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Carl Zeiss laser scanning confocal microscope
<t>Confocal</t> <t>laser</t> <t>scanning</t> microscopy images of DiD-loaded vc378 distribution in the microneedle. Representative confocal images of microneedles coated with DiD-loaded vc378 (left, DiD-loaded vc378; middle, DIC; right, merge). The scale bars are 200 μm.
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1) Product Images from "Patchless administration of canine influenza vaccine on dog’s ear using insertion-responsive microneedles (IRMN) without removal of hair and its in vivo efficacy evaluation"

Article Title: Patchless administration of canine influenza vaccine on dog’s ear using insertion-responsive microneedles (IRMN) without removal of hair and its in vivo efficacy evaluation

Journal: European Journal of Pharmaceutics and Biopharmaceutics

doi: 10.1016/j.ejpb.2020.06.006

Confocal laser scanning microscopy images of DiD-loaded vc378 distribution in the microneedle. Representative confocal images of microneedles coated with DiD-loaded vc378 (left, DiD-loaded vc378; middle, DIC; right, merge). The scale bars are 200 μm.
Figure Legend Snippet: Confocal laser scanning microscopy images of DiD-loaded vc378 distribution in the microneedle. Representative confocal images of microneedles coated with DiD-loaded vc378 (left, DiD-loaded vc378; middle, DIC; right, merge). The scale bars are 200 μm.

Techniques Used: Confocal Laser Scanning Microscopy

2) Product Images from "OSMR controls glioma stem cell respiration and confers resistance of glioblastoma to ionizing radiation"

Article Title: OSMR controls glioma stem cell respiration and confers resistance of glioblastoma to ionizing radiation

Journal: Nature Communications

doi: 10.1038/s41467-020-17885-z

Presence of mitochondrial OSMR in human BTSCs. a – d Four different patient-derived BTSC lines were subjected to subcellular fractionation, and the lysates for each fraction were analyzed by immunoblotting using antibodies to OSMR. α-Tubulin, H3K4me3, BCL2/TOM20, Na+/K+ ATPase, and calnexin. WCL: Whole-cell lysates; Cyto: cytoplasmic; Mito: mitochondrial. The Western blots represent a minimum of three replicates from different passage numbers for each BTSC. e , f BTSC73 and BTSC147 were subjected to immunostaining using antibodies to OSMR (green) and the mitochondrial matrix protein ATP synthase inhibitor F1 (ATPIF1, red). Nuclei were stained with DAPI. White rectangles mark the inset to demonstrate the co-localization of OSMR with ATPIF1. g , h PLA of OSMR and ATPIF1 were performed in BTSC73 ( g ) and BTSC147 ( h ). Primary antibodies were omitted as controls. i Double labeling of the PLA signal (red) and the MitoTracker (green) in BTSC73 is shown. j A FRAP assay was performed on BTSC73 transduced with GFP-OSMR and stained with MitoTracker (red). Different regions of interest (ROIs) containing GFP-OSMR in the mitochondria were defined. ROI1 indicates a non-bleached area and ROI2, a photobleached area. The fluorescence recovery was monitored over time following photobleaching. Images were obtained on a laser scanning confocal microscope (ZEISS LSM 800). Scale bar = 10 μm; Inset scale bar = 1 μm. Representative images of three independent experiments are shown.
Figure Legend Snippet: Presence of mitochondrial OSMR in human BTSCs. a – d Four different patient-derived BTSC lines were subjected to subcellular fractionation, and the lysates for each fraction were analyzed by immunoblotting using antibodies to OSMR. α-Tubulin, H3K4me3, BCL2/TOM20, Na+/K+ ATPase, and calnexin. WCL: Whole-cell lysates; Cyto: cytoplasmic; Mito: mitochondrial. The Western blots represent a minimum of three replicates from different passage numbers for each BTSC. e , f BTSC73 and BTSC147 were subjected to immunostaining using antibodies to OSMR (green) and the mitochondrial matrix protein ATP synthase inhibitor F1 (ATPIF1, red). Nuclei were stained with DAPI. White rectangles mark the inset to demonstrate the co-localization of OSMR with ATPIF1. g , h PLA of OSMR and ATPIF1 were performed in BTSC73 ( g ) and BTSC147 ( h ). Primary antibodies were omitted as controls. i Double labeling of the PLA signal (red) and the MitoTracker (green) in BTSC73 is shown. j A FRAP assay was performed on BTSC73 transduced with GFP-OSMR and stained with MitoTracker (red). Different regions of interest (ROIs) containing GFP-OSMR in the mitochondria were defined. ROI1 indicates a non-bleached area and ROI2, a photobleached area. The fluorescence recovery was monitored over time following photobleaching. Images were obtained on a laser scanning confocal microscope (ZEISS LSM 800). Scale bar = 10 μm; Inset scale bar = 1 μm. Representative images of three independent experiments are shown.

Techniques Used: Derivative Assay, Fractionation, Western Blot, Immunostaining, Staining, Proximity Ligation Assay, Labeling, FRAP Assay, Transduction, Fluorescence, Microscopy

OSMR interacts with different components of ETC in human BTSCs. a , b Mitochondrial fractions from BTSC73 ( a ) and BTSC147 ( b ) were treated with 0.5 mg/mL proteinase K or proteinase K and 1% Triton X-100. Lysates were analyzed by immunoblotting using indicated antibodies. c – f WCL and mitochondrial fractions from BTSC73 ( c , d ) and BTSC147 ( e , f ) were subjected to immunoprecipitation using antibodies to OSMR or mouse IgG control, followed by immunoblotting with mtHSP70 and TIM44 antibodies. g , h PLA of OSMR and mtHSP70 were performed in BTSC73 ( g ) and BTSC147 ( h ). Primary antibodies were omitted for the controls. i Double labeling of the PLA signal (red) from the OSMR/mtHSP70 interaction and MitoTracker (green) is shown. j OSMR protein expression level was assessed in the mitochondrial fractions obtained from BTSC73 electroporated with siRNA control (siCTL) or siRNA against mtHSP70 (si mtHSP70 ). BLC2 was used as a loading control. k OSMR protein expression level was assessed in the mitochondrial fractions obtained from BTSC73 electroporated with siCTL or siRNA against TIM44 (si TIM44 ). BCL2 was used as a loading control. l – o WCL or mitochondrial fractions from BTSC73 ( l , m ) and BTSC147 ( n , o ) were subjected to immunoprecipitation using an antibody to OSMR or mouse IgG control followed by immunoblotting with NDUFS1 and NDUFS2 antibodies. p , q PLA analyses of OSMR/NDUFS1 and OSMR/NDUFS2 were carried out in BTSC73 ( p ) and BTSC147 ( q ). r , s Double labeling of the PLA signal (red) and the MitoTracker (green) is shown. Images were obtained with a 63X objectives on a laser scanning confocal microscope (ZEISS LSM 800). Scale bar = 10 μm. Inset scale bar = 1 μm. Representative images of three independent experiments are shown. The Western blots represent a minimum of three replicates from different passage numbers for each BTSC.
Figure Legend Snippet: OSMR interacts with different components of ETC in human BTSCs. a , b Mitochondrial fractions from BTSC73 ( a ) and BTSC147 ( b ) were treated with 0.5 mg/mL proteinase K or proteinase K and 1% Triton X-100. Lysates were analyzed by immunoblotting using indicated antibodies. c – f WCL and mitochondrial fractions from BTSC73 ( c , d ) and BTSC147 ( e , f ) were subjected to immunoprecipitation using antibodies to OSMR or mouse IgG control, followed by immunoblotting with mtHSP70 and TIM44 antibodies. g , h PLA of OSMR and mtHSP70 were performed in BTSC73 ( g ) and BTSC147 ( h ). Primary antibodies were omitted for the controls. i Double labeling of the PLA signal (red) from the OSMR/mtHSP70 interaction and MitoTracker (green) is shown. j OSMR protein expression level was assessed in the mitochondrial fractions obtained from BTSC73 electroporated with siRNA control (siCTL) or siRNA against mtHSP70 (si mtHSP70 ). BLC2 was used as a loading control. k OSMR protein expression level was assessed in the mitochondrial fractions obtained from BTSC73 electroporated with siCTL or siRNA against TIM44 (si TIM44 ). BCL2 was used as a loading control. l – o WCL or mitochondrial fractions from BTSC73 ( l , m ) and BTSC147 ( n , o ) were subjected to immunoprecipitation using an antibody to OSMR or mouse IgG control followed by immunoblotting with NDUFS1 and NDUFS2 antibodies. p , q PLA analyses of OSMR/NDUFS1 and OSMR/NDUFS2 were carried out in BTSC73 ( p ) and BTSC147 ( q ). r , s Double labeling of the PLA signal (red) and the MitoTracker (green) is shown. Images were obtained with a 63X objectives on a laser scanning confocal microscope (ZEISS LSM 800). Scale bar = 10 μm. Inset scale bar = 1 μm. Representative images of three independent experiments are shown. The Western blots represent a minimum of three replicates from different passage numbers for each BTSC.

Techniques Used: Immunoprecipitation, Proximity Ligation Assay, Labeling, Expressing, Microscopy, Western Blot

3) Product Images from "A Fish Leukocyte Immune-Type Receptor Uses a Novel Intracytoplasmic Tail Networking Mechanism to Cross-Inhibit the Phagocytic Response"

Article Title: A Fish Leukocyte Immune-Type Receptor Uses a Novel Intracytoplasmic Tail Networking Mechanism to Cross-Inhibit the Phagocytic Response

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms21145146

Confocal microscopic analysis of phosphotyrosine staining levels within phagocytic cups. 2.6b ITAM CYT /1.1b WT CYT co-expressing AD293 cells ( A ) were grown on coverslips and incubated with 4.5 µm non-fluorescent (NF) beads opsonized with α-HA mAb and mouse isotype IgG1. After 8 min of incubation at 37 °C, cells were fixed with 4% PFA for 10 min and non-phagocytosed beads were stained using Alexa 647-conjugated goat-α-mouse secondary pAb (red). Cells were then permeabilized and specifically stained for intracellular phosphotyrosine molecules by first incubating them with a rabbit α-phosphotyrosine mAb and then with a secondary goat-α-rabbit pAb conjugated to Alexa 488 (green). Z-stack images were obtained at a magnification of 63X using a Zeiss LSM 710 scanning confocal microscope. Representative images from z-stack acquisitions are shown as images with phosphotyrosine (green) staining, surface-exposed bead (red) staining, and merged-fluorescence images or brightfield-fluorescence merged images. Yellow arrowheads show the positions of representative phagocytic cups and the target bead of interest is indicated by an asterisk (*). ( B ) Qualitative analysis of bead and phosphotyrosine molecule staining intensities was performed using ImageJ software (NIH, Bathesda, Maryland, DC, USA) by calculating the MFI (y-axis) of the bead (red line) and phosphotyrosine molecule staining (green line) across the dash arrowed line. ( C ) To further quantify the phosphotyrosine molecules recruited to phagocytic cups under different activation conditions, 2.6b ITAM CYT /1.1b WT CYT co-expressing cells were incubated with NF beads opsonized with indicated mAbs (’+’ and ‘−’ indicate the presence and absence of corresponding mAbs, respectively). For analyses, a region of interest (ROI, indicated by dashed circles) was first drawn that includes phagocytic cups so phosphotyrosine signal intensities in this area could be calculated. As summarized in ( D ), at least 50 phagocytic cups from three independent experiments were pooled and the data is represented as mean integrated fluorescent intensity ± SEM. Differing letters indicate statistical significance ( p ≤ 0.05) between means. Experimental groups were compared using a one-way ANOVA, followed by the Tukey test using Prism 6 software (GraphPad Software, La Jolla, CA, USA).
Figure Legend Snippet: Confocal microscopic analysis of phosphotyrosine staining levels within phagocytic cups. 2.6b ITAM CYT /1.1b WT CYT co-expressing AD293 cells ( A ) were grown on coverslips and incubated with 4.5 µm non-fluorescent (NF) beads opsonized with α-HA mAb and mouse isotype IgG1. After 8 min of incubation at 37 °C, cells were fixed with 4% PFA for 10 min and non-phagocytosed beads were stained using Alexa 647-conjugated goat-α-mouse secondary pAb (red). Cells were then permeabilized and specifically stained for intracellular phosphotyrosine molecules by first incubating them with a rabbit α-phosphotyrosine mAb and then with a secondary goat-α-rabbit pAb conjugated to Alexa 488 (green). Z-stack images were obtained at a magnification of 63X using a Zeiss LSM 710 scanning confocal microscope. Representative images from z-stack acquisitions are shown as images with phosphotyrosine (green) staining, surface-exposed bead (red) staining, and merged-fluorescence images or brightfield-fluorescence merged images. Yellow arrowheads show the positions of representative phagocytic cups and the target bead of interest is indicated by an asterisk (*). ( B ) Qualitative analysis of bead and phosphotyrosine molecule staining intensities was performed using ImageJ software (NIH, Bathesda, Maryland, DC, USA) by calculating the MFI (y-axis) of the bead (red line) and phosphotyrosine molecule staining (green line) across the dash arrowed line. ( C ) To further quantify the phosphotyrosine molecules recruited to phagocytic cups under different activation conditions, 2.6b ITAM CYT /1.1b WT CYT co-expressing cells were incubated with NF beads opsonized with indicated mAbs (’+’ and ‘−’ indicate the presence and absence of corresponding mAbs, respectively). For analyses, a region of interest (ROI, indicated by dashed circles) was first drawn that includes phagocytic cups so phosphotyrosine signal intensities in this area could be calculated. As summarized in ( D ), at least 50 phagocytic cups from three independent experiments were pooled and the data is represented as mean integrated fluorescent intensity ± SEM. Differing letters indicate statistical significance ( p ≤ 0.05) between means. Experimental groups were compared using a one-way ANOVA, followed by the Tukey test using Prism 6 software (GraphPad Software, La Jolla, CA, USA).

Techniques Used: Staining, Expressing, Incubation, Microscopy, Fluorescence, Software, Activation Assay

4) Product Images from "OSMR controls glioma stem cell respiration and confers resistance of glioblastoma to ionizing radiation"

Article Title: OSMR controls glioma stem cell respiration and confers resistance of glioblastoma to ionizing radiation

Journal: Nature Communications

doi: 10.1038/s41467-020-17885-z

Presence of mitochondrial OSMR in human BTSCs. a – d Four different patient-derived BTSC lines were subjected to subcellular fractionation, and the lysates for each fraction were analyzed by immunoblotting using antibodies to OSMR. α-Tubulin, H3K4me3, BCL2/TOM20, Na+/K+ ATPase, and calnexin. WCL: Whole-cell lysates; Cyto: cytoplasmic; Mito: mitochondrial. The Western blots represent a minimum of three replicates from different passage numbers for each BTSC. e , f BTSC73 and BTSC147 were subjected to immunostaining using antibodies to OSMR (green) and the mitochondrial matrix protein ATP synthase inhibitor F1 (ATPIF1, red). Nuclei were stained with DAPI. White rectangles mark the inset to demonstrate the co-localization of OSMR with ATPIF1. g , h PLA of OSMR and ATPIF1 were performed in BTSC73 ( g ) and BTSC147 ( h ). Primary antibodies were omitted as controls. i Double labeling of the PLA signal (red) and the MitoTracker (green) in BTSC73 is shown. j A FRAP assay was performed on BTSC73 transduced with GFP-OSMR and stained with MitoTracker (red). Different regions of interest (ROIs) containing GFP-OSMR in the mitochondria were defined. ROI1 indicates a non-bleached area and ROI2, a photobleached area. The fluorescence recovery was monitored over time following photobleaching. Images were obtained on a laser scanning confocal microscope (ZEISS LSM 800). Scale bar = 10 μm; Inset scale bar = 1 μm. Representative images of three independent experiments are shown.
Figure Legend Snippet: Presence of mitochondrial OSMR in human BTSCs. a – d Four different patient-derived BTSC lines were subjected to subcellular fractionation, and the lysates for each fraction were analyzed by immunoblotting using antibodies to OSMR. α-Tubulin, H3K4me3, BCL2/TOM20, Na+/K+ ATPase, and calnexin. WCL: Whole-cell lysates; Cyto: cytoplasmic; Mito: mitochondrial. The Western blots represent a minimum of three replicates from different passage numbers for each BTSC. e , f BTSC73 and BTSC147 were subjected to immunostaining using antibodies to OSMR (green) and the mitochondrial matrix protein ATP synthase inhibitor F1 (ATPIF1, red). Nuclei were stained with DAPI. White rectangles mark the inset to demonstrate the co-localization of OSMR with ATPIF1. g , h PLA of OSMR and ATPIF1 were performed in BTSC73 ( g ) and BTSC147 ( h ). Primary antibodies were omitted as controls. i Double labeling of the PLA signal (red) and the MitoTracker (green) in BTSC73 is shown. j A FRAP assay was performed on BTSC73 transduced with GFP-OSMR and stained with MitoTracker (red). Different regions of interest (ROIs) containing GFP-OSMR in the mitochondria were defined. ROI1 indicates a non-bleached area and ROI2, a photobleached area. The fluorescence recovery was monitored over time following photobleaching. Images were obtained on a laser scanning confocal microscope (ZEISS LSM 800). Scale bar = 10 μm; Inset scale bar = 1 μm. Representative images of three independent experiments are shown.

Techniques Used: Derivative Assay, Fractionation, Western Blot, Immunostaining, Staining, Proximity Ligation Assay, Labeling, FRAP Assay, Transduction, Fluorescence, Microscopy

OSMR interacts with different components of ETC in human BTSCs. a , b Mitochondrial fractions from BTSC73 ( a ) and BTSC147 ( b ) were treated with 0.5 mg/mL proteinase K or proteinase K and 1% Triton X-100. Lysates were analyzed by immunoblotting using indicated antibodies. c – f WCL and mitochondrial fractions from BTSC73 ( c , d ) and BTSC147 ( e , f ) were subjected to immunoprecipitation using antibodies to OSMR or mouse IgG control, followed by immunoblotting with mtHSP70 and TIM44 antibodies. g , h PLA of OSMR and mtHSP70 were performed in BTSC73 ( g ) and BTSC147 ( h ). Primary antibodies were omitted for the controls. i Double labeling of the PLA signal (red) from the OSMR/mtHSP70 interaction and MitoTracker (green) is shown. j OSMR protein expression level was assessed in the mitochondrial fractions obtained from BTSC73 electroporated with siRNA control (siCTL) or siRNA against mtHSP70 (si mtHSP70 ). BLC2 was used as a loading control. k OSMR protein expression level was assessed in the mitochondrial fractions obtained from BTSC73 electroporated with siCTL or siRNA against TIM44 (si TIM44 ). BCL2 was used as a loading control. l – o WCL or mitochondrial fractions from BTSC73 ( l , m ) and BTSC147 ( n , o ) were subjected to immunoprecipitation using an antibody to OSMR or mouse IgG control followed by immunoblotting with NDUFS1 and NDUFS2 antibodies. p , q PLA analyses of OSMR/NDUFS1 and OSMR/NDUFS2 were carried out in BTSC73 ( p ) and BTSC147 ( q ). r , s Double labeling of the PLA signal (red) and the MitoTracker (green) is shown. Images were obtained with a 63X objectives on a laser scanning confocal microscope (ZEISS LSM 800). Scale bar = 10 μm. Inset scale bar = 1 μm. Representative images of three independent experiments are shown. The Western blots represent a minimum of three replicates from different passage numbers for each BTSC.
Figure Legend Snippet: OSMR interacts with different components of ETC in human BTSCs. a , b Mitochondrial fractions from BTSC73 ( a ) and BTSC147 ( b ) were treated with 0.5 mg/mL proteinase K or proteinase K and 1% Triton X-100. Lysates were analyzed by immunoblotting using indicated antibodies. c – f WCL and mitochondrial fractions from BTSC73 ( c , d ) and BTSC147 ( e , f ) were subjected to immunoprecipitation using antibodies to OSMR or mouse IgG control, followed by immunoblotting with mtHSP70 and TIM44 antibodies. g , h PLA of OSMR and mtHSP70 were performed in BTSC73 ( g ) and BTSC147 ( h ). Primary antibodies were omitted for the controls. i Double labeling of the PLA signal (red) from the OSMR/mtHSP70 interaction and MitoTracker (green) is shown. j OSMR protein expression level was assessed in the mitochondrial fractions obtained from BTSC73 electroporated with siRNA control (siCTL) or siRNA against mtHSP70 (si mtHSP70 ). BLC2 was used as a loading control. k OSMR protein expression level was assessed in the mitochondrial fractions obtained from BTSC73 electroporated with siCTL or siRNA against TIM44 (si TIM44 ). BCL2 was used as a loading control. l – o WCL or mitochondrial fractions from BTSC73 ( l , m ) and BTSC147 ( n , o ) were subjected to immunoprecipitation using an antibody to OSMR or mouse IgG control followed by immunoblotting with NDUFS1 and NDUFS2 antibodies. p , q PLA analyses of OSMR/NDUFS1 and OSMR/NDUFS2 were carried out in BTSC73 ( p ) and BTSC147 ( q ). r , s Double labeling of the PLA signal (red) and the MitoTracker (green) is shown. Images were obtained with a 63X objectives on a laser scanning confocal microscope (ZEISS LSM 800). Scale bar = 10 μm. Inset scale bar = 1 μm. Representative images of three independent experiments are shown. The Western blots represent a minimum of three replicates from different passage numbers for each BTSC.

Techniques Used: Immunoprecipitation, Proximity Ligation Assay, Labeling, Expressing, Microscopy, Western Blot

5) Product Images from "Tubeimoside I-induced lung cancer cell death and the underlying crosstalk between lysosomes and mitochondria"

Article Title: Tubeimoside I-induced lung cancer cell death and the underlying crosstalk between lysosomes and mitochondria

Journal: Cell Death & Disease

doi: 10.1038/s41419-020-02915-x

Tub induced blocking of late-stage autophagic flux in lung cancer cells. a Tub induced an increase in the number of GFP-LC3 puncta. GFP-LC3-overexpressing stable cell lines were treated with the vehicle, rapamycin (Rapa, 0.5 μM), bafilomycin A1 (Baf, 0.1 μM) or Tub (20 μM) for 24 h. Images of the cells were captured with a laser-scanning confocal microscope (scale bar = 20 µm). b Tub induced the upregulation of LC3-II and p62. c Lung cancer cells transfected with mCherry-GFP-LC3 tandem plasmids were treated with the vehicle, HBSS, Baf (0.1 μM) or Tub (20 μM) for 24 h. Like Baf treatment, Tub treatment also caused an increase in yellow fluorescence (creating by the merging of red and green fluorescence emitted by mCherry and GFP, respectively). The images were captured by a laser-scanning confocal microscope. The bar chart (right) represents the colocalization rate of GFP and mCherry, which was calculated with the Image J software (Scale bar = 5 µm). *** p
Figure Legend Snippet: Tub induced blocking of late-stage autophagic flux in lung cancer cells. a Tub induced an increase in the number of GFP-LC3 puncta. GFP-LC3-overexpressing stable cell lines were treated with the vehicle, rapamycin (Rapa, 0.5 μM), bafilomycin A1 (Baf, 0.1 μM) or Tub (20 μM) for 24 h. Images of the cells were captured with a laser-scanning confocal microscope (scale bar = 20 µm). b Tub induced the upregulation of LC3-II and p62. c Lung cancer cells transfected with mCherry-GFP-LC3 tandem plasmids were treated with the vehicle, HBSS, Baf (0.1 μM) or Tub (20 μM) for 24 h. Like Baf treatment, Tub treatment also caused an increase in yellow fluorescence (creating by the merging of red and green fluorescence emitted by mCherry and GFP, respectively). The images were captured by a laser-scanning confocal microscope. The bar chart (right) represents the colocalization rate of GFP and mCherry, which was calculated with the Image J software (Scale bar = 5 µm). *** p

Techniques Used: Blocking Assay, Stable Transfection, Microscopy, Transfection, Fluorescence, Software

6) Product Images from "RepA Promotes the Nucleolar Exclusion of the V2 Protein of Mulberry Mosaic Dwarf-Associated Virus"

Article Title: RepA Promotes the Nucleolar Exclusion of the V2 Protein of Mulberry Mosaic Dwarf-Associated Virus

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2020.01828

RepA-dependent nucleolar exclusion of V2. RFP-H2B plant leaves were infiltrated with Agrobacterium tumefaciens cultures carrying constructs to express GFP-V2 and the other six individual ORFs (V1, V3, V4, V5, RepA, and Rep) of MMDaV. Fluorescence was visualized under a Zeiss LSM 880 confocal laser scanning microscope at 36 hpi. The corresponding region in the white box in column 1 is magnified and shown from Column 2 to Column 5. RFP-H2B was used as a nuclear marker. This experiment was done three times and more than 20 cells were observed per sample and replicate. Scale bars correspond to 10 μm.
Figure Legend Snippet: RepA-dependent nucleolar exclusion of V2. RFP-H2B plant leaves were infiltrated with Agrobacterium tumefaciens cultures carrying constructs to express GFP-V2 and the other six individual ORFs (V1, V3, V4, V5, RepA, and Rep) of MMDaV. Fluorescence was visualized under a Zeiss LSM 880 confocal laser scanning microscope at 36 hpi. The corresponding region in the white box in column 1 is magnified and shown from Column 2 to Column 5. RFP-H2B was used as a nuclear marker. This experiment was done three times and more than 20 cells were observed per sample and replicate. Scale bars correspond to 10 μm.

Techniques Used: Construct, Fluorescence, Laser-Scanning Microscopy, Marker

V2 interacts with NbFib2. (A) Yeast two-hybrid assay showing the interaction between V2 and NbFib2 in yeast cells. Full-length NbFib2 was expressed as GAL4 DNA-binding domain fusion (BD, bait) and V2 was expressed as GAL4 activation domain fusion (AD, prey) in yeast cells of the strain Y2H Gold. The interaction of p53 and T was used as a positive control, and cotransformation of Lam and T was used as a negative control. Growth on the plates lacking leucine and tryptophan (SD/-LT) indicates successful transformation of both prey and bait vectors, respectively. Interaction between NbFib2 and V2 is indicated by growth of yeast cells on media also lacking histidine supplementing with 5 mM 3-amino-1,2,4-triazole (SD/-LTH + 3-AT). (B) Bimolecular fluorescence complementation assay (BiFC) assay showing the interaction between NbFib2 and V2 in plant cells. Constructs containing N-terminal YFP fusion (nYFP) and C-terminal YFP fusion (cYFP) fusions were infiltrated into RFP-H2B plant leaves. Combinations of BiFC constructs are shown at the top of each panel. Images were taken using a Zeiss LSM 880 confocal laser scanning microscope at 48 hpi. Reconstituted YFP signals resulting from V2-NbFib2 interaction are displayed as a false-green color. RFP-H2B served as a nuclear marker. Note that deletion of the predicted nuclear localization signal (from amino acid 61–76) of V2 abolishes its interaction with NbFib2. (C) Colocalization analysis of NbFib2 with V2 and V2 mutant in the epidermal cells of N. benthamiana by Zeiss LSM 880 confocal laser scanning microscope at 36 hpi. At least 60 cells from three repeats were examined. Scale bars correspond to 10 μm. (D) Immunoblot of proteins from RFP-H2B plant leaves infiltrated with construct as indicated using anti-GFP antibody. Ponceau staining of the large subunit of Rubisco serves as a loading control.
Figure Legend Snippet: V2 interacts with NbFib2. (A) Yeast two-hybrid assay showing the interaction between V2 and NbFib2 in yeast cells. Full-length NbFib2 was expressed as GAL4 DNA-binding domain fusion (BD, bait) and V2 was expressed as GAL4 activation domain fusion (AD, prey) in yeast cells of the strain Y2H Gold. The interaction of p53 and T was used as a positive control, and cotransformation of Lam and T was used as a negative control. Growth on the plates lacking leucine and tryptophan (SD/-LT) indicates successful transformation of both prey and bait vectors, respectively. Interaction between NbFib2 and V2 is indicated by growth of yeast cells on media also lacking histidine supplementing with 5 mM 3-amino-1,2,4-triazole (SD/-LTH + 3-AT). (B) Bimolecular fluorescence complementation assay (BiFC) assay showing the interaction between NbFib2 and V2 in plant cells. Constructs containing N-terminal YFP fusion (nYFP) and C-terminal YFP fusion (cYFP) fusions were infiltrated into RFP-H2B plant leaves. Combinations of BiFC constructs are shown at the top of each panel. Images were taken using a Zeiss LSM 880 confocal laser scanning microscope at 48 hpi. Reconstituted YFP signals resulting from V2-NbFib2 interaction are displayed as a false-green color. RFP-H2B served as a nuclear marker. Note that deletion of the predicted nuclear localization signal (from amino acid 61–76) of V2 abolishes its interaction with NbFib2. (C) Colocalization analysis of NbFib2 with V2 and V2 mutant in the epidermal cells of N. benthamiana by Zeiss LSM 880 confocal laser scanning microscope at 36 hpi. At least 60 cells from three repeats were examined. Scale bars correspond to 10 μm. (D) Immunoblot of proteins from RFP-H2B plant leaves infiltrated with construct as indicated using anti-GFP antibody. Ponceau staining of the large subunit of Rubisco serves as a loading control.

Techniques Used: Y2H Assay, Binding Assay, Activation Assay, Positive Control, Laser Capture Microdissection, Negative Control, Transformation Assay, Bimolecular Fluorescence Complementation Assay, Construct, Laser-Scanning Microscopy, Marker, Mutagenesis, Staining

RepA interacts with V2 in yeast and plant cells. (A) Yeast two-hybrid assay showing the interaction between RepA and V2 in yeast cells. Growth of yeast cotransformants containing the BD-RepA and AD-V2 fusions on the plates lacking leucine, tryptophan, histidine, and adenine (SD/-LTHA) indicates specific interaction between RepA and V2. (B) BiFC assay showing the interaction between RepA and V2 in plant cells. Combinations of BiFC constructs are shown at the top of each panel. Images were taken using a Zeiss LSM 880 confocal laser scanning microscope at 48 hpi. Reconstituted YFP signals as a consequence of V2-RepA interaction are depicted as a false-green color. RFP-H2B served as a nuclear marker.
Figure Legend Snippet: RepA interacts with V2 in yeast and plant cells. (A) Yeast two-hybrid assay showing the interaction between RepA and V2 in yeast cells. Growth of yeast cotransformants containing the BD-RepA and AD-V2 fusions on the plates lacking leucine, tryptophan, histidine, and adenine (SD/-LTHA) indicates specific interaction between RepA and V2. (B) BiFC assay showing the interaction between RepA and V2 in plant cells. Combinations of BiFC constructs are shown at the top of each panel. Images were taken using a Zeiss LSM 880 confocal laser scanning microscope at 48 hpi. Reconstituted YFP signals as a consequence of V2-RepA interaction are depicted as a false-green color. RFP-H2B served as a nuclear marker.

Techniques Used: Y2H Assay, Bimolecular Fluorescence Complementation Assay, Construct, Laser-Scanning Microscopy, Marker

V2 localization within the nucleolus is modulated in the presence of mulberry mosaic dwarf-associated geminivirus (MMDaV). (A) Subcellular localization of green fluorescent protein (GFP) or GFP-V2 fusion in the absence or presence of MMDaV infection in transgenic Nicotiana benthamian a plants expressing red fluorescent protein (RFP)-tagged histone 2B (RFP-H2B). RFP-H2B was used as a nuclear marker. (B) Colocalization analysis of V2 and fibrillarin 2 (NbFib2) in the absence or presence of MMDaV in N. benthamiana plants. To create an environment mimicking MMDaV infection, the infectious clone of MMDaV was infiltrated into RFP-H2B or N. benthamiana leaves 12 h prior to the infiltration of GFP or GFP-V2. Images were taken using Zeiss LSM 880 confocal laser scanning microscope at 36 hours post infiltration (hpi) of GFP or GFP-V2. This experiment was done three times and more than 20 cells were observed per sample and replicate. A representative image is shown for each set. The corresponding region in the white box in column 1 is magnified and shown from Column 2 to Column 5. Scale bars correspond to 10 μm.
Figure Legend Snippet: V2 localization within the nucleolus is modulated in the presence of mulberry mosaic dwarf-associated geminivirus (MMDaV). (A) Subcellular localization of green fluorescent protein (GFP) or GFP-V2 fusion in the absence or presence of MMDaV infection in transgenic Nicotiana benthamian a plants expressing red fluorescent protein (RFP)-tagged histone 2B (RFP-H2B). RFP-H2B was used as a nuclear marker. (B) Colocalization analysis of V2 and fibrillarin 2 (NbFib2) in the absence or presence of MMDaV in N. benthamiana plants. To create an environment mimicking MMDaV infection, the infectious clone of MMDaV was infiltrated into RFP-H2B or N. benthamiana leaves 12 h prior to the infiltration of GFP or GFP-V2. Images were taken using Zeiss LSM 880 confocal laser scanning microscope at 36 hours post infiltration (hpi) of GFP or GFP-V2. This experiment was done three times and more than 20 cells were observed per sample and replicate. A representative image is shown for each set. The corresponding region in the white box in column 1 is magnified and shown from Column 2 to Column 5. Scale bars correspond to 10 μm.

Techniques Used: Infection, Transgenic Assay, Expressing, Marker, Laser-Scanning Microscopy

7) Product Images from "OSMR controls glioma stem cell respiration and confers resistance of glioblastoma to ionizing radiation"

Article Title: OSMR controls glioma stem cell respiration and confers resistance of glioblastoma to ionizing radiation

Journal: Nature Communications

doi: 10.1038/s41467-020-17885-z

Presence of mitochondrial OSMR in human BTSCs. a – d Four different patient-derived BTSC lines were subjected to subcellular fractionation, and the lysates for each fraction were analyzed by immunoblotting using antibodies to OSMR. α-Tubulin, H3K4me3, BCL2/TOM20, Na+/K+ ATPase, and calnexin. WCL: Whole-cell lysates; Cyto: cytoplasmic; Mito: mitochondrial. The Western blots represent a minimum of three replicates from different passage numbers for each BTSC. e , f BTSC73 and BTSC147 were subjected to immunostaining using antibodies to OSMR (green) and the mitochondrial matrix protein ATP synthase inhibitor F1 (ATPIF1, red). Nuclei were stained with DAPI. White rectangles mark the inset to demonstrate the co-localization of OSMR with ATPIF1. g , h PLA of OSMR and ATPIF1 were performed in BTSC73 ( g ) and BTSC147 ( h ). Primary antibodies were omitted as controls. i Double labeling of the PLA signal (red) and the MitoTracker (green) in BTSC73 is shown. j A FRAP assay was performed on BTSC73 transduced with GFP-OSMR and stained with MitoTracker (red). Different regions of interest (ROIs) containing GFP-OSMR in the mitochondria were defined. ROI1 indicates a non-bleached area and ROI2, a photobleached area. The fluorescence recovery was monitored over time following photobleaching. Images were obtained on a laser scanning confocal microscope (ZEISS LSM 800). Scale bar = 10 μm; Inset scale bar = 1 μm. Representative images of three independent experiments are shown.
Figure Legend Snippet: Presence of mitochondrial OSMR in human BTSCs. a – d Four different patient-derived BTSC lines were subjected to subcellular fractionation, and the lysates for each fraction were analyzed by immunoblotting using antibodies to OSMR. α-Tubulin, H3K4me3, BCL2/TOM20, Na+/K+ ATPase, and calnexin. WCL: Whole-cell lysates; Cyto: cytoplasmic; Mito: mitochondrial. The Western blots represent a minimum of three replicates from different passage numbers for each BTSC. e , f BTSC73 and BTSC147 were subjected to immunostaining using antibodies to OSMR (green) and the mitochondrial matrix protein ATP synthase inhibitor F1 (ATPIF1, red). Nuclei were stained with DAPI. White rectangles mark the inset to demonstrate the co-localization of OSMR with ATPIF1. g , h PLA of OSMR and ATPIF1 were performed in BTSC73 ( g ) and BTSC147 ( h ). Primary antibodies were omitted as controls. i Double labeling of the PLA signal (red) and the MitoTracker (green) in BTSC73 is shown. j A FRAP assay was performed on BTSC73 transduced with GFP-OSMR and stained with MitoTracker (red). Different regions of interest (ROIs) containing GFP-OSMR in the mitochondria were defined. ROI1 indicates a non-bleached area and ROI2, a photobleached area. The fluorescence recovery was monitored over time following photobleaching. Images were obtained on a laser scanning confocal microscope (ZEISS LSM 800). Scale bar = 10 μm; Inset scale bar = 1 μm. Representative images of three independent experiments are shown.

Techniques Used: Derivative Assay, Fractionation, Western Blot, Immunostaining, Staining, Proximity Ligation Assay, Labeling, FRAP Assay, Transduction, Fluorescence, Microscopy

OSMR interacts with different components of ETC in human BTSCs. a , b Mitochondrial fractions from BTSC73 ( a ) and BTSC147 ( b ) were treated with 0.5 mg/mL proteinase K or proteinase K and 1% Triton X-100. Lysates were analyzed by immunoblotting using indicated antibodies. c – f WCL and mitochondrial fractions from BTSC73 ( c , d ) and BTSC147 ( e , f ) were subjected to immunoprecipitation using antibodies to OSMR or mouse IgG control, followed by immunoblotting with mtHSP70 and TIM44 antibodies. g , h PLA of OSMR and mtHSP70 were performed in BTSC73 ( g ) and BTSC147 ( h ). Primary antibodies were omitted for the controls. i Double labeling of the PLA signal (red) from the OSMR/mtHSP70 interaction and MitoTracker (green) is shown. j OSMR protein expression level was assessed in the mitochondrial fractions obtained from BTSC73 electroporated with siRNA control (siCTL) or siRNA against mtHSP70 (si mtHSP70 ). BLC2 was used as a loading control. k OSMR protein expression level was assessed in the mitochondrial fractions obtained from BTSC73 electroporated with siCTL or siRNA against TIM44 (si TIM44 ). BCL2 was used as a loading control. l – o WCL or mitochondrial fractions from BTSC73 ( l , m ) and BTSC147 ( n , o ) were subjected to immunoprecipitation using an antibody to OSMR or mouse IgG control followed by immunoblotting with NDUFS1 and NDUFS2 antibodies. p , q PLA analyses of OSMR/NDUFS1 and OSMR/NDUFS2 were carried out in BTSC73 ( p ) and BTSC147 ( q ). r , s Double labeling of the PLA signal (red) and the MitoTracker (green) is shown. Images were obtained with a 63X objectives on a laser scanning confocal microscope (ZEISS LSM 800). Scale bar = 10 μm. Inset scale bar = 1 μm. Representative images of three independent experiments are shown. The Western blots represent a minimum of three replicates from different passage numbers for each BTSC.
Figure Legend Snippet: OSMR interacts with different components of ETC in human BTSCs. a , b Mitochondrial fractions from BTSC73 ( a ) and BTSC147 ( b ) were treated with 0.5 mg/mL proteinase K or proteinase K and 1% Triton X-100. Lysates were analyzed by immunoblotting using indicated antibodies. c – f WCL and mitochondrial fractions from BTSC73 ( c , d ) and BTSC147 ( e , f ) were subjected to immunoprecipitation using antibodies to OSMR or mouse IgG control, followed by immunoblotting with mtHSP70 and TIM44 antibodies. g , h PLA of OSMR and mtHSP70 were performed in BTSC73 ( g ) and BTSC147 ( h ). Primary antibodies were omitted for the controls. i Double labeling of the PLA signal (red) from the OSMR/mtHSP70 interaction and MitoTracker (green) is shown. j OSMR protein expression level was assessed in the mitochondrial fractions obtained from BTSC73 electroporated with siRNA control (siCTL) or siRNA against mtHSP70 (si mtHSP70 ). BLC2 was used as a loading control. k OSMR protein expression level was assessed in the mitochondrial fractions obtained from BTSC73 electroporated with siCTL or siRNA against TIM44 (si TIM44 ). BCL2 was used as a loading control. l – o WCL or mitochondrial fractions from BTSC73 ( l , m ) and BTSC147 ( n , o ) were subjected to immunoprecipitation using an antibody to OSMR or mouse IgG control followed by immunoblotting with NDUFS1 and NDUFS2 antibodies. p , q PLA analyses of OSMR/NDUFS1 and OSMR/NDUFS2 were carried out in BTSC73 ( p ) and BTSC147 ( q ). r , s Double labeling of the PLA signal (red) and the MitoTracker (green) is shown. Images were obtained with a 63X objectives on a laser scanning confocal microscope (ZEISS LSM 800). Scale bar = 10 μm. Inset scale bar = 1 μm. Representative images of three independent experiments are shown. The Western blots represent a minimum of three replicates from different passage numbers for each BTSC.

Techniques Used: Immunoprecipitation, Proximity Ligation Assay, Labeling, Expressing, Microscopy, Western Blot

8) Product Images from "Capturing Amyloid-β Oligomers by Stirring with Microscaled Iron Oxide Stir Bars into Magnetic Plaques to Reduce Cytotoxicity toward Neuronal Cells"

Article Title: Capturing Amyloid-β Oligomers by Stirring with Microscaled Iron Oxide Stir Bars into Magnetic Plaques to Reduce Cytotoxicity toward Neuronal Cells

Journal: Nanomaterials

doi: 10.3390/nano10071284

Phagocytic action of BV-2 cells. ( a ) Laser scanning confocal microscope (LSCM) images of BV-2 cells after co-incubation with oAβ 42 , npAβ 42 , and mpAβ 42 , respectively ( n = 8). Aβ 42 in different forms was IHC stained using 6E10 as the primary antibody (λ ex = 565 nm, λ em = 680–730 nm). Scale bar: 50 μm. ( b ) Relative uptake index of Aβ 42 by BV-2 cells. One-way ANOVA was used to examine the mean differences between the end points of the data groups. ** p
Figure Legend Snippet: Phagocytic action of BV-2 cells. ( a ) Laser scanning confocal microscope (LSCM) images of BV-2 cells after co-incubation with oAβ 42 , npAβ 42 , and mpAβ 42 , respectively ( n = 8). Aβ 42 in different forms was IHC stained using 6E10 as the primary antibody (λ ex = 565 nm, λ em = 680–730 nm). Scale bar: 50 μm. ( b ) Relative uptake index of Aβ 42 by BV-2 cells. One-way ANOVA was used to examine the mean differences between the end points of the data groups. ** p

Techniques Used: Microscopy, Incubation, Immunohistochemistry, Staining

9) Product Images from "Patchless administration of canine influenza vaccine on dog’s ear using insertion-responsive microneedles (IRMN) without removal of hair and its in vivo efficacy evaluation"

Article Title: Patchless administration of canine influenza vaccine on dog’s ear using insertion-responsive microneedles (IRMN) without removal of hair and its in vivo efficacy evaluation

Journal: European Journal of Pharmaceutics and Biopharmaceutics

doi: 10.1016/j.ejpb.2020.06.006

Confocal laser scanning microscopy images of DiD-loaded vc378 distribution in the microneedle. Representative confocal images of microneedles coated with DiD-loaded vc378 (left, DiD-loaded vc378; middle, DIC; right, merge). The scale bars are 200 μm.
Figure Legend Snippet: Confocal laser scanning microscopy images of DiD-loaded vc378 distribution in the microneedle. Representative confocal images of microneedles coated with DiD-loaded vc378 (left, DiD-loaded vc378; middle, DIC; right, merge). The scale bars are 200 μm.

Techniques Used: Confocal Laser Scanning Microscopy

10) Product Images from "Micro Versus Macro – The Effect of Environmental Confinement on Cellular Nanoparticle Uptake"

Article Title: Micro Versus Macro – The Effect of Environmental Confinement on Cellular Nanoparticle Uptake

Journal: Frontiers in Bioengineering and Biotechnology

doi: 10.3389/fbioe.2020.00869

Schematic of the experimental methodology. (A) Seeding equal number of cells in the microfluidic device and the petri dish and allowing cells 4–16 h to attach to the glass bottom of the device. (B) FND suspension incubation for 4 h. (C) Fixed and stained macrophages are imaged in a laser scanning confocal microscope. Red dots in the image are FNDs where nucleus and cytoskeleton are indicated with blue and green color respectively. In every experiment, FNDs/cell are quantified in 50 cells per group. Each experiment is repeated three independent times.
Figure Legend Snippet: Schematic of the experimental methodology. (A) Seeding equal number of cells in the microfluidic device and the petri dish and allowing cells 4–16 h to attach to the glass bottom of the device. (B) FND suspension incubation for 4 h. (C) Fixed and stained macrophages are imaged in a laser scanning confocal microscope. Red dots in the image are FNDs where nucleus and cytoskeleton are indicated with blue and green color respectively. In every experiment, FNDs/cell are quantified in 50 cells per group. Each experiment is repeated three independent times.

Techniques Used: Incubation, Staining, Microscopy

11) Product Images from "Global transcriptional regulation by cell-free supernatant of Salmonella Typhimurium peptide transporter mutant leads to inhibition of intra-species biofilm initiation"

Article Title: Global transcriptional regulation by cell-free supernatant of Salmonella Typhimurium peptide transporter mutant leads to inhibition of intra-species biofilm initiation

Journal: bioRxiv

doi: 10.1101/2020.07.15.204859

Salmonella ΔyjiY cell free supernatant significantly reduces the biofilm biomass by reducing cell-cell adhesion A. Representative images of biofilm formed on coverslips that were stained with Congo red and imaged using a confocal microscope to generate 3D images and quantify cellulose biomass. Scale is shown on the X- and Y-axes. B. The tensile strength of the biofilm was measured by glass bead assay. Weight of glass beads required to just sink the biofilm to bottom, was plotted (Data are presented as mean + SEM of 5 independent experiments). C. Representative scanning electron micrograph of biofilm formed on a coverslip. Scale bar is 10 μm. D. Cell length of the biofilm inoculated treated or untreated STM WT cells was measured using ImageJ, and plotted (Data are presented as mean + SEM of 1200 cells were measured from 3 independent experiments). E. Representative confocal images of biofilm cells showing a difference in cell length. Scale bar is 5 μm (1000-1200 cells were measured from 3 independent experiments for each treatment). Student’s t-test was used to analyze the data; p values ****
Figure Legend Snippet: Salmonella ΔyjiY cell free supernatant significantly reduces the biofilm biomass by reducing cell-cell adhesion A. Representative images of biofilm formed on coverslips that were stained with Congo red and imaged using a confocal microscope to generate 3D images and quantify cellulose biomass. Scale is shown on the X- and Y-axes. B. The tensile strength of the biofilm was measured by glass bead assay. Weight of glass beads required to just sink the biofilm to bottom, was plotted (Data are presented as mean + SEM of 5 independent experiments). C. Representative scanning electron micrograph of biofilm formed on a coverslip. Scale bar is 10 μm. D. Cell length of the biofilm inoculated treated or untreated STM WT cells was measured using ImageJ, and plotted (Data are presented as mean + SEM of 1200 cells were measured from 3 independent experiments). E. Representative confocal images of biofilm cells showing a difference in cell length. Scale bar is 5 μm (1000-1200 cells were measured from 3 independent experiments for each treatment). Student’s t-test was used to analyze the data; p values ****

Techniques Used: Staining, Microscopy

12) Product Images from "Preservation of hydrogen peroxide-induced oxidative damage in HepG-2 cells by rice protein hydrolysates pretreated with electron beams"

Article Title: Preservation of hydrogen peroxide-induced oxidative damage in HepG-2 cells by rice protein hydrolysates pretreated with electron beams

Journal: Scientific Reports

doi: 10.1038/s41598-020-64814-7

Effect of ERPHs on the intracellular ROS level. HepG-2 cells were pretreated with NRPHs and ERPHs for 48 h before treatment with 0.4 mM H 2 O 2 for 4 h. Then the cells were exposed to DCFH-DA for 30 min. DCF fluorescence of the treated cells were measured by using a laser scanning confocal microscope. Data are shown as means ± S.D.
Figure Legend Snippet: Effect of ERPHs on the intracellular ROS level. HepG-2 cells were pretreated with NRPHs and ERPHs for 48 h before treatment with 0.4 mM H 2 O 2 for 4 h. Then the cells were exposed to DCFH-DA for 30 min. DCF fluorescence of the treated cells were measured by using a laser scanning confocal microscope. Data are shown as means ± S.D.

Techniques Used: Fluorescence, Microscopy

13) Product Images from "Effect of Astaxanthin on Activation of Autophagy and Inhibition of Apoptosis in Helicobacter pylori-Infected Gastric Epithelial Cell Line AGS"

Article Title: Effect of Astaxanthin on Activation of Autophagy and Inhibition of Apoptosis in Helicobacter pylori-Infected Gastric Epithelial Cell Line AGS

Journal: Nutrients

doi: 10.3390/nu12061750

Effect of astaxanthin on autophagy activation in H. pylori -stimulated AGS cells. The cells were pre-treated with 50 nM astaxanthin for 3 h and then stimulated with H. pylori (cell to H. pylori ratio of 1:50) for 24 h. ( A ) Cells were stained with acridine orange (AO) dye and visualized under a confocal laser scanning microscope (left panel). AO-positive cells were quantified and expressed as % of cells with AO-positive cells/total number of cells (right panel). ( B ) The cells were stained with anti-LC3B antibody and rhodamine-labeled mouse anti-rabbit IgG antibody. Immunocytochemical staining for LC3B (red) and DNA counterstaining with DAPI (blue) are shown in the left panel. Each sample was analyzed using a threshold of > 7 dots/cell. LC3B puncta-positive cells were quantified and expressed as % of cells with > 7 LC3B puncta/total number of cells (right panel). * p
Figure Legend Snippet: Effect of astaxanthin on autophagy activation in H. pylori -stimulated AGS cells. The cells were pre-treated with 50 nM astaxanthin for 3 h and then stimulated with H. pylori (cell to H. pylori ratio of 1:50) for 24 h. ( A ) Cells were stained with acridine orange (AO) dye and visualized under a confocal laser scanning microscope (left panel). AO-positive cells were quantified and expressed as % of cells with AO-positive cells/total number of cells (right panel). ( B ) The cells were stained with anti-LC3B antibody and rhodamine-labeled mouse anti-rabbit IgG antibody. Immunocytochemical staining for LC3B (red) and DNA counterstaining with DAPI (blue) are shown in the left panel. Each sample was analyzed using a threshold of > 7 dots/cell. LC3B puncta-positive cells were quantified and expressed as % of cells with > 7 LC3B puncta/total number of cells (right panel). * p

Techniques Used: Activation Assay, Staining, Laser-Scanning Microscopy, Labeling

14) Product Images from "Efficient miRNA Inhibitor with GO-PEI Nanosheets for Osteosarcoma Suppression by Targeting PTEN"

Article Title: Efficient miRNA Inhibitor with GO-PEI Nanosheets for Osteosarcoma Suppression by Targeting PTEN

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S257084

Uptake of GO-PEI into osteosarcoma cells. ( A ) TEM images of GO-PEI-incubated and non-incubated cells (3 μg/mL GO-PEI in the cell culture medium). The right panel is enlarged images of white squares in the image of GO-PEI-incubated cells. Scale bars: 1 μm. ( B ) FITC-BSA-labeled GO-PEI (green) is visualized in cells through confocal laser scanning microscopy. Fluorescence images of FITC-labeled GO-PEI (green) within MG63 and U2OS cells are shown. The cell cytoskeleton was stained with α-tubulin (red) for MG63 cells and rhodamine-phalloidin (red) for U2OS cells, and the nuclei were stained with DAPI (blue). The right panel is enlarged images of white squares in the image of GO-PEI-incubated cells. Scale bars: 2 μm. ( C ) Cell viability of MG63 and U2OS cells exposed to different concentrations of GO-PEI was measured by CCK8 assays. * p
Figure Legend Snippet: Uptake of GO-PEI into osteosarcoma cells. ( A ) TEM images of GO-PEI-incubated and non-incubated cells (3 μg/mL GO-PEI in the cell culture medium). The right panel is enlarged images of white squares in the image of GO-PEI-incubated cells. Scale bars: 1 μm. ( B ) FITC-BSA-labeled GO-PEI (green) is visualized in cells through confocal laser scanning microscopy. Fluorescence images of FITC-labeled GO-PEI (green) within MG63 and U2OS cells are shown. The cell cytoskeleton was stained with α-tubulin (red) for MG63 cells and rhodamine-phalloidin (red) for U2OS cells, and the nuclei were stained with DAPI (blue). The right panel is enlarged images of white squares in the image of GO-PEI-incubated cells. Scale bars: 2 μm. ( C ) Cell viability of MG63 and U2OS cells exposed to different concentrations of GO-PEI was measured by CCK8 assays. * p

Techniques Used: Transmission Electron Microscopy, Incubation, Cell Culture, Labeling, Confocal Laser Scanning Microscopy, Fluorescence, Staining

Related Articles

Microscopy:

Article Title: Capturing Amyloid-β Oligomers by Stirring with Microscaled Iron Oxide Stir Bars into Magnetic Plaques to Reduce Cytotoxicity toward Neuronal Cells
Article Snippet: .. The neurite outgrowth was examined by laser scanning confocal microscope (LSCM, ZEISS LSM-780, Jena, Germany). .. The cell nuclei and cytoskeletons were stained with Hoechst 33342 and CytoPainter F-actin Staining Kit-Green Fluorescence, respectively.

Article Title: A Fish Leukocyte Immune-Type Receptor Uses a Novel Intracytoplasmic Tail Networking Mechanism to Cross-Inhibit the Phagocytic Response
Article Snippet: .. Imaging was performed as previously described [ , ] using a Laser Scanning Confocal Microscope (LSCM; Zeiss LSM 710, objective 60× 1.3 oil plan-Apochromat, Munich, Germany). .. All images were collected and analyzed using Zen 2011 software and ImageJ for calculating fluorescent intensities.

Article Title: OSMR controls glioma stem cell respiration and confers resistance of glioblastoma to ionizing radiation
Article Snippet: .. FRAP was performed using a laser scanning confocal microscope (ZEISS LSM 800). ..

Article Title: Tubeimoside I-induced lung cancer cell death and the underlying crosstalk between lysosomes and mitochondria
Article Snippet: .. Images were captured with a laser-scanning confocal microscope equipped with an argon laser (excitation wavelength: 555 nm) and a ×63 objective lens (LSM 800; Carl Zeiss, Jena, Germany). .. Intracellular ROS detectionNCI-H1299 or NCI-H1975 cells were plated in 6-well plates, cultured overnight, and treated with different concentrations of Tub for 24 h. The cells were collected, washed with PBS, and stained with 10 µM H2DCFDA (D399; Thermo Fisher Scientific, Waltham, MA, USA) for 20 min at 37 °C in the dark.

Article Title: Patchless administration of canine influenza vaccine on dog’s ear using insertion-responsive microneedles (IRMN) without removal of hair and its in vivo efficacy evaluation
Article Snippet: .. The cells were then washed with PBS and fluorescent images were collected using a laser scanning confocal microscope (LSM700, Carl Zeiss, Jena, Germany). .. 2.6 Vaccination in dogsSeven-week-old male Beagle dogs were purchased from CLSbio (Bucheon, South Korea) with each dog being serologically negative for influenza viruses and housed in isolation cages within the BSL-2 facility at Green Cross Veterinary Products (Yongin, South Korea) for the study.

Article Title: RepA Promotes the Nucleolar Exclusion of the V2 Protein of Mulberry Mosaic Dwarf-Associated Virus
Article Snippet: .. Confocal Microscopy Imaging of fluorescent proteins in the epidermal cells of agroinfiltrated N. benthamiana or RFP-H2B plants was performed on a laser-scanning confocal microscope (LSM880; Carl Zeiss, Jena, Germany) at 36–48 hours post-infiltration (hpi). .. Leaf tissues were examined using a Zeiss c-Apochromat 40 × 1.2 water immersion Korr objective.

Article Title: OSMR controls glioma stem cell respiration and confers resistance of glioblastoma to ionizing radiation
Article Snippet: .. Images were captured using a 63X objective on a laser scanning confocal microscope (ZEISS LSM 800). .. Duolink proximity ligation assayPLAs were performed using a Duolink In Situ Red Starter Kit (Sigma, #DUO92101) according to the manufacturer’s instructions.

Article Title: OSMR controls glioma stem cell respiration and confers resistance of glioblastoma to ionizing radiation
Article Snippet: .. The visualized fluorescence PLA signals were captured using a 63X objective on a laser scanning confocal microscope (ZEISS LSM 800). .. Fluorescence recovery after photobleaching assayGFP-OSMR expressing BTSC73 were cultured on coverslip II cell culture chamber slides (Sarstedt, #946190802).

Proximity Ligation Assay:

Article Title: OSMR controls glioma stem cell respiration and confers resistance of glioblastoma to ionizing radiation
Article Snippet: .. The visualized fluorescence PLA signals were captured using a 63X objective on a laser scanning confocal microscope (ZEISS LSM 800). .. Fluorescence recovery after photobleaching assayGFP-OSMR expressing BTSC73 were cultured on coverslip II cell culture chamber slides (Sarstedt, #946190802).

Confocal Microscopy:

Article Title: RepA Promotes the Nucleolar Exclusion of the V2 Protein of Mulberry Mosaic Dwarf-Associated Virus
Article Snippet: .. Confocal Microscopy Imaging of fluorescent proteins in the epidermal cells of agroinfiltrated N. benthamiana or RFP-H2B plants was performed on a laser-scanning confocal microscope (LSM880; Carl Zeiss, Jena, Germany) at 36–48 hours post-infiltration (hpi). .. Leaf tissues were examined using a Zeiss c-Apochromat 40 × 1.2 water immersion Korr objective.

Fluorescence:

Article Title: OSMR controls glioma stem cell respiration and confers resistance of glioblastoma to ionizing radiation
Article Snippet: .. The visualized fluorescence PLA signals were captured using a 63X objective on a laser scanning confocal microscope (ZEISS LSM 800). .. Fluorescence recovery after photobleaching assayGFP-OSMR expressing BTSC73 were cultured on coverslip II cell culture chamber slides (Sarstedt, #946190802).

Imaging:

Article Title: A Fish Leukocyte Immune-Type Receptor Uses a Novel Intracytoplasmic Tail Networking Mechanism to Cross-Inhibit the Phagocytic Response
Article Snippet: .. Imaging was performed as previously described [ , ] using a Laser Scanning Confocal Microscope (LSCM; Zeiss LSM 710, objective 60× 1.3 oil plan-Apochromat, Munich, Germany). .. All images were collected and analyzed using Zen 2011 software and ImageJ for calculating fluorescent intensities.

Article Title: RepA Promotes the Nucleolar Exclusion of the V2 Protein of Mulberry Mosaic Dwarf-Associated Virus
Article Snippet: .. Confocal Microscopy Imaging of fluorescent proteins in the epidermal cells of agroinfiltrated N. benthamiana or RFP-H2B plants was performed on a laser-scanning confocal microscope (LSM880; Carl Zeiss, Jena, Germany) at 36–48 hours post-infiltration (hpi). .. Leaf tissues were examined using a Zeiss c-Apochromat 40 × 1.2 water immersion Korr objective.

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    Carl Zeiss lsm700 confocal laser scanning microscope
    STAT3 enhances V-ATPase activity. a Representative immunoblots (left) and quantification (right) of the indicated V-ATPase subunits from lysates of HeLa CRISPR control clone (C-4) and STAT3-KO clones (KO-1 and −11). TUBA1A served as a loading control. b Numbers of lysosomal genes whose expression analyzed by RNA-Seq was decreased or increased over ≥ 1.5-fold ( P ≤ 0.05) in HeLa-STAT3-KO cells as compared to HeLa-C4 control cells. Lysosomal genes were defined as genes whose protein products localize to lysosomes according to either Gene Ontology or Kyoto Encyclopedia of Genes and Genomes databases. See Supplementary information, Fig. S5b for the list of altered genes. c Representative immunoblots of the indicted proteins from lysates of HeLa cells transfected with the indicated siRNAs 72 h earlier. n = 3. d Representative images (left) and quantification (right) of PLA puncta with antibodies against ATP6V1A (V1A) and ATP6V0D1 (V0D1) in HeLa CRISPR control (C-4) and STAT3-KO (STAT3-KO-11) cells, as well as in HeLa cells transfected with the indicated siRNAs 72 h earlier. DNA was stained with DAPI. Images were taken with 60× magnification using Zeiss <t>LSM700</t> confocal microscope. The optimal slice thickness (∼350 nm) was defined by the Zeiss zen software. Scale bar, 10 µm. e Quantification of PLA puncta with antibodies against STAT3 and V1A in HeLa cells (left) or Flag and V0D1 in HeLa-STAT3-Flag cells (right). Cells were transfected with the indicated siRNAs 72 h earlier. f Activity of V-ATPase in the presence of 30 µg/mL superfolder-GFP (sfGFP; control) or ΔN-STAT3-sfGFP. V-ATPase was immunoprecipitated with anti-HA magnetic beads from lysosomal lysates of HeLa cells transiently transfected with pCDNA3.1-HA-ATP6V1A. When indicated, the samples were treated with 100 nM bafilomycin A1. Right, standard curve for the measurement of the free phosphate ion used to estimate the ATP consumption. Protein blot for recombinant proteins is shown in Supplementary information, Fig. S5c . Error bars, SD of ≥ 3 independent experiments. A minimum of ten cells/sample were analyzed in d , e . P -values were calculated by one-way ANOVA combined with Dunnett’s multiple comparisons test ( a , d ), DEseq2 ( b ), or by two-tailed, homoscedastic Student’s t -test ( e , f )
    Lsm700 Confocal Laser Scanning Microscope, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 92/100, based on 190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lsm700 confocal laser scanning microscope/product/Carl Zeiss
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    Carl Zeiss lsm 710 laser scanning confocal microscope
    Live-cell imaging of IpLITR 1.1b-mediated target interactions. Rat basophilic leukemia-2H3 cells (3 × 10 5 ) stably co-expressing IpLITR 1.1b and LifeAct-GFP were incubated at 37°C with 9 × 10 5 αHA monoclonal antibody-coated 4.5 µm microspheres. Immediately after the addition of target beads, images were collected at 10 s intervals for ~8 min using a Zeiss <t>LSM</t> 710 laser scanning confocal microscope (objective 60×, 1.3 oil plan-Apochromat; Munich, Germany). Both the brightfield-LifeAct-GFP merged views (top panels) and the LifeAct-GFP views alone (bottom panels) are shown for two representative (A,B) IpLITR 1.1b-mediated target interactions with the location of the target microsphere indicated with an asterisk. Representative time-stamps in (A,B) were extracted from Videos S6 and S8 in Presentation 2 of Supplementary Material, respectively.
    Lsm 710 Laser Scanning Confocal Microscope, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 92/100, based on 512 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lsm 710 laser scanning confocal microscope/product/Carl Zeiss
    Average 92 stars, based on 512 article reviews
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    STAT3 enhances V-ATPase activity. a Representative immunoblots (left) and quantification (right) of the indicated V-ATPase subunits from lysates of HeLa CRISPR control clone (C-4) and STAT3-KO clones (KO-1 and −11). TUBA1A served as a loading control. b Numbers of lysosomal genes whose expression analyzed by RNA-Seq was decreased or increased over ≥ 1.5-fold ( P ≤ 0.05) in HeLa-STAT3-KO cells as compared to HeLa-C4 control cells. Lysosomal genes were defined as genes whose protein products localize to lysosomes according to either Gene Ontology or Kyoto Encyclopedia of Genes and Genomes databases. See Supplementary information, Fig. S5b for the list of altered genes. c Representative immunoblots of the indicted proteins from lysates of HeLa cells transfected with the indicated siRNAs 72 h earlier. n = 3. d Representative images (left) and quantification (right) of PLA puncta with antibodies against ATP6V1A (V1A) and ATP6V0D1 (V0D1) in HeLa CRISPR control (C-4) and STAT3-KO (STAT3-KO-11) cells, as well as in HeLa cells transfected with the indicated siRNAs 72 h earlier. DNA was stained with DAPI. Images were taken with 60× magnification using Zeiss LSM700 confocal microscope. The optimal slice thickness (∼350 nm) was defined by the Zeiss zen software. Scale bar, 10 µm. e Quantification of PLA puncta with antibodies against STAT3 and V1A in HeLa cells (left) or Flag and V0D1 in HeLa-STAT3-Flag cells (right). Cells were transfected with the indicated siRNAs 72 h earlier. f Activity of V-ATPase in the presence of 30 µg/mL superfolder-GFP (sfGFP; control) or ΔN-STAT3-sfGFP. V-ATPase was immunoprecipitated with anti-HA magnetic beads from lysosomal lysates of HeLa cells transiently transfected with pCDNA3.1-HA-ATP6V1A. When indicated, the samples were treated with 100 nM bafilomycin A1. Right, standard curve for the measurement of the free phosphate ion used to estimate the ATP consumption. Protein blot for recombinant proteins is shown in Supplementary information, Fig. S5c . Error bars, SD of ≥ 3 independent experiments. A minimum of ten cells/sample were analyzed in d , e . P -values were calculated by one-way ANOVA combined with Dunnett’s multiple comparisons test ( a , d ), DEseq2 ( b ), or by two-tailed, homoscedastic Student’s t -test ( e , f )

    Journal: Cell Research

    Article Title: STAT3 associates with vacuolar H+-ATPase and regulates cytosolic and lysosomal pH

    doi: 10.1038/s41422-018-0080-0

    Figure Lengend Snippet: STAT3 enhances V-ATPase activity. a Representative immunoblots (left) and quantification (right) of the indicated V-ATPase subunits from lysates of HeLa CRISPR control clone (C-4) and STAT3-KO clones (KO-1 and −11). TUBA1A served as a loading control. b Numbers of lysosomal genes whose expression analyzed by RNA-Seq was decreased or increased over ≥ 1.5-fold ( P ≤ 0.05) in HeLa-STAT3-KO cells as compared to HeLa-C4 control cells. Lysosomal genes were defined as genes whose protein products localize to lysosomes according to either Gene Ontology or Kyoto Encyclopedia of Genes and Genomes databases. See Supplementary information, Fig. S5b for the list of altered genes. c Representative immunoblots of the indicted proteins from lysates of HeLa cells transfected with the indicated siRNAs 72 h earlier. n = 3. d Representative images (left) and quantification (right) of PLA puncta with antibodies against ATP6V1A (V1A) and ATP6V0D1 (V0D1) in HeLa CRISPR control (C-4) and STAT3-KO (STAT3-KO-11) cells, as well as in HeLa cells transfected with the indicated siRNAs 72 h earlier. DNA was stained with DAPI. Images were taken with 60× magnification using Zeiss LSM700 confocal microscope. The optimal slice thickness (∼350 nm) was defined by the Zeiss zen software. Scale bar, 10 µm. e Quantification of PLA puncta with antibodies against STAT3 and V1A in HeLa cells (left) or Flag and V0D1 in HeLa-STAT3-Flag cells (right). Cells were transfected with the indicated siRNAs 72 h earlier. f Activity of V-ATPase in the presence of 30 µg/mL superfolder-GFP (sfGFP; control) or ΔN-STAT3-sfGFP. V-ATPase was immunoprecipitated with anti-HA magnetic beads from lysosomal lysates of HeLa cells transiently transfected with pCDNA3.1-HA-ATP6V1A. When indicated, the samples were treated with 100 nM bafilomycin A1. Right, standard curve for the measurement of the free phosphate ion used to estimate the ATP consumption. Protein blot for recombinant proteins is shown in Supplementary information, Fig. S5c . Error bars, SD of ≥ 3 independent experiments. A minimum of ten cells/sample were analyzed in d , e . P -values were calculated by one-way ANOVA combined with Dunnett’s multiple comparisons test ( a , d ), DEseq2 ( b ), or by two-tailed, homoscedastic Student’s t -test ( e , f )

    Article Snippet: To estimate the cytosolic pH, cells washed with Live Cell Imaging Solution were incubated for 30 min in 37 °C in the same solution containing 1:1,000 dilution of pHrodo™ Green AM Intracellular pH Indicator and 1:100 dilution of PowerLoad™ concentrate, washed with Live Cell Imaging Solution, and analyzed by LSM700 confocal laser scanning microscope.

    Techniques: Activity Assay, Western Blot, CRISPR, Clone Assay, Expressing, RNA Sequencing Assay, Transfection, Proximity Ligation Assay, Staining, Microscopy, Software, Immunoprecipitation, Magnetic Beads, Recombinant, Two Tailed Test

    STAT3 regulates cytosolic pH. a Intensity of lysosomal RFP-STAT3 in A549-RFP-STAT3 cells loaded with 0.4 mg/ml cascade blue dextran for 1 h, chased for 5 h, and treated with EBSS for 4 h, 0.1 µM bafilomycin A1 or 10 µM niclosamide for 1 h, 25 µM EIPA in the absence of NaHCO 3 or the presence of 50 mM propionate for 0.5 h, or with 1 mM LLOMe for 15 min. Histograms show mean lysosomal RFP intensities/cell (top) and distribution of lysosomal RFP intensities in a cell population (bottom). Representative images of live cells taken with 60× magnification using Zeiss LSM700 confocal microscope are shown on the right. Scale bar, 10 µm. b Representative immunoblots of LAMP1 and STAT3 in lysosomal lysates of HeLa cells left untreated or treated as in a , except for LLOMe treatment that was for 1 h. The histogram shows relative ratios of STAT3/LAMP1. Cytosolic acidification caused by these treatments is shown in Supplementary information, Fig. S6a . c Representative immunoblots of the indicated proteins in lysates of HeLa cells treated as in b . The histogram shows relative ratios of P-Y705-STAT3 and P-S727-STAT3. d Representative immunoblots of the indicated proteins in lysates of HeLa cells treated with 25 µM EIPA + 50 mM propionate for the indicated times. CCND1, cyclin D1; BIRC5, survivin. n = 3. e Quantitative PCR analysis of CCND1 mRNA levels in HeLa cells left untreated or treated with 25 µM EIPA + 50 mM propionate for 4 h. ACTA1 mRNA served as an internal control. f Quantification of STAT3/LAMP2 ratios in immunoblots of proteins from lysosomes immunopurified with anti-LAMP1. See Supplementary information, Fig. S6d for a representative blot. g Quantification of PLA (anti-STAT3 and anti-ATP6V1A) puncta in MCF7-vector and MCF7-p95DNErbB2 cells. Error bars, SD of ≥ 3 independent experiments. P -values were calculated by one-way ANOVA combined with Dunnett’s multiple comparisons test ( b , c , d , f and g ) or by two-tailed, homoscedastic Student’s t -test ( e ) in comparison with the untreated cells

    Journal: Cell Research

    Article Title: STAT3 associates with vacuolar H+-ATPase and regulates cytosolic and lysosomal pH

    doi: 10.1038/s41422-018-0080-0

    Figure Lengend Snippet: STAT3 regulates cytosolic pH. a Intensity of lysosomal RFP-STAT3 in A549-RFP-STAT3 cells loaded with 0.4 mg/ml cascade blue dextran for 1 h, chased for 5 h, and treated with EBSS for 4 h, 0.1 µM bafilomycin A1 or 10 µM niclosamide for 1 h, 25 µM EIPA in the absence of NaHCO 3 or the presence of 50 mM propionate for 0.5 h, or with 1 mM LLOMe for 15 min. Histograms show mean lysosomal RFP intensities/cell (top) and distribution of lysosomal RFP intensities in a cell population (bottom). Representative images of live cells taken with 60× magnification using Zeiss LSM700 confocal microscope are shown on the right. Scale bar, 10 µm. b Representative immunoblots of LAMP1 and STAT3 in lysosomal lysates of HeLa cells left untreated or treated as in a , except for LLOMe treatment that was for 1 h. The histogram shows relative ratios of STAT3/LAMP1. Cytosolic acidification caused by these treatments is shown in Supplementary information, Fig. S6a . c Representative immunoblots of the indicated proteins in lysates of HeLa cells treated as in b . The histogram shows relative ratios of P-Y705-STAT3 and P-S727-STAT3. d Representative immunoblots of the indicated proteins in lysates of HeLa cells treated with 25 µM EIPA + 50 mM propionate for the indicated times. CCND1, cyclin D1; BIRC5, survivin. n = 3. e Quantitative PCR analysis of CCND1 mRNA levels in HeLa cells left untreated or treated with 25 µM EIPA + 50 mM propionate for 4 h. ACTA1 mRNA served as an internal control. f Quantification of STAT3/LAMP2 ratios in immunoblots of proteins from lysosomes immunopurified with anti-LAMP1. See Supplementary information, Fig. S6d for a representative blot. g Quantification of PLA (anti-STAT3 and anti-ATP6V1A) puncta in MCF7-vector and MCF7-p95DNErbB2 cells. Error bars, SD of ≥ 3 independent experiments. P -values were calculated by one-way ANOVA combined with Dunnett’s multiple comparisons test ( b , c , d , f and g ) or by two-tailed, homoscedastic Student’s t -test ( e ) in comparison with the untreated cells

    Article Snippet: To estimate the cytosolic pH, cells washed with Live Cell Imaging Solution were incubated for 30 min in 37 °C in the same solution containing 1:1,000 dilution of pHrodo™ Green AM Intracellular pH Indicator and 1:100 dilution of PowerLoad™ concentrate, washed with Live Cell Imaging Solution, and analyzed by LSM700 confocal laser scanning microscope.

    Techniques: Microscopy, Western Blot, Real-time Polymerase Chain Reaction, Proximity Ligation Assay, Plasmid Preparation, Two Tailed Test

    STAT3 localizes to the lysosomal membrane. a Representative images of A549-RFP-STAT3 cells labeled with the indicated organelle markers (blue). Values, mean percentage of RFP-STAT3 puncta colocalizing with the indicated organelle marker ± SD of three independent experiments with ≥ 10 cells/sample analyzed in each. Colocalization analysis was not applicable (NA) in KDEL-BFP-labeled cells due to the diffuse staining. The areas marked with white squares are magnified in upper right corners. Images of live cells were taken with 60× magnification using Zeiss LSM700 confocal microscope. See Supplementary information, Fig. S1 for colocalization of STAT3 and lysosomes in other cells. b Representative immunoblots of STAT3 and the indicated organelle markers in total cell lysates or the indicated flow throughs (FT) and immunoprecipitates (eluate) from HeLa cells. n = 3. c Representative immunoblots of the indicated proteins in total cell lysates or the indicated fractions of HeLa cells (left) and quantification of P-Y705-STAT3 and P-S727-STAT3 levels relative to total STAT3. d Representative immunoblots (top) and quantification (bottom) of the indicated proteins in lysates of HeLa cell lysosomes purified by iron-dextran method and left untreated or treated with 10 µg/ml proteinase K and 100 µg/ml digitonin for 10 min at 25 °C when indicated. CTSD, cathepsin D. e Representative images (left) and quantification of cytosolic RFP-STAT3 puncta (right) in A549-RFP-STAT3 cells left untreated or treated with 100 ng/mL IL6 for 30 min and stained with Hoechst. Error bars, SD of three independent experiments, with ≥ 10 cells analyzed/sample. P - values were calculated by one-way ANOVA combined with Dunnett’s multiple comparisons test ( c ) or two-tailed, homoscedastic Student’s t -test ( e ). The optimal slice thickness (∼350 nm) of confocal images ( a , e ) was defined by the Zeiss zen software. Scale bar, 10 µm

    Journal: Cell Research

    Article Title: STAT3 associates with vacuolar H+-ATPase and regulates cytosolic and lysosomal pH

    doi: 10.1038/s41422-018-0080-0

    Figure Lengend Snippet: STAT3 localizes to the lysosomal membrane. a Representative images of A549-RFP-STAT3 cells labeled with the indicated organelle markers (blue). Values, mean percentage of RFP-STAT3 puncta colocalizing with the indicated organelle marker ± SD of three independent experiments with ≥ 10 cells/sample analyzed in each. Colocalization analysis was not applicable (NA) in KDEL-BFP-labeled cells due to the diffuse staining. The areas marked with white squares are magnified in upper right corners. Images of live cells were taken with 60× magnification using Zeiss LSM700 confocal microscope. See Supplementary information, Fig. S1 for colocalization of STAT3 and lysosomes in other cells. b Representative immunoblots of STAT3 and the indicated organelle markers in total cell lysates or the indicated flow throughs (FT) and immunoprecipitates (eluate) from HeLa cells. n = 3. c Representative immunoblots of the indicated proteins in total cell lysates or the indicated fractions of HeLa cells (left) and quantification of P-Y705-STAT3 and P-S727-STAT3 levels relative to total STAT3. d Representative immunoblots (top) and quantification (bottom) of the indicated proteins in lysates of HeLa cell lysosomes purified by iron-dextran method and left untreated or treated with 10 µg/ml proteinase K and 100 µg/ml digitonin for 10 min at 25 °C when indicated. CTSD, cathepsin D. e Representative images (left) and quantification of cytosolic RFP-STAT3 puncta (right) in A549-RFP-STAT3 cells left untreated or treated with 100 ng/mL IL6 for 30 min and stained with Hoechst. Error bars, SD of three independent experiments, with ≥ 10 cells analyzed/sample. P - values were calculated by one-way ANOVA combined with Dunnett’s multiple comparisons test ( c ) or two-tailed, homoscedastic Student’s t -test ( e ). The optimal slice thickness (∼350 nm) of confocal images ( a , e ) was defined by the Zeiss zen software. Scale bar, 10 µm

    Article Snippet: To estimate the cytosolic pH, cells washed with Live Cell Imaging Solution were incubated for 30 min in 37 °C in the same solution containing 1:1,000 dilution of pHrodo™ Green AM Intracellular pH Indicator and 1:100 dilution of PowerLoad™ concentrate, washed with Live Cell Imaging Solution, and analyzed by LSM700 confocal laser scanning microscope.

    Techniques: Labeling, Marker, Staining, Microscopy, Western Blot, Flow Cytometry, Purification, Two Tailed Test, Software

    STAT3 regulates lysosomal pH and activity. a Lysosomal pH determined by FITC/TMR ratio in a HeLa CRISPR control cell clone (C-4), STAT3-KO clones (KO-1 and −11), and KO-11 clone reconstituted with wild-type (WT) or mutated (Y705F, DBM, S727A) STAT3 constructs. Representative immunoblots show STAT3 and ACTB (loading control) protein levels in the clones. Standard curve for pH measurements is shown in Supplementary information, Fig. S4a . b Lysosomal pH determined as in a in CRISPR control and STAT3-KO HMF3 and H6C7 cells. Standard curve for pH measurements is shown in Supplementary information, Fig. S4a . c Volume of acidic compartment (VAC) in HeLa cell clones described in a analyzed by flow cytometer after 5 min staining with 75 nM Lysotracker Green. Relative fluorescence intensities are shown on the left. A representative flow cytometry profile is shown on the right. For other flow cytometry profiles and gating of the cells, see Supplementary information, Fig. S4c and d . d Representative immunoblots of LAMP1, STAT3, and cathepsin B (CTSB) in lysosomal lysates of the indicated HeLa cell clones. The histogram shows ratios between the active (25 kDa) and inactive (31 kDa) CTSB as percentages of the value in C4 control clone. e AlexaFluor 488-Dextran degradation in the indicated HeLa clones loaded with 0.4 mg/ml AlexaFluor 488-dextran for 20 min, washed, and fixed with or without a 4 h chase period. Representative images taken with 60× magnification using Zeiss LSM700 confocal microscope are shown on the right. Error bars, SD of ≥ 3 independent experiments. A minimum of 10 cells/sample were analyzed in a , b , and e . P -values were calculated by one-way ANOVA combined with Dunnett’s multiple comparisons test ( a , c ) or two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli ( b , d ) for multiple comparisons, or by two-tailed, homoscedastic Student’s t -test ( e )

    Journal: Cell Research

    Article Title: STAT3 associates with vacuolar H+-ATPase and regulates cytosolic and lysosomal pH

    doi: 10.1038/s41422-018-0080-0

    Figure Lengend Snippet: STAT3 regulates lysosomal pH and activity. a Lysosomal pH determined by FITC/TMR ratio in a HeLa CRISPR control cell clone (C-4), STAT3-KO clones (KO-1 and −11), and KO-11 clone reconstituted with wild-type (WT) or mutated (Y705F, DBM, S727A) STAT3 constructs. Representative immunoblots show STAT3 and ACTB (loading control) protein levels in the clones. Standard curve for pH measurements is shown in Supplementary information, Fig. S4a . b Lysosomal pH determined as in a in CRISPR control and STAT3-KO HMF3 and H6C7 cells. Standard curve for pH measurements is shown in Supplementary information, Fig. S4a . c Volume of acidic compartment (VAC) in HeLa cell clones described in a analyzed by flow cytometer after 5 min staining with 75 nM Lysotracker Green. Relative fluorescence intensities are shown on the left. A representative flow cytometry profile is shown on the right. For other flow cytometry profiles and gating of the cells, see Supplementary information, Fig. S4c and d . d Representative immunoblots of LAMP1, STAT3, and cathepsin B (CTSB) in lysosomal lysates of the indicated HeLa cell clones. The histogram shows ratios between the active (25 kDa) and inactive (31 kDa) CTSB as percentages of the value in C4 control clone. e AlexaFluor 488-Dextran degradation in the indicated HeLa clones loaded with 0.4 mg/ml AlexaFluor 488-dextran for 20 min, washed, and fixed with or without a 4 h chase period. Representative images taken with 60× magnification using Zeiss LSM700 confocal microscope are shown on the right. Error bars, SD of ≥ 3 independent experiments. A minimum of 10 cells/sample were analyzed in a , b , and e . P -values were calculated by one-way ANOVA combined with Dunnett’s multiple comparisons test ( a , c ) or two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli ( b , d ) for multiple comparisons, or by two-tailed, homoscedastic Student’s t -test ( e )

    Article Snippet: To estimate the cytosolic pH, cells washed with Live Cell Imaging Solution were incubated for 30 min in 37 °C in the same solution containing 1:1,000 dilution of pHrodo™ Green AM Intracellular pH Indicator and 1:100 dilution of PowerLoad™ concentrate, washed with Live Cell Imaging Solution, and analyzed by LSM700 confocal laser scanning microscope.

    Techniques: Activity Assay, CRISPR, Clone Assay, Construct, Western Blot, Flow Cytometry, Cytometry, Staining, Fluorescence, Microscopy, Two Tailed Test

    STAT3 interacts with V-ATPase via its coiled-coil domain. a Domain structure of STAT3 with mutations used in b indicated below. SH2, Src homology 2 domain; TAD, transactivation domain. b Quantification of PLA (anti-Flag and anti-ATP6V1A) puncta in HeLa cells expressing the indicated Flag-tagged STAT3 constructs (top). Error bars, SD of three independent experiments with ≥ 10 cells analyzed/sample. P -values were calculated by one-way ANOVA combined with Dunnett’s multiple comparisons test. Rough estimates of the relative expression levels of Flag-tagged STAT3 constructs are shown below the histogram as percentages of the expression of the wild-type STAT3. See Supplementary information, Fig. S3b for representative immunoblots. Bottom, representative images of PLAs taken with 60× magnification using Zeiss LSM700 confocal microscope. The optimal slice thickness (∼350 nm) was defined by the Zeiss zen software. Lysosomes were visualized with cascade blue dextran. Scale bar, 10 µm. c Representative immunoblots of the indicated proteins in total cell lysates or the indicated fractions of HeLa-STAT3-KO cells reconstituted with STAT3-Y705F (20 µg protein/lane). n = 3

    Journal: Cell Research

    Article Title: STAT3 associates with vacuolar H+-ATPase and regulates cytosolic and lysosomal pH

    doi: 10.1038/s41422-018-0080-0

    Figure Lengend Snippet: STAT3 interacts with V-ATPase via its coiled-coil domain. a Domain structure of STAT3 with mutations used in b indicated below. SH2, Src homology 2 domain; TAD, transactivation domain. b Quantification of PLA (anti-Flag and anti-ATP6V1A) puncta in HeLa cells expressing the indicated Flag-tagged STAT3 constructs (top). Error bars, SD of three independent experiments with ≥ 10 cells analyzed/sample. P -values were calculated by one-way ANOVA combined with Dunnett’s multiple comparisons test. Rough estimates of the relative expression levels of Flag-tagged STAT3 constructs are shown below the histogram as percentages of the expression of the wild-type STAT3. See Supplementary information, Fig. S3b for representative immunoblots. Bottom, representative images of PLAs taken with 60× magnification using Zeiss LSM700 confocal microscope. The optimal slice thickness (∼350 nm) was defined by the Zeiss zen software. Lysosomes were visualized with cascade blue dextran. Scale bar, 10 µm. c Representative immunoblots of the indicated proteins in total cell lysates or the indicated fractions of HeLa-STAT3-KO cells reconstituted with STAT3-Y705F (20 µg protein/lane). n = 3

    Article Snippet: To estimate the cytosolic pH, cells washed with Live Cell Imaging Solution were incubated for 30 min in 37 °C in the same solution containing 1:1,000 dilution of pHrodo™ Green AM Intracellular pH Indicator and 1:100 dilution of PowerLoad™ concentrate, washed with Live Cell Imaging Solution, and analyzed by LSM700 confocal laser scanning microscope.

    Techniques: Proximity Ligation Assay, Expressing, Construct, Western Blot, Microscopy, Software

    Live-cell imaging of IpLITR 1.1b-mediated target interactions. Rat basophilic leukemia-2H3 cells (3 × 10 5 ) stably co-expressing IpLITR 1.1b and LifeAct-GFP were incubated at 37°C with 9 × 10 5 αHA monoclonal antibody-coated 4.5 µm microspheres. Immediately after the addition of target beads, images were collected at 10 s intervals for ~8 min using a Zeiss LSM 710 laser scanning confocal microscope (objective 60×, 1.3 oil plan-Apochromat; Munich, Germany). Both the brightfield-LifeAct-GFP merged views (top panels) and the LifeAct-GFP views alone (bottom panels) are shown for two representative (A,B) IpLITR 1.1b-mediated target interactions with the location of the target microsphere indicated with an asterisk. Representative time-stamps in (A,B) were extracted from Videos S6 and S8 in Presentation 2 of Supplementary Material, respectively.

    Journal: Frontiers in Immunology

    Article Title: Selective Regulation of Cytoskeletal Dynamics and Filopodia Formation by Teleost Leukocyte Immune-Type Receptors Differentially Contributes to Target Capture During the Phagocytic Process

    doi: 10.3389/fimmu.2018.01144

    Figure Lengend Snippet: Live-cell imaging of IpLITR 1.1b-mediated target interactions. Rat basophilic leukemia-2H3 cells (3 × 10 5 ) stably co-expressing IpLITR 1.1b and LifeAct-GFP were incubated at 37°C with 9 × 10 5 αHA monoclonal antibody-coated 4.5 µm microspheres. Immediately after the addition of target beads, images were collected at 10 s intervals for ~8 min using a Zeiss LSM 710 laser scanning confocal microscope (objective 60×, 1.3 oil plan-Apochromat; Munich, Germany). Both the brightfield-LifeAct-GFP merged views (top panels) and the LifeAct-GFP views alone (bottom panels) are shown for two representative (A,B) IpLITR 1.1b-mediated target interactions with the location of the target microsphere indicated with an asterisk. Representative time-stamps in (A,B) were extracted from Videos S6 and S8 in Presentation 2 of Supplementary Material, respectively.

    Article Snippet: Immediately after the addition of target beads, images were collected at 10 s intervals for ~8 min using a Zeiss LSM 710 laser scanning confocal microscope.

    Techniques: Live Cell Imaging, Stable Transfection, Expressing, Incubation, Microscopy

    Live-cell imaging of IpLITR 2.6b/IpFcRγ-L-mediated phagocytosis. Rat basophilic leukemia-2H3 cells (3 × 10 5 ) stably co-expressing IpLITR 2.6b/IpFcRγ-L and LifeAct-GFP were incubated at 37°C with 9 × 10 5 αHA monoclonal antibody-coated 4.5 µm microspheres. Immediately after the addition of target beads, images were collected at 10 s intervals for ~8 min using a Zeiss LSM 710 laser scanning confocal microscope (objective 60×, 1.3 oil plan-Apochromat; Munich, Germany). Both the brightfield-LifeAct-GFP merged views (top panels) and the LifeAct-GFP views alone (bottom panels) are shown with the location of the target microsphere indicated with an asterisk. Representative time-stamps were extracted from Video S1 in Presentation 2 of Supplementary Material.

    Journal: Frontiers in Immunology

    Article Title: Selective Regulation of Cytoskeletal Dynamics and Filopodia Formation by Teleost Leukocyte Immune-Type Receptors Differentially Contributes to Target Capture During the Phagocytic Process

    doi: 10.3389/fimmu.2018.01144

    Figure Lengend Snippet: Live-cell imaging of IpLITR 2.6b/IpFcRγ-L-mediated phagocytosis. Rat basophilic leukemia-2H3 cells (3 × 10 5 ) stably co-expressing IpLITR 2.6b/IpFcRγ-L and LifeAct-GFP were incubated at 37°C with 9 × 10 5 αHA monoclonal antibody-coated 4.5 µm microspheres. Immediately after the addition of target beads, images were collected at 10 s intervals for ~8 min using a Zeiss LSM 710 laser scanning confocal microscope (objective 60×, 1.3 oil plan-Apochromat; Munich, Germany). Both the brightfield-LifeAct-GFP merged views (top panels) and the LifeAct-GFP views alone (bottom panels) are shown with the location of the target microsphere indicated with an asterisk. Representative time-stamps were extracted from Video S1 in Presentation 2 of Supplementary Material.

    Article Snippet: Immediately after the addition of target beads, images were collected at 10 s intervals for ~8 min using a Zeiss LSM 710 laser scanning confocal microscope.

    Techniques: Live Cell Imaging, Stable Transfection, Expressing, Incubation, Microscopy

    Live-cell imaging of IpLITR 2.6b/IpFcRγ-L-mediated phagocytosis at different incubation temperatures. Rat basophilic leukemia-2H3 cells (3 × 10 5 ) stably co-expressing IpLITR 2.6b/IpFcRγ-L and LifeAct-GFP were incubated at 37°C (A) or at 27°C (B) with 9 × 10 5 αHA monoclonal antibody-coated 4.5 µm bright blue microspheres. Immediately after the addition of target beads, images were collected at 10 s intervals for ~8 min using a Zeiss LSM 710 laser scanning confocal microscope (objective 60×, 1.3 oil plan-Apochromat; Munich, Germany). Representative time-stamps in (A) were extracted from Video S3 in Presentation 2 of Supplementary Material and the time-stamps in (B) were from Video S4 in Presentation 2 of Supplementary Material. In (A) , the target microsphere of interest is indicated with an arrowhead.

    Journal: Frontiers in Immunology

    Article Title: Selective Regulation of Cytoskeletal Dynamics and Filopodia Formation by Teleost Leukocyte Immune-Type Receptors Differentially Contributes to Target Capture During the Phagocytic Process

    doi: 10.3389/fimmu.2018.01144

    Figure Lengend Snippet: Live-cell imaging of IpLITR 2.6b/IpFcRγ-L-mediated phagocytosis at different incubation temperatures. Rat basophilic leukemia-2H3 cells (3 × 10 5 ) stably co-expressing IpLITR 2.6b/IpFcRγ-L and LifeAct-GFP were incubated at 37°C (A) or at 27°C (B) with 9 × 10 5 αHA monoclonal antibody-coated 4.5 µm bright blue microspheres. Immediately after the addition of target beads, images were collected at 10 s intervals for ~8 min using a Zeiss LSM 710 laser scanning confocal microscope (objective 60×, 1.3 oil plan-Apochromat; Munich, Germany). Representative time-stamps in (A) were extracted from Video S3 in Presentation 2 of Supplementary Material and the time-stamps in (B) were from Video S4 in Presentation 2 of Supplementary Material. In (A) , the target microsphere of interest is indicated with an arrowhead.

    Article Snippet: Immediately after the addition of target beads, images were collected at 10 s intervals for ~8 min using a Zeiss LSM 710 laser scanning confocal microscope.

    Techniques: Live Cell Imaging, Incubation, Stable Transfection, Expressing, Microscopy

    Live-cell imaging of IpLITR 1.1b-mediated target interactions at different incubation temperatures. Rat basophilic leukemia-2H3 cells (3 × 10 5 ) stably co-expressing IpLITR 1.1b and LifeAct-GFP were incubated at 37°C (A,B) or at 27°C (C–E) with 9 × 10 5 αHA monoclonal antibody-coated 4.5 µm bright blue microspheres. Immediately after the addition of target beads, images were collected at 10 s intervals for ~8 min using a Zeiss LSM 710 laser scanning confocal microscope (objective 60×, 1.3 oil plan-Apochromat; Munich, Germany). Representative time-stamps in (A,B) were extracted from Videos S12 in Presentation 2 and S15 in Presentation 3 of Supplementary Material, respectively, and the time-stamps in (C–E) were from Videos S16 – S18 in Presentation 3 of Supplementary Material, respectively. In all time-stamps, target beads are indicated with an arrowhead and in (b; 130 s) a second cell with a pre-captured target bead is indicated with an arrow.

    Journal: Frontiers in Immunology

    Article Title: Selective Regulation of Cytoskeletal Dynamics and Filopodia Formation by Teleost Leukocyte Immune-Type Receptors Differentially Contributes to Target Capture During the Phagocytic Process

    doi: 10.3389/fimmu.2018.01144

    Figure Lengend Snippet: Live-cell imaging of IpLITR 1.1b-mediated target interactions at different incubation temperatures. Rat basophilic leukemia-2H3 cells (3 × 10 5 ) stably co-expressing IpLITR 1.1b and LifeAct-GFP were incubated at 37°C (A,B) or at 27°C (C–E) with 9 × 10 5 αHA monoclonal antibody-coated 4.5 µm bright blue microspheres. Immediately after the addition of target beads, images were collected at 10 s intervals for ~8 min using a Zeiss LSM 710 laser scanning confocal microscope (objective 60×, 1.3 oil plan-Apochromat; Munich, Germany). Representative time-stamps in (A,B) were extracted from Videos S12 in Presentation 2 and S15 in Presentation 3 of Supplementary Material, respectively, and the time-stamps in (C–E) were from Videos S16 – S18 in Presentation 3 of Supplementary Material, respectively. In all time-stamps, target beads are indicated with an arrowhead and in (b; 130 s) a second cell with a pre-captured target bead is indicated with an arrow.

    Article Snippet: Immediately after the addition of target beads, images were collected at 10 s intervals for ~8 min using a Zeiss LSM 710 laser scanning confocal microscope.

    Techniques: Live Cell Imaging, Incubation, Stable Transfection, Expressing, Microscopy