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Carl Zeiss confocal microscope
Confocal Microscope, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 99/100, based on 9214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/confocal microscope/product/Carl Zeiss
Average 99 stars, based on 9214 article reviews
Price from $9.99 to $1999.99
confocal microscope - by Bioz Stars, 2020-09
99/100 stars

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Related Articles

Microscopy:

Article Title: Notoginsenoside Fc Accelerates Reendothelialization following Vascular Injury in Diabetic Rats by Promoting Endothelial Cell Autophagy
Article Snippet: .. Samples were observed and photographed with a confocal microscope (LSM 710 META; Zeiss, Oberkochen, Germany). ..

Article Title: PNPLA3, CGI‐58, and Inhibition of Hepatic Triglyceride Hydrolysis in Mice
Article Snippet: .. Cells on coverslips were rinsed two to three times with PBS, immersed in a drop of Vectashield antifade mounting medium (H‐1400, Vector Laboratories, Burlingame, CA), and visualized using a confocal microscope (Leica TCS SP5 and Zeiss LSM 880). .. For Oil Red O staining, liver samples were fixed in 4% PFA and neutral lipids were visualized using light microscopy (DM2000, Leica) as described.

Article Title: Controlled Release of Vancomycin From a Thermoresponsive Hydrogel System for the Prophylactic Treatment of Postoperative Acute Endophthalmitis
Article Snippet: .. Each well was imaged (10×, 1.79 microns/pixel resolution) using a confocal microscope (FITc/TRITc; 488 nm/543 nm) (LSM 5 Pascal, Zeiss Microscopy, Thornwood, NY). .. Cells appearing green (alive) were quantified using the ImageJ cell counting software and compared with the red cells (dead) to determine percent cell viability.

Article Title: Differentiation of Embryonic Stem Cells 1 (Dies1) Is a Component of Bone Morphogenetic Protein 4 (BMP4) Signaling Pathway Required for Proper Differentiation of Mouse Embryonic Stem Cells *
Article Snippet: .. Images were captured with an inverted microscope (DMI4000, Leica Microsystems) and with a confocal microscope (LSM 510 META, Zeiss). .. Nude mice were injected subcutaneously with 2 × 106 ESC cells transfected with Dies1 shRNA (left side) or NS shRNA (right side).

Article Title: Regulation of Cigarette Smoke (CS)-Induced Autophagy by Nrf2
Article Snippet: .. Fluorescence Images were acquired by using of a confocal microscope (LSM 510 meta, Carl Zeiss, Thornwood, NY). ..

Article Title: Bcl-2-functionalized ultrasmall superparamagnetic iron oxide nanoparticles coated with amphiphilic polymer enhance the labeling efficiency of islets for detection by magnetic resonance imaging
Article Snippet: .. Observation and image acquisition were performed with a confocal microscope (LSM 710 system, Carl Zeiss Meditec, Jena, Germany). .. In vitro Prussian blue staining and in vivo immunohistochemistry of Bcl-2-USPIO-labeled primary islet cells The primary islet cells that were isolated from the ICR mice, as described above, were labeled with Bcl-2-USPIO (10 or 30 μg/mL Fe concentration) and cultured in complete RPMI 1640 medium for 24 hours.

Article Title: New insights into Fe localization in plant tissues
Article Snippet: .. Laser scanning confocal microscopy The microscope imaging was performed in Montpellier RIO Imaging Platform ( http://www/mri/cnrs.fr ) with a confocal microscope (LSM 510, Meta; Carl Zeiss MicroImaging, http://www.zeiss.de ). .. An Argon laser at 488 nm provided excitation for the Alexa 488, and 405 nm was used for DAPI staining.

Article Title: The scaffold protein EPG-7 links cargo-receptor complexes with the autophagic assembly machinery
Article Snippet: .. Slides were viewed using a confocal microscope (LSM 510 Meta; Carl Zeiss) with a 63×/1.40 oil-immersion objective lens (Plan-Apochromat; Carl Zeiss) and a camera (AxioCam HRm; Carl Zeiss) at RT. .. Images were processed and viewed using LSM Image Browser software (Carl Zeiss).

Inverted Microscopy:

Article Title: Differentiation of Embryonic Stem Cells 1 (Dies1) Is a Component of Bone Morphogenetic Protein 4 (BMP4) Signaling Pathway Required for Proper Differentiation of Mouse Embryonic Stem Cells *
Article Snippet: .. Images were captured with an inverted microscope (DMI4000, Leica Microsystems) and with a confocal microscope (LSM 510 META, Zeiss). .. Nude mice were injected subcutaneously with 2 × 106 ESC cells transfected with Dies1 shRNA (left side) or NS shRNA (right side).

Confocal Microscopy:

Article Title: New insights into Fe localization in plant tissues
Article Snippet: .. Laser scanning confocal microscopy The microscope imaging was performed in Montpellier RIO Imaging Platform ( http://www/mri/cnrs.fr ) with a confocal microscope (LSM 510, Meta; Carl Zeiss MicroImaging, http://www.zeiss.de ). .. An Argon laser at 488 nm provided excitation for the Alexa 488, and 405 nm was used for DAPI staining.

Imaging:

Article Title: New insights into Fe localization in plant tissues
Article Snippet: .. Laser scanning confocal microscopy The microscope imaging was performed in Montpellier RIO Imaging Platform ( http://www/mri/cnrs.fr ) with a confocal microscope (LSM 510, Meta; Carl Zeiss MicroImaging, http://www.zeiss.de ). .. An Argon laser at 488 nm provided excitation for the Alexa 488, and 405 nm was used for DAPI staining.

Fluorescence:

Article Title: Regulation of Cigarette Smoke (CS)-Induced Autophagy by Nrf2
Article Snippet: .. Fluorescence Images were acquired by using of a confocal microscope (LSM 510 meta, Carl Zeiss, Thornwood, NY). ..

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  • 90
    Carl Zeiss lsm 700 inverted confocal microscope
    Confocal images of transgenic OPA1wt and OPA1 Q285STOP mouse retina sections expressing LC3-GFP and RedMIT Representative images of the retina from the RedMIT-GFP-LC3-OPA1 +/+ (Left) and the RedMIT-GFP-LC3-OPA1 Q285STOP (Right) mice sections show autophagosomes with GFP-tagged LC3 and RedMIT tagged mitochondria. Sections were cut at 10 μm and visualized using a Zeiss <t>LSM</t> 700 inverted confocal microscope with a plan-Apo 63x NA 1.4 oil-immersion objective. Red boxes indicate the area magnified in each inset. RedMIT were observed from the ONL toward the IPL in both OPA1 wt and OPA1 Q285STOP . No difference in GFP-LC3 or mitochondrial mRFP is seen between OPA wild type and mutant (zoomed insets, bottom panels). In particular, no colocalization was observed between GFP-LC3 and RedMIT in both OPA1 wt and OPA1 Q285STOP (Pearson correlation coefficient of +0.22 and −0.16; n = 4). Scale bars: 20 μm OS, outer segments; IS, inner segments; ONL, outer nucleus layer; OPL, outer plexiform layer; INL, inner nucleus layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
    Lsm 700 Inverted Confocal Microscope, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 90/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lsm 700 inverted confocal microscope/product/Carl Zeiss
    Average 90 stars, based on 41 article reviews
    Price from $9.99 to $1999.99
    lsm 700 inverted confocal microscope - by Bioz Stars, 2020-09
    90/100 stars
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    92
    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
    Average 92 stars, based on 190 article reviews
    Price from $9.99 to $1999.99
    lsm700 confocal laser scanning microscope - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    92
    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
    Price from $9.99 to $1999.99
    lsm 710 laser scanning confocal microscope - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    Confocal images of transgenic OPA1wt and OPA1 Q285STOP mouse retina sections expressing LC3-GFP and RedMIT Representative images of the retina from the RedMIT-GFP-LC3-OPA1 +/+ (Left) and the RedMIT-GFP-LC3-OPA1 Q285STOP (Right) mice sections show autophagosomes with GFP-tagged LC3 and RedMIT tagged mitochondria. Sections were cut at 10 μm and visualized using a Zeiss LSM 700 inverted confocal microscope with a plan-Apo 63x NA 1.4 oil-immersion objective. Red boxes indicate the area magnified in each inset. RedMIT were observed from the ONL toward the IPL in both OPA1 wt and OPA1 Q285STOP . No difference in GFP-LC3 or mitochondrial mRFP is seen between OPA wild type and mutant (zoomed insets, bottom panels). In particular, no colocalization was observed between GFP-LC3 and RedMIT in both OPA1 wt and OPA1 Q285STOP (Pearson correlation coefficient of +0.22 and −0.16; n = 4). Scale bars: 20 μm OS, outer segments; IS, inner segments; ONL, outer nucleus layer; OPL, outer plexiform layer; INL, inner nucleus layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Validating the RedMIT/GFP-LC3 Mouse Model by Studying Mitophagy in Autosomal Dominant Optic Atrophy Due to the OPA1Q285STOP Mutation

    doi: 10.3389/fcell.2018.00103

    Figure Lengend Snippet: Confocal images of transgenic OPA1wt and OPA1 Q285STOP mouse retina sections expressing LC3-GFP and RedMIT Representative images of the retina from the RedMIT-GFP-LC3-OPA1 +/+ (Left) and the RedMIT-GFP-LC3-OPA1 Q285STOP (Right) mice sections show autophagosomes with GFP-tagged LC3 and RedMIT tagged mitochondria. Sections were cut at 10 μm and visualized using a Zeiss LSM 700 inverted confocal microscope with a plan-Apo 63x NA 1.4 oil-immersion objective. Red boxes indicate the area magnified in each inset. RedMIT were observed from the ONL toward the IPL in both OPA1 wt and OPA1 Q285STOP . No difference in GFP-LC3 or mitochondrial mRFP is seen between OPA wild type and mutant (zoomed insets, bottom panels). In particular, no colocalization was observed between GFP-LC3 and RedMIT in both OPA1 wt and OPA1 Q285STOP (Pearson correlation coefficient of +0.22 and −0.16; n = 4). Scale bars: 20 μm OS, outer segments; IS, inner segments; ONL, outer nucleus layer; OPL, outer plexiform layer; INL, inner nucleus layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

    Article Snippet: Cryostat eye sections were cut at 10 μm and examined using a Zeiss LSM 700 inverted confocal microscope with a plan-Apo 63x NA 1.4 oil-immersion objective.

    Techniques: Transgenic Assay, Expressing, Mouse Assay, Microscopy, Mutagenesis

    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