Journal: Cell

Article Title: Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins

doi: 10.1016/j.cell.2021.12.021

Figure Lengend Snippet:

Article Snippet: HEK293T cells (ATCC; CRL-3216), Gesicle Producer 293T cells (Takara; 632617), 3T3 cells (ATCC; CRL-1658), and Neuro-2a cells (ATCC; CCL-131) were maintained in DMEM + GlutaMAX (Life Technologies) supplemented with 10% (v/v) fetal bovine serum.

Techniques: Recombinant, Transfection, SYBR Green Assay, DNA Extraction, Multiplex Assay, Purification, Gel Extraction, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Cell Isolation, Titration, Amplification, Sequencing, Software

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: Scientific reports

Article Title: HIV-1 enhances mTORC1 activity and repositions lysosomes to the periphery by co-opting Rag GTPases.

doi: 10.1038/s41598-017-05410-0

Figure Lengend Snippet: Figure 2. mTOR activity is required for optimal viral-protein synthesis. (a) Schematic of experimental protocol. (b) HeLa cells were transfected with pNL4-3, left untreated or treated with 250 nM Torin1 in medium containing AHA. At the indicated times, cell lysates were collected and Click-it chemistry was performed followed by immunoprecipitation with rabbit anti-p24 antibody. Membranes were probed with the indicated antibodies. (c) Densitometry quantification of de novo synthesized, biotinylated Gag from panel b was determined by ImageJ analysis. For each time point, the values presented in the graph are normalized against the total amount of Gag in the cell lysate (refer to Supplementary Fig. 1 for total protein synthesis). Full-length blots are shown in Supplementary Fig. 5. (d) HeLa cells were transfected with either pcDNA3.1 or pNL4-3 and infected with L. major (lmj) WT or GP63 KO. Cell lysates were subjected to SDS-PAGE, immunoblotted and probed with the indicated antibodies against host (left panels) and viral (right panels) antigens.

Article Snippet: The labeled material was precleared with normal rabbit serum and 25 μL of a 50:50 slurry of protein G-Sepharose (Thermo Scientific), incubated with rabbit anti-p24 (Fitzgerald) for 16 h at 4 °C and with 30 μL of a 50:50 slurry of protein G-Sepharose for 3 h at room temperature.

Techniques: Activity Assay, Transfection, Immunoprecipitation, Synthesized, Infection, SDS Page

Journal: Scientific reports

Article Title: HIV-1 enhances mTORC1 activity and repositions lysosomes to the periphery by co-opting Rag GTPases.

doi: 10.1038/s41598-017-05410-0

Figure Lengend Snippet: Figure 5. RagA and RagB are necessary for HIV-1 to control LEL trafficking and for the viral budding and release. (a) HeLa cells, treated with non-silencing (NS) siRNA or siRNAs targeting RagA and RagB mRNAs, were transfected with empty vector control, pCDNA3.1 or HIV-1 using pNL4-3/GagmRFP, and then mock treated or treated with Ars. Cells were stained for mTOR (Cell Signaling 7C10) and LAMP-1. Light blue arrowheads identify HIV-1-expressing cells, while HIV-1-negative cells are indicated with green arrowheads. A dashed line contours individual cells. Scale bars are 10 μm. (b) The graphs represent the fluorescence intensities of mTOR (green) and LAMP-1 (blue) across the dashed lines from panel a. (c) The percentage of mock or HIV-1-transfected cells showing perinuclear clustering of LEL from panel a. The results are presented as the mean ± S.D. from three different experiments. P value is indicated by *(p < 0.05); n.s.: no signifcant difference between the means. (d) HeLa cells were co-transfected with the HIV-1 pNL4-3 and either non- sense (NS) siRNA or siRNA against RagA and RagB. HIV-1 particles were collected from the supernatant after filtration and ultracentrifugation. Cell lysates and virus particles were separated by SDS-PAGE, transferred to nitrocellulose and probed with the indicated antibodies. siRNA-mediated fold changes in RagA, RagB and virus production are indicated below insets in panel d in this representative experiment. Full-length blots are shown in Supplementary Fig. 5. (e) HeLa cells were co-transfected with the HIV-1 pNL4-3 and either non-sense (NS) siRNA or siRNA against RagA and RagB. Cells were fixed 24 h after transfection and stained for p24. (f) The percentage of HIV-1 and siRagA/B transfected cells showing peripheral accumulation of Gag from e. The results are presented as the mean ± S.D. from three different experiments. (g) HeLa cells were transfected as in e and subjected to transmission electron microscopy.

Article Snippet: The labeled material was precleared with normal rabbit serum and 25 μL of a 50:50 slurry of protein G-Sepharose (Thermo Scientific), incubated with rabbit anti-p24 (Fitzgerald) for 16 h at 4 °C and with 30 μL of a 50:50 slurry of protein G-Sepharose for 3 h at room temperature.

Techniques: Control, Transfection, Plasmid Preparation, Staining, Expressing, Fluorescence, Filtration, Virus, SDS Page, Transmission Assay, Electron Microscopy

Journal: Scientific reports

Article Title: HIV-1 enhances mTORC1 activity and repositions lysosomes to the periphery by co-opting Rag GTPases.

doi: 10.1038/s41598-017-05410-0

Figure Lengend Snippet: Figure 6. RagA and RagB interact with Gag and Vif. (a) Lysates from control pcDNA3.1- or pNL4-3- transfected HeLa cells were subjected to immunoprecipitation with RagA antibodies. Samples were subjected to SDS-PAGE followed by transfer to nitrocellulose membranes and probed with the indicated antibodies. Full- length blots are shown in Supplementary Fig. 5. (b) Lysate from pcDNA3.1-, pNL4-3-, pNLXX−, pNLXX+ pSVGag− and pNLVif–transfected HeLa cells were subjected to immunoprecipitation with RagA and RagB antibodies or isotype rabbit IgG control. The blots were probed with anti-p24, anti-Vif, anti-RagA and anti- RagB antibodies. Full-length blots are shown in Supplementary Fig. 5. (c) HeLa cells were transfected with either pNLXX or pNLVif- and left untreated (Unt) or stressed with 500 µM Ars for 1 h. HIV-1-expressing cells were identified using FISH to localize vRNA and stained using antibodies against mTOR (Cell Signaling) and LAMP-1. Light blue arrowheads identify pNLXX or pNLVif- expressing cells, while pNLXX or pNLVif- negative cells in the same field are indicated with green arrowheads. A dashed line contours individual cells. Scale bars are 10 μm. (d) The percentage of mock, pNLXX or pNLVif- transfected cells showing perinuclear clustering of mTOR/LAMP-1 from panel c. The results are presented as the mean ± S.D. from three different experiments. P value is indicated by *(p < 0.05).

Article Snippet: The labeled material was precleared with normal rabbit serum and 25 μL of a 50:50 slurry of protein G-Sepharose (Thermo Scientific), incubated with rabbit anti-p24 (Fitzgerald) for 16 h at 4 °C and with 30 μL of a 50:50 slurry of protein G-Sepharose for 3 h at room temperature.

Techniques: Control, Transfection, Immunoprecipitation, SDS Page, Expressing, Staining