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

Journal: PLoS ONE

Article Title: Fluorofenidone Offers Improved Renoprotection at Early Interventions during the Course of Diabetic Nephropathy in db/db Mice via Multiple Pathways

doi: 10.1371/journal.pone.0111242

Figure Lengend Snippet: A) RNA was purified from the indicated mice (n = 3 per group). TGF-β1 mRNA abundance was subsequently determined by real-time PCR following our published methodology , , . Real-time PCR was performed 3 times in triplicate. TGF-β1 mRNA levels were normalized to actin, and expressed as the fold change in reference to TGF-β1 mRNA in db/m mice. *: p <0.05 in comparison to db/m ; #: p <0.05 in comparison to the respective Ctrl mice; other comparison are also presented. B) TGF-β1 protein in the indicated mice (n = 6 per group) was quantified using ELISA. Assay was repeated three times in triplicate. TGF-β1 protein was expressed as ng per mg of total protein (ng/mg total protein). Statistical analysis was performed as detailed above.

Article Snippet: Kidney-associated TGF-β1 protein abundance was measured using an ELISA kit (Boster Biological Technology, Wuhan, China) according to the manufacturers' instructions.

Techniques: Purification, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay