Journal: Journal of Nanobiotechnology

Article Title: Doxorubicin-polyglycerol-nanodiamond conjugate is a cytostatic agent that evades chemoresistance and reverses cancer-induced immunosuppression in triple-negative breast cancer

doi: 10.1186/s12951-019-0541-8

Figure Lengend Snippet: M2–M1 phenotypic shift of tumor-associated macrophages induced by Nano-DOX-treated 4T1 cells. a , b Immunostaining of CD86 (an M1 marker) in type-2 macrophages (M2) assayed by FACS. c Expression of GBP5 (an M1 marker) in M2 assayed by western blotting. d mRNA expression of iNOS (an M1 marker) in M2 assayed by RT-PCR. e , f mRNA expression of CD206 and TGF-β (M2 markers), respectively in M2 assayed by RT-PCR. g Immunohistochemical staining of CD80, CD86, GBP5, MHC-II (M1 markers), and CD206 (an M2 marker) in 4T1 tumor xenograts at the end of 3-week treatment of Nano-DOX or DOX. In the ex vivo cell experiments, type-1 and type-2 macrophages (M1 and M2) were activated from mouse bone marrow-derived macrophages according to published protocols. M2 were treated with Nano-DOX (2 μg/mL) or culture medium conditioned by Nano-DOX (2 μg/mL)-treated 4T1 cells (ND-CM). Duration of Nano-DOX or Nano-DOX-CM treatment was 24 h. In FACS analysis, geometric means were used to quantify fluorescence intensity. Values were mean ± SD (n = 3, *p < 0.05, **p < 0.01)

Article Snippet: Antibodies for immunohistochemical analysis included rabbit anti-Ki67 (Abcam, ab15580), mouse anti-PCNA (BOSTER, BM0104), rabbit anti-Caspase-3 (Proteintech, 19677-1-AP), rabbit anti-CD3 (Abcam, ab5690), rabbit anti-F4/80 (Abcam, Ab100790), mouse anti-MDR-1 (SantaCruz, sc-55510), rabbit anti-IL-6 (Bioss, bs-0782R), Rabbit anti-G-CSF (Bioss, bs-1023R), rabbit anti-HSP90 (Abcam, ab13495), rabbit anti-CRT (Abcam, ab92516), rabbit anti-HMGB1 (Abcam, ab79823), rabbit anti-CD80 (Bioss, bs-2211R), mouse anti-CD86 (Abcam, ab213044), rabbit anti-GBP5 (Proteintech, 13220-1-AP), rabbit anti-MHC-II (Bioss, bs-4298R), rabbit anti-CD206 (Abcam, ab64693), rabbit anti-CD4 (Bioss, bs-0766R), rabbit anti-CD8 (Bioss, bs-0648R), rabbit anti-CD69 (MULTI SCIENCES, ab2304), rabbit anti-foxp3 (Bioss, bs-10211R).

Techniques: Immunostaining, Marker, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Staining, Ex Vivo, Derivative Assay, Fluorescence

Journal: Journal of Nanobiotechnology

Article Title: Doxorubicin-polyglycerol-nanodiamond conjugate is a cytostatic agent that evades chemoresistance and reverses cancer-induced immunosuppression in triple-negative breast cancer

doi: 10.1186/s12951-019-0541-8

Figure Lengend Snippet: Activation of DC and T lymphocytes by Nano-DOX-treated 4T1 cells. a – d Immunostaining of CD40, CD80, CD83 and MHC-II (markers of DC activation) in bone marrow-derived DC (BMDC) assayed by FACS. Representative FACS histograms are presented in Additional file : Fig. S6. e Spleen-derived CD4 + and CD8 + T lymphocytes characterized by immunofluorescent staining and FACS. f , g Proliferation of CD4 + and CD8 + T lymphocytes, assayed by CFSE staining and FACS. Representative FACS histograms are presented in Additional file : Fig. S7. h , i Immunostaining of CD69 (a marker of T lymphocyte activation) in CD4 + and CD8 + T lymphocytes, assayed by FACS. Representative FACS dot plots are presented in Additional file : Fig. S8 . j Immunohistochemical staining of CD4, CD8, CD69 and foxp3 (a marker of Treg) in 4T1 tumor xenografts at the end of 3-week treatment of Nano-DOX or DOX. In the ex vivo cell experiments, BMDC were first treated with Nano-DOX (2 μg/mL) or culture medium conditioned by Nano-DOX (2 μg/mL)-treated 4T1 cells (ND-CM) for 24 h. Spleen-derived lymphocytes were then co-cultured with the BMDC in the same system for another 24 h. In FACS analysis, geometric means were used to quantify fluorescence intensity. Values were mean ± SD (n = 3, *p < 0.05, **p < 0.01)

Article Snippet: Antibodies for immunohistochemical analysis included rabbit anti-Ki67 (Abcam, ab15580), mouse anti-PCNA (BOSTER, BM0104), rabbit anti-Caspase-3 (Proteintech, 19677-1-AP), rabbit anti-CD3 (Abcam, ab5690), rabbit anti-F4/80 (Abcam, Ab100790), mouse anti-MDR-1 (SantaCruz, sc-55510), rabbit anti-IL-6 (Bioss, bs-0782R), Rabbit anti-G-CSF (Bioss, bs-1023R), rabbit anti-HSP90 (Abcam, ab13495), rabbit anti-CRT (Abcam, ab92516), rabbit anti-HMGB1 (Abcam, ab79823), rabbit anti-CD80 (Bioss, bs-2211R), mouse anti-CD86 (Abcam, ab213044), rabbit anti-GBP5 (Proteintech, 13220-1-AP), rabbit anti-MHC-II (Bioss, bs-4298R), rabbit anti-CD206 (Abcam, ab64693), rabbit anti-CD4 (Bioss, bs-0766R), rabbit anti-CD8 (Bioss, bs-0648R), rabbit anti-CD69 (MULTI SCIENCES, ab2304), rabbit anti-foxp3 (Bioss, bs-10211R).

Techniques: Activation Assay, Immunostaining, Derivative Assay, Staining, Marker, Immunohistochemical staining, Ex Vivo, Cell Culture, Fluorescence

Journal: Cells

Article Title: Astrocytes Derived from Familial and Sporadic Alzheimer’s Disease iPSCs Show Altered Calcium Signaling and Respond Differently to Misfolded Protein Tau

doi: 10.3390/cells11091429

Figure Lengend Snippet: Deregulation of astrocytic protein upon exposure to tau. ( a ) Immunoblots using antibody against proteins EAAT-2, phosphoPKC II (pPKCα/β), total PKC II (tPKCα/β), phosphoERK1/2 (pERK1/2), total ERK1/2 (tERK1/2), pro- and active MMP-9 and MMP-2. Actin was used as loading control. Graph showing levels of ( b ) EAAT-2, ( c ) phospho/total PKCα/β, ( d ) phospho/total ERK1/2, ( e ) pro-MMP9, ( f ) active-MMP9, ( g ) pro-MMP2, and ( h ) active-MMP2 in astrocytes between the untreated and monomeric and aggregated tau-treated groups. One-way ANOVA followed by Tukey’s test for multiple analysis was used for statistical analysis ( n = 3/group; n = 3 experimental repeats), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The following antibodies were used: DC25 (1:1*; Axon Neuroscience, Bratislava, Slovakia; *supernatant from hybridoma cells), GFAP (1:1000; ab7260; Abcam), Connexin-43 (1:2000; ab11370; Abcam), Aquaporin-4 (1:1000; AB3594; Millipore), EAAT-2 (1:1000; AB1783; Millipore), Phospho-ERK1/2 (1:2000; 28733-1-AP; Proteintech), Total-ERK1/2 (1:2000; 16443-1-AP; Proteintech), Phospho-PKCα/β II (1:1000; #9375S; Cell Signaling Technology), Total-PKCα/β II (1:1000; #2056S; Cell Signaling Technology), MMP-9 (1:1000; #819701; Biolegend), MMP-2 (1:1000; #679902; Biolegend; Amsterdam, Netherlands), GluN1 (1:1000; #114011; SYSY), GAP-43 (1:1000; NBP1-71668; Novus biologicals), Drebrin (1:2000; ab178408; Abcam), and Actin (1:2500; ab76548; Abcam).

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: Sestrin2 protects dendrite cells against ferroptosis induced by sepsis

doi: 10.1038/s41419-021-04122-8

Figure Lengend Snippet: DCs were stimulated with LPS at various concentrations (40 ng/ml, 200 ng/ml, 1 μg/ml, 5 μg/ml) for different intervals (0, 3, 6, 12, 48 h). DCs cultured without LPS were defined as controls. A , B Representative flow cytometric analysis was performed for CD80, CD86, and MHC-II expression on DCs. C – E , G – I Intracellular levels of GSH, Fe 2+ , and ROS in DCs were measured by detection kits. F , J Ferroptosis-related proteins were analyzed as presented in the methods. Data were presented as the mean ± SD of three replicates ( n = 3 per group). Statistical significance: * P < 0.05, ** P < 0.01 vs. the control group.

Article Snippet: Fluorescent antibodies were applied to flow cytometry analysis, e.g., major CD80 conjugated to phycoerythrin (PE)-Cyanine 5 was obtained from eBioscience, San Diego, CA, and CD86 conjugated to allophycocyanin (APC), as well as major histocompatibility complex (MHC)-II conjugated to fluorescein isothioctante (FITC) were purchased from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.

Techniques: Cell Culture, Expressing

Journal: Journal of Orthopaedic Surgery and Research

Article Title: The study of targeted blocking SDF-1/CXCR4 signaling pathway with three antagonists on MMPs, type II collagen, and aggrecan levels in articular cartilage of guinea pigs

doi: 10.1186/s13018-020-01646-1

Figure Lengend Snippet: The expression of Col II and aggrecan measured using RT-PCR

Article Snippet: Membranes were probed by a human anti-Col II monoclonal antibody (1:1000 dilution; Proteintech Group, Chicago, USA) and anti-β-actin polyclonal antibody (1:1000 dilution; Cell Signaling Technology, Danvers, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction