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Addgene inc rfp lamp1
Rfp Lamp1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rfp lamp1/product/Addgene inc
Average 92 stars, based on 4 article reviews
Price from $9.99 to $1999.99
rfp lamp1 - by Bioz Stars, 2020-04
92/100 stars

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Related Articles

Clone Assay:

Article Title: ADAP2 Is an Interferon Stimulated Gene That Restricts RNA Virus Entry
Article Snippet: GFP-ADAP2-ΔArfGAP and GFP-ADAP2-ΔPH2 were constructed by amplification of ADAP2 cDNA with primers beginning at residue 132 or ending at residue 254, respectively, followed by cloning into NT-GFP Fusion TOPO TA according to the manufacturer’s protocol (Invitrogen). .. RFP-LAMP1 (plasmid #1817), RFP-EEA1 (plasmid #42635), GFP-Rab8a (plasmid #24898), GFP-Rab8a[Q67L] (plasmid #24900), and pcDNA-HA-Arf6 (plasmid # 10834) were obtained from Addgene.

Article Title: Identification of a Phosphorylation-Dependent Nuclear Localization Motif in Interferon Regulatory Factor 2 Binding Protein 2
Article Snippet: For generating mutant constructs, the IRF2BP2 mutants – 355 GAC GAC GAC GAC 358 (aspartic acids), TCT 360 TAT (S360A), and TCT 360 GAT (S360D) - flanked between the endogenous PstI and NcoI in IRF2BP2 were ordered from Integrated DNA Technologies (San Diego, CA) as pIDTSMART-IRF2BP2 and were subcloned in pEGFP-IRF2BP2a (Cloning details will be provided upon inquiry). .. DsRed-Rab11 WT (Addgene plasmid 12679) and RFP-LAMP1 (Addgene plasmid 1817) expression vectors were obtained from Addgene (Cambridge, MA).

Transfection:

Article Title: Sensitization of tamoxifen-resistant breast cancer cells by Z-ligustilide through inhibiting autophagy and accumulating DNA damages
Article Snippet: .. Plasmids transfection The GFP-LC3, RFP-LC3, RFP-LAMP1 and mRFP-LC3 tandem fluorescence-tagged LC3 construct (tfLC3) were provided by Addgene. .. MCF-7 and MCF-7TR5 cells were transfected with GFP-LC3B or RFP-LC3 plasmid using lipofectamine 2000 (Invitrogen, 11668–019).

Article Title: ADAP2 Is an Interferon Stimulated Gene That Restricts RNA Virus Entry
Article Snippet: Paragraph title: Plasmids, siRNAs, and transfections ... RFP-LAMP1 (plasmid #1817), RFP-EEA1 (plasmid #42635), GFP-Rab8a (plasmid #24898), GFP-Rab8a[Q67L] (plasmid #24900), and pcDNA-HA-Arf6 (plasmid # 10834) were obtained from Addgene.

Article Title: Rab Family Proteins Regulate the Endosomal Trafficking and Function of RGS4
Article Snippet: All tissue culture media and transfection reagents were purchased from Invitrogen and Roche Applied Science, respectively. .. CFP-Arf6, CFP-Arf6TN, RFP-Lamp1, and RFP-Rab7 were obtained from addgene.org.

Amplification:

Article Title: ADAP2 Is an Interferon Stimulated Gene That Restricts RNA Virus Entry
Article Snippet: GFP-ADAP2-ΔArfGAP and GFP-ADAP2-ΔPH2 were constructed by amplification of ADAP2 cDNA with primers beginning at residue 132 or ending at residue 254, respectively, followed by cloning into NT-GFP Fusion TOPO TA according to the manufacturer’s protocol (Invitrogen). .. RFP-LAMP1 (plasmid #1817), RFP-EEA1 (plasmid #42635), GFP-Rab8a (plasmid #24898), GFP-Rab8a[Q67L] (plasmid #24900), and pcDNA-HA-Arf6 (plasmid # 10834) were obtained from Addgene.

Article Title: Identification of a Phosphorylation-Dependent Nuclear Localization Motif in Interferon Regulatory Factor 2 Binding Protein 2
Article Snippet: To generate the GFP-TEAD4 expression construct, TEAD4 was first amplified by PCR with the following primers: 5′- GAA TTC TGG AGC CTT GGA GGG CAC GGC - 3′ ( EcoRI ) and 5′- GGA TCC CCG AGT CTC TCA TTC TTT CAC CAG -3′ ( BamHI ) and subcloned into pEGFP-C1 vector between EcoRI and BamHI sites. .. DsRed-Rab11 WT (Addgene plasmid 12679) and RFP-LAMP1 (Addgene plasmid 1817) expression vectors were obtained from Addgene (Cambridge, MA).

Fluorescence:

Article Title: Sensitization of tamoxifen-resistant breast cancer cells by Z-ligustilide through inhibiting autophagy and accumulating DNA damages
Article Snippet: .. Plasmids transfection The GFP-LC3, RFP-LC3, RFP-LAMP1 and mRFP-LC3 tandem fluorescence-tagged LC3 construct (tfLC3) were provided by Addgene. .. MCF-7 and MCF-7TR5 cells were transfected with GFP-LC3B or RFP-LC3 plasmid using lipofectamine 2000 (Invitrogen, 11668–019).

Mutagenesis:

Article Title: Amyotrophic lateral sclerosis-linked mutations increase the viscosity of liquid-like TDP-43 RNP granules in neurons
Article Snippet: Constructs include EGFP–TDP-43 WT (a gift from Virginia Lee, University of Pennsylvania, Philadelphia), TDP-43 5FL (a gift from Aaron Gitler, Stanford University, Stanford, CA), DsRed2-mito (a gift from Thomas Schwarz, Boston Children's Hospital, Boston), PAGFP-Synapsin 1a (a gift from Paul Greengard, The Rockefeller University, New York), and RFP-LAMP1 (Addgene; no. 1817). .. A site-directed mutagenesis kit (Stratagene) was used to generate EGFP–TDP-43 M337V and G298S from the WT construct.

Article Title: ADAP2 Is an Interferon Stimulated Gene That Restricts RNA Virus Entry
Article Snippet: Mutagenesis was performed using Quikchange (Stratagene) according to the manufacturer’s protocol. .. RFP-LAMP1 (plasmid #1817), RFP-EEA1 (plasmid #42635), GFP-Rab8a (plasmid #24898), GFP-Rab8a[Q67L] (plasmid #24900), and pcDNA-HA-Arf6 (plasmid # 10834) were obtained from Addgene.

Article Title: Identification of a Phosphorylation-Dependent Nuclear Localization Motif in Interferon Regulatory Factor 2 Binding Protein 2
Article Snippet: For generating mutant constructs, the IRF2BP2 mutants – 355 GAC GAC GAC GAC 358 (aspartic acids), TCT 360 TAT (S360A), and TCT 360 GAT (S360D) - flanked between the endogenous PstI and NcoI in IRF2BP2 were ordered from Integrated DNA Technologies (San Diego, CA) as pIDTSMART-IRF2BP2 and were subcloned in pEGFP-IRF2BP2a (Cloning details will be provided upon inquiry). .. DsRed-Rab11 WT (Addgene plasmid 12679) and RFP-LAMP1 (Addgene plasmid 1817) expression vectors were obtained from Addgene (Cambridge, MA).

Subcloning:

Article Title: Identification of a Phosphorylation-Dependent Nuclear Localization Motif in Interferon Regulatory Factor 2 Binding Protein 2
Article Snippet: DsRed-Rab11 WT (Addgene plasmid 12679) and RFP-LAMP1 (Addgene plasmid 1817) expression vectors were obtained from Addgene (Cambridge, MA). .. Expression vectors for wild type IRF2BP2, S360A and S360D mutants were generated by subcloning a full-length EcoR1 fragment from pEGFP-IRF2BP2 into the pXJ40-CMVHA vector.

Marker:

Article Title: Rab Family Proteins Regulate the Endosomal Trafficking and Function of RGS4
Article Snippet: Fluorescent-tagged versions of the trans-Golgi network marker protein TGN38 were from J. Lippincott-Schwartz (National Institutes of Health, Bethesda), and CFP-Rab5a, CFP-Rab5SN, and CFP-Rab11 were generously provided by Marino Zerial (Max Planck Institute). .. CFP-Arf6, CFP-Arf6TN, RFP-Lamp1, and RFP-Rab7 were obtained from addgene.org.

Incubation:

Article Title: Sensitization of tamoxifen-resistant breast cancer cells by Z-ligustilide through inhibiting autophagy and accumulating DNA damages
Article Snippet: Plasmids transfection The GFP-LC3, RFP-LC3, RFP-LAMP1 and mRFP-LC3 tandem fluorescence-tagged LC3 construct (tfLC3) were provided by Addgene. .. After a 6 hour incubation, the transfection medium was removed, and the cells were incubated in fresh medium for 24 h. Then, cells were treated with TAM (5 μM) or Z-LIG (50 μM) for 24 h prior to fixation.

Microscopy:

Article Title: Sensitization of tamoxifen-resistant breast cancer cells by Z-ligustilide through inhibiting autophagy and accumulating DNA damages
Article Snippet: Plasmids transfection The GFP-LC3, RFP-LC3, RFP-LAMP1 and mRFP-LC3 tandem fluorescence-tagged LC3 construct (tfLC3) were provided by Addgene. .. Image acquisition was done using an Olympus FV1000 (Olympus) confocal microscope.

Purification:

Article Title: Identification of a Phosphorylation-Dependent Nuclear Localization Motif in Interferon Regulatory Factor 2 Binding Protein 2
Article Snippet: All plasmid constructs were purified by CsCl-banding and their reading frames were verified by sequencing. .. DsRed-Rab11 WT (Addgene plasmid 12679) and RFP-LAMP1 (Addgene plasmid 1817) expression vectors were obtained from Addgene (Cambridge, MA).

Sequencing:

Article Title: Identification of a Phosphorylation-Dependent Nuclear Localization Motif in Interferon Regulatory Factor 2 Binding Protein 2
Article Snippet: All plasmid constructs were purified by CsCl-banding and their reading frames were verified by sequencing. .. DsRed-Rab11 WT (Addgene plasmid 12679) and RFP-LAMP1 (Addgene plasmid 1817) expression vectors were obtained from Addgene (Cambridge, MA).

Generated:

Article Title: Identification of a Phosphorylation-Dependent Nuclear Localization Motif in Interferon Regulatory Factor 2 Binding Protein 2
Article Snippet: Similarly, a pEGFP-IRF2BP2b construct was generated by swapping a PstI/NcoI fragment of IRF2BP2b into the pEGFP-IRF2BP2a clone. .. DsRed-Rab11 WT (Addgene plasmid 12679) and RFP-LAMP1 (Addgene plasmid 1817) expression vectors were obtained from Addgene (Cambridge, MA).

Construct:

Article Title: Sensitization of tamoxifen-resistant breast cancer cells by Z-ligustilide through inhibiting autophagy and accumulating DNA damages
Article Snippet: .. Plasmids transfection The GFP-LC3, RFP-LC3, RFP-LAMP1 and mRFP-LC3 tandem fluorescence-tagged LC3 construct (tfLC3) were provided by Addgene. .. MCF-7 and MCF-7TR5 cells were transfected with GFP-LC3B or RFP-LC3 plasmid using lipofectamine 2000 (Invitrogen, 11668–019).

Article Title: Amyotrophic lateral sclerosis-linked mutations increase the viscosity of liquid-like TDP-43 RNP granules in neurons
Article Snippet: .. Constructs include EGFP–TDP-43 WT (a gift from Virginia Lee, University of Pennsylvania, Philadelphia), TDP-43 5FL (a gift from Aaron Gitler, Stanford University, Stanford, CA), DsRed2-mito (a gift from Thomas Schwarz, Boston Children's Hospital, Boston), PAGFP-Synapsin 1a (a gift from Paul Greengard, The Rockefeller University, New York), and RFP-LAMP1 (Addgene; no. 1817). .. The EGFP tag on the TDP-43 construct was replaced with either Halo tag or mEos3.2 tag (Addgene; no. 54550) to generate Halo– and mEos3.2–TDP-43 WT constructs, respectively.

Article Title: ADAP2 Is an Interferon Stimulated Gene That Restricts RNA Virus Entry
Article Snippet: GFP-ADAP2-ΔArfGAP and GFP-ADAP2-ΔPH2 were constructed by amplification of ADAP2 cDNA with primers beginning at residue 132 or ending at residue 254, respectively, followed by cloning into NT-GFP Fusion TOPO TA according to the manufacturer’s protocol (Invitrogen). .. RFP-LAMP1 (plasmid #1817), RFP-EEA1 (plasmid #42635), GFP-Rab8a (plasmid #24898), GFP-Rab8a[Q67L] (plasmid #24900), and pcDNA-HA-Arf6 (plasmid # 10834) were obtained from Addgene.

Article Title: Identification of a Phosphorylation-Dependent Nuclear Localization Motif in Interferon Regulatory Factor 2 Binding Protein 2
Article Snippet: Paragraph title: Plasmid Constructs ... DsRed-Rab11 WT (Addgene plasmid 12679) and RFP-LAMP1 (Addgene plasmid 1817) expression vectors were obtained from Addgene (Cambridge, MA).

Expressing:

Article Title: Amyotrophic lateral sclerosis-linked mutations increase the viscosity of liquid-like TDP-43 RNP granules in neurons
Article Snippet: Constructs include EGFP–TDP-43 WT (a gift from Virginia Lee, University of Pennsylvania, Philadelphia), TDP-43 5FL (a gift from Aaron Gitler, Stanford University, Stanford, CA), DsRed2-mito (a gift from Thomas Schwarz, Boston Children's Hospital, Boston), PAGFP-Synapsin 1a (a gift from Paul Greengard, The Rockefeller University, New York), and RFP-LAMP1 (Addgene; no. 1817). .. Of note, the position of the tag on the N terminus vs. C terminus affected the expression pattern of TDP-43 in neurons.

Article Title: ADAP2 Is an Interferon Stimulated Gene That Restricts RNA Virus Entry
Article Snippet: Plasmids, siRNAs, and transfections ADAP2-V5 and GFP-ADAP2 were constructed by amplification of human ADAP2 cDNA (clone Id: 5214358, Thermo Scientific) and cloning into pcDNA 3.1/V5-His TOPO TA Expression Kit or NT-GFP Fusion TOPO TA according to the manufacturer’s protocol (Invitrogen). .. RFP-LAMP1 (plasmid #1817), RFP-EEA1 (plasmid #42635), GFP-Rab8a (plasmid #24898), GFP-Rab8a[Q67L] (plasmid #24900), and pcDNA-HA-Arf6 (plasmid # 10834) were obtained from Addgene.

Article Title: Identification of a Phosphorylation-Dependent Nuclear Localization Motif in Interferon Regulatory Factor 2 Binding Protein 2
Article Snippet: .. DsRed-Rab11 WT (Addgene plasmid 12679) and RFP-LAMP1 (Addgene plasmid 1817) expression vectors were obtained from Addgene (Cambridge, MA). .. Expression vectors for wild type IRF2BP2, S360A and S360D mutants were generated by subcloning a full-length EcoR1 fragment from pEGFP-IRF2BP2 into the pXJ40-CMVHA vector.

Polymerase Chain Reaction:

Article Title: Identification of a Phosphorylation-Dependent Nuclear Localization Motif in Interferon Regulatory Factor 2 Binding Protein 2
Article Snippet: To generate the GFP-TEAD4 expression construct, TEAD4 was first amplified by PCR with the following primers: 5′- GAA TTC TGG AGC CTT GGA GGG CAC GGC - 3′ ( EcoRI ) and 5′- GGA TCC CCG AGT CTC TCA TTC TTT CAC CAG -3′ ( BamHI ) and subcloned into pEGFP-C1 vector between EcoRI and BamHI sites. .. DsRed-Rab11 WT (Addgene plasmid 12679) and RFP-LAMP1 (Addgene plasmid 1817) expression vectors were obtained from Addgene (Cambridge, MA).

Plasmid Preparation:

Article Title: Sensitization of tamoxifen-resistant breast cancer cells by Z-ligustilide through inhibiting autophagy and accumulating DNA damages
Article Snippet: Plasmids transfection The GFP-LC3, RFP-LC3, RFP-LAMP1 and mRFP-LC3 tandem fluorescence-tagged LC3 construct (tfLC3) were provided by Addgene. .. MCF-7 and MCF-7TR5 cells were transfected with GFP-LC3B or RFP-LC3 plasmid using lipofectamine 2000 (Invitrogen, 11668–019).

Article Title: ADAP2 Is an Interferon Stimulated Gene That Restricts RNA Virus Entry
Article Snippet: .. RFP-LAMP1 (plasmid #1817), RFP-EEA1 (plasmid #42635), GFP-Rab8a (plasmid #24898), GFP-Rab8a[Q67L] (plasmid #24900), and pcDNA-HA-Arf6 (plasmid # 10834) were obtained from Addgene. .. EGFP-tagged Rab34 and Rab7 constructs have been described previously [ ].

Article Title: Rab Family Proteins Regulate the Endosomal Trafficking and Function of RGS4
Article Snippet: The pIRESpuro-GLUE plasmid, used to generate the hemagglutinin (HA)-tagged RGS4-HA, RGS4-HA, was a kind gift from Stephane Anger (University of Toronto). .. CFP-Arf6, CFP-Arf6TN, RFP-Lamp1, and RFP-Rab7 were obtained from addgene.org.

Article Title: Identification of a Phosphorylation-Dependent Nuclear Localization Motif in Interferon Regulatory Factor 2 Binding Protein 2
Article Snippet: .. DsRed-Rab11 WT (Addgene plasmid 12679) and RFP-LAMP1 (Addgene plasmid 1817) expression vectors were obtained from Addgene (Cambridge, MA). .. Expression vectors for wild type IRF2BP2, S360A and S360D mutants were generated by subcloning a full-length EcoR1 fragment from pEGFP-IRF2BP2 into the pXJ40-CMVHA vector.

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    Addgene inc lamp1 rfp
    TAPE is localized in endolysosomes. A and B , 293 cells were transfected with EGFP-tagged hTAPE and its deletion mutants, together with an endosomal protein Rab5-DsRed ( A ) or a lysosomal protein <t>Lamp1-RFP</t> ( B ). Transfected cells were then passed into a
    Lamp1 Rfp, supplied by Addgene inc, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lamp1 rfp/product/Addgene inc
    Average 99 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    lamp1 rfp - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

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    TAPE is localized in endolysosomes. A and B , 293 cells were transfected with EGFP-tagged hTAPE and its deletion mutants, together with an endosomal protein Rab5-DsRed ( A ) or a lysosomal protein Lamp1-RFP ( B ). Transfected cells were then passed into a

    Journal: The Journal of Biological Chemistry

    Article Title: TBK1-associated Protein in Endolysosomes (TAPE) Is an Innate Immune Regulator Modulating the TLR3 and TLR4 Signaling Pathways *

    doi: 10.1074/jbc.M110.164632

    Figure Lengend Snippet: TAPE is localized in endolysosomes. A and B , 293 cells were transfected with EGFP-tagged hTAPE and its deletion mutants, together with an endosomal protein Rab5-DsRed ( A ) or a lysosomal protein Lamp1-RFP ( B ). Transfected cells were then passed into a

    Article Snippet: HEK293 cells in the 6-well plate were transfected with TAPE-EGFP together with Rab5-DsRed (an early endosomal marker) or Lamp1-RFP (a lysosomal marker).

    Techniques: Transfection

    Time-lapse imaging after infection of U251 glioma cells with a VP26-GFP–expressing HSV-1 in tubacin-treated and/or IFN-β–treated RFP-LAMP1–expressing U251 cells.

    Journal: The Journal of Clinical Investigation

    Article Title: Histone deacetylase 6 inhibition enhances oncolytic viral replication in glioma

    doi: 10.1172/JCI80713

    Figure Lengend Snippet: Time-lapse imaging after infection of U251 glioma cells with a VP26-GFP–expressing HSV-1 in tubacin-treated and/or IFN-β–treated RFP-LAMP1–expressing U251 cells.

    Article Snippet: HDAC6 flag (plasmid #13823), pmCherry_a_tubulin_IRES_puro2 (plasmid#21043), and LAMP1-RFP (plasmid #1817) were obtained from Addgene.

    Techniques: Imaging, Infection, Expressing

    Percentage colocalization of GFP-VP26 intracellular capsids in RFP-LAMP1–expressing U251 cells.

    Journal: The Journal of Clinical Investigation

    Article Title: Histone deacetylase 6 inhibition enhances oncolytic viral replication in glioma

    doi: 10.1172/JCI80713

    Figure Lengend Snippet: Percentage colocalization of GFP-VP26 intracellular capsids in RFP-LAMP1–expressing U251 cells.

    Article Snippet: HDAC6 flag (plasmid #13823), pmCherry_a_tubulin_IRES_puro2 (plasmid#21043), and LAMP1-RFP (plasmid #1817) were obtained from Addgene.

    Techniques: Expressing

    Subcellular co-localization of GFP-hRFT1, -2, or -3 with LAMP1-RFP in MDCK cells MDCK cells were transiently co-transfected with GFP-hRFT1, -2, or -3 and LAMP1-RFP. Representative images from at least three separate experiments were shown for indicated constructs.

    Journal: Biochimica et biophysica acta

    Article Title: Differential expression of human riboflavin transporters -1, -2, -3 in polarized epithelia: a key role for hRFT-2 in intestinal riboflavin uptake

    doi: 10.1016/j.bbamem.2011.08.004

    Figure Lengend Snippet: Subcellular co-localization of GFP-hRFT1, -2, or -3 with LAMP1-RFP in MDCK cells MDCK cells were transiently co-transfected with GFP-hRFT1, -2, or -3 and LAMP1-RFP. Representative images from at least three separate experiments were shown for indicated constructs.

    Article Snippet: LAMP1-RFP was obtained from Addgene Inc (Cambridge, MA).

    Techniques: Transfection, Construct

    Effects of Tioconazole on Autophagic Activity in Cancer Cells. (A) Human glioma H4 cells that stably express GFP-LC3 were transfected with 5 nM non-targeting siRNA (Ctrl) or siRNA against ATG4 family members (ATG4) for 48 h, and the knockdown efficiency of ATG4 was verified with real time PCR. ( B) The knocked-down cells were fixed for observation via fluorescence microscopy, and the number of GFP-LC3 puncta is quantified in the right panel. Bar: 20 μm. (C) The cells that stably express GFP-LC3 were treated with tioconazole for 8 h and fixed to observe the GFP-LC3 puncta, which were ( D) quantified via fluorescence microscopy. The autophagy inhibitor CQ was used as a positive control. Bar: 20 μm. (E) HCT16 cells treated with tioconazole (Tc, 40 μM) or CQ (20 μM) for 6 h were fixed and imaged with TEM. Representative autophagic vacuoles (AVs) are shown. Arrowhead: autophagosome. Bar: 200 nm. ( F) The numbers of AVs for each optical section (9 μm 2 ) are quantified (n=8). ( G ) HCT116 cells expressing GFP-LC3 and RFP-Lamp1 were treated with Tc for 6 h and fixed to observe colocalization of GFP-LC3 and RFP-Lamp1 with confocal microscopy. GFP-LC3 that had colocalized with or was surrounded by RFP-Lamp1 was identified as fusion between autophagosomes and lysosomes. The colocalization coefficients of images were quantified by the Ziess LSM 710 Software and are shown in the right panel. Bar: 20 μm. (H) HCT16 cells were transfected with siRNA (5 nM) for 66 h and then treated with CQ (20 μM) for 2 h. Cells were harvested for immunoblotting using antibodies against LC3, ATG4B or ACTB, and autophagy flux was quantified in untreated cells and cells treated with CQ based on changes in LC3-II. (I) H4, HCT116 or MDA-MB-231 cells were treated with Tc for 6 h in the presence (+) or absence (-) of CQ (20 μM) and harvested for immunoblotting. (J) The autophagic flux was quantified as described in (I). Representative data are shown, and the quantified results are expressed as the mean ± SEM from at least 3 individual experiments. *p

    Journal: Theranostics

    Article Title: Drug Repurposing Screening Identifies Tioconazole as an ATG4 Inhibitor that Suppresses Autophagy and Sensitizes Cancer Cells to Chemotherapy

    doi: 10.7150/thno.22012

    Figure Lengend Snippet: Effects of Tioconazole on Autophagic Activity in Cancer Cells. (A) Human glioma H4 cells that stably express GFP-LC3 were transfected with 5 nM non-targeting siRNA (Ctrl) or siRNA against ATG4 family members (ATG4) for 48 h, and the knockdown efficiency of ATG4 was verified with real time PCR. ( B) The knocked-down cells were fixed for observation via fluorescence microscopy, and the number of GFP-LC3 puncta is quantified in the right panel. Bar: 20 μm. (C) The cells that stably express GFP-LC3 were treated with tioconazole for 8 h and fixed to observe the GFP-LC3 puncta, which were ( D) quantified via fluorescence microscopy. The autophagy inhibitor CQ was used as a positive control. Bar: 20 μm. (E) HCT16 cells treated with tioconazole (Tc, 40 μM) or CQ (20 μM) for 6 h were fixed and imaged with TEM. Representative autophagic vacuoles (AVs) are shown. Arrowhead: autophagosome. Bar: 200 nm. ( F) The numbers of AVs for each optical section (9 μm 2 ) are quantified (n=8). ( G ) HCT116 cells expressing GFP-LC3 and RFP-Lamp1 were treated with Tc for 6 h and fixed to observe colocalization of GFP-LC3 and RFP-Lamp1 with confocal microscopy. GFP-LC3 that had colocalized with or was surrounded by RFP-Lamp1 was identified as fusion between autophagosomes and lysosomes. The colocalization coefficients of images were quantified by the Ziess LSM 710 Software and are shown in the right panel. Bar: 20 μm. (H) HCT16 cells were transfected with siRNA (5 nM) for 66 h and then treated with CQ (20 μM) for 2 h. Cells were harvested for immunoblotting using antibodies against LC3, ATG4B or ACTB, and autophagy flux was quantified in untreated cells and cells treated with CQ based on changes in LC3-II. (I) H4, HCT116 or MDA-MB-231 cells were treated with Tc for 6 h in the presence (+) or absence (-) of CQ (20 μM) and harvested for immunoblotting. (J) The autophagic flux was quantified as described in (I). Representative data are shown, and the quantified results are expressed as the mean ± SEM from at least 3 individual experiments. *p

    Article Snippet: For protein colocalization, the H4 stable cells previously described were transfected with 1 μg plasmids Cherry tagged ATG4B or Lamp1-RFP (Addgene, 1817) for 36 h using Lipofectamine 2000 (Invitrogen).

    Techniques: Activity Assay, Stable Transfection, Transfection, Real-time Polymerase Chain Reaction, Fluorescence, Microscopy, Positive Control, Transmission Electron Microscopy, Expressing, Confocal Microscopy, Software, Multiple Displacement Amplification