monodansylcadaverine mdc Millipore Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore monodansylcadaverine mdc
    <t>Monodansylcadaverine</t> <t>(MDC)</t> stains autophagic K562 cells with sensitivity equivalent to LC3B immunoblotting and GFP-LC3B fluorescence microscopy. ( A ) Fluorescence microscopy. K562 cells were treated with dimethyl sulfoxide (DMSO) or 1 μM imatinib (IM) overnight followed by MDC staining. Images of live cells were taken at different exposure times (milliseconds, ms) using an inverted fluorescence microscope. Scale bar: 25 μm. ( B ) MDC Fluorescence spectrophotometry. K562 cells treated with DMSO or IM were stained with MDC. MDC fluorescence of live K562 cells was quantified using a micro-plate reader at an excitation wavelength of 335 nm and an emission wavelength of 525 nm. The relative MDC levels were obtained by dividing MDC fluorescence of IM-treated cells to that of DMSO-treated cells. Error bars represent three independent experiments. ( C ) LC3B immunoblotting. Cropped images are shown and full images are included in supplemental materials. ACTB (β actin) is the loading control. The intensities of LC3B-II or ACTB were quantified using Image J. The fold changes of LC3B-II/ACTB were obtained by dividing the ratio of LC3B-II/ACTB in IM-treated cells with that of DMSO-treated cells. ( D ) MDC staining of K562 cells treated with IM and/or bafilomycin A1 (BFA1). K562 cells were treated with a combination of 1 μM IM (10 hours) and 5 nM of BFA1 (48 hours). Cells were imaged using a fluorescence confocal microscope. ( E ) Quantification of MDC intensities. Fluorescence intensities of MDC in 10 different cells from three images were quantified using Image J. ( F ) GFP-LC3B fluorescence microscopy. K562 cells were stably transfected with a construct encoding GFP-LC3B. Cells were treated with a combination of IM and BFA1 and then imaged. ( G ) Quantification of cells with GFP-LC3B puncta. Four to five images of more than thirty K562 cells from each treatment were randomly selected for the following quantification analyses. GFP-expressing K562 cells or K562 cells with GFP puncta (indicating autophagy) were counted by three persons. Percentages of K562 cells with puncta were obtained by dividing numbers of K562 cells with GFP puncta with those of GFP-expressing cells. Scale bar: 25 nm.
    Monodansylcadaverine Mdc, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 908 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monodansylcadaverine mdc/product/Millipore
    Average 99 stars, based on 908 article reviews
    Price from $9.99 to $1999.99
    monodansylcadaverine mdc - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore 3 methyladenine
    Effects of autophagy inhibitors on intracellular PNP content in A549 cells at 24 h post-exposure to PNP. Intracellular PNP content in A549 cells was reduced by 38% and 64%, respectively, when autophagosome formation was inhibited with <t>3-methyladenine</t> (3-MA) or autophagosome-lysosome fusion was inhibited with bafilomycin. Data are normalized to control. * p
    3 Methyladenine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4065 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/3 methyladenine/product/Millipore
    Average 99 stars, based on 4065 article reviews
    Price from $9.99 to $1999.99
    3 methyladenine - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore propidium iodide pi
    Effect of psoralidin in cell cycle distribution of A549 cells. (A) Cells were treated with 5, 10 and 20 µM psoralidin for 24 h, and then stained with <t>propidium</t> iodide. The DNA content was measured by flow cytometry. (B) The cell cycle distributions were analyzed and presented as mean ± SD of three independent experiments. ∗ P
    Propidium Iodide Pi, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16503 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/propidium iodide pi/product/Millipore
    Average 99 stars, based on 16503 article reviews
    Price from $9.99 to $1999.99
    propidium iodide pi - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore rapamycin
    The effect of chloroquine on autophagic flux in vivo. Transgenic mice expressing mCherry-LC3 mice were randomly assigned to be exposed to chloroquine (Cq, 10 mg/kg), or <t>rapamycin</t> (2 mg/kg) or rapamycin plus chloroquine by i.p. injection. (A) Representative photographs of myocardium in mCherry-LC3 mice 4 hr after the indicated treatments. (B) Represented is the percentage surface area of mCherry-LC3 fluorescence per microscopic field. Results are mean ± SE of three independent experiments. *p
    Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rapamycin/product/Millipore
    Average 99 stars, based on 13301 article reviews
    Price from $9.99 to $1999.99
    rapamycin - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore acridine orange ao
    The effect of chloroquine on autophagic flux in vivo. Transgenic mice expressing mCherry-LC3 mice were randomly assigned to be exposed to chloroquine (Cq, 10 mg/kg), or <t>rapamycin</t> (2 mg/kg) or rapamycin plus chloroquine by i.p. injection. (A) Representative photographs of myocardium in mCherry-LC3 mice 4 hr after the indicated treatments. (B) Represented is the percentage surface area of mCherry-LC3 fluorescence per microscopic field. Results are mean ± SE of three independent experiments. *p
    Acridine Orange Ao, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1099 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/acridine orange ao/product/Millipore
    Average 99 stars, based on 1099 article reviews
    Price from $9.99 to $1999.99
    acridine orange ao - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore dimethyl sulfoxide dmso
    <t>PTE</t> treatment induces autophagy. (A) Transmission electronic microscopy analyzed the autophagy-associated morphological change in RPMI-8226 cells following exposure to PTE. Upper panel, ×1,700 magnification; lower panel, ×5,000 magnification. (B) Monodansylcadaverine-labeled vacuoles were observed in RPMI-8226 cells using a fluorescence microscope. (C) RPMI-8226 and (D) ARH-77 cells were treated with <t>DMSO</t> (control) or different concentrations of PTE for 48 h, western blotting was applied to investigate the levels of the autophagy markers beclin1, ATG5 and LC3. β-actin was used as a loading control. (E) Effect of PTE treatment on RPMI-8226 cells with or without 3MA was analyzed by flow cytometry using an Annexin V-FITC/PI kit. * P
    Dimethyl Sulfoxide Dmso, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20238 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dimethyl sulfoxide dmso/product/Millipore
    Average 99 stars, based on 20238 article reviews
    Price from $9.99 to $1999.99
    dimethyl sulfoxide dmso - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore bafilomycin a1
    RhoA and ROCK1 are essential for ARV S1133-induced autophagy, apoptosis, and the conversion of autophagy to apoptosis. (A) Vero cells transfected with RhoA-T19N and incubated for 24 hr, before being transfected with siRNA for 72 hr, pre-treated with 5 μM Y-27632, 10 μM Z-VAD-FMK, 50 μM 3-MA and 0.1 μM <t>Bafilomycin</t> A1 at non-toxic concentrations for 4 hr, and then infected with ARV S1133 at an MOI of 5 for an additional 18-hr incubation. Autophagic vacuoles were stained with MDC, and the percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (black bar). Vero cells transfected with LC3-GFP, together with RhoA-T19N, and incubated for 24 hr, or transfected with siRNA for 48 hr then transfected with LC3-GFP, or pretreated with inhibitor for 4 hr then transfected with LC3-GFP. 24 hr after LC3-GFP transfection, ARV S1133 was added for an additional 18-hr incubation period. GFP-positive cells containing more than 3 dots were counted as positive LC3-GFP cells. The percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (white bar). (B) ARV S1133 infected cells at an MOI of 5 with an incubation period of 36 hr with identical treatment timings to those described in (A) . Three apoptosis assays were performed. The left y-axis represents the percentage of Hoechst 33258 positive cells and the caspase-3 activity in relative light units (RLUs). The right y-axis represents the OD 405 nm values from an apoptotic ELISA assay. All experiments were performed three times, each in duplicate. The data are presented as the mean ± SD. (C) Punctate dots of GFP indicating autophagosomes are shown by the white arrows (400x magnification).
    Bafilomycin A1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7507 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bafilomycin a1/product/Millipore
    Average 99 stars, based on 7507 article reviews
    Price from $9.99 to $1999.99
    bafilomycin a1 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore 3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide
    RhoA and ROCK1 are essential for ARV S1133-induced autophagy, apoptosis, and the conversion of autophagy to apoptosis. (A) Vero cells transfected with RhoA-T19N and incubated for 24 hr, before being transfected with siRNA for 72 hr, pre-treated with 5 μM Y-27632, 10 μM Z-VAD-FMK, 50 μM 3-MA and 0.1 μM <t>Bafilomycin</t> A1 at non-toxic concentrations for 4 hr, and then infected with ARV S1133 at an MOI of 5 for an additional 18-hr incubation. Autophagic vacuoles were stained with MDC, and the percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (black bar). Vero cells transfected with LC3-GFP, together with RhoA-T19N, and incubated for 24 hr, or transfected with siRNA for 48 hr then transfected with LC3-GFP, or pretreated with inhibitor for 4 hr then transfected with LC3-GFP. 24 hr after LC3-GFP transfection, ARV S1133 was added for an additional 18-hr incubation period. GFP-positive cells containing more than 3 dots were counted as positive LC3-GFP cells. The percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (white bar). (B) ARV S1133 infected cells at an MOI of 5 with an incubation period of 36 hr with identical treatment timings to those described in (A) . Three apoptosis assays were performed. The left y-axis represents the percentage of Hoechst 33258 positive cells and the caspase-3 activity in relative light units (RLUs). The right y-axis represents the OD 405 nm values from an apoptotic ELISA assay. All experiments were performed three times, each in duplicate. The data are presented as the mean ± SD. (C) Punctate dots of GFP indicating autophagosomes are shown by the white arrows (400x magnification).
    3 4 5 Dimethylthiazol 2 Yl 2 5 Diphenyltetrazolium Bromide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13080 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide/product/Millipore
    Average 99 stars, based on 13080 article reviews
    Price from $9.99 to $1999.99
    3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore wortmannin
    Effect of miR-497 mimic and sponge on the expression of Bcl2 and LC3B protein in neonatal rat cardiomyocytes (NRCs) a. NRCs were transfected with miR-497 mimic or negative control (NC). Western blot shows the effects of miR-497 overexpression on Bcl-2, Bax, LC3B-II and beclin-1. b. Quantitation for panel A. NRCs were transfected with miR-497 sponge or vector adenovirus. Western blot shows the protein expression of Bcl-2, Bax and LC3B-II c. beclin-1 and P62 d. which were significantly changed in miR-497 sponge-treated cells e, f. Western blotting of LC3B-II in NRCs treated with/without miR-497 sponge (S), vector (V), Ad-sh-beclin-1 (sh-b), Ad-sh-control (sh-c), bafilomycin A1 (Baf), <t>wortmannin</t> (W). Data are mean ± SEM, n = 5–7 for each group; for panel b and e, * P
    Wortmannin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9936 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/wortmannin/product/Millipore
    Average 99 stars, based on 9936 article reviews
    Price from $9.99 to $1999.99
    wortmannin - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore hoechst 33258
    Cell and nuclear morphology were visualized following PAB treatment. (A) After 24 and 48 h of PAB treatment, morphological changes in SW579 cells were visualized by phase contrast microscopy. (B) Cells were stained using <t>Hoechst</t> 33258 to observe nuclear changes and imaged via fluorescence microscopy. Data are representative of three individual experiments; n=3. Scale bar, 30 µ m. PAB, pseudolaric acid B; Con, control.
    Hoechst 33258, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16587 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hoechst 33258/product/Millipore
    Average 99 stars, based on 16587 article reviews
    Price from $9.99 to $1999.99
    hoechst 33258 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore cytochalasin d
    Differential endocytosis of primary and secondary CPP in human monocyte-derived macrophages and aortic endothelial cells. Human monocyte-derived macrophages (hMDM) or human aortic endothelial cells (haEC) were treated with Alexa488-labeled primary or secondary CPP, both 100 μg/mL calcium content, synthesized from FCS/DMEM supplemented with phosphate (final conc. 3.5 mM). Monomeric labeled-fetuin-A was included as a comparator (1 mg/ml). Cell-associated fluorescence was measured by flow cytometry in fixed cells at the stated timepoints.  (A)  Endocyosis observed in hMDM and  (B)  haEC, respectively.  (C,D)  hMDM were pre-treated for 30–60 min with one of several inhibitors (vs. vehicle) or receptor blocking antibodies (vs. isotype control).  (C)  Pre-treatment with cytochalasin  (D) , an inhibitor of actin polymerization, markedly attenuated uptake of both primary and secondary CPP and (
    Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cytochalasin d/product/Millipore
    Average 99 stars, based on 9195 article reviews
    Price from $9.99 to $1999.99
    cytochalasin d - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore rnase a
    Differential endocytosis of primary and secondary CPP in human monocyte-derived macrophages and aortic endothelial cells. Human monocyte-derived macrophages (hMDM) or human aortic endothelial cells (haEC) were treated with Alexa488-labeled primary or secondary CPP, both 100 μg/mL calcium content, synthesized from FCS/DMEM supplemented with phosphate (final conc. 3.5 mM). Monomeric labeled-fetuin-A was included as a comparator (1 mg/ml). Cell-associated fluorescence was measured by flow cytometry in fixed cells at the stated timepoints.  (A)  Endocyosis observed in hMDM and  (B)  haEC, respectively.  (C,D)  hMDM were pre-treated for 30–60 min with one of several inhibitors (vs. vehicle) or receptor blocking antibodies (vs. isotype control).  (C)  Pre-treatment with cytochalasin  (D) , an inhibitor of actin polymerization, markedly attenuated uptake of both primary and secondary CPP and (
    Rnase A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 29470 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase a/product/Millipore
    Average 99 stars, based on 29470 article reviews
    Price from $9.99 to $1999.99
    rnase a - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore filipin
    Caveolin-mediated endocytic mechanisms are implicated in internalization of rMGA_0676. (A) Internalization of rMGA_0676 was blocked by caveolin-mediated endocytic inhibitor <t>filipin.</t> DF-1 cells were treated with filipin followed by rMGA_0676 (40 μg/ml) for 24 h at 37°C to detect the inhibition of rMGA_0676 internalization. The samples underwent IFA and were examined with a laser confocal scanning microscope. Cellular F-actin was stained with Alexa Fluor 555-conjugated phalloidin. The nuclei were counterstained with DAPI (blue). The rMGA_0676 was hybridized with mouse anti-His monoclonal antibody and labeled with FITC-conjugated goat anti mouse IgG antibody green fluorescence (GF). The normal DF-1 cells as the negative control (Con), cells were treated with Cholera toxin-FITC as the caveolin-mediated endocytic positive control (Cholera toxin). (B) The mean value of GF was used to quantify the positive staining for rMGA_0676 in filipin treated DF-1 cells (A) , the data were analyzed using SPSS software, and the graph was made using GraphPad Prism 5.0. “ ** ”represented statistically significant differences ( P
    Filipin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2595 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/filipin/product/Millipore
    Average 99 stars, based on 2595 article reviews
    Price from $9.99 to $1999.99
    filipin - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore methyl β cyclodextrin mβcd
    Effect of endocytosis inhibition on the VEGF-A-regulated cleavage of Flt1. A–D : HEK293 cells ( A and B ) transfected with hemagglutinin (HA)-Flt1-Myc-Flag (Flt1) or FLT1ΔCTD (deleted COOH-terminal domain, ΔCTD) and AG1-G1-FLT1 ( C and D ) were incubated with and without 100 ng/ml VEGF-A for 24 h with dynasore (80 μM) or <t>methyl-β-cyclodextrin</t> <t>(MβCD;</t> 50 μg/ml) for 5 h and then immunoblotted for HA, tubulin, or AF321 (Flt1) antibodies. Full-length Flt1 (FL) and the NH 2 -terminal fragment (NTF) are seen in lysates and conditioned media (CM), respectively. Neither dynasore nor MβCD have an impact on VEGF-A mediated inhibition of FLT1 and FLT1ΔCTD cleavage. A representative immunoblot in seen in A and C , and the dynasore experiments were repeated, and the pooled NTF data quantified by densitometry is shown in panel B and D . Means ± SD, n = 3. * P
    Methyl β Cyclodextrin Mβcd, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 802 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/methyl β cyclodextrin mβcd/product/Millipore
    Average 99 stars, based on 802 article reviews
    Price from $9.99 to $1999.99
    methyl β cyclodextrin mβcd - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore z vad fmk
    RhoA and ROCK1 are essential for ARV S1133-induced autophagy, apoptosis, and the conversion of autophagy to apoptosis. (A) Vero cells transfected with RhoA-T19N and incubated for 24 hr, before being transfected with siRNA for 72 hr, pre-treated with 5 μM Y-27632, 10 μM <t>Z-VAD-FMK,</t> 50 μM 3-MA and 0.1 μM Bafilomycin A1 at non-toxic concentrations for 4 hr, and then infected with ARV S1133 at an MOI of 5 for an additional 18-hr incubation. Autophagic vacuoles were stained with MDC, and the percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (black bar). Vero cells transfected with LC3-GFP, together with RhoA-T19N, and incubated for 24 hr, or transfected with siRNA for 48 hr then transfected with LC3-GFP, or pretreated with inhibitor for 4 hr then transfected with LC3-GFP. 24 hr after LC3-GFP transfection, ARV S1133 was added for an additional 18-hr incubation period. GFP-positive cells containing more than 3 dots were counted as positive LC3-GFP cells. The percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (white bar). (B) ARV S1133 infected cells at an MOI of 5 with an incubation period of 36 hr with identical treatment timings to those described in (A) . Three apoptosis assays were performed. The left y-axis represents the percentage of Hoechst 33258 positive cells and the caspase-3 activity in relative light units (RLUs). The right y-axis represents the OD 405 nm values from an apoptotic ELISA assay. All experiments were performed three times, each in duplicate. The data are presented as the mean ± SD. (C) Punctate dots of GFP indicating autophagosomes are shown by the white arrows (400x magnification).
    Z Vad Fmk, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1921 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/z vad fmk/product/Millipore
    Average 99 stars, based on 1921 article reviews
    Price from $9.99 to $1999.99
    z vad fmk - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore hoechst 33342
    PD induces apoptosis in BEL-7402 cells. BEL-7402 cells were treated with different concentrations of PD for 24 h. (A) Apoptotic cells were detected by Annexin V/PI staining using flow cytometry. (B) Quantitation of the results obtained from (A). (C) The apoptotic cells were evaluated with <t>Hoechst</t> 33342 staining and imaged using the In Cell Analyzer 2000 System. (D) The expression of apoptosis-related proteins, including cleaved PARP, cleaved caspase-3, Bcl-2, and Bax, were analyzed by Western blot analysis. (E) Quantitation of the results obtained from (D). b P
    Hoechst 33342, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 27427 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hoechst 33342/product/Millipore
    Average 99 stars, based on 27427 article reviews
    Price from $9.99 to $1999.99
    hoechst 33342 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore 4 6 diamidino 2 phenylindole
    PD induces apoptosis in BEL-7402 cells. BEL-7402 cells were treated with different concentrations of PD for 24 h. (A) Apoptotic cells were detected by Annexin V/PI staining using flow cytometry. (B) Quantitation of the results obtained from (A). (C) The apoptotic cells were evaluated with <t>Hoechst</t> 33342 staining and imaged using the In Cell Analyzer 2000 System. (D) The expression of apoptosis-related proteins, including cleaved PARP, cleaved caspase-3, Bcl-2, and Bax, were analyzed by Western blot analysis. (E) Quantitation of the results obtained from (D). b P
    4 6 Diamidino 2 Phenylindole, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 25556 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/4 6 diamidino 2 phenylindole/product/Millipore
    Average 99 stars, based on 25556 article reviews
    Price from $9.99 to $1999.99
    4 6 diamidino 2 phenylindole - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore n acetylcysteine nac
    PD induces apoptosis in BEL-7402 cells. BEL-7402 cells were treated with different concentrations of PD for 24 h. (A) Apoptotic cells were detected by Annexin V/PI staining using flow cytometry. (B) Quantitation of the results obtained from (A). (C) The apoptotic cells were evaluated with <t>Hoechst</t> 33342 staining and imaged using the In Cell Analyzer 2000 System. (D) The expression of apoptosis-related proteins, including cleaved PARP, cleaved caspase-3, Bcl-2, and Bax, were analyzed by Western blot analysis. (E) Quantitation of the results obtained from (D). b P
    N Acetylcysteine Nac, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1554 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/n acetylcysteine nac/product/Millipore
    Average 99 stars, based on 1554 article reviews
    Price from $9.99 to $1999.99
    n acetylcysteine nac - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore triton x 100
    PD induces apoptosis in BEL-7402 cells. BEL-7402 cells were treated with different concentrations of PD for 24 h. (A) Apoptotic cells were detected by Annexin V/PI staining using flow cytometry. (B) Quantitation of the results obtained from (A). (C) The apoptotic cells were evaluated with <t>Hoechst</t> 33342 staining and imaged using the In Cell Analyzer 2000 System. (D) The expression of apoptosis-related proteins, including cleaved PARP, cleaved caspase-3, Bcl-2, and Bax, were analyzed by Western blot analysis. (E) Quantitation of the results obtained from (D). b P
    Triton X 100, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 65173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/triton x 100/product/Millipore
    Average 99 stars, based on 65173 article reviews
    Price from $9.99 to $1999.99
    triton x 100 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore 3 4 5 dimethyl 2 thiazolyl 2 5 diphenyl 2 h tetrazolium bromide
    PD induces apoptosis in BEL-7402 cells. BEL-7402 cells were treated with different concentrations of PD for 24 h. (A) Apoptotic cells were detected by Annexin V/PI staining using flow cytometry. (B) Quantitation of the results obtained from (A). (C) The apoptotic cells were evaluated with <t>Hoechst</t> 33342 staining and imaged using the In Cell Analyzer 2000 System. (D) The expression of apoptosis-related proteins, including cleaved PARP, cleaved caspase-3, Bcl-2, and Bax, were analyzed by Western blot analysis. (E) Quantitation of the results obtained from (D). b P
    3 4 5 Dimethyl 2 Thiazolyl 2 5 Diphenyl 2 H Tetrazolium Bromide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 310 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/3 4 5 dimethyl 2 thiazolyl 2 5 diphenyl 2 h tetrazolium bromide/product/Millipore
    Average 99 stars, based on 310 article reviews
    Price from $9.99 to $1999.99
    3 4 5 dimethyl 2 thiazolyl 2 5 diphenyl 2 h tetrazolium bromide - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Monodansylcadaverine (MDC) stains autophagic K562 cells with sensitivity equivalent to LC3B immunoblotting and GFP-LC3B fluorescence microscopy. ( A ) Fluorescence microscopy. K562 cells were treated with dimethyl sulfoxide (DMSO) or 1 μM imatinib (IM) overnight followed by MDC staining. Images of live cells were taken at different exposure times (milliseconds, ms) using an inverted fluorescence microscope. Scale bar: 25 μm. ( B ) MDC Fluorescence spectrophotometry. K562 cells treated with DMSO or IM were stained with MDC. MDC fluorescence of live K562 cells was quantified using a micro-plate reader at an excitation wavelength of 335 nm and an emission wavelength of 525 nm. The relative MDC levels were obtained by dividing MDC fluorescence of IM-treated cells to that of DMSO-treated cells. Error bars represent three independent experiments. ( C ) LC3B immunoblotting. Cropped images are shown and full images are included in supplemental materials. ACTB (β actin) is the loading control. The intensities of LC3B-II or ACTB were quantified using Image J. The fold changes of LC3B-II/ACTB were obtained by dividing the ratio of LC3B-II/ACTB in IM-treated cells with that of DMSO-treated cells. ( D ) MDC staining of K562 cells treated with IM and/or bafilomycin A1 (BFA1). K562 cells were treated with a combination of 1 μM IM (10 hours) and 5 nM of BFA1 (48 hours). Cells were imaged using a fluorescence confocal microscope. ( E ) Quantification of MDC intensities. Fluorescence intensities of MDC in 10 different cells from three images were quantified using Image J. ( F ) GFP-LC3B fluorescence microscopy. K562 cells were stably transfected with a construct encoding GFP-LC3B. Cells were treated with a combination of IM and BFA1 and then imaged. ( G ) Quantification of cells with GFP-LC3B puncta. Four to five images of more than thirty K562 cells from each treatment were randomly selected for the following quantification analyses. GFP-expressing K562 cells or K562 cells with GFP puncta (indicating autophagy) were counted by three persons. Percentages of K562 cells with puncta were obtained by dividing numbers of K562 cells with GFP puncta with those of GFP-expressing cells. Scale bar: 25 nm.

    Journal: Scientific Reports

    Article Title: A large-scale RNA interference screen identifies genes that regulate autophagy at different stages

    doi: 10.1038/s41598-018-21106-5

    Figure Lengend Snippet: Monodansylcadaverine (MDC) stains autophagic K562 cells with sensitivity equivalent to LC3B immunoblotting and GFP-LC3B fluorescence microscopy. ( A ) Fluorescence microscopy. K562 cells were treated with dimethyl sulfoxide (DMSO) or 1 μM imatinib (IM) overnight followed by MDC staining. Images of live cells were taken at different exposure times (milliseconds, ms) using an inverted fluorescence microscope. Scale bar: 25 μm. ( B ) MDC Fluorescence spectrophotometry. K562 cells treated with DMSO or IM were stained with MDC. MDC fluorescence of live K562 cells was quantified using a micro-plate reader at an excitation wavelength of 335 nm and an emission wavelength of 525 nm. The relative MDC levels were obtained by dividing MDC fluorescence of IM-treated cells to that of DMSO-treated cells. Error bars represent three independent experiments. ( C ) LC3B immunoblotting. Cropped images are shown and full images are included in supplemental materials. ACTB (β actin) is the loading control. The intensities of LC3B-II or ACTB were quantified using Image J. The fold changes of LC3B-II/ACTB were obtained by dividing the ratio of LC3B-II/ACTB in IM-treated cells with that of DMSO-treated cells. ( D ) MDC staining of K562 cells treated with IM and/or bafilomycin A1 (BFA1). K562 cells were treated with a combination of 1 μM IM (10 hours) and 5 nM of BFA1 (48 hours). Cells were imaged using a fluorescence confocal microscope. ( E ) Quantification of MDC intensities. Fluorescence intensities of MDC in 10 different cells from three images were quantified using Image J. ( F ) GFP-LC3B fluorescence microscopy. K562 cells were stably transfected with a construct encoding GFP-LC3B. Cells were treated with a combination of IM and BFA1 and then imaged. ( G ) Quantification of cells with GFP-LC3B puncta. Four to five images of more than thirty K562 cells from each treatment were randomly selected for the following quantification analyses. GFP-expressing K562 cells or K562 cells with GFP puncta (indicating autophagy) were counted by three persons. Percentages of K562 cells with puncta were obtained by dividing numbers of K562 cells with GFP puncta with those of GFP-expressing cells. Scale bar: 25 nm.

    Article Snippet: PP242, chloroquine, bafilomycin A1 and monodansylcadaverine (MDC) were purchased from Sigma-Aldrich.

    Techniques: Fluorescence, Microscopy, Staining, Mass Spectrometry, Spectrophotometry, Stable Transfection, Transfection, Construct, Expressing

    No transamidase activity is detected in caveolae. (A) In situ transglutaminase (TG) activity was analyzed in differentiating osteoblast cultures by growing the cells in the presence of monodansylcadaverine (MDC), as described in the Materials

    Journal: Journal of Histochemistry and Cytochemistry

    Article Title: Cellular Factor XIIIA Transglutaminase Localizes in Caveolae and Regulates Caveolin-1 Phosphorylation, Homo-oligomerization and c-Src Signaling in Osteoblasts

    doi: 10.1369/0022155415597964

    Figure Lengend Snippet: No transamidase activity is detected in caveolae. (A) In situ transglutaminase (TG) activity was analyzed in differentiating osteoblast cultures by growing the cells in the presence of monodansylcadaverine (MDC), as described in the Materials

    Article Snippet: Methyl-β-cyclodextrin, monodansylcadaverine and 2-(N-morpholino)ethanesulfonic acid (MES) were from Sigma-Aldrich (St. Louis, MO).

    Techniques: Activity Assay, In Situ

    Autophagy is upstream of apoptosis in VacA-induced cell death ( a ) Measurement of MAP1LC3B-II conversion following VacA treatment using western blot analysis. AGS cells were treated with 50 ng/ml VacA for 4 h in the presence of 2 mM 3-MA. ( b ) Detection of AO staining by flow cytometry following VacA treatment. AGS cells were treated as above. ( c ) Detection of active CASP3 following 3-MA pretreatment in cells. ( d ) The inhibition efficiency of siRNAs against ATG12 and BECN1. AGS cells were transfected with siRNAs targeting ATG12 and BECN1 (100 nM each) for 24 h, and the protein levels of the two targets were evaluated using western blot analysis. ( e ) The effect of 50 ng/ml VacA on PARP1 cleavage in AGS cells transfected with siC, siATG12, or siBECN1. ( f ) Detection of cell death by flow cytometry in cells transfected with siC, siATG12, or siBECN1 for 24 h. ( g , h ) Detection of MDC and AO staining of cells treated with 50 mM Z-VAD or 50 ng/ml VacA using flow cytometry analysis. ( i ) The MAP1LC3B-II conversion was detected using western blot analysis. AGS cells were treated as above

    Journal: Cell Death & Disease

    Article Title: Helicobacter pylori VacA induces autophagic cell death in gastric epithelial cells via the endoplasmic reticulum stress pathway

    doi: 10.1038/s41419-017-0011-x

    Figure Lengend Snippet: Autophagy is upstream of apoptosis in VacA-induced cell death ( a ) Measurement of MAP1LC3B-II conversion following VacA treatment using western blot analysis. AGS cells were treated with 50 ng/ml VacA for 4 h in the presence of 2 mM 3-MA. ( b ) Detection of AO staining by flow cytometry following VacA treatment. AGS cells were treated as above. ( c ) Detection of active CASP3 following 3-MA pretreatment in cells. ( d ) The inhibition efficiency of siRNAs against ATG12 and BECN1. AGS cells were transfected with siRNAs targeting ATG12 and BECN1 (100 nM each) for 24 h, and the protein levels of the two targets were evaluated using western blot analysis. ( e ) The effect of 50 ng/ml VacA on PARP1 cleavage in AGS cells transfected with siC, siATG12, or siBECN1. ( f ) Detection of cell death by flow cytometry in cells transfected with siC, siATG12, or siBECN1 for 24 h. ( g , h ) Detection of MDC and AO staining of cells treated with 50 mM Z-VAD or 50 ng/ml VacA using flow cytometry analysis. ( i ) The MAP1LC3B-II conversion was detected using western blot analysis. AGS cells were treated as above

    Article Snippet: Some chemical reagents were purchased from Sigma, including 3-MA (M9281), Baf A1 (B1793), thapsigargin (T9033), AO (A8097), MDC (30432), and carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethyl ketone (Z-VAD-FMK, V116); antibodies against autophagy-related protein 12 (ATG12, WH0009140m1), TRIB3 (WH0057761M3), and MAP1LC3B (L7543) were also obtained from Sigma.

    Techniques: Western Blot, Staining, Flow Cytometry, Cytometry, Inhibition, Transfection

    VacA toxin induces autophagy in AGS cells ( a , b ) AGS cells were subjected to the indicated treatments for 6 h and were stained with acridine orange or MDC. After incubation, the cells were immediately analyzed by flow cytometry. ( c ) Measurement of the MAP1LC3B-II conversion in AGS cells subjected to the indicated treatments using western blot analysis. ( d , e ) VacA increased the conversion of MAP1LC3B-I to MAP1LC3B-II in AGS cells. AGS cells were treated with a gradually increasing concentration of VacA or were treated with 50 ng/ml VacA for different times. ( f ) VacA induced complete autophagic flux in AGS cells. AGS cells were treated with 50 ng/ml VacA for 6 h in the presence of 10 nM Baf A1. ( g ) The number of GFP-MAP1LC3B puncta in each cell was counted using a confocal microscope. ( h ) Representative TEM images of AGS cells treated with VacA treatment for 6 or 24 h. The white arrows indicate the autophagosomes, and the black arrows indicate the autolysosomes. The experiments were performed in triplicate, and all replicates showed similar results. * P

    Journal: Cell Death & Disease

    Article Title: Helicobacter pylori VacA induces autophagic cell death in gastric epithelial cells via the endoplasmic reticulum stress pathway

    doi: 10.1038/s41419-017-0011-x

    Figure Lengend Snippet: VacA toxin induces autophagy in AGS cells ( a , b ) AGS cells were subjected to the indicated treatments for 6 h and were stained with acridine orange or MDC. After incubation, the cells were immediately analyzed by flow cytometry. ( c ) Measurement of the MAP1LC3B-II conversion in AGS cells subjected to the indicated treatments using western blot analysis. ( d , e ) VacA increased the conversion of MAP1LC3B-I to MAP1LC3B-II in AGS cells. AGS cells were treated with a gradually increasing concentration of VacA or were treated with 50 ng/ml VacA for different times. ( f ) VacA induced complete autophagic flux in AGS cells. AGS cells were treated with 50 ng/ml VacA for 6 h in the presence of 10 nM Baf A1. ( g ) The number of GFP-MAP1LC3B puncta in each cell was counted using a confocal microscope. ( h ) Representative TEM images of AGS cells treated with VacA treatment for 6 or 24 h. The white arrows indicate the autophagosomes, and the black arrows indicate the autolysosomes. The experiments were performed in triplicate, and all replicates showed similar results. * P

    Article Snippet: Some chemical reagents were purchased from Sigma, including 3-MA (M9281), Baf A1 (B1793), thapsigargin (T9033), AO (A8097), MDC (30432), and carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethyl ketone (Z-VAD-FMK, V116); antibodies against autophagy-related protein 12 (ATG12, WH0009140m1), TRIB3 (WH0057761M3), and MAP1LC3B (L7543) were also obtained from Sigma.

    Techniques: Staining, Incubation, Flow Cytometry, Cytometry, Western Blot, Concentration Assay, Microscopy, Transmission Electron Microscopy

    Autophagic morphological changes were evaluated by fluorescence microscopy using monodansylcadaverine (MDC) staining. MG-63 cells were treated with 10 µ M aloe-emodin (AE) for 6 h and then irradiated with light (4.8 J/cm 2 ). At 3, 6 and 12 h after irradiation, autophagic vacuoles were detected using MDC staining.

    Journal: Oncology Reports

    Article Title: Aloe-emodin-mediated photodynamic therapy induces autophagy and apoptosis in human osteosarcoma cell line MG-63 through the ROS/JNK signaling pathway

    doi: 10.3892/or.2016.4703

    Figure Lengend Snippet: Autophagic morphological changes were evaluated by fluorescence microscopy using monodansylcadaverine (MDC) staining. MG-63 cells were treated with 10 µ M aloe-emodin (AE) for 6 h and then irradiated with light (4.8 J/cm 2 ). At 3, 6 and 12 h after irradiation, autophagic vacuoles were detected using MDC staining.

    Article Snippet: Monodansylcadaverine (MDC) staining After the corresponding treatment, the cells were rinsed with PBS for 3 times and incubated in 200 µ l of 0.05 mM MDC (Sigma) at 37°C for 30 min. Then, the cells were rinsed with PBS for 2 times and the aggregation of autophagic vacuoles was observed under a fluorescence microscope (DMI4000 B; Leica Microsystems, Wetzlar, Germany) with an excitation wavelength of 460–500 nm and an emission wavelength of 512–542 nm.

    Techniques: Fluorescence, Microscopy, Staining, Irradiation

    Effects of autophagy inhibitors on intracellular PNP content in A549 cells at 24 h post-exposure to PNP. Intracellular PNP content in A549 cells was reduced by 38% and 64%, respectively, when autophagosome formation was inhibited with 3-methyladenine (3-MA) or autophagosome-lysosome fusion was inhibited with bafilomycin. Data are normalized to control. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Evidence for Nanoparticle-Induced Lysosomal Dysfunction in Lung Adenocarcinoma (A549) Cells

    doi: 10.3390/ijms20215253

    Figure Lengend Snippet: Effects of autophagy inhibitors on intracellular PNP content in A549 cells at 24 h post-exposure to PNP. Intracellular PNP content in A549 cells was reduced by 38% and 64%, respectively, when autophagosome formation was inhibited with 3-methyladenine (3-MA) or autophagosome-lysosome fusion was inhibited with bafilomycin. Data are normalized to control. * p

    Article Snippet: A 1:1 mixture of phenol red-free Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (DME/F-12), nonessential amino acid solution (NEAA), N -(2-hydroxyethyl)piperazine-N ′-(2-ethanesulfonic acid) hemisodium salt (HEPES), 2-(N -morpholino)ethanesulfonic acid sodium salt (MES), monodansylcadaverine (MDC), cytochalasin D (CCD), adenosine triphosphate (ATP), dimethylsulfoxide (DMSO), l -glutamine, 3-methyladenine (3-MA), bafilomycin, nocodazole, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), ciprofloxacin, trypsin-EDTA, Triton X-100, paraformaldehyde, chloroquine, and acridine orange (AO) were all obtained from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques:

    Effect of psoralidin in cell cycle distribution of A549 cells. (A) Cells were treated with 5, 10 and 20 µM psoralidin for 24 h, and then stained with propidium iodide. The DNA content was measured by flow cytometry. (B) The cell cycle distributions were analyzed and presented as mean ± SD of three independent experiments. ∗ P

    Journal: PeerJ

    Article Title: Psoralidin induces autophagy through ROS generation which inhibits the proliferation of human lung cancer A549 cells

    doi: 10.7717/peerj.555

    Figure Lengend Snippet: Effect of psoralidin in cell cycle distribution of A549 cells. (A) Cells were treated with 5, 10 and 20 µM psoralidin for 24 h, and then stained with propidium iodide. The DNA content was measured by flow cytometry. (B) The cell cycle distributions were analyzed and presented as mean ± SD of three independent experiments. ∗ P

    Article Snippet: Dimethyl sulfoxide (DMSO), MTT, Hoechst 33342, monodansylcadaverine (MDC), propidium iodide (PI), 3-methyladenine (3-MA), Ac-DEVD-CHO, z-VAD-FMK, DAPI, CM-H2 DCF-DA, N-acetyl cysteine (NAC), Annexin V-FITC were from Sigma Aldrich (St. Louis, MO, USA).

    Techniques: Staining, Flow Cytometry, Cytometry

    The effect of chloroquine on autophagic flux in vivo. Transgenic mice expressing mCherry-LC3 mice were randomly assigned to be exposed to chloroquine (Cq, 10 mg/kg), or rapamycin (2 mg/kg) or rapamycin plus chloroquine by i.p. injection. (A) Representative photographs of myocardium in mCherry-LC3 mice 4 hr after the indicated treatments. (B) Represented is the percentage surface area of mCherry-LC3 fluorescence per microscopic field. Results are mean ± SE of three independent experiments. *p

    Journal: Autophagy

    Article Title: A Method to Measure Cardiac Autophagic Flux in vivo

    doi:

    Figure Lengend Snippet: The effect of chloroquine on autophagic flux in vivo. Transgenic mice expressing mCherry-LC3 mice were randomly assigned to be exposed to chloroquine (Cq, 10 mg/kg), or rapamycin (2 mg/kg) or rapamycin plus chloroquine by i.p. injection. (A) Representative photographs of myocardium in mCherry-LC3 mice 4 hr after the indicated treatments. (B) Represented is the percentage surface area of mCherry-LC3 fluorescence per microscopic field. Results are mean ± SE of three independent experiments. *p

    Article Snippet: Chloroquine, rapamycin, Bafilomycin A1, and 3-Methyladenine (3-MA) were purchased from EMD Biosciences.

    Techniques: In Vivo, Transgenic Assay, Mouse Assay, Expressing, Injection, Fluorescence

    Dose-response and time-course dependency of chloroquine effects on autophagy in cardiac myocytes. GFP-LC3-expressing HL-1 cardiac myocytes were incubated for 2 hr in the indicated conditions in serum free media. Represented is the percentage of cells with numerous punctate GFP-LC3 labeled vacuoles per total cells scored. Results are mean ± SE of 3 independent experiments. rapamycin (Rp), chloroquine (Cq), saline (control). (A) Dose-response curve of chloroquine effects on AV accumulation. Cardiac myocytes stimulated with rapamycin were treated with indicated dose of chloroquine. *p

    Journal: Autophagy

    Article Title: A Method to Measure Cardiac Autophagic Flux in vivo

    doi:

    Figure Lengend Snippet: Dose-response and time-course dependency of chloroquine effects on autophagy in cardiac myocytes. GFP-LC3-expressing HL-1 cardiac myocytes were incubated for 2 hr in the indicated conditions in serum free media. Represented is the percentage of cells with numerous punctate GFP-LC3 labeled vacuoles per total cells scored. Results are mean ± SE of 3 independent experiments. rapamycin (Rp), chloroquine (Cq), saline (control). (A) Dose-response curve of chloroquine effects on AV accumulation. Cardiac myocytes stimulated with rapamycin were treated with indicated dose of chloroquine. *p

    Article Snippet: Chloroquine, rapamycin, Bafilomycin A1, and 3-Methyladenine (3-MA) were purchased from EMD Biosciences.

    Techniques: Expressing, Incubation, Labeling

    The effect of chloroquine on lysosomal activity in HL-1 cardiac myocytes. (A) HL-1 cardiac myocytes were incubated with saline (Control), rapamycin (Rap, 5 μM), chloroquine (Cq, 3 μM), rapamycin plus chloroquine (Rap+Cq, 5 μM and 3 μM, respectively), or Bafilomycin A1 (Baf, 50 nM), in serum free media for 2 hr. Following treatment, cells were loaded with 50 nM LysoTracker Red for 5 min in the culture medium and analyzed by fluorescence microscopy. LysoTracker Red assessments were performed in three independent experiments; results presented are representative. (B) Higher magnification image of cells transfected with GFP-LC3 and loaded with LysoTracker Red, showing colocalization of GFP-LC3 and LysoTracker Red in a subset of structures (arrows).

    Journal: Autophagy

    Article Title: A Method to Measure Cardiac Autophagic Flux in vivo

    doi:

    Figure Lengend Snippet: The effect of chloroquine on lysosomal activity in HL-1 cardiac myocytes. (A) HL-1 cardiac myocytes were incubated with saline (Control), rapamycin (Rap, 5 μM), chloroquine (Cq, 3 μM), rapamycin plus chloroquine (Rap+Cq, 5 μM and 3 μM, respectively), or Bafilomycin A1 (Baf, 50 nM), in serum free media for 2 hr. Following treatment, cells were loaded with 50 nM LysoTracker Red for 5 min in the culture medium and analyzed by fluorescence microscopy. LysoTracker Red assessments were performed in three independent experiments; results presented are representative. (B) Higher magnification image of cells transfected with GFP-LC3 and loaded with LysoTracker Red, showing colocalization of GFP-LC3 and LysoTracker Red in a subset of structures (arrows).

    Article Snippet: Chloroquine, rapamycin, Bafilomycin A1, and 3-Methyladenine (3-MA) were purchased from EMD Biosciences.

    Techniques: Activity Assay, Incubation, Fluorescence, Microscopy, Transfection

    Comparison of chloroquine and Bafilomycin A1 effects on autophagosome accumulation in cardiac myocytes. GFP-LC3-expressing HL-1 cardiac myocytes were incubated in the indicated conditions in serum free media. Represented is the percentage of cells with numerous punctate GFP-LC3 labeled vacuoles per total cells scored. Results are mean ± SE of three or more independent experiments. Saline (control), rapamycin (Rp, 5 μM), H 2 O 2 (H 2 O 2 , 10 −6 M), Bafilomycin A1(Baf, 50 nM), chloroquine (Cq, 3 μM), 3-MA (10 mM). *p

    Journal: Autophagy

    Article Title: A Method to Measure Cardiac Autophagic Flux in vivo

    doi:

    Figure Lengend Snippet: Comparison of chloroquine and Bafilomycin A1 effects on autophagosome accumulation in cardiac myocytes. GFP-LC3-expressing HL-1 cardiac myocytes were incubated in the indicated conditions in serum free media. Represented is the percentage of cells with numerous punctate GFP-LC3 labeled vacuoles per total cells scored. Results are mean ± SE of three or more independent experiments. Saline (control), rapamycin (Rp, 5 μM), H 2 O 2 (H 2 O 2 , 10 −6 M), Bafilomycin A1(Baf, 50 nM), chloroquine (Cq, 3 μM), 3-MA (10 mM). *p

    Article Snippet: Chloroquine, rapamycin, Bafilomycin A1, and 3-Methyladenine (3-MA) were purchased from EMD Biosciences.

    Techniques: Expressing, Incubation, Labeling

    Effect of chloroquine on autophagosome accumulation in cardiac myocytes. GFP-LC3-expressing HL-1 cardiac myocytes were treated with saline (Control), rapamycin (Rap, 5 μM), chloroquine (Cq, 3 μM), or rapamycin plus chloroquine (Rap+Cq) in serum free media for 2 hr. (A) Shown are maximum projection images of Z-stacks taken of GFP-LC3 fluorescence in HL-1 cardiac myocytes treated with the indicated conditions. Scale bar, 25 mm. (B) Autophagic flux was determined via fluorescent imaging of GFP-LC3 without and with chloroquine. Represented is the percentage of cells with numerous punctate GFP-LC3 labeled vacuoles per total cells scored. Results are mean ± SE of three independent experiments. *p

    Journal: Autophagy

    Article Title: A Method to Measure Cardiac Autophagic Flux in vivo

    doi:

    Figure Lengend Snippet: Effect of chloroquine on autophagosome accumulation in cardiac myocytes. GFP-LC3-expressing HL-1 cardiac myocytes were treated with saline (Control), rapamycin (Rap, 5 μM), chloroquine (Cq, 3 μM), or rapamycin plus chloroquine (Rap+Cq) in serum free media for 2 hr. (A) Shown are maximum projection images of Z-stacks taken of GFP-LC3 fluorescence in HL-1 cardiac myocytes treated with the indicated conditions. Scale bar, 25 mm. (B) Autophagic flux was determined via fluorescent imaging of GFP-LC3 without and with chloroquine. Represented is the percentage of cells with numerous punctate GFP-LC3 labeled vacuoles per total cells scored. Results are mean ± SE of three independent experiments. *p

    Article Snippet: Chloroquine, rapamycin, Bafilomycin A1, and 3-Methyladenine (3-MA) were purchased from EMD Biosciences.

    Techniques: Expressing, Fluorescence, Imaging, Labeling

    PTE treatment induces autophagy. (A) Transmission electronic microscopy analyzed the autophagy-associated morphological change in RPMI-8226 cells following exposure to PTE. Upper panel, ×1,700 magnification; lower panel, ×5,000 magnification. (B) Monodansylcadaverine-labeled vacuoles were observed in RPMI-8226 cells using a fluorescence microscope. (C) RPMI-8226 and (D) ARH-77 cells were treated with DMSO (control) or different concentrations of PTE for 48 h, western blotting was applied to investigate the levels of the autophagy markers beclin1, ATG5 and LC3. β-actin was used as a loading control. (E) Effect of PTE treatment on RPMI-8226 cells with or without 3MA was analyzed by flow cytometry using an Annexin V-FITC/PI kit. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Pterostilbene inhibits nutrient metabolism and induces apoptosis through AMPK activation in multiple myeloma cells

    doi: 10.3892/ijmm.2018.3857

    Figure Lengend Snippet: PTE treatment induces autophagy. (A) Transmission electronic microscopy analyzed the autophagy-associated morphological change in RPMI-8226 cells following exposure to PTE. Upper panel, ×1,700 magnification; lower panel, ×5,000 magnification. (B) Monodansylcadaverine-labeled vacuoles were observed in RPMI-8226 cells using a fluorescence microscope. (C) RPMI-8226 and (D) ARH-77 cells were treated with DMSO (control) or different concentrations of PTE for 48 h, western blotting was applied to investigate the levels of the autophagy markers beclin1, ATG5 and LC3. β-actin was used as a loading control. (E) Effect of PTE treatment on RPMI-8226 cells with or without 3MA was analyzed by flow cytometry using an Annexin V-FITC/PI kit. * P

    Article Snippet: PTE, 3-methyladenine (3-MA), monodansylcadaverine (MDC) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).

    Techniques: Transmission Assay, Microscopy, Labeling, Fluorescence, Western Blot, Flow Cytometry, Cytometry

    PTE suppresses the expression of metabolic proteins in MM cells. RPMI-8226 (left) and ARH-77 (right) cells were treated with DMSO (control) or different concentrations of PTE for 48 h. Western blotting was used to analyze the protein expression levels of (A) p-AMPK (Thr 172); (B) FASN, p-ACC (Ser 79) and ACC; (C) p-mTOR (Ser 2448), mTOR, p-4E-BP1 (Thr 37/46), 4E-BP1, eIF2α and p-eIF2α. β-actin served as a loading control. The presented western blotting data are representative of those obtained in at least three separate experiments. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Pterostilbene inhibits nutrient metabolism and induces apoptosis through AMPK activation in multiple myeloma cells

    doi: 10.3892/ijmm.2018.3857

    Figure Lengend Snippet: PTE suppresses the expression of metabolic proteins in MM cells. RPMI-8226 (left) and ARH-77 (right) cells were treated with DMSO (control) or different concentrations of PTE for 48 h. Western blotting was used to analyze the protein expression levels of (A) p-AMPK (Thr 172); (B) FASN, p-ACC (Ser 79) and ACC; (C) p-mTOR (Ser 2448), mTOR, p-4E-BP1 (Thr 37/46), 4E-BP1, eIF2α and p-eIF2α. β-actin served as a loading control. The presented western blotting data are representative of those obtained in at least three separate experiments. * P

    Article Snippet: PTE, 3-methyladenine (3-MA), monodansylcadaverine (MDC) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).

    Techniques: Expressing, Western Blot

    PTE is a safe drug for the treatment of MM. (A) Liver damage markers, including ALT, AST, ALP and TBIL were measured in serum samples from NOD/SCID mice treated with 5% DMSO or PTE with an automatic biochemical analyzer. (B) Kidney function markers BUN and Scr were measured in serum samples from NOD/SCID mice treated with 5% DMSO or PTE by an automatic biochemical analyzer. (C) Cardiac injury markers cTnT and CK-MB were measured with an ELISA kit. The values are expressed as means ± standard deviation. PTE, pterostilbene; MM, multiple myeloma; ALT, alanine aminotransferase; AST, aspartate amino transferase; ALP, alkaline phosphatase; TBIL, total bilirubin; DMSO, dimethyl sulfoxide; BUN, urea nitrogen; Scr, serum creatinine; cTnT, cardiac troponins T; CK-MB, creatine kinase-MB.

    Journal: International Journal of Molecular Medicine

    Article Title: Pterostilbene inhibits nutrient metabolism and induces apoptosis through AMPK activation in multiple myeloma cells

    doi: 10.3892/ijmm.2018.3857

    Figure Lengend Snippet: PTE is a safe drug for the treatment of MM. (A) Liver damage markers, including ALT, AST, ALP and TBIL were measured in serum samples from NOD/SCID mice treated with 5% DMSO or PTE with an automatic biochemical analyzer. (B) Kidney function markers BUN and Scr were measured in serum samples from NOD/SCID mice treated with 5% DMSO or PTE by an automatic biochemical analyzer. (C) Cardiac injury markers cTnT and CK-MB were measured with an ELISA kit. The values are expressed as means ± standard deviation. PTE, pterostilbene; MM, multiple myeloma; ALT, alanine aminotransferase; AST, aspartate amino transferase; ALP, alkaline phosphatase; TBIL, total bilirubin; DMSO, dimethyl sulfoxide; BUN, urea nitrogen; Scr, serum creatinine; cTnT, cardiac troponins T; CK-MB, creatine kinase-MB.

    Article Snippet: PTE, 3-methyladenine (3-MA), monodansylcadaverine (MDC) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).

    Techniques: AST Assay, ALP Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

    PTE reduces MM cell viability and induces cell apoptosis. (A) RPMI-8226, ARH-77, U266 and NCI-H929 cells were treated with PTE and their viability was measured using a Cell Counting kit-8 assay. The IC 50 value is shown. (B) Flow cytometric analysis of the effect of PTE on apoptosis using an Annexin V-FITC/PI kit. DMSO served as the control. (C) RPMI-8226 and (D) ARH-77 cells were treated with DMSO (control) or different concentrations of PTE for 48 h, the expression levels of cleaved caspase 3, cleaved caspase 9 and cleaved PARP were determined using western blotting. β-actin served as a loading control. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Pterostilbene inhibits nutrient metabolism and induces apoptosis through AMPK activation in multiple myeloma cells

    doi: 10.3892/ijmm.2018.3857

    Figure Lengend Snippet: PTE reduces MM cell viability and induces cell apoptosis. (A) RPMI-8226, ARH-77, U266 and NCI-H929 cells were treated with PTE and their viability was measured using a Cell Counting kit-8 assay. The IC 50 value is shown. (B) Flow cytometric analysis of the effect of PTE on apoptosis using an Annexin V-FITC/PI kit. DMSO served as the control. (C) RPMI-8226 and (D) ARH-77 cells were treated with DMSO (control) or different concentrations of PTE for 48 h, the expression levels of cleaved caspase 3, cleaved caspase 9 and cleaved PARP were determined using western blotting. β-actin served as a loading control. * P

    Article Snippet: PTE, 3-methyladenine (3-MA), monodansylcadaverine (MDC) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).

    Techniques: Cell Counting, Flow Cytometry, Expressing, Western Blot

    PTE inhibits MM progression in vivo . (A) Representative image (left) and growth curves (right) of xenograft tumors from NOD/SCID mice subcutaneously injected with RPMI-8226 cells and treated with 5% DMSO or PTE. (B) Immunohistochemical analysis of terminal deoxynucleotidyl-transferasemediated dUTP nick-end labeling and cleaved caspase 3 on tumor tissue sections from mice injected with RPMI-8226 cells and treated with 5% DMSO or PTE. (C) Immunohistochemical analysis of p-AMPK (Thr 172), FASN and p-mTOR (Ser 2448) on tumor tissue sections from mice injected with RPMI-8226 cells and treated with 5% DMSO or PTE (n=5). ** P

    Journal: International Journal of Molecular Medicine

    Article Title: Pterostilbene inhibits nutrient metabolism and induces apoptosis through AMPK activation in multiple myeloma cells

    doi: 10.3892/ijmm.2018.3857

    Figure Lengend Snippet: PTE inhibits MM progression in vivo . (A) Representative image (left) and growth curves (right) of xenograft tumors from NOD/SCID mice subcutaneously injected with RPMI-8226 cells and treated with 5% DMSO or PTE. (B) Immunohistochemical analysis of terminal deoxynucleotidyl-transferasemediated dUTP nick-end labeling and cleaved caspase 3 on tumor tissue sections from mice injected with RPMI-8226 cells and treated with 5% DMSO or PTE. (C) Immunohistochemical analysis of p-AMPK (Thr 172), FASN and p-mTOR (Ser 2448) on tumor tissue sections from mice injected with RPMI-8226 cells and treated with 5% DMSO or PTE (n=5). ** P

    Article Snippet: PTE, 3-methyladenine (3-MA), monodansylcadaverine (MDC) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).

    Techniques: In Vivo, Mouse Assay, Injection, Immunohistochemistry, End Labeling

    RhoA and ROCK1 are essential for ARV S1133-induced autophagy, apoptosis, and the conversion of autophagy to apoptosis. (A) Vero cells transfected with RhoA-T19N and incubated for 24 hr, before being transfected with siRNA for 72 hr, pre-treated with 5 μM Y-27632, 10 μM Z-VAD-FMK, 50 μM 3-MA and 0.1 μM Bafilomycin A1 at non-toxic concentrations for 4 hr, and then infected with ARV S1133 at an MOI of 5 for an additional 18-hr incubation. Autophagic vacuoles were stained with MDC, and the percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (black bar). Vero cells transfected with LC3-GFP, together with RhoA-T19N, and incubated for 24 hr, or transfected with siRNA for 48 hr then transfected with LC3-GFP, or pretreated with inhibitor for 4 hr then transfected with LC3-GFP. 24 hr after LC3-GFP transfection, ARV S1133 was added for an additional 18-hr incubation period. GFP-positive cells containing more than 3 dots were counted as positive LC3-GFP cells. The percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (white bar). (B) ARV S1133 infected cells at an MOI of 5 with an incubation period of 36 hr with identical treatment timings to those described in (A) . Three apoptosis assays were performed. The left y-axis represents the percentage of Hoechst 33258 positive cells and the caspase-3 activity in relative light units (RLUs). The right y-axis represents the OD 405 nm values from an apoptotic ELISA assay. All experiments were performed three times, each in duplicate. The data are presented as the mean ± SD. (C) Punctate dots of GFP indicating autophagosomes are shown by the white arrows (400x magnification).

    Journal: BMC Veterinary Research

    Article Title: RhoA/ROCK1 regulates Avian Reovirus S1133-induced switch from autophagy to apoptosis

    doi: 10.1186/s12917-015-0417-6

    Figure Lengend Snippet: RhoA and ROCK1 are essential for ARV S1133-induced autophagy, apoptosis, and the conversion of autophagy to apoptosis. (A) Vero cells transfected with RhoA-T19N and incubated for 24 hr, before being transfected with siRNA for 72 hr, pre-treated with 5 μM Y-27632, 10 μM Z-VAD-FMK, 50 μM 3-MA and 0.1 μM Bafilomycin A1 at non-toxic concentrations for 4 hr, and then infected with ARV S1133 at an MOI of 5 for an additional 18-hr incubation. Autophagic vacuoles were stained with MDC, and the percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (black bar). Vero cells transfected with LC3-GFP, together with RhoA-T19N, and incubated for 24 hr, or transfected with siRNA for 48 hr then transfected with LC3-GFP, or pretreated with inhibitor for 4 hr then transfected with LC3-GFP. 24 hr after LC3-GFP transfection, ARV S1133 was added for an additional 18-hr incubation period. GFP-positive cells containing more than 3 dots were counted as positive LC3-GFP cells. The percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (white bar). (B) ARV S1133 infected cells at an MOI of 5 with an incubation period of 36 hr with identical treatment timings to those described in (A) . Three apoptosis assays were performed. The left y-axis represents the percentage of Hoechst 33258 positive cells and the caspase-3 activity in relative light units (RLUs). The right y-axis represents the OD 405 nm values from an apoptotic ELISA assay. All experiments were performed three times, each in duplicate. The data are presented as the mean ± SD. (C) Punctate dots of GFP indicating autophagosomes are shown by the white arrows (400x magnification).

    Article Snippet: Y-27632, which selectively inhibits p160ROCK [ ], 3-MA, Bafilomycin A1, and Z-VAD-FMK were obtained from Calbiochem (San Diego, CA, USA).

    Techniques: Transfection, Incubation, Infection, Staining, Activity Assay, Enzyme-linked Immunosorbent Assay

    Effect of miR-497 mimic and sponge on the expression of Bcl2 and LC3B protein in neonatal rat cardiomyocytes (NRCs) a. NRCs were transfected with miR-497 mimic or negative control (NC). Western blot shows the effects of miR-497 overexpression on Bcl-2, Bax, LC3B-II and beclin-1. b. Quantitation for panel A. NRCs were transfected with miR-497 sponge or vector adenovirus. Western blot shows the protein expression of Bcl-2, Bax and LC3B-II c. beclin-1 and P62 d. which were significantly changed in miR-497 sponge-treated cells e, f. Western blotting of LC3B-II in NRCs treated with/without miR-497 sponge (S), vector (V), Ad-sh-beclin-1 (sh-b), Ad-sh-control (sh-c), bafilomycin A1 (Baf), wortmannin (W). Data are mean ± SEM, n = 5–7 for each group; for panel b and e, * P

    Journal: Oncotarget

    Article Title: Inhibition of microRNA-497 ameliorates anoxia/reoxygenation injury in cardiomyocytes by suppressing cell apoptosis and enhancing autophagy

    doi:

    Figure Lengend Snippet: Effect of miR-497 mimic and sponge on the expression of Bcl2 and LC3B protein in neonatal rat cardiomyocytes (NRCs) a. NRCs were transfected with miR-497 mimic or negative control (NC). Western blot shows the effects of miR-497 overexpression on Bcl-2, Bax, LC3B-II and beclin-1. b. Quantitation for panel A. NRCs were transfected with miR-497 sponge or vector adenovirus. Western blot shows the protein expression of Bcl-2, Bax and LC3B-II c. beclin-1 and P62 d. which were significantly changed in miR-497 sponge-treated cells e, f. Western blotting of LC3B-II in NRCs treated with/without miR-497 sponge (S), vector (V), Ad-sh-beclin-1 (sh-b), Ad-sh-control (sh-c), bafilomycin A1 (Baf), wortmannin (W). Data are mean ± SEM, n = 5–7 for each group; for panel b and e, * P

    Article Snippet: Agents 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), bafilomycin A1 (Baf), wortmannin, monodansylcadaverine (MDC), 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), triphenyltetrazolium chloride (TTC) were purchased from Sigma.

    Techniques: Expressing, Transfection, Negative Control, Western Blot, Over Expression, Quantitation Assay, Plasmid Preparation

    Cell and nuclear morphology were visualized following PAB treatment. (A) After 24 and 48 h of PAB treatment, morphological changes in SW579 cells were visualized by phase contrast microscopy. (B) Cells were stained using Hoechst 33258 to observe nuclear changes and imaged via fluorescence microscopy. Data are representative of three individual experiments; n=3. Scale bar, 30 µ m. PAB, pseudolaric acid B; Con, control.

    Journal: Molecular Medicine Reports

    Article Title: Pseudolaric acid B inhibits proliferation in SW579 human thyroid squamous cell carcinoma

    doi: 10.3892/mmr.2015.4418

    Figure Lengend Snippet: Cell and nuclear morphology were visualized following PAB treatment. (A) After 24 and 48 h of PAB treatment, morphological changes in SW579 cells were visualized by phase contrast microscopy. (B) Cells were stained using Hoechst 33258 to observe nuclear changes and imaged via fluorescence microscopy. Data are representative of three individual experiments; n=3. Scale bar, 30 µ m. PAB, pseudolaric acid B; Con, control.

    Article Snippet: Propidium iodode (PI), monodansylcadaverine (MDC), rhodamine 123, 3-methyladenine (3-MA), Hoechst 33258, RNase A and MTT were purchased from Sigma-Aldrich.

    Techniques: Microscopy, Staining, Fluorescence

    α-Tubulin distribution analysis of PAB-treated SW579 cells by fluorescence microscopy. SW579 cells were treated with PAB (4 µ M) for 24 h. Control cells were treated with dimethyl sulfoxide. Cells were fixed in 4% paraformaldehyde, and stained with anti-α-tubulin antibodies prior to being co-stained with fluorescein isothiocyanate-labeled secondary antibody and Hoechst 33258 (blue). Merged images of α-tubulin and DNA staining are presented. Arrows indicate aggregated α-tubulin. Data are representative of three individual experiments; n=3. Scale bar=15 µ m. PAB, pseudolaric acid B.

    Journal: Molecular Medicine Reports

    Article Title: Pseudolaric acid B inhibits proliferation in SW579 human thyroid squamous cell carcinoma

    doi: 10.3892/mmr.2015.4418

    Figure Lengend Snippet: α-Tubulin distribution analysis of PAB-treated SW579 cells by fluorescence microscopy. SW579 cells were treated with PAB (4 µ M) for 24 h. Control cells were treated with dimethyl sulfoxide. Cells were fixed in 4% paraformaldehyde, and stained with anti-α-tubulin antibodies prior to being co-stained with fluorescein isothiocyanate-labeled secondary antibody and Hoechst 33258 (blue). Merged images of α-tubulin and DNA staining are presented. Arrows indicate aggregated α-tubulin. Data are representative of three individual experiments; n=3. Scale bar=15 µ m. PAB, pseudolaric acid B.

    Article Snippet: Propidium iodode (PI), monodansylcadaverine (MDC), rhodamine 123, 3-methyladenine (3-MA), Hoechst 33258, RNase A and MTT were purchased from Sigma-Aldrich.

    Techniques: Fluorescence, Microscopy, Staining, Labeling

    Differential endocytosis of primary and secondary CPP in human monocyte-derived macrophages and aortic endothelial cells. Human monocyte-derived macrophages (hMDM) or human aortic endothelial cells (haEC) were treated with Alexa488-labeled primary or secondary CPP, both 100 μg/mL calcium content, synthesized from FCS/DMEM supplemented with phosphate (final conc. 3.5 mM). Monomeric labeled-fetuin-A was included as a comparator (1 mg/ml). Cell-associated fluorescence was measured by flow cytometry in fixed cells at the stated timepoints.  (A)  Endocyosis observed in hMDM and  (B)  haEC, respectively.  (C,D)  hMDM were pre-treated for 30–60 min with one of several inhibitors (vs. vehicle) or receptor blocking antibodies (vs. isotype control).  (C)  Pre-treatment with cytochalasin  (D) , an inhibitor of actin polymerization, markedly attenuated uptake of both primary and secondary CPP and (

    Journal: Frontiers in Immunology

    Article Title: Cellular Clearance and Biological Activity of Calciprotein Particles Depend on Their Maturation State and Crystallinity

    doi: 10.3389/fimmu.2018.01991

    Figure Lengend Snippet: Differential endocytosis of primary and secondary CPP in human monocyte-derived macrophages and aortic endothelial cells. Human monocyte-derived macrophages (hMDM) or human aortic endothelial cells (haEC) were treated with Alexa488-labeled primary or secondary CPP, both 100 μg/mL calcium content, synthesized from FCS/DMEM supplemented with phosphate (final conc. 3.5 mM). Monomeric labeled-fetuin-A was included as a comparator (1 mg/ml). Cell-associated fluorescence was measured by flow cytometry in fixed cells at the stated timepoints. (A) Endocyosis observed in hMDM and (B) haEC, respectively. (C,D) hMDM were pre-treated for 30–60 min with one of several inhibitors (vs. vehicle) or receptor blocking antibodies (vs. isotype control). (C) Pre-treatment with cytochalasin (D) , an inhibitor of actin polymerization, markedly attenuated uptake of both primary and secondary CPP and (

    Article Snippet: While in others, cells were pre-treated for 30 min with one of several chemical inhibitors at previously defined concentrations as detailed in Supplementary Table (all from Sigma): cytochalasin D (10 μM), chlorpromazine (10 μg/ml), filipin (2 μg/ml), genestein (200 μM), 5-(N,N-dimethyl)amiloride hydrochloride (10 μM; 5-DMA), MβCD (2 mM), Ly294002 (100 μM), monodansylcadaverine (100 μM; MDC) polyinosinic acid (10 μg/ml) or vehicle (DMSO/media); then washed and switched to media containing AF488-CPP (100 μg/ml) and incubated for 60 min at 37°C; or were pre-treated with blocking antibodies (60 min at 4°C) directed against SRA (AbD Serotec, 10 μg/ml;), TLR2/4/6 (10 μg/ml; Invivogen), CD36 (clone FA6-152; 10 μg/ml; Hycult Biotech), or annexin-2 (20 μg/ml; BD Biosciences) or relevant isotype IgG control (all 10 μg/ml; eBioscience) before incubation with CPP-containing media for 60 min at 37°C.

    Techniques: Conditioned Place Preference, Derivative Assay, Labeling, Synthesized, Fluorescence, Flow Cytometry, Cytometry, Blocking Assay

    Caveolin-mediated endocytic mechanisms are implicated in internalization of rMGA_0676. (A) Internalization of rMGA_0676 was blocked by caveolin-mediated endocytic inhibitor filipin. DF-1 cells were treated with filipin followed by rMGA_0676 (40 μg/ml) for 24 h at 37°C to detect the inhibition of rMGA_0676 internalization. The samples underwent IFA and were examined with a laser confocal scanning microscope. Cellular F-actin was stained with Alexa Fluor 555-conjugated phalloidin. The nuclei were counterstained with DAPI (blue). The rMGA_0676 was hybridized with mouse anti-His monoclonal antibody and labeled with FITC-conjugated goat anti mouse IgG antibody green fluorescence (GF). The normal DF-1 cells as the negative control (Con), cells were treated with Cholera toxin-FITC as the caveolin-mediated endocytic positive control (Cholera toxin). (B) The mean value of GF was used to quantify the positive staining for rMGA_0676 in filipin treated DF-1 cells (A) , the data were analyzed using SPSS software, and the graph was made using GraphPad Prism 5.0. “ ** ”represented statistically significant differences ( P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Mechanism of Apoptosis Induction by Mycoplasmal Nuclease MGA_0676 in Chicken Embryo Fibroblasts

    doi: 10.3389/fcimb.2018.00105

    Figure Lengend Snippet: Caveolin-mediated endocytic mechanisms are implicated in internalization of rMGA_0676. (A) Internalization of rMGA_0676 was blocked by caveolin-mediated endocytic inhibitor filipin. DF-1 cells were treated with filipin followed by rMGA_0676 (40 μg/ml) for 24 h at 37°C to detect the inhibition of rMGA_0676 internalization. The samples underwent IFA and were examined with a laser confocal scanning microscope. Cellular F-actin was stained with Alexa Fluor 555-conjugated phalloidin. The nuclei were counterstained with DAPI (blue). The rMGA_0676 was hybridized with mouse anti-His monoclonal antibody and labeled with FITC-conjugated goat anti mouse IgG antibody green fluorescence (GF). The normal DF-1 cells as the negative control (Con), cells were treated with Cholera toxin-FITC as the caveolin-mediated endocytic positive control (Cholera toxin). (B) The mean value of GF was used to quantify the positive staining for rMGA_0676 in filipin treated DF-1 cells (A) , the data were analyzed using SPSS software, and the graph was made using GraphPad Prism 5.0. “ ** ”represented statistically significant differences ( P

    Article Snippet: Human holo-transferrin (Tf), cholera toxin-FITC, monodansylcadaverine (MDC), and filipin were purchased from Sigma-Aldrich (Louis, MO, USA).

    Techniques: Inhibition, Immunofluorescence, Microscopy, Staining, Labeling, Fluorescence, Negative Control, Positive Control, Software

    Effect of endocytosis inhibition on the VEGF-A-regulated cleavage of Flt1. A–D : HEK293 cells ( A and B ) transfected with hemagglutinin (HA)-Flt1-Myc-Flag (Flt1) or FLT1ΔCTD (deleted COOH-terminal domain, ΔCTD) and AG1-G1-FLT1 ( C and D ) were incubated with and without 100 ng/ml VEGF-A for 24 h with dynasore (80 μM) or methyl-β-cyclodextrin (MβCD; 50 μg/ml) for 5 h and then immunoblotted for HA, tubulin, or AF321 (Flt1) antibodies. Full-length Flt1 (FL) and the NH 2 -terminal fragment (NTF) are seen in lysates and conditioned media (CM), respectively. Neither dynasore nor MβCD have an impact on VEGF-A mediated inhibition of FLT1 and FLT1ΔCTD cleavage. A representative immunoblot in seen in A and C , and the dynasore experiments were repeated, and the pooled NTF data quantified by densitometry is shown in panel B and D . Means ± SD, n = 3. * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: VEGF-A selectively inhibits FLT1 ectodomain shedding independent of receptor activation and receptor endocytosis

    doi: 10.1152/ajpcell.00247.2017

    Figure Lengend Snippet: Effect of endocytosis inhibition on the VEGF-A-regulated cleavage of Flt1. A–D : HEK293 cells ( A and B ) transfected with hemagglutinin (HA)-Flt1-Myc-Flag (Flt1) or FLT1ΔCTD (deleted COOH-terminal domain, ΔCTD) and AG1-G1-FLT1 ( C and D ) were incubated with and without 100 ng/ml VEGF-A for 24 h with dynasore (80 μM) or methyl-β-cyclodextrin (MβCD; 50 μg/ml) for 5 h and then immunoblotted for HA, tubulin, or AF321 (Flt1) antibodies. Full-length Flt1 (FL) and the NH 2 -terminal fragment (NTF) are seen in lysates and conditioned media (CM), respectively. Neither dynasore nor MβCD have an impact on VEGF-A mediated inhibition of FLT1 and FLT1ΔCTD cleavage. A representative immunoblot in seen in A and C , and the dynasore experiments were repeated, and the pooled NTF data quantified by densitometry is shown in panel B and D . Means ± SD, n = 3. * P

    Article Snippet: Bafilomycin A1, GF109203X hydrochloride, genistein, monodansylcadaverine (MDC), dynasore hydrate, methyl-β-cyclodextrin (MβCD), and N-acetylleucylleucylnorleucinal (ALLN) were purchased from Sigma-Aldrich (St. Louis, MO), and dl -dithiothreitol (DTT) was from EMD Millipore (Billerica, MA).

    Techniques: Inhibition, Transfection, Incubation

    RhoA and ROCK1 are essential for ARV S1133-induced autophagy, apoptosis, and the conversion of autophagy to apoptosis. (A) Vero cells transfected with RhoA-T19N and incubated for 24 hr, before being transfected with siRNA for 72 hr, pre-treated with 5 μM Y-27632, 10 μM Z-VAD-FMK, 50 μM 3-MA and 0.1 μM Bafilomycin A1 at non-toxic concentrations for 4 hr, and then infected with ARV S1133 at an MOI of 5 for an additional 18-hr incubation. Autophagic vacuoles were stained with MDC, and the percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (black bar). Vero cells transfected with LC3-GFP, together with RhoA-T19N, and incubated for 24 hr, or transfected with siRNA for 48 hr then transfected with LC3-GFP, or pretreated with inhibitor for 4 hr then transfected with LC3-GFP. 24 hr after LC3-GFP transfection, ARV S1133 was added for an additional 18-hr incubation period. GFP-positive cells containing more than 3 dots were counted as positive LC3-GFP cells. The percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (white bar). (B) ARV S1133 infected cells at an MOI of 5 with an incubation period of 36 hr with identical treatment timings to those described in (A) . Three apoptosis assays were performed. The left y-axis represents the percentage of Hoechst 33258 positive cells and the caspase-3 activity in relative light units (RLUs). The right y-axis represents the OD 405 nm values from an apoptotic ELISA assay. All experiments were performed three times, each in duplicate. The data are presented as the mean ± SD. (C) Punctate dots of GFP indicating autophagosomes are shown by the white arrows (400x magnification).

    Journal: BMC Veterinary Research

    Article Title: RhoA/ROCK1 regulates Avian Reovirus S1133-induced switch from autophagy to apoptosis

    doi: 10.1186/s12917-015-0417-6

    Figure Lengend Snippet: RhoA and ROCK1 are essential for ARV S1133-induced autophagy, apoptosis, and the conversion of autophagy to apoptosis. (A) Vero cells transfected with RhoA-T19N and incubated for 24 hr, before being transfected with siRNA for 72 hr, pre-treated with 5 μM Y-27632, 10 μM Z-VAD-FMK, 50 μM 3-MA and 0.1 μM Bafilomycin A1 at non-toxic concentrations for 4 hr, and then infected with ARV S1133 at an MOI of 5 for an additional 18-hr incubation. Autophagic vacuoles were stained with MDC, and the percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (black bar). Vero cells transfected with LC3-GFP, together with RhoA-T19N, and incubated for 24 hr, or transfected with siRNA for 48 hr then transfected with LC3-GFP, or pretreated with inhibitor for 4 hr then transfected with LC3-GFP. 24 hr after LC3-GFP transfection, ARV S1133 was added for an additional 18-hr incubation period. GFP-positive cells containing more than 3 dots were counted as positive LC3-GFP cells. The percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (white bar). (B) ARV S1133 infected cells at an MOI of 5 with an incubation period of 36 hr with identical treatment timings to those described in (A) . Three apoptosis assays were performed. The left y-axis represents the percentage of Hoechst 33258 positive cells and the caspase-3 activity in relative light units (RLUs). The right y-axis represents the OD 405 nm values from an apoptotic ELISA assay. All experiments were performed three times, each in duplicate. The data are presented as the mean ± SD. (C) Punctate dots of GFP indicating autophagosomes are shown by the white arrows (400x magnification).

    Article Snippet: Y-27632, which selectively inhibits p160ROCK [ ], 3-MA, Bafilomycin A1, and Z-VAD-FMK were obtained from Calbiochem (San Diego, CA, USA).

    Techniques: Transfection, Incubation, Infection, Staining, Activity Assay, Enzyme-linked Immunosorbent Assay

    PD induces apoptosis in BEL-7402 cells. BEL-7402 cells were treated with different concentrations of PD for 24 h. (A) Apoptotic cells were detected by Annexin V/PI staining using flow cytometry. (B) Quantitation of the results obtained from (A). (C) The apoptotic cells were evaluated with Hoechst 33342 staining and imaged using the In Cell Analyzer 2000 System. (D) The expression of apoptosis-related proteins, including cleaved PARP, cleaved caspase-3, Bcl-2, and Bax, were analyzed by Western blot analysis. (E) Quantitation of the results obtained from (D). b P

    Journal: Acta Pharmacologica Sinica

    Article Title: Platycodin D induces apoptosis and triggers ERK- and JNK-mediated autophagy in human hepatocellular carcinoma BEL-7402 cells

    doi: 10.1038/aps.2015.99

    Figure Lengend Snippet: PD induces apoptosis in BEL-7402 cells. BEL-7402 cells were treated with different concentrations of PD for 24 h. (A) Apoptotic cells were detected by Annexin V/PI staining using flow cytometry. (B) Quantitation of the results obtained from (A). (C) The apoptotic cells were evaluated with Hoechst 33342 staining and imaged using the In Cell Analyzer 2000 System. (D) The expression of apoptosis-related proteins, including cleaved PARP, cleaved caspase-3, Bcl-2, and Bax, were analyzed by Western blot analysis. (E) Quantitation of the results obtained from (D). b P

    Article Snippet: 3-[4,5-Dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide (MTT), DMSO, CQ, BAF, phenylmethanesulfonyl fluoride, monodansylcadaverine (MDC), and Hoechst 33342 were obtained from Sigma (St Louis, MO, USA).

    Techniques: Staining, Flow Cytometry, Cytometry, Quantitation Assay, Expressing, Western Blot