( e ) hESC-MSC-IMRC-CM increased glutathione peroxidase (GSH-PX) production in A549 cells compared to untreated controls. Data in ( b , d , e ) represent mean ± SD of three independent experiments (N = 3), as analyzed by GraphPad prism Two-way ANOVA Tukey’s multiple comparisons test. Scale bars in ( c ), 100 μm.

Article Snippet: After blocking with 5% non-fat milk or 3% BSA in PBS for 1 h at RT, the membrane was incubated overnight at 4 °C with primary antibodies: rabbit monoclonal IgG HO-1 (27282-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NRF2 (Proteintech, 16396-1-AP), rabbit monoclonal IgG SMA (14395-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX4 (BA2813, Boster, Wuhan, China), mouse monoclonal IgG NOX2 (BA2811, Boster, Wuhan, China), mouse monoclonal IgG anti-α-tubulin (T5168, SIGMA, St. Louis, MO, USA), rabbit monoclonal IgG ATF6 (24169-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG N-cadherin (22018-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX3 (20065-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG Toll-like receptor 4 (TLR4) (BS20594R, Bioss, Woburn, MA, USA), and rabbit monoclonal IgG Myeloid differentiation primary response 88 (MyD88) (NB100-56698, Novusbio, Centennial, CO, USA).

Techniques: Western Blot, Expressing, Staining

Journal: Biomedicines

Article Title: Secretome of hESC-Derived MSC-like Immune and Matrix Regulatory Cells Mitigate Pulmonary Fibrosis through Antioxidant and Anti-Inflammatory Effects

doi: 10.3390/biomedicines11020463

Figure Lengend Snippet: hESC-MSC-IMRCs and hESC-MSC-IMRC-CM inhibit the development of pulmonary fibrosis in mice. ( a ) Schematic illustration shows the experimental workflow for the treatment of the BLM-induced IPF mouse model at an early stage of PF development. C57BL/6 mice were challenged with BLM via the laryngotracheal route at day ( d ) 0 and d7. A 200 μL sample of saline containing 3 × 10 6 cells of hESC-MSC-IMRCs and 200 μL of hESC-MSC-IMRC-CM were delivered via the tail vein injection at d3, d7, and d14 after the first dose of BLM challenge. The lung tissues were harvested at d28 for analysis. ( b ) The effect of hESC-MSC-IMRC-CM on lung index (ratio of lung/body weights). ( c ) Representative histochemical images of H&E and Masson staining demonstrate that both hESC-MSC-IMRC-CM and hESC-MSC-IMRCs can ameliorate the pathogenesis of pulmonary fibrosis (H&E staining) with ECM deposition (Masson staining) compared to animals challenged with BLM alone. ( d ) Histological analysis of the fibrotic area. ( e , f ) The levels of hydroxyproline (Hyp) and malondialdehyde (MDA) contents. ( g ) Representative immunoblots show a reduction in indicated molecules of oxidative stress-signaling pathways and α-SMA in BLM-induced fibrotic lung administrated with hESC-MSC-IMRC-CM or hESC-MSC-IMRCs compared to the lungs of BLM-challenged mice alone. ( h ) The relative levels of Nox4, Nrf2, Ho-1 and α-SMA proteins as evaluated by a densitometric analysis in ( g ). Data in ( b , d – f , h ) represent mean ± SD of three independent experiments (N = 3), as analyzed by GraphPad prism two-way ANOVA Tukey’s multiple comparisons test. Scale bars in ( c ), 200 µm in the first and third panels, 20 µm in the second and fourth rows.

Article Snippet: After blocking with 5% non-fat milk or 3% BSA in PBS for 1 h at RT, the membrane was incubated overnight at 4 °C with primary antibodies: rabbit monoclonal IgG HO-1 (27282-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NRF2 (Proteintech, 16396-1-AP), rabbit monoclonal IgG SMA (14395-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX4 (BA2813, Boster, Wuhan, China), mouse monoclonal IgG NOX2 (BA2811, Boster, Wuhan, China), mouse monoclonal IgG anti-α-tubulin (T5168, SIGMA, St. Louis, MO, USA), rabbit monoclonal IgG ATF6 (24169-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG N-cadherin (22018-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX3 (20065-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG Toll-like receptor 4 (TLR4) (BS20594R, Bioss, Woburn, MA, USA), and rabbit monoclonal IgG Myeloid differentiation primary response 88 (MyD88) (NB100-56698, Novusbio, Centennial, CO, USA).

Techniques: Saline, Injection, Staining, Western Blot, Protein-Protein interactions

Journal: Biomedicines

Article Title: Secretome of hESC-Derived MSC-like Immune and Matrix Regulatory Cells Mitigate Pulmonary Fibrosis through Antioxidant and Anti-Inflammatory Effects

doi: 10.3390/biomedicines11020463

Figure Lengend Snippet: hESC-MSC-IMRC-CM alleviates the progression of pulmonary fibrosis in mice. ( a ) Schematic illustration shows the experimental workflow for treatments of late-stage of PF disease in the BLM-induced PF mouse model. C57BL/6 mice were challenged with BLM via the laryngotracheal route at day ( d ) 0 and d7. A 200 μL sample of saline containing 3 × 10 6 cells of hESC-MSC-IMRCs or 200 μL hESC-MSC-IMRC-CM was administrated via tail vein injection at d7, d14, and d21 after the first dose of BLM challenge. The lung tissues were harvested at d28 for analysis. ( b ) The ratio of lung weight/body weight (LW/BW). ( c ) Representative histochemical images of H&E and Masson staining demonstrate that both hESC-MSC-IMRC-CM and hESC-MSC-IMRCs can ameliorate the progression of pulmonary fibrosis (H&E staining) with ECM deposition (Masson staining) compared to the BLM-group. ( d ) Histological analysis of the fibrotic area. ( e , f ) The level of hydroxyproline (Hyp) and malondialdehyde (MDA) contents. ( g ) Representative immunoblots show a decreased expression of indicated components of oxidative stress signaling cascade and α-SMA in BLM-induced lung treated with hESC-MSC-IMRC-CM or hESC-MSC-IMRCs compared to the untreated BLM group. ( h ) The relative levels of Nox4, Nrf2, Ho-1, and α-SMA proteins as evaluated by a densitometric analysis in ( g ). Data in ( b , d – f , h ) represent mean ± SD of three independent experiments (N = 3), as analyzed by GraphPad prism two-way ANOVA Tukey’s multiple comparisons test. Scale bars in ( c ), 200 µm in the first and third rows, 20 µm in the second and fourth rows.

Article Snippet: After blocking with 5% non-fat milk or 3% BSA in PBS for 1 h at RT, the membrane was incubated overnight at 4 °C with primary antibodies: rabbit monoclonal IgG HO-1 (27282-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NRF2 (Proteintech, 16396-1-AP), rabbit monoclonal IgG SMA (14395-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX4 (BA2813, Boster, Wuhan, China), mouse monoclonal IgG NOX2 (BA2811, Boster, Wuhan, China), mouse monoclonal IgG anti-α-tubulin (T5168, SIGMA, St. Louis, MO, USA), rabbit monoclonal IgG ATF6 (24169-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG N-cadherin (22018-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX3 (20065-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG Toll-like receptor 4 (TLR4) (BS20594R, Bioss, Woburn, MA, USA), and rabbit monoclonal IgG Myeloid differentiation primary response 88 (MyD88) (NB100-56698, Novusbio, Centennial, CO, USA).

Techniques: Saline, Injection, Staining, Western Blot, Expressing