Journal: Cellular and Molecular Immunology

Article Title: Chronic activation of 4-1BB signaling induces granuloma development in tumor-draining lymph nodes that is detrimental to subsequent CD8 + T cell responses

doi: 10.1038/s41423-020-00533-3

Figure Lengend Snippet: Granulomas of TDLNs following the repeated injection of anti-4-1BB mAb in vivo. a C57BL/6 mice were injected subcutaneously with MC38 tumor cells and received anti-4-1BB mAb or rat IgG every 5 days, for a total of four times, from day 10. b Growth rate of MC38 tumors. c Inguinal TDLNs on day 14. d Frozen sections were prepared from TDLNs on day 14 and stained with anti-mouse CD8β-Alexa 647 and anti-B220-Alexa 594 antibodies. Slides were mounted with DAPI-containing solution. e Three days after 4th mAb injection, TDLNs were collected, and the paraffin sections of TDLNs were stained with anti-PCNA mAb and HRP-conjugated 2nd Ab. f Percentages of B220 + B cells in TDLNs on day 28. g H&E staining of paraffin sections from rat IgG- or anti-4-1BB-treated TDLNs on day 28. h Frozen sections of inguinal TDLNs on day 28 were stained with anti-mouse CD68-biotin and detected using streptavidin-Cy3. Slides were mounted with DAPI-containing solution. Confocal images were captured by a laser scanning microscope (Zeiss LSM780, Carl Zeiss). Data are from two ( c , d ) and three ( b , e – h ) independent experiments with 3–5 mice per experiment. Student’s t test was performed in b and f , and the results are shown as the means ± SDs (* p < 0.05; ** p < 0.01)

Article Snippet: CD8 microbeads were purchased from Miltenyi Biotec (Auburn, CA).

Techniques: Injection, In Vivo, Staining, Laser-Scanning Microscopy

Journal: Cellular and Molecular Immunology

Article Title: Chronic activation of 4-1BB signaling induces granuloma development in tumor-draining lymph nodes that is detrimental to subsequent CD8 + T cell responses

doi: 10.1038/s41423-020-00533-3

Figure Lengend Snippet: 4-1BB-induced LN granulomas in the absence of CD8 + T cells or IFN-γ. a MC38 tumor-bearing mice were injected intraperitoneally with rat IgG or anti-4-1BB mAb, and half of the mice were further treated with depleting anti-CD8 mAb (dCD8) 10 and 15 days after injection of MC38 tumor cells. On day 23, frozen sections of TDLNs were stained with anti-mouse CD68-biotin and detected using streptavidin-Cy3. b CD68 + areas were calculated from ( a ) using ImageJ. c IFN-γ expression of rat IgG- or anti-4-1BB-treated CD8 + T cells on day 18. d – g WT and IFN-γR KO (gR −/− ) B6 mice were injected subcutaneously with MC38 tumor cells and treated with rat IgG or anti-4-1BB mAb every 5 days from day 10 as described above ( d ). e On day 23, LN longitudinal length was calculated using a stereomicroscope (Zeiss Stereo Discovery V20) with a camera (Zeiss AxioCamHRc camera). f Frozen sections of TDLNs on day 23 were stained with anti-mouse CD68-biotin and detected using streptavidin-Cy3. Slides were mounted with DAPI-containing solution. Confocal images were captured by a laser scanning microscope (Zeiss LSM780, Carl Zeiss). g CD68 + areas were calculated from ( f ) using ImageJ. Data are from two ( c – g ) and three ( a , b ) independent experiments with 3–5 mice per experiment. Student’s t test was performed in b , c , e , and g and is shown as the means ± SDs (* p < 0.05; ** p < 0.01)

Article Snippet: CD8 microbeads were purchased from Miltenyi Biotec (Auburn, CA).

Techniques: Injection, Staining, Expressing, Laser-Scanning Microscopy

Journal: Cellular and Molecular Immunology

Article Title: Chronic activation of 4-1BB signaling induces granuloma development in tumor-draining lymph nodes that is detrimental to subsequent CD8 + T cell responses

doi: 10.1038/s41423-020-00533-3

Figure Lengend Snippet: 4-1BB triggering in vivo increases PD-1 + CD8 + T cells in the medulla of TDLNs. a – d Rat IgG or anti-4-1BB mAb was injected i.p. to the mice 5 and 10 days after the challenge of MC38 tumor cells. Inguinal TDLN cells were collected from the mice on day 13. PD-1 expression on CD4 + T and CD8 + T cells in inguinal TDLNs and the spleen was analyzed by flow cytometry ( a ). Total live cell numbers and the percentage and absolute number of PD-1 + CD8 + T cells in inguinal TDLNs were calculated ( b ). The phenotype of PD-1 + CD8 + T cells in inguinal TDLNs and tumor tissues ( c ). IFN-γ expression of PD-1 − and PD-1 + CD8 + T cells in TDLNs of anti-4-1BB-treated mice ( d ). e – i C57BL/6 mice were single- or multiple-injected intraperitoneally with rat IgG or anti-4-1BB mAb from 10 days after MC38 tumor challenge ( e ). f Inguinal TDLNs collected and photographed on the indicated days. g TDLN cells were stained with anti-PD-1-PE and anti-CD8β-PE-Cy5 at the indicated days, and percentages of PD-1 + CD8 + T cells were calculated. h Inguinal TDLNs were collected from each group of mice 5 days after a single injection of Ab. Frozen sections of TDLNs were stained with anti-PD-1 or anti-ER-TR7 mAb, followed by fluorescence-conjugated secondary mAb. Images were captured with a laser scanning microscope (Zeiss LSM780, Carl Zeiss). T T cell zone, B B cell zone, IFR interfollicular region, MR medullar region. i ER-TR7 + areas in TDLNs were calculated from ( h ) with the ImageJ program. Data are from three ( a – c , h , i ) or two ( d , f , g ) independent experiments with five mice per experiment. Student’s t test was performed in b , g , and i and is shown as the means ± SDs (* p < 0.05; ** p < 0.01)

Article Snippet: CD8 microbeads were purchased from Miltenyi Biotec (Auburn, CA).

Techniques: In Vivo, Injection, Expressing, Flow Cytometry, Staining, Fluorescence, Laser-Scanning Microscopy

Journal: Cellular and Molecular Immunology

Article Title: Chronic activation of 4-1BB signaling induces granuloma development in tumor-draining lymph nodes that is detrimental to subsequent CD8 + T cell responses

doi: 10.1038/s41423-020-00533-3

Figure Lengend Snippet: Normal trafficking of pmel-1 CD8 + T cells following 4-1BB triggering in vivo. C57BL/6 mice were injected intravenously with CFSE-labeled pmel-1 Thy1.1 + CD8 + T cells ( a , c , d ) or CellTrace Violet-labeled S1PR1-GFP × pmel-1 Thy1.1 + CD8 + T cells ( b ), immunized with hgp100 peptide in IFA, and further stimulated with rat IgG or anti-4-1BB mAb at days 0 and 2. Inguinal LN cells of the mice that received pmel-1 Thy1.1 + CD8 + T cells were stained with the indicated mAbs at day 5, and gated Thy1.1 + cells are plotted CFSE vs. PD-1, LAG3, KLRG-1, CD62L or CCR7 ( a ). Inguinal LN cells of the mice that received S1PR1-GFP × pmel-1 Thy1.1 + CD8 + T cells were stained with anti-Thy1.1-PE on day 5, and gated Thy1.1 + cells were plotted CellTrace Violet vs. S1PR1-GFP ( b ). Percentage and absolute number of pmel-1 Thy1.1 + CD8 + T cells in inguinal LNs of the mice that received pmel-1 Thy1.1 + CD8 + T cells at day 5 ( c ). Division rates of pmel-1 Thy1.1 + CD8 + T cells of rat IgG- or anti-4-1BB-treated mice ( d ). e – g CFSE-labeled naive pmel-1 Thy1.1 + CD8 + T cells were adoptively transferred to B6 mice 7 days after the B16-F10 challenge and simultaneously immunized with hgp100 peptide in IFA. Rat IgG or anti-4-1BB mAb was intraperitoneally injected into the mice on days 7 and 9. The TDLNs and tumor tissues were collected from the mice 4 or 7 days after peptide immunization, counted, and stained with fluorochrome-conjugated anti-Thy1.1, anti-CD8, and anti-CD45 mAbs. Gated CD8 + T cells were plotted as Thy1.1 vs. CFSE ( f ). The percentage and absolute number of Thy1.1 + CD8 + T cells in the TDLNs ( g ). The percentage of transferred Thy1.1 + and endogenous Thy1.1 − CD8 + T cells in the tumor tissues ( h ). Data are from three ( a – d ) and two ( e – h ) independent experiments with 5–6 mice per experiment. Student’s t test was performed in b and shown as the means ± SDs (* p < 0.05; ** p < 0.01; *** p < 0.005)

Article Snippet: CD8 microbeads were purchased from Miltenyi Biotec (Auburn, CA).

Techniques: In Vivo, Injection, Labeling, Staining

Journal: Cellular and Molecular Immunology

Article Title: Chronic activation of 4-1BB signaling induces granuloma development in tumor-draining lymph nodes that is detrimental to subsequent CD8 + T cell responses

doi: 10.1038/s41423-020-00533-3

Figure Lengend Snippet: Proliferation and trafficking of pmel-1 CD8 + T cells and tumor growth in mice pretreated with anti-4-1BB mAb. a – f B16-bearing C57BL/6 mice were treated with rat IgG or anti-4-1BB mAb at days 5 and 10 and received CFSE-labeled naive Thy1.1 + CD8 + T cells at day 14. TLDNs and tumor tissues were analyzed at days 15 and 21. a Schematic diagram of the experiment. b The inguinal TDLNs were collected from the mice 1 or 7 days after CD8 + T cell transfer, stained with fluorochrome-conjugated anti-CD8 and anti-Thy1.1 mAb, and further stained with 7-AAD. Single-cell suspensions of tumor tissues were stained with fluorochrome-conjugated anti-CD45, anti-CD8, and anti-Thy1.1 mAbs. All samples were subsequently analyzed by FACSCalibur (BD Bioscience). c Total cell numbers at day 21. d Percentages of dividing and nondividing pmel-1 Thy1.1 + CD8 + T cells in TDLNs at day 21. e Absolute numbers of dividing and nondividing pmel-1 Thy1.1 + CD8 + T cells in TDLNs at day 21. f Percentages of Thy1.1 − CD8 + TILs in CD45 + cells from tumor tissues at day 21. g – h C57BL/6 mice were immunized with 20 μg OVA in IFA and injected with anti-4-1BB mAb at days 3, 6, and 9. On day 20, the untreated (UnTx) or the OVA + mAb-treated (Ab-Tx) mice were injected subcutaneously with MC38 tumor cells and further received rat IgG or anti-4-1BB mAb every 5 days from day 3 after the tumor challenge. Tumor growth rates were monitored every 3–4 days. Data are from two ( b – f ) or three ( h ) independent experiments with five mice per experiment. Student’s t test was performed in c – f and shown as the means ± SDs (* p < 0.05; ** p < 0.01)

Article Snippet: CD8 microbeads were purchased from Miltenyi Biotec (Auburn, CA).

Techniques: Labeling, Staining, Injection

Journal: Cellular and Molecular Immunology

Article Title: Chronic activation of 4-1BB signaling induces granuloma development in tumor-draining lymph nodes that is detrimental to subsequent CD8 + T cell responses

doi: 10.1038/s41423-020-00533-3

Figure Lengend Snippet: Combined 4-1BB agonist and PD-1 antagonist treatment of MC38 tumor-bearing mice. MC38 tumor-bearing C57BL/6 mice were treated with anti-4-1BB mAb and/or anti-PD-1 mAb every 5 days from day 7 ( n = 5). a Growth rate of MC38 tumors ( n = 5). b The inguinal TDLNs were collected from each group of mice and photographed at day 14 ( n = 3–4). c PD-1 expression on CD4 + T and CD8 + T cells in the inguinal TDLNs and spleens at day 14. d The absolute number of inguinal TDLN cells on day 14. Percentage and absolute number of PD + CD4 + T cells ( e ), or PD + CD8 + T cells ( f ) in the inguinal TDLNs. Frozen sections of the inguinal TDLNs at day 14 were stained with anti-mouse CD68-biotin and detected using streptavidin-Cy3 ( g ), or anti-ER-TR7 (BMA) and anti-rat IgG-Cy3 ( h ). Slides were mounted with DAPI-containing solution. Images were captured by a laser scanning microscope (Zeiss LSM780, Carl Zeiss). i MC38 tumor-bearing C57BL/6 mice were treated with rat IgG or anti-PD-1 mAb every 5 days from day 18 or first treated with 50 μg of anti-4-1BB mAb every 3 days, for a total of three times, from day 10 and further injected with rat IgG or anti-PD-1 mAb every 5 days, three times, from day 23. All mice were routinely monitored for tumor growth. Data are from two ( g – i ) or three ( a – f ) independent experiments with five mice ( a , g , h , i ) or 3–4 mice ( b – f ) per experiment. Student’s t test was performed in d – f and represented as the mean ± SD (* p < 0.05; ** p < 0.01; *** p < 0.005)

Article Snippet: CD8 microbeads were purchased from Miltenyi Biotec (Auburn, CA).

Techniques: Expressing, Staining, Laser-Scanning Microscopy, Injection

Journal:

Article Title: Identification of Novel Principles of Keratin Filament Network Turnover in Living Cells V⃞

doi: 10.1091/mbc.E03-09-0707

Figure Lengend Snippet: Details of KF network formation in living SW13 cells synthesizing HK8-CFP and HK18-YFP fluorescent hybrids. (A) Epifluorescence microscopy (detection of HK18-YFP) of a characteristic peripheral segment (position of plasma membrane indicated at 3.5 min) highlighting intermediate steps of KF network production. Two KF precursors are marked by color and followed until integration into the filament system. Note the formation of small granules below the cell surface that elongate, fuse, and become part of the KF network. The corresponding movie 5 shows further details. (B) Size changes of a single particle that was tracked from its first detection until integration into the network. For clarity's sake only the pixels associated with that particular particle are shown. Sampling frequency, 30 s. (C) The graph is derived from B, showing a surface view of the altering particle dimensions demonstrating a continuous, nonincremental particle elongation (l) before fusion with the KF network over time (t). (D) 3D reconstruction (voltex presentation) of the HK18-YFP fluorescence as recorded by confocal laser scanning microscopy of a peripheral region revealing spatial characteristics of various intermediates of filament assembly in relationship to intracellular topology (position of cell border demarcated). The volume of the top segment has the dimensions 20 × 20 × 3.5 μm. The bottom cube is a cross section to specifically highlight the increase in diameter of KF precursors only after integration into the peripheral KF network. (E) Plot summarizing the quantitative analysis of 10 time-space diagrams (see, e.g., C) depicting the increase in length (top graph) and diameter (bottom graph). Note the continuous elongation of particles during the entire 20 min in contrast to the increase in diameter that is limited to the first 5 min. Bars, SD. Bars, 1 μm in A and D.

Article Snippet: Cell Lines and cDNA Transfection The following cell lines were used and cultured according to the specifications given by ATCC or the respective references: rat kangaroo kidney PtK2 cells (ATCC CCL-56), Madin-Darby canine kidney (MDCK) cells (clone 20; ATCC CCL-34), bovine kidney-derived MDBK cells (ATCC CCL-22), human mammary adenocarcinoma-derived MCF7 cells (ATCC HTB-22), human hepatocellular carcinoma-derived PLC cells (ATCC CRL8024), human colon carcinoma CaCo-2 cells (ATCC HTB-37), human immortalized HaCaT keratinocytes ( Boukamp et al., 1988 ), and human small-cell carcinoma SW13 cells derived from the adrenal cortex (ATCC CCL-105).

Techniques: Epifluorescence Microscopy, Clinical Proteomics, Membrane, Single Particle, Sampling, Derivative Assay, Fluorescence, Confocal Laser Scanning Microscopy

Journal: Autophagy

Article Title: Microglial phagocytosis dysfunction in stroke is driven by energy depletion and induction of autophagy

doi: 10.1080/15548627.2023.2165313

Figure Lengend Snippet: Autophagy flux is induced in microglia after tMCAo and OND. ( A ) Representative confocal images of the DG of 2 mo fms -EGFP mice exposed to sham and tMCAo surgery at 6 h and 1 d. LC3 present in autophagosomes was immunostained and observed as puncta (yellow). Microglia were visualized by the transgenic expression of fms -EGFP (cyan) and cellular nuclei were identified by DAPI (white). SQSTM1 ( Ai ) and LAMP1 ( Aii ) were immunostained and visualized as puncta (magenta). ( B ) Number of LC3 puncta normalized to microglial cytoplasmic area (LC3 puncta number/µm 2 ) in sham and tMCAo (6 h and 1 d) mice. ( C ) Total area of LC3 puncta normalized to microglial cytoplasmic area (LC3 puncta area/µm 2 ) in sham and tMCAo (6 h and 1 d) mice. ( D ) Number of LC3 and SQSTM1 puncta that colocalize normalized to microglial cytoplasmic area (LC3-SQSTM1 puncta number colocalization/µm 2 ) in sham and tMCAo (6 h and 1 d) mice. ( E ) Number of LC3 and LAMP1 puncta that colocalize normalized to microglial cytoplasmic area (LC3-LAMP1 puncta number colocalization/µm 2 ) in sham and tMCAo (6 h and 1 d) mice. ( F ) Number of SQSTM1 puncta normalized to microglial cytoplasmic area (SQSTM1 puncta number/µm 2 ) in sham and tMCAo (6 h and 1 d) mice. ( G ) Number of LAMP1 puncta normalized to microglial cytoplasmic area (LAMP1 puncta number/µm 2 ) in sham and tMCAo (6 h and 1 d) mice. ( H ) Quantified total SQSTM1 puncta area normalized by microglial cytoplasmic area (SQSTM1 puncta area/µm 2 ) ( Hi ), the colocalized area of LC3 and SQSTM1 puncta normalized by microglial cytoplasmic area (colocalized LC3-SQSTM1 puncta area/µm 2 ) ( Hii ), total LAMP1 puncta area normalized by microglial cytoplasmic area (LAMP1 puncta area/µm 2 ) ( Hiii ), and the colocalized area of LC3 and LAMP1 puncta normalized by microglial cytoplasmic area (colocalized LC3-LAMP1 puncta area/µm 2 ) ( Hiv ). ( I ) Experimental design used to transfect BV2 microglia-like cells with the fluorescent tandem mRFP-GFP-LC3 to assess autophagy flux in control conditions (C) and after OND (3 h) or rapamycin (Rapa, 100 nM, 6 h) treatments. Representative confocal images of control, OND and rapamycin treated microglia. Nuclei are stained with DAPI (white), autophagosomes and autolysosomes are differentiated according to the tandem expression (yellow and red, respectively). ( J ) GFP:RFP mean fluorescence intensity ratio, indicative of autophagy flux. ( K ) Mean RFP fluorescence intensity ( Ki ), mean GFP fluorescence intensity ( Kii ), total number of puncta ( Kiii ), and area occupied by puncta ( Kiv ) per cell and normalized to control conditions (expressed as % change versus control conditions). Bars show mean ± SEM ( B-H ). Violin plots show the data distribution, including extreme values; lower and upper hinges correspond to the first and third quartile, respectively ( J, K ). n = 4 mice per experimental condition ( B-H ); n = 12–17 cells from 3 independent experiments ( J, K ). Data were analyzed by one-way ANOVA followed by Holm-Sidak post hoc test ( B-H ), Bonferroni post hoc test ( J ); by Kruskal-Wallis one- way ANOVA on ranks followed by Dunn’s multiple comparisons ( K ). * represents significance between sham or control and tMCAo, OND or rapamycin: one symbol represents p < 0.05, two symbols represent p < 0.01 and three symbols represent p < 0.001. Scale bars: 20 µm, z = 20 µm ( A ); 10 µm, z = 1.9 µm (control), 3.3 µm (OND), and 3.9 µm (rapamycin) ( I ).

Article Snippet: SQSTM1 (rabbit) , 1:200 , Novus Biologicals , NBP1-48,320.

Techniques: Transgenic Assay, Expressing, Control, Staining, Fluorescence

Journal: Autophagy

Article Title: Microglial phagocytosis dysfunction in stroke is driven by energy depletion and induction of autophagy

doi: 10.1080/15548627.2023.2165313

Figure Lengend Snippet: List of reagents and/or resources used in this article.

Article Snippet: SQSTM1 (rabbit) , 1:200 , Novus Biologicals , NBP1-48,320.

Techniques: Western Blot, Activity Assay, Software, Laser-Scanning Microscopy, Microscopy, Irradiation

Journal: International Journal of Molecular Sciences

Article Title: Bionanofactories for Green Synthesis of Silver Nanoparticles: Toward Antimicrobial Applications

doi: 10.3390/ijms222111993

Figure Lengend Snippet: Use of bionanofactories in the synthesis of silver nanoparticles, along with a brief outline of their physicochemical and antimicrobial properties.

Article Snippet: Other biosynthesizing Agents , Gum kondagogu ( Cochlospermum gossypium ) , 416 nm , 3443, 2916, 2850, 1727, 1630, 1597, 1384, 1351, 1254, 1148, 1043 , TEM ▪ Shape—anisotropic nanostructures such as nanotriangles, a few nanorods, hexagonal and polygonal nanoprisms, and abundant unevenly shaped nanoparticles were observed ▪ Nature—polydisperse ▪ For 30 min of reaction time, size of 55.0 nm; for 60 min of reaction time, size of 18.9 nm SAED ▪ Nature—crystalline ▪ Structure—face-centered cubic (FCC) TEM ▪ Shape—spherical ▪ Average particle size: for 30 min of reaction time, 11.2 nm; for 60 min of reaction time, 4.5nm XRD ▪ Nature—crystalline ▪ Structure—face-centered cubic , ▪ Antibacterial activity was observed against S. aureus ATCC 25923, E. coli ATCC 25922, E. coli ATCC 35218, and P. aeruginosa ATCC 27853 , [ ] .

Techniques: In Vitro, Activity Assay, Algae, Bioassay, Concentration Assay, Inhibition, Synthesized, Particle Size Analysis, Bacteria, Kinetic Assay, Purification, Plasmid Preparation, Diffusion-based Assay, Confocal Laser Scanning Microscopy

Journal: European journal of immunology

Article Title: Rapid translocation of intracellular toll-like receptors depends on endosomal NADPH oxidase.

doi: 10.1002/eji.202250271

Figure Lengend Snippet: Figure 1. Role of endosomal NADPH-oxidase 2 (NOX2) and UNC93b1 in endosomal TLR signaling. CD115+ splenocytes were isolated from indicated mouse strains and stimulated with TLR3 (A + D), TLR7/8 (B + E), and TLR9 ligands (C + F) for up to 24 h. (A–C) Expression of TNF-α mRNA by TLR ligand-stimulated CD115+ splenocytes relative to unstimulated (A + B) or control ODN-stimulated cells (C). TNF-α secretion was measured by ELISA in the cell culture supernatants at indicated time points (D and E). n = 6 biological replicates, *p < 0.005; two-way ANOVA, Sidak’s multiple comparisons test. Gp91phox−/−versus WT. (G + H) Representative confocal microscopy images (single confocal plane) showing TLR9 translocation: Monocytes isolated from the spleen of WT C57BL/6J mice (G) or gp91phox−/−mice (H) were stimulated with CpG for 30 min. After fixation, cells were stained with anti-(α)-TLR9 antibodies (green), anti-(α)-calnexin, or anti-(α)-EEA1 (red) and 4,6-diamidino-2-phenylindole (DAPI) (blue) and visualized by confocal laser scanning microscopy. In WT monocytes, stimulation with CpG leads to the colocalization of TLR9 and the endosomal marker EEA1, whereas in monocytes from gp91phox−/−mice, no translocation is observed. Bar = 100 μm. Quantification of colocalization; n = 3 regions of interest (ROIs) consisting of at least three cells, generated by three independent experiments. *p < 0.0001; two-tailed t-test.

Article Snippet: Material and methods Reagents Reagents were purchased as follows: TLR3 ligand Poly I:C, TLR7/8 ligand R848, and TLR9 ligand CpG C (ODN 2395 and respective ODN control) (all Invivogen, San Diego, CA, USA); NFA (Sigma-Aldrich, St. Louis, MO, USA); mouse TNF-α ELISA kit (R&D Systems, Wiesbaden, Germany); rabbit anti-mouse TLR9 (Acris/Origene, Rockville, MD, USA); rabbit antihuman TLR3 and rabbit antihuman TLR9 (both ThermoFisher, Dreieich, Germany); biotin-labeled monoclonal antihuman TLR8 (Origene, Rockville, MD, USA), anti-Calnexin-Biotin (Novus Biologicals, Centennial, CO, USA); goat anti-EEA1 (Abcam, Cambridge, UK); Streptavidin DyLight 549 and DyLight 488 (both Biozol, Eching, Germany); donkey anti-Goat IgG DyLight 550 and anti-rabbit Alexa Fluor 488 (both ThermoFisher, Dreieich, Germany); H2DCFDA ROS indicator (ThermoFisher, Dreieich, Germany); and CD115 MicroBead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Isolation, Expressing, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Confocal Microscopy, Translocation Assay, Staining, Confocal Laser Scanning Microscopy, Marker, Generated, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Bionanofactories for Green Synthesis of Silver Nanoparticles: Toward Antimicrobial Applications

doi: 10.3390/ijms222111993

Figure Lengend Snippet: Use of bionanofactories in the synthesis of silver nanoparticles, along with a brief outline of their physicochemical and antimicrobial properties.

Article Snippet: Other biosynthesizing Agents , Gum kondagogu ( Cochlospermum gossypium ) , 416 nm , 3443, 2916, 2850, 1727, 1630, 1597, 1384, 1351, 1254, 1148, 1043 , TEM ▪ Shape—anisotropic nanostructures such as nanotriangles, a few nanorods, hexagonal and polygonal nanoprisms, and abundant unevenly shaped nanoparticles were observed ▪ Nature—polydisperse ▪ For 30 min of reaction time, size of 55.0 nm; for 60 min of reaction time, size of 18.9 nm SAED ▪ Nature—crystalline ▪ Structure—face-centered cubic (FCC) TEM ▪ Shape—spherical ▪ Average particle size: for 30 min of reaction time, 11.2 nm; for 60 min of reaction time, 4.5nm XRD ▪ Nature—crystalline ▪ Structure—face-centered cubic , ▪ Antibacterial activity was observed against S. aureus ATCC 25923, E. coli ATCC 25922, E. coli ATCC 35218, and P. aeruginosa ATCC 27853 , [ ] .

Techniques: In Vitro, Activity Assay, Algae, Bioassay, Concentration Assay, Inhibition, Synthesized, Particle Size Analysis, Bacteria, Kinetic Assay, Purification, Plasmid Preparation, Diffusion-based Assay, Confocal Laser Scanning Microscopy

Journal: Oncotarget

Article Title: Endoplasmic reticulum-Golgi intermediate compartment protein 3 knockdown suppresses lung cancer through endoplasmic reticulum stress-induced autophagy.

doi: 10.18632/oncotarget.11678

Figure Lengend Snippet: Figure 1: ERGIC3 expression in tumor tissues and effect of shERGIC3 downregulation. (A) Densitometric analysis of ERGIC3 protein on Western blot in human normal and adenocarcinoma lung tissues. Each bar indicates mean ± standard error of mean (SEM; n = 5). N, normal lung tissues; GI, grade I adenocarcinoma tissues; GII, grade II adenocarcinoma tissues; GIII, grade III adenocarcinoma tissues (*P < 0.05, compared to normal lung tissues). (B) Quantitative PCR (qPCR) analysis of ERGIC3. ERGIC3 downregulated stable cell line (shERGIC3) showed significant differences compared to control (***P < 0.001) or shScr (###P < 0.001) (n = 4). (C) Real-time cell proliferation analysis using xCELLigence RTCA DP system; 1 × 103 cells from control A549, shScr and shERGIC3 stable cell lines were seeded in 16-well E-plates, and incubated for 72 h (*P < 0.05, compared to control and ##P < 0.01, compared to shScr, n = 3). (D) Western blot and densitometric analyses of PCNA in A549 control, shScr and shERGIC3 stable cells (n = 4). (E) Western blot and densitometric analyses of p-Akt1 (Thr308 and Ser473). Significant differences are indicated by *P < 0.05, **P < 0.01 and ***P < 0.001 (*: compared to control, #: compared to shScr, n = 3).

Article Snippet: ERGIC3 shRNAs were purchased from OriGene.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Stable Transfection, Control, Incubation

Journal: Oncotarget

Article Title: Endoplasmic reticulum-Golgi intermediate compartment protein 3 knockdown suppresses lung cancer through endoplasmic reticulum stress-induced autophagy.

doi: 10.18632/oncotarget.11678

Figure Lengend Snippet: Figure 2: Suppression of ERGIC3 induces ER stress-induced autophagy in A549 cells. (A) Western blot analyses of ERGIC3, CHOP, Calnexin, IRE1α, p62/SQSTM1 and LC3 in A549 control, shScr and shERGIC3 groups. Cells were cultured for 2 days and then collected for Western blot analysis. Significant differences are indicated by *P < 0.05, **P < 0.01 and ***P < 0.001 (*: compared to control, #: compared to shScr, n = 4). (B) Subcellular morphological changes in shERGIC3 stable cells were revealed by transmission electron microscopy (TEM) analysis. Cells were cultured for 2 days then were fixed and observed using TEM. In each group of figures, calibration bars in the left figure of each group = 2 μm, while those in the right figure of each group = 0.5 μm. Arrow indicates distorted nucleus. N, nucleus; G, Golgi apparatus; ER, endoplasmic reticulum; M, mitochondria; A, autophagosome; AL, Autolysosomes; shScr, small hairpin scramble; shERGIC3, small hairpin ERGIC3.

Article Snippet: ERGIC3 shRNAs were purchased from OriGene.

Techniques: Western Blot, Control, Cell Culture, Transmission Assay, Electron Microscopy

Journal: Oncotarget

Article Title: Endoplasmic reticulum-Golgi intermediate compartment protein 3 knockdown suppresses lung cancer through endoplasmic reticulum stress-induced autophagy.

doi: 10.18632/oncotarget.11678

Figure Lengend Snippet: Figure 3: Subcellular localization of ERGIC3 and relation to ER stress. (A) Confocal laser scanning microscope (CLSM) data analysis of ERGIC3 (Green) and an ER marker (Red). After transfection of the ER marker, tunicamycin (1 μg/mL) was applied for 24 h to A549 cells. After fixation, incubation with an anti-ERGIC3 antibody was performed for 24 h. Calibration bars = 10 μm. Green, ERGIC3; Red, ER marker; Blue, nucleus. (B) ERGIC3-tGFP was transfected into A549 cells for 24 h, followed by incubation with anti-GM130 antibody for 24 h, and nuclei staining (DAPI). Calibration bars = 10 μm. Green, ERGIC3; Red, GM130; Blue: nucleus (C) Western blot analysis of ERGIC3 and Calnexin expression depending on tunicamycin concentration (0.1 to 5 μg/mL) for 24 h. (D) Pre-treatment with 5 mg/mL tauroursodeoxycholic acid (TUDCA) was performed for 2 h, followed by 5 μg/mL tunicamycin for 24 h. Western blot assays of ERGIC3, Calnexin and GAPDH were performed after sampling cell lysates. Con, control; Tuni only, tunicamycin treated; TUDCA, TUDCA pretreated and tunicamycin treated).

Article Snippet: ERGIC3 shRNAs were purchased from OriGene.

Techniques: Laser-Scanning Microscopy, Marker, Transfection, Incubation, Staining, Western Blot, Expressing, Concentration Assay, Sampling, Control

Journal: Oncotarget

Article Title: Endoplasmic reticulum-Golgi intermediate compartment protein 3 knockdown suppresses lung cancer through endoplasmic reticulum stress-induced autophagy.

doi: 10.18632/oncotarget.11678

Figure Lengend Snippet: Figure 5: Aerosol delivery of shERGIC3 triggers ER stress-induced autophagy in K-rasLA1 mice. (A) Quantitative PCR (qPCR) analysis of ERGIC3 in lung tissue. Aerosol delivery of shERGIC3 significantly decreased ERGIC3 expression in the lungs compared to control (***P < 0.001) and shScr (###P < 0.001) (n = 4). (B) Western blot and densitometric analyses of ERGIC3, CHOP, IRE1α, LC3 and p62/SQSTM1 in the lungs of control, shScr and shERGIC3-treated mice. Significant differences are indicated by *P < 0.05, **P < 0.01 and ***P < 0.001 (*: compared to control, #: compared to shScr, n = 4).

Article Snippet: ERGIC3 shRNAs were purchased from OriGene.

Techniques: Aerosol, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot