oligonucleotide duplexes Search Results


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  • 99
    Thermo Fisher sirna oligonucleotide duplexes
    hGAAP expression alters the number and size of focal adhesions. U2-OS cells <t>transfected</t> with <t>siRNA</t> (24 h) or plasmids encoding hGAAP were seeded onto fibronectin-coated slides. (A) Confocal images show cells transfected with vinculin-GFP (14 h), fixed, and stained with phalloidin–Alexa Fluor 568. (B) Summary results (means ± SEM from ≥25 cells for each condition) show numbers of focal adhesions per cell, determined by counting vinculin-GFP spots. (C) IRM images of U2-OS cells overexpressing hGAAP or hGAAP Ctmut, or transfected with the indicated siRNAs. Images are typical of three independent experiments. (D) Example of the image analysis used to determine focal adhesion number and size. Individual cells were imaged for IRM (a) and phalloidin staining (b). IRM images were then band pass filtered (c), and a threshold was imposed (d) to obtain the final images used to determine the number and size of the adhesions. (E and F) Summary results (means ± SEM from ≥20 cells for each condition) show numbers of adhesions per cell (E) and their areas (F). *, P
    Sirna Oligonucleotide Duplexes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 147 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore sirna oligonucleotide duplexes
    Cetuximab sensitizes cancer cells to ROS-elevating agent-induced apoptosis in an <t>EGFR-expression-dependent</t> manner. ( A ) HN5 cells were subjected to knockdown with each of two different EGFR siRNAs or control <t>siRNA</t> for 72 h. During the last 24 h of siRNA transfection, the cells were either untreated or treated with 10 mM DCA, 20 nM cetuximab, or both. Cell lysates were subjected to Western blotting with the indicated antibodies. ( B ) HN5 and FaDu cells were subjected to knockdown of Rab5 or Rab11 with specific siRNAs or control siRNA for 72 h. During the last 24 h of siRNA transfection, the cells were either untreated or treated with 10 mM DCA, 20 nM cetuximab, or both. Cell lysates were subjected to Western blotting with the indicated antibodies.
    Sirna Oligonucleotide Duplexes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Horizon Discovery sirna duplex oligonucleotides
    Cdc7 codepletion rescues fork slowing in <t>Chk1-depleted</t> cells. ( A ) Protein levels of Cdc7, Chk1, and β-Actin (loading control) in U2OS cells after 48 hours depletion with Cdc7, Chk1, Cdc7, and Chk1 or control <t>siRNA.</t> ( B ) Quantification of origin firing in Cdc7-, Chk1- or control-depleted cells. First label origins (green-red-green) are shown as percentage of all red (CldU) labeled tracks. ( C ) Distribution of replication fork speeds in Cdc7- or control-depleted cells. ( D ) Distribution of replication fork speeds in Chk1- or control-depleted cells. ( E ) Distribution of replication fork speeds in Cdc7- or Cdc7 and Chk1-depleted cells. ( F ) Average replication fork speeds in Cdc7-, Chk1-, or control-depleted cells. Means and standard deviation (S.D.) (bars) of three independent experiments are shown. Values marked with asterisks are significantly different (student’s t -test, * p
    Sirna Duplex Oligonucleotides, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 89/100, based on 82 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Horizon Discovery sirna oligonucleotide duplexes
    WRN regulates mRNA levels of <t>XLF.</t> (A) XLF protein levels decreased in WRN -deficient fibroblasts. Whole cell lysates were prepared from human fibroblasts GM00637G (normal) and AG11395A ( WRN deficient) and subjected to immunoblotting analysis for XLF and β-actin. (B) Inhibition of WRN expression by <t>siRNA</t> resulted in a decrease of XLF protein levels. U2OS cells were transfected with si-CTR, XLF siRNA (si1-XLF or si2-XLF) or WRN siRNA (si1-WRN or si2-WRN). Total cell lysates were harvested 48 h following transfection and subjected to immunoblotting analysis with the antibodies indicated. (C) MG132 treatment did not restore the decrease of XLF protein levels caused by the depletion of WRN. U2OS cells were treated as described in (B) except that MG132 was added to cells 4 h prior to harvest. (D) Inhibition of WRN expression led to a decrease of XLF mRNA. U2OS cells were treated as described in (B). Total RNA was extracted and subjected to real-time RT-PCR assays for XLF mRNA. XLF, XRCC4-like factor; si-CTR, control siRNA; RT-PCR, reverse transcription PCR.
    Sirna Oligonucleotide Duplexes, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 89/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher sirna duplex oligonucleotides
    WRN regulates mRNA levels of <t>XLF.</t> (A) XLF protein levels decreased in WRN -deficient fibroblasts. Whole cell lysates were prepared from human fibroblasts GM00637G (normal) and AG11395A ( WRN deficient) and subjected to immunoblotting analysis for XLF and β-actin. (B) Inhibition of WRN expression by <t>siRNA</t> resulted in a decrease of XLF protein levels. U2OS cells were transfected with si-CTR, XLF siRNA (si1-XLF or si2-XLF) or WRN siRNA (si1-WRN or si2-WRN). Total cell lysates were harvested 48 h following transfection and subjected to immunoblotting analysis with the antibodies indicated. (C) MG132 treatment did not restore the decrease of XLF protein levels caused by the depletion of WRN. U2OS cells were treated as described in (B) except that MG132 was added to cells 4 h prior to harvest. (D) Inhibition of WRN expression led to a decrease of XLF mRNA. U2OS cells were treated as described in (B). Total RNA was extracted and subjected to real-time RT-PCR assays for XLF mRNA. XLF, XRCC4-like factor; si-CTR, control siRNA; RT-PCR, reverse transcription PCR.
    Sirna Duplex Oligonucleotides, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery oligonucleotide duplexes
    WRN regulates mRNA levels of <t>XLF.</t> (A) XLF protein levels decreased in WRN -deficient fibroblasts. Whole cell lysates were prepared from human fibroblasts GM00637G (normal) and AG11395A ( WRN deficient) and subjected to immunoblotting analysis for XLF and β-actin. (B) Inhibition of WRN expression by <t>siRNA</t> resulted in a decrease of XLF protein levels. U2OS cells were transfected with si-CTR, XLF siRNA (si1-XLF or si2-XLF) or WRN siRNA (si1-WRN or si2-WRN). Total cell lysates were harvested 48 h following transfection and subjected to immunoblotting analysis with the antibodies indicated. (C) MG132 treatment did not restore the decrease of XLF protein levels caused by the depletion of WRN. U2OS cells were treated as described in (B) except that MG132 was added to cells 4 h prior to harvest. (D) Inhibition of WRN expression led to a decrease of XLF mRNA. U2OS cells were treated as described in (B). Total RNA was extracted and subjected to real-time RT-PCR assays for XLF mRNA. XLF, XRCC4-like factor; si-CTR, control siRNA; RT-PCR, reverse transcription PCR.
    Oligonucleotide Duplexes, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 90/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher synthetic oligonucleotide duplexes
    WRN regulates mRNA levels of <t>XLF.</t> (A) XLF protein levels decreased in WRN -deficient fibroblasts. Whole cell lysates were prepared from human fibroblasts GM00637G (normal) and AG11395A ( WRN deficient) and subjected to immunoblotting analysis for XLF and β-actin. (B) Inhibition of WRN expression by <t>siRNA</t> resulted in a decrease of XLF protein levels. U2OS cells were transfected with si-CTR, XLF siRNA (si1-XLF or si2-XLF) or WRN siRNA (si1-WRN or si2-WRN). Total cell lysates were harvested 48 h following transfection and subjected to immunoblotting analysis with the antibodies indicated. (C) MG132 treatment did not restore the decrease of XLF protein levels caused by the depletion of WRN. U2OS cells were treated as described in (B) except that MG132 was added to cells 4 h prior to harvest. (D) Inhibition of WRN expression led to a decrease of XLF mRNA. U2OS cells were treated as described in (B). Total RNA was extracted and subjected to real-time RT-PCR assays for XLF mRNA. XLF, XRCC4-like factor; si-CTR, control siRNA; RT-PCR, reverse transcription PCR.
    Synthetic Oligonucleotide Duplexes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    GenePharma Company sirna oligonucleotide duplexes
    WRN regulates mRNA levels of <t>XLF.</t> (A) XLF protein levels decreased in WRN -deficient fibroblasts. Whole cell lysates were prepared from human fibroblasts GM00637G (normal) and AG11395A ( WRN deficient) and subjected to immunoblotting analysis for XLF and β-actin. (B) Inhibition of WRN expression by <t>siRNA</t> resulted in a decrease of XLF protein levels. U2OS cells were transfected with si-CTR, XLF siRNA (si1-XLF or si2-XLF) or WRN siRNA (si1-WRN or si2-WRN). Total cell lysates were harvested 48 h following transfection and subjected to immunoblotting analysis with the antibodies indicated. (C) MG132 treatment did not restore the decrease of XLF protein levels caused by the depletion of WRN. U2OS cells were treated as described in (B) except that MG132 was added to cells 4 h prior to harvest. (D) Inhibition of WRN expression led to a decrease of XLF mRNA. U2OS cells were treated as described in (B). Total RNA was extracted and subjected to real-time RT-PCR assays for XLF mRNA. XLF, XRCC4-like factor; si-CTR, control siRNA; RT-PCR, reverse transcription PCR.
    Sirna Oligonucleotide Duplexes, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 89/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenePharma Company small interfering rna sirna duplex oligonucleotides
    Effect of LPS (75 µg/ml) treatment on SOD activity in HUVECs. LPS treatment significantly decreased the inhibition of SOD in HUVECs/si-NC and HUVECs/siCav-1. *P≤0.05 and **P≤0.01. LPS, lipopolysaccharide; SOD, superoxide dismutase; HUVECs, human umbilical vein endothelial cells; Cav-1, caveolin-1; si, small interfering <t>RNA;</t> NC, negative control.
    Small Interfering Rna Sirna Duplex Oligonucleotides, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 88/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Horizon Discovery duplex rna oligonucleotides
    (A) Schematic illustration of the sequence motifs in the helicase core of DEAD box <t>RNA</t> helicase and the <t>p68</t> LGLD and HLIGR mutants. (B) Coomassie blue staining of SDS-PAGE of recombinant p68 wt (lane 1), LGLD mutant (lane 2), or HILGR mutant (lane 3) that were expressed and purified from bacterial E. coli. (C) Cross-links of p68 wt (lane 1), LGLD mutant (lane 2), or HILGR mutant (lane 3) to [α- 32 P]ATP were analyzed by SDS-PAGE followed by autoradiography. (D) RNA unwinding by p68 wt (lane 3), LGLD mutant (lane 4), or HILGR mutant (lane 5) was analyzed by SDS-PAGE followed by autoradiography. Lane 1 is duplex RNA denatured by heating to 95°C. Lane 2 is the duplex RNA. (E) ATPase activities of p68 wt/mutant (indicated) were measured by colorimetric assay. The ATPase activity ( y axis) is expressed as μM P i /mg of p68 or mutants.
    Duplex Rna Oligonucleotides, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Kaneka Corp oligonucleotide duplexes
    (A) Schematic illustration of the sequence motifs in the helicase core of DEAD box <t>RNA</t> helicase and the <t>p68</t> LGLD and HLIGR mutants. (B) Coomassie blue staining of SDS-PAGE of recombinant p68 wt (lane 1), LGLD mutant (lane 2), or HILGR mutant (lane 3) that were expressed and purified from bacterial E. coli. (C) Cross-links of p68 wt (lane 1), LGLD mutant (lane 2), or HILGR mutant (lane 3) to [α- 32 P]ATP were analyzed by SDS-PAGE followed by autoradiography. (D) RNA unwinding by p68 wt (lane 3), LGLD mutant (lane 4), or HILGR mutant (lane 5) was analyzed by SDS-PAGE followed by autoradiography. Lane 1 is duplex RNA denatured by heating to 95°C. Lane 2 is the duplex RNA. (E) ATPase activities of p68 wt/mutant (indicated) were measured by colorimetric assay. The ATPase activity ( y axis) is expressed as μM P i /mg of p68 or mutants.
    Oligonucleotide Duplexes, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Trevigen biotin labeled oligonucleotide duplex
    (A) Schematic illustration of the sequence motifs in the helicase core of DEAD box <t>RNA</t> helicase and the <t>p68</t> LGLD and HLIGR mutants. (B) Coomassie blue staining of SDS-PAGE of recombinant p68 wt (lane 1), LGLD mutant (lane 2), or HILGR mutant (lane 3) that were expressed and purified from bacterial E. coli. (C) Cross-links of p68 wt (lane 1), LGLD mutant (lane 2), or HILGR mutant (lane 3) to [α- 32 P]ATP were analyzed by SDS-PAGE followed by autoradiography. (D) RNA unwinding by p68 wt (lane 3), LGLD mutant (lane 4), or HILGR mutant (lane 5) was analyzed by SDS-PAGE followed by autoradiography. Lane 1 is duplex RNA denatured by heating to 95°C. Lane 2 is the duplex RNA. (E) ATPase activities of p68 wt/mutant (indicated) were measured by colorimetric assay. The ATPase activity ( y axis) is expressed as μM P i /mg of p68 or mutants.
    Biotin Labeled Oligonucleotide Duplex, supplied by Trevigen, used in various techniques. Bioz Stars score: 85/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher rna oligonucleotide duplexes
    (A) Schematic illustration of the sequence motifs in the helicase core of DEAD box <t>RNA</t> helicase and the <t>p68</t> LGLD and HLIGR mutants. (B) Coomassie blue staining of SDS-PAGE of recombinant p68 wt (lane 1), LGLD mutant (lane 2), or HILGR mutant (lane 3) that were expressed and purified from bacterial E. coli. (C) Cross-links of p68 wt (lane 1), LGLD mutant (lane 2), or HILGR mutant (lane 3) to [α- 32 P]ATP were analyzed by SDS-PAGE followed by autoradiography. (D) RNA unwinding by p68 wt (lane 3), LGLD mutant (lane 4), or HILGR mutant (lane 5) was analyzed by SDS-PAGE followed by autoradiography. Lane 1 is duplex RNA denatured by heating to 95°C. Lane 2 is the duplex RNA. (E) ATPase activities of p68 wt/mutant (indicated) were measured by colorimetric assay. The ATPase activity ( y axis) is expressed as μM P i /mg of p68 or mutants.
    Rna Oligonucleotide Duplexes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Kaneka Corp sirna oligonucleotide duplexes
    Flotillin upregulation stimulates AXL endocytosis towards non-degradative late endosomes and promotes its stabilization. A) AXL knock-down in MCF10AF1F2 cells decreases ZEB1 and N-cadherin expression . MCF10AmCh and MCF10AF1F2 cells were <t>transfected</t> with control <t>siRNA</t> (CTL) or against AXL and probed by western blotting with antibodies for the indicated proteins. The quantification results are the mean ± SEM of 4 independent experiment, and are expressed as fold-increase compared with control (MCF10AmCh). B) ZEB1 nuclear localization analyzed by immunocytochemistry and co-staining with Hoechst (Hst) and C) E-cadherin distribution analyzed by immunocytochemistry in MCF10AmCh and MCF10AF1F2 cells transfected with control (CTL) or anti-AXL siRNAs. Results shown are representative of 4 independent experiments. Scale bar: 10 µm. C) Flotillin-oligomerization induced by a CRY2-CIBN-based optogenetic approach promotes co-clustering of AXL . Still images from a TIRF-microscopy movie of a MCF10A cell that co-expresses CRY2-mCitrin, flotillin 2-CIBN-mCherry, and AXL-Halo labeled with Halo-Tag-Janelia Fluor 646. Representative image of a whole cell 15s after starting the 488 nm illumination to perform CRY2 binding to flotillin 2-CIBN-mCh and subsequent oligomerization (see also supplementary Fig. S6 for the details regarding the validation of the constructs and the optogenetic approach). Magnified images from the boxed area taken before and after 488 nm illumination. Arrowheads indicate clusters of CRY2-mCitrin and flotillin 2-CIBN-mCherry where AXL-Halo accumulates (see also video 4). Scale bars: 10 µm for the main image and 2 µm for the images from the boxed area. D) Co-accumulation of AXL and flotillin 2 at the PM prior to their co-endocytosis . Still images of a time-lapse series of one MCF10AF1F2 cell that expresses AXL-GFP to show flotillin 2/AXL co-localization at the PM and their co-endocytosis at PM sites (showed by arrowheads). Kymographs show the temporal variation of the GFP and mCherry signals along two line-scans (blue at the PM endocytic site, and yellow close to the PM endocytic site). Graphs show the intensity variation of both signals along the dotted white lines on the kymographs. The left kymograph shows the accumulation of AXL-GFP and flotillin 2-mCherry at the PM followed by a sudden concomitant drop of both signals (around 83s, indicated by a star). The right kymograph shows that in the meantime, both AXL-GFP and flotillin 2-mCherry signals remained stable at the PM outside and close to this endocytic site (See also video 5). E) Flotillin 1 co-precipitates with AXL. AXL was immunoprecipitated from MCF10AF1F2 cell lysates, and flotillin 1 co-immunoprecipitation was assessed by immunoblotting. Control immunoprecipitation was performed with non-relevant immunoglobulins (Ig). F) Flotillin upregulation accelerates AXL endocytosis. Kinetics of AXL internalization in MCF10AmCh and MCF10AF1F2 cells (left), and in MDA-MB-231shLuci and MDA-MB-231shFlot2 cells (right). Surface proteins were labeled with biotin at 4°C, and cells were incubated at 37°C for the indicated times to allow endocytosis. AXL presence in the internalized biotinylated fraction was probed by western blotting using relevant antibodies and quantified as the percentage of the maximum level of internalized protein (reached at 12 and 8 min, respectively, in MCF10AmCh and MCF10AF1F2 cells). Results are expressed as the mean ± SEM of 4 independent experiments. The AXL internalization rates (dashed lines) were 8.48 ± 1.28 and 12.43 ± 0.48 %/min in MCF10AmCh and MCF10AF1F2 cells, respectively, revealing a significant 1.45-fold increase (p=0.0088) upon flotillin upregulation. Comparison of the AXL internalization rates (dashed lines) in MDA-MB-231shLuci and MDA-MB-231shFlot2 cells showed a significant 3.66-fold decrease (p=0.0009) upon flotillin downregulation. Results are expressed as the percentage of the maximum level of surface labeled AXL at T0 and are the mean ± SEM of 4 independent experiments. G) Flotillin upregulation promotes AXL protein level increase . Cell lysates from MCF10AmCh and MCF10AF1F2 cells were probed by western blotting with antibodies against AXL phosphorylated on Y702, AXL and tubulin. Results are expressed as fold-increase compared with MCF10AmCh cells and are the mean ± SEM of 6 independent experiments. H) Flotillin upregulation does not increase AXL mRNA level. RT-qPCR analysis of AXL expression in MCF10AmCh and MCF10AF1F2 cells. Results are expressed relative to the level in MCF10AmCh cells and are the mean ± SEM of 4 independent experiments. I) Flotillin upregulation stabilizes AXL. MCF10A and MCF10AF1F2 cells were incubated with cycloheximide (CHX, 100µg/ml) and cell lysates collected at the indicated time points. AXL and tubulin levels were assessed by western blotting. The graph shows the normalized AXL levels in each cell line during CHX incubation. CHX treatment was stopped after 9 hours because MCF10Amch cells did not survive longer treatment. *P
    Sirna Oligonucleotide Duplexes, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Sigma-Genosys sirna oligonucleotide duplexes
    Flotillin upregulation stimulates AXL endocytosis towards non-degradative late endosomes and promotes its stabilization. A) AXL knock-down in MCF10AF1F2 cells decreases ZEB1 and N-cadherin expression . MCF10AmCh and MCF10AF1F2 cells were <t>transfected</t> with control <t>siRNA</t> (CTL) or against AXL and probed by western blotting with antibodies for the indicated proteins. The quantification results are the mean ± SEM of 4 independent experiment, and are expressed as fold-increase compared with control (MCF10AmCh). B) ZEB1 nuclear localization analyzed by immunocytochemistry and co-staining with Hoechst (Hst) and C) E-cadherin distribution analyzed by immunocytochemistry in MCF10AmCh and MCF10AF1F2 cells transfected with control (CTL) or anti-AXL siRNAs. Results shown are representative of 4 independent experiments. Scale bar: 10 µm. C) Flotillin-oligomerization induced by a CRY2-CIBN-based optogenetic approach promotes co-clustering of AXL . Still images from a TIRF-microscopy movie of a MCF10A cell that co-expresses CRY2-mCitrin, flotillin 2-CIBN-mCherry, and AXL-Halo labeled with Halo-Tag-Janelia Fluor 646. Representative image of a whole cell 15s after starting the 488 nm illumination to perform CRY2 binding to flotillin 2-CIBN-mCh and subsequent oligomerization (see also supplementary Fig. S6 for the details regarding the validation of the constructs and the optogenetic approach). Magnified images from the boxed area taken before and after 488 nm illumination. Arrowheads indicate clusters of CRY2-mCitrin and flotillin 2-CIBN-mCherry where AXL-Halo accumulates (see also video 4). Scale bars: 10 µm for the main image and 2 µm for the images from the boxed area. D) Co-accumulation of AXL and flotillin 2 at the PM prior to their co-endocytosis . Still images of a time-lapse series of one MCF10AF1F2 cell that expresses AXL-GFP to show flotillin 2/AXL co-localization at the PM and their co-endocytosis at PM sites (showed by arrowheads). Kymographs show the temporal variation of the GFP and mCherry signals along two line-scans (blue at the PM endocytic site, and yellow close to the PM endocytic site). Graphs show the intensity variation of both signals along the dotted white lines on the kymographs. The left kymograph shows the accumulation of AXL-GFP and flotillin 2-mCherry at the PM followed by a sudden concomitant drop of both signals (around 83s, indicated by a star). The right kymograph shows that in the meantime, both AXL-GFP and flotillin 2-mCherry signals remained stable at the PM outside and close to this endocytic site (See also video 5). E) Flotillin 1 co-precipitates with AXL. AXL was immunoprecipitated from MCF10AF1F2 cell lysates, and flotillin 1 co-immunoprecipitation was assessed by immunoblotting. Control immunoprecipitation was performed with non-relevant immunoglobulins (Ig). F) Flotillin upregulation accelerates AXL endocytosis. Kinetics of AXL internalization in MCF10AmCh and MCF10AF1F2 cells (left), and in MDA-MB-231shLuci and MDA-MB-231shFlot2 cells (right). Surface proteins were labeled with biotin at 4°C, and cells were incubated at 37°C for the indicated times to allow endocytosis. AXL presence in the internalized biotinylated fraction was probed by western blotting using relevant antibodies and quantified as the percentage of the maximum level of internalized protein (reached at 12 and 8 min, respectively, in MCF10AmCh and MCF10AF1F2 cells). Results are expressed as the mean ± SEM of 4 independent experiments. The AXL internalization rates (dashed lines) were 8.48 ± 1.28 and 12.43 ± 0.48 %/min in MCF10AmCh and MCF10AF1F2 cells, respectively, revealing a significant 1.45-fold increase (p=0.0088) upon flotillin upregulation. Comparison of the AXL internalization rates (dashed lines) in MDA-MB-231shLuci and MDA-MB-231shFlot2 cells showed a significant 3.66-fold decrease (p=0.0009) upon flotillin downregulation. Results are expressed as the percentage of the maximum level of surface labeled AXL at T0 and are the mean ± SEM of 4 independent experiments. G) Flotillin upregulation promotes AXL protein level increase . Cell lysates from MCF10AmCh and MCF10AF1F2 cells were probed by western blotting with antibodies against AXL phosphorylated on Y702, AXL and tubulin. Results are expressed as fold-increase compared with MCF10AmCh cells and are the mean ± SEM of 6 independent experiments. H) Flotillin upregulation does not increase AXL mRNA level. RT-qPCR analysis of AXL expression in MCF10AmCh and MCF10AF1F2 cells. Results are expressed relative to the level in MCF10AmCh cells and are the mean ± SEM of 4 independent experiments. I) Flotillin upregulation stabilizes AXL. MCF10A and MCF10AF1F2 cells were incubated with cycloheximide (CHX, 100µg/ml) and cell lysates collected at the indicated time points. AXL and tubulin levels were assessed by western blotting. The graph shows the normalized AXL levels in each cell line during CHX incubation. CHX treatment was stopped after 9 hours because MCF10Amch cells did not survive longer treatment. *P
    Sirna Oligonucleotide Duplexes, supplied by Sigma-Genosys, used in various techniques. Bioz Stars score: 89/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hGAAP expression alters the number and size of focal adhesions. U2-OS cells transfected with siRNA (24 h) or plasmids encoding hGAAP were seeded onto fibronectin-coated slides. (A) Confocal images show cells transfected with vinculin-GFP (14 h), fixed, and stained with phalloidin–Alexa Fluor 568. (B) Summary results (means ± SEM from ≥25 cells for each condition) show numbers of focal adhesions per cell, determined by counting vinculin-GFP spots. (C) IRM images of U2-OS cells overexpressing hGAAP or hGAAP Ctmut, or transfected with the indicated siRNAs. Images are typical of three independent experiments. (D) Example of the image analysis used to determine focal adhesion number and size. Individual cells were imaged for IRM (a) and phalloidin staining (b). IRM images were then band pass filtered (c), and a threshold was imposed (d) to obtain the final images used to determine the number and size of the adhesions. (E and F) Summary results (means ± SEM from ≥20 cells for each condition) show numbers of adhesions per cell (E) and their areas (F). *, P

    Journal: The Journal of Cell Biology

    Article Title: hGAAP promotes cell adhesion and migration via the stimulation of store-operated Ca2+ entry and calpain 2

    doi: 10.1083/jcb.201301016

    Figure Lengend Snippet: hGAAP expression alters the number and size of focal adhesions. U2-OS cells transfected with siRNA (24 h) or plasmids encoding hGAAP were seeded onto fibronectin-coated slides. (A) Confocal images show cells transfected with vinculin-GFP (14 h), fixed, and stained with phalloidin–Alexa Fluor 568. (B) Summary results (means ± SEM from ≥25 cells for each condition) show numbers of focal adhesions per cell, determined by counting vinculin-GFP spots. (C) IRM images of U2-OS cells overexpressing hGAAP or hGAAP Ctmut, or transfected with the indicated siRNAs. Images are typical of three independent experiments. (D) Example of the image analysis used to determine focal adhesion number and size. Individual cells were imaged for IRM (a) and phalloidin staining (b). IRM images were then band pass filtered (c), and a threshold was imposed (d) to obtain the final images used to determine the number and size of the adhesions. (E and F) Summary results (means ± SEM from ≥20 cells for each condition) show numbers of adhesions per cell (E) and their areas (F). *, P

    Article Snippet: Cells were seeded to achieve 20–30% confluence in 24 h. The siRNA oligonucleotide duplexes (Invitrogen) were transfected using Oligofectamine (Invitrogen) according to the manufacturer’s instructions.

    Techniques: Expressing, Transfection, Staining

    Overexpression of hGAAP increases the speed of cell migration. (A–F) U2-OS cells overexpressing hGAAP, hGAAP Ctmut, or control neo (A–C) or cells transfected with siRNA (D–F) were seeded at low density on fibronectin-coated dishes. Individual cells were imaged at 5-min intervals for 18 h. Tracks of individual cells ( n = 30) are shown in A and D. Migration rates (B and E) and persistence (where 1 represents a straight line of migration from start to finish; C and F) are shown as means ± SEM from 30 cells. **, P

    Journal: The Journal of Cell Biology

    Article Title: hGAAP promotes cell adhesion and migration via the stimulation of store-operated Ca2+ entry and calpain 2

    doi: 10.1083/jcb.201301016

    Figure Lengend Snippet: Overexpression of hGAAP increases the speed of cell migration. (A–F) U2-OS cells overexpressing hGAAP, hGAAP Ctmut, or control neo (A–C) or cells transfected with siRNA (D–F) were seeded at low density on fibronectin-coated dishes. Individual cells were imaged at 5-min intervals for 18 h. Tracks of individual cells ( n = 30) are shown in A and D. Migration rates (B and E) and persistence (where 1 represents a straight line of migration from start to finish; C and F) are shown as means ± SEM from 30 cells. **, P

    Article Snippet: Cells were seeded to achieve 20–30% confluence in 24 h. The siRNA oligonucleotide duplexes (Invitrogen) were transfected using Oligofectamine (Invitrogen) according to the manufacturer’s instructions.

    Techniques: Over Expression, Migration, Transfection

    Overexpression or knockdown of hGAAP affects cell adhesion, cell detachment, and cell spreading. (A and B) Immunoblot (IB) of U2-OS cells with anti-HA Ab (A) or HeLa cells with anti-V5 Ab (B) shows expression of tagged hGAAP and hGAAP Ctmut in stable cell lines but not in cells expressing control plasmids (neo and puro). (C) Levels of endogenous hGAAP mRNA were determined by RT-PCR in U2-OS cells transfected with siRNAs for hGAAP (siRNA 1 and 2) or control siRNA. An analysis without reverse transcription (−RT) was included to control for DNA contamination. (D) U2-OS cells overexpressing hGAAP-HA were transfected with the indicated siRNAs, and hGAAP-HA expression was measured using IB with an anti-HA Ab. (E) Summary results (means ± SEM, from three independent experiments) show relative expression of hGAAP protein quantified from IB as in D. (F and G) Adherence of U2-OS (F) or HeLa cells (G) expressing the indicated hGAAPs was determined after washing cells with PBS at intervals after seeding. Results are means ± SEM, from three independent experiments. *, P

    Journal: The Journal of Cell Biology

    Article Title: hGAAP promotes cell adhesion and migration via the stimulation of store-operated Ca2+ entry and calpain 2

    doi: 10.1083/jcb.201301016

    Figure Lengend Snippet: Overexpression or knockdown of hGAAP affects cell adhesion, cell detachment, and cell spreading. (A and B) Immunoblot (IB) of U2-OS cells with anti-HA Ab (A) or HeLa cells with anti-V5 Ab (B) shows expression of tagged hGAAP and hGAAP Ctmut in stable cell lines but not in cells expressing control plasmids (neo and puro). (C) Levels of endogenous hGAAP mRNA were determined by RT-PCR in U2-OS cells transfected with siRNAs for hGAAP (siRNA 1 and 2) or control siRNA. An analysis without reverse transcription (−RT) was included to control for DNA contamination. (D) U2-OS cells overexpressing hGAAP-HA were transfected with the indicated siRNAs, and hGAAP-HA expression was measured using IB with an anti-HA Ab. (E) Summary results (means ± SEM, from three independent experiments) show relative expression of hGAAP protein quantified from IB as in D. (F and G) Adherence of U2-OS (F) or HeLa cells (G) expressing the indicated hGAAPs was determined after washing cells with PBS at intervals after seeding. Results are means ± SEM, from three independent experiments. *, P

    Article Snippet: Cells were seeded to achieve 20–30% confluence in 24 h. The siRNA oligonucleotide duplexes (Invitrogen) were transfected using Oligofectamine (Invitrogen) according to the manufacturer’s instructions.

    Techniques: Over Expression, Expressing, Stable Transfection, Reverse Transcription Polymerase Chain Reaction, Transfection

    Cetuximab sensitizes cancer cells to ROS-elevating agent-induced apoptosis in an EGFR-expression-dependent manner. ( A ) HN5 cells were subjected to knockdown with each of two different EGFR siRNAs or control siRNA for 72 h. During the last 24 h of siRNA transfection, the cells were either untreated or treated with 10 mM DCA, 20 nM cetuximab, or both. Cell lysates were subjected to Western blotting with the indicated antibodies. ( B ) HN5 and FaDu cells were subjected to knockdown of Rab5 or Rab11 with specific siRNAs or control siRNA for 72 h. During the last 24 h of siRNA transfection, the cells were either untreated or treated with 10 mM DCA, 20 nM cetuximab, or both. Cell lysates were subjected to Western blotting with the indicated antibodies.

    Journal: Cancer letters

    Article Title: ASCT2 (SLC1A5) is an EGFR-associated protein that can be co-targeted by cetuximab to sensitize cancer cells to ROS-induced apoptosis

    doi: 10.1016/j.canlet.2016.07.020

    Figure Lengend Snippet: Cetuximab sensitizes cancer cells to ROS-elevating agent-induced apoptosis in an EGFR-expression-dependent manner. ( A ) HN5 cells were subjected to knockdown with each of two different EGFR siRNAs or control siRNA for 72 h. During the last 24 h of siRNA transfection, the cells were either untreated or treated with 10 mM DCA, 20 nM cetuximab, or both. Cell lysates were subjected to Western blotting with the indicated antibodies. ( B ) HN5 and FaDu cells were subjected to knockdown of Rab5 or Rab11 with specific siRNAs or control siRNA for 72 h. During the last 24 h of siRNA transfection, the cells were either untreated or treated with 10 mM DCA, 20 nM cetuximab, or both. Cell lysates were subjected to Western blotting with the indicated antibodies.

    Article Snippet: siRNA oligonucleotide duplexes for PDK1 and EGFR were purchased from Sigma-Aldrich Co.

    Techniques: Expressing, Transfection, Western Blot

    ASCT2 is physically associated with EGFR. ( A ) HN5 cell lysates were subjected to EGFR immunoprecipitation (IP) with cetuximab or a control antibody, or subjected to ASCT2 immunoprecipitation with an anti-ASCT2 rabbit monoclonal antibody (D7C12, Cell Signaling) or a control rabbit anti-mouse IgG antibody, followed by Western blotting (WB) of the immunoprecipitates with an anti-ASCT2 rabbit polyclonal antibody (H-52, Santa Cruz Biotechnology) and with an anti-EGFR mouse monoclonal antibody (F4, Sigma-Aldrich), respectively. HC, heavy chain. ( B ) ASCT2 co-immunoprecipitation by cetuximab was validated by Western blotting using different ASCT2 (D7C12) and EGFR (D38B1, Cell Signaling) antibodies as indicated. ( C ) HN5 cells were untransfected or transfected with control siRNA, ASCT2 siRNA, or EGFR siRNA for 72 h before being plated on coverslips for overnight. The cells were then incubated with blocking buffer only or with an ASCT2 antibody (H-52) and an EGFR antibody (F4), alone and together, as indicated, and then subjected to Duolink proximity ligation assay as described in Materials and methods. Scale bars, 25 μm. ( D ) Lysates from the indicated HNSCC cell lines were subjected to EGFR immunoprecipitation with cetuximab or ASCT2 immunoprecipitation with an ASCT2 antibody (H-52), along with a control IgG immunoprecipitation. The immunoprecipitates were then analyzed by Western blotting with the indicated antibodies.

    Journal: Cancer letters

    Article Title: ASCT2 (SLC1A5) is an EGFR-associated protein that can be co-targeted by cetuximab to sensitize cancer cells to ROS-induced apoptosis

    doi: 10.1016/j.canlet.2016.07.020

    Figure Lengend Snippet: ASCT2 is physically associated with EGFR. ( A ) HN5 cell lysates were subjected to EGFR immunoprecipitation (IP) with cetuximab or a control antibody, or subjected to ASCT2 immunoprecipitation with an anti-ASCT2 rabbit monoclonal antibody (D7C12, Cell Signaling) or a control rabbit anti-mouse IgG antibody, followed by Western blotting (WB) of the immunoprecipitates with an anti-ASCT2 rabbit polyclonal antibody (H-52, Santa Cruz Biotechnology) and with an anti-EGFR mouse monoclonal antibody (F4, Sigma-Aldrich), respectively. HC, heavy chain. ( B ) ASCT2 co-immunoprecipitation by cetuximab was validated by Western blotting using different ASCT2 (D7C12) and EGFR (D38B1, Cell Signaling) antibodies as indicated. ( C ) HN5 cells were untransfected or transfected with control siRNA, ASCT2 siRNA, or EGFR siRNA for 72 h before being plated on coverslips for overnight. The cells were then incubated with blocking buffer only or with an ASCT2 antibody (H-52) and an EGFR antibody (F4), alone and together, as indicated, and then subjected to Duolink proximity ligation assay as described in Materials and methods. Scale bars, 25 μm. ( D ) Lysates from the indicated HNSCC cell lines were subjected to EGFR immunoprecipitation with cetuximab or ASCT2 immunoprecipitation with an ASCT2 antibody (H-52), along with a control IgG immunoprecipitation. The immunoprecipitates were then analyzed by Western blotting with the indicated antibodies.

    Article Snippet: siRNA oligonucleotide duplexes for PDK1 and EGFR were purchased from Sigma-Aldrich Co.

    Techniques: Immunoprecipitation, Western Blot, Transfection, Incubation, Blocking Assay, Proximity Ligation Assay

    Cdc7 codepletion rescues fork slowing in Chk1-depleted cells. ( A ) Protein levels of Cdc7, Chk1, and β-Actin (loading control) in U2OS cells after 48 hours depletion with Cdc7, Chk1, Cdc7, and Chk1 or control siRNA. ( B ) Quantification of origin firing in Cdc7-, Chk1- or control-depleted cells. First label origins (green-red-green) are shown as percentage of all red (CldU) labeled tracks. ( C ) Distribution of replication fork speeds in Cdc7- or control-depleted cells. ( D ) Distribution of replication fork speeds in Chk1- or control-depleted cells. ( E ) Distribution of replication fork speeds in Cdc7- or Cdc7 and Chk1-depleted cells. ( F ) Average replication fork speeds in Cdc7-, Chk1-, or control-depleted cells. Means and standard deviation (S.D.) (bars) of three independent experiments are shown. Values marked with asterisks are significantly different (student’s t -test, * p

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Chk1 promotes replication fork progression by controlling replication initiation

    doi: 10.1073/pnas.1005031107

    Figure Lengend Snippet: Cdc7 codepletion rescues fork slowing in Chk1-depleted cells. ( A ) Protein levels of Cdc7, Chk1, and β-Actin (loading control) in U2OS cells after 48 hours depletion with Cdc7, Chk1, Cdc7, and Chk1 or control siRNA. ( B ) Quantification of origin firing in Cdc7-, Chk1- or control-depleted cells. First label origins (green-red-green) are shown as percentage of all red (CldU) labeled tracks. ( C ) Distribution of replication fork speeds in Cdc7- or control-depleted cells. ( D ) Distribution of replication fork speeds in Chk1- or control-depleted cells. ( E ) Distribution of replication fork speeds in Cdc7- or Cdc7 and Chk1-depleted cells. ( F ) Average replication fork speeds in Cdc7-, Chk1-, or control-depleted cells. Means and standard deviation (S.D.) (bars) of three independent experiments are shown. Values marked with asterisks are significantly different (student’s t -test, * p

    Article Snippet: To knock down human Chk1 and Cdc7 we employed siRNA duplex oligonucleotides (Dharmacon) directed against the Chk1 target sequence (sense): UCGUGAGCGUUUGUUGAAC ( ) and the Cdc7 target sequence (sense): GCAGUCAAAGACUGUGGAU ( ).

    Techniques: Labeling, Standard Deviation

    WRN regulates mRNA levels of XLF. (A) XLF protein levels decreased in WRN -deficient fibroblasts. Whole cell lysates were prepared from human fibroblasts GM00637G (normal) and AG11395A ( WRN deficient) and subjected to immunoblotting analysis for XLF and β-actin. (B) Inhibition of WRN expression by siRNA resulted in a decrease of XLF protein levels. U2OS cells were transfected with si-CTR, XLF siRNA (si1-XLF or si2-XLF) or WRN siRNA (si1-WRN or si2-WRN). Total cell lysates were harvested 48 h following transfection and subjected to immunoblotting analysis with the antibodies indicated. (C) MG132 treatment did not restore the decrease of XLF protein levels caused by the depletion of WRN. U2OS cells were treated as described in (B) except that MG132 was added to cells 4 h prior to harvest. (D) Inhibition of WRN expression led to a decrease of XLF mRNA. U2OS cells were treated as described in (B). Total RNA was extracted and subjected to real-time RT-PCR assays for XLF mRNA. XLF, XRCC4-like factor; si-CTR, control siRNA; RT-PCR, reverse transcription PCR.

    Journal: Molecular Medicine Reports

    Article Title: Werner syndrome protein positively regulates XRCC4-like factor transcription

    doi: 10.3892/mmr.2014.2030

    Figure Lengend Snippet: WRN regulates mRNA levels of XLF. (A) XLF protein levels decreased in WRN -deficient fibroblasts. Whole cell lysates were prepared from human fibroblasts GM00637G (normal) and AG11395A ( WRN deficient) and subjected to immunoblotting analysis for XLF and β-actin. (B) Inhibition of WRN expression by siRNA resulted in a decrease of XLF protein levels. U2OS cells were transfected with si-CTR, XLF siRNA (si1-XLF or si2-XLF) or WRN siRNA (si1-WRN or si2-WRN). Total cell lysates were harvested 48 h following transfection and subjected to immunoblotting analysis with the antibodies indicated. (C) MG132 treatment did not restore the decrease of XLF protein levels caused by the depletion of WRN. U2OS cells were treated as described in (B) except that MG132 was added to cells 4 h prior to harvest. (D) Inhibition of WRN expression led to a decrease of XLF mRNA. U2OS cells were treated as described in (B). Total RNA was extracted and subjected to real-time RT-PCR assays for XLF mRNA. XLF, XRCC4-like factor; si-CTR, control siRNA; RT-PCR, reverse transcription PCR.

    Article Snippet: All predesigned siRNA oligonucleotide duplexes (OnTarget plus option) directed against human XLF or WRN (si-XLF or si-WRN) were purchased from Dharmacon, Inc. (Lafayette, CO, USA).

    Techniques: Inhibition, Expressing, Transfection, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    WRN positively regulates the XLF promoter activity in U2OS cells. (A) Inhibition of WRN expression decreased the XLF promoter activity. The putative XLF promoter containing a sequence from −596 to +169, relative to the transcription start site of XLF , was subcloned into the PGL3-basic luciferase vector, and the resulting reporter construct was transfected into WRN -depleted U2OS cells. Triplicate samples were analyzed. (B) WRN bound the XLF promoter region. ChIP assays were performed using two different anti-WRN antibodies. ChIP with α1-WRN antibody did not yield any PCR product, which also served as a negative control. The PCR product corresponds to the position from −360 to −90 of the promoter relative to the transcription start site. XLF, XRCC4-like factor; ChIP, chromatin immunoprecipitation; si-CTR, control siRNA.

    Journal: Molecular Medicine Reports

    Article Title: Werner syndrome protein positively regulates XRCC4-like factor transcription

    doi: 10.3892/mmr.2014.2030

    Figure Lengend Snippet: WRN positively regulates the XLF promoter activity in U2OS cells. (A) Inhibition of WRN expression decreased the XLF promoter activity. The putative XLF promoter containing a sequence from −596 to +169, relative to the transcription start site of XLF , was subcloned into the PGL3-basic luciferase vector, and the resulting reporter construct was transfected into WRN -depleted U2OS cells. Triplicate samples were analyzed. (B) WRN bound the XLF promoter region. ChIP assays were performed using two different anti-WRN antibodies. ChIP with α1-WRN antibody did not yield any PCR product, which also served as a negative control. The PCR product corresponds to the position from −360 to −90 of the promoter relative to the transcription start site. XLF, XRCC4-like factor; ChIP, chromatin immunoprecipitation; si-CTR, control siRNA.

    Article Snippet: All predesigned siRNA oligonucleotide duplexes (OnTarget plus option) directed against human XLF or WRN (si-XLF or si-WRN) were purchased from Dharmacon, Inc. (Lafayette, CO, USA).

    Techniques: Activity Assay, Inhibition, Expressing, Sequencing, Luciferase, Plasmid Preparation, Construct, Transfection, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Negative Control

    XLF suppresses cellular senescence. (A) Inhibition of XLF expression in WI38 cells. WI38 cells at passage 39 PD were transfected with si-CTR or si-XLF. Total cell lysates were extracted 48 h following transfection and subjected to immunoblotting analysis with the antibodies indicated. (B) Depletion of XLF inhibited cell proliferation. XLF expression was inhibited by transfection with si-XLF as described in (A) and cell numbers were determined every 24 h. Six duplicate samples were analyzed at each time point. (C and D) Inhibition of XLF expression accelerated cellular senescence. XLF-depleted WI38 cells were assayed for β-gal. Representative images of stained cells are shown in (C) and quantitative data in (D). PD, population doubling; XLF, XRCC4-like factor; β-gal, β-galactosidase; si-CTR, control siRNA.

    Journal: Molecular Medicine Reports

    Article Title: Werner syndrome protein positively regulates XRCC4-like factor transcription

    doi: 10.3892/mmr.2014.2030

    Figure Lengend Snippet: XLF suppresses cellular senescence. (A) Inhibition of XLF expression in WI38 cells. WI38 cells at passage 39 PD were transfected with si-CTR or si-XLF. Total cell lysates were extracted 48 h following transfection and subjected to immunoblotting analysis with the antibodies indicated. (B) Depletion of XLF inhibited cell proliferation. XLF expression was inhibited by transfection with si-XLF as described in (A) and cell numbers were determined every 24 h. Six duplicate samples were analyzed at each time point. (C and D) Inhibition of XLF expression accelerated cellular senescence. XLF-depleted WI38 cells were assayed for β-gal. Representative images of stained cells are shown in (C) and quantitative data in (D). PD, population doubling; XLF, XRCC4-like factor; β-gal, β-galactosidase; si-CTR, control siRNA.

    Article Snippet: All predesigned siRNA oligonucleotide duplexes (OnTarget plus option) directed against human XLF or WRN (si-XLF or si-WRN) were purchased from Dharmacon, Inc. (Lafayette, CO, USA).

    Techniques: Inhibition, Expressing, Transfection, Staining

    Effect of LPS (75 µg/ml) treatment on SOD activity in HUVECs. LPS treatment significantly decreased the inhibition of SOD in HUVECs/si-NC and HUVECs/siCav-1. *P≤0.05 and **P≤0.01. LPS, lipopolysaccharide; SOD, superoxide dismutase; HUVECs, human umbilical vein endothelial cells; Cav-1, caveolin-1; si, small interfering RNA; NC, negative control.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Downregulated caveolin-1 expression serves a potential role in coronary artery spasm by inducing nitric oxide production in vitro

    doi: 10.3892/etm.2018.6646

    Figure Lengend Snippet: Effect of LPS (75 µg/ml) treatment on SOD activity in HUVECs. LPS treatment significantly decreased the inhibition of SOD in HUVECs/si-NC and HUVECs/siCav-1. *P≤0.05 and **P≤0.01. LPS, lipopolysaccharide; SOD, superoxide dismutase; HUVECs, human umbilical vein endothelial cells; Cav-1, caveolin-1; si, small interfering RNA; NC, negative control.

    Article Snippet: Small interfering RNA (siRNA) duplex oligonucleotides that were specific for Cav-1 were synthesized by Suzhou GenePharma Co., Ltd. (Suzhou; China).

    Techniques: Activity Assay, Inhibition, Small Interfering RNA, Negative Control

    Effects of 10 µM Ach added at 10 sec on the [Ca 2+ ]i responses in Fluo4-acetoxymethyl ester-loaded HUVECs/si-NC with or without LPS treatment. The responses in the eight cell groups were evaluated. The ACh-stimulated changes in the [Ca 2+ ]i fluorescence of HUVECs/si-NC are shown in the (A) absence and (B) presence of LPS, at the following time points: (a) 1 sec; (b) 17 sec; (c) 35 sec; (d) 70 sec; (e) 99 sec; (f) 122 sec; (g) 158 sec; (h) 199 sec; (i) 212 sec; (j) 232 sec; (k) 268 sec; and (l) 334 sec. In HUVECs/si-NC without LPS treatment, a characteristic biphasic [Ca 2+ ]i response was observed following the addition of ACh (10 µM) at 10 sec, with an initial rapid increase in [Ca 2+ ]i, reaching a maximum level at a different time-points for every cell, followed by a sustained plateau in [Ca 2+ ]i that declined slowly toward the baseline. In addition, a [Ca 2+ ]i oscillation phenomenon was observed with peak and plateau levels occurring regularly. In the HUVECs/si-NC group with LPS treatment, the addition of ACh (10 µM) at 10 sec resulted in a smaller peak and plateau compared with those observed in the group without LPS treatment, with certain cells not presenting any response. [Ca 2+ ]i, intracellular Ca 2+ ; LPS, lipopolysaccharide; ACh, acetylcholine; HUVECs, human umbilical vein endothelial cells; Cav-1, caveolin-1; si, small interfering RNA; NC, negative control.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Downregulated caveolin-1 expression serves a potential role in coronary artery spasm by inducing nitric oxide production in vitro

    doi: 10.3892/etm.2018.6646

    Figure Lengend Snippet: Effects of 10 µM Ach added at 10 sec on the [Ca 2+ ]i responses in Fluo4-acetoxymethyl ester-loaded HUVECs/si-NC with or without LPS treatment. The responses in the eight cell groups were evaluated. The ACh-stimulated changes in the [Ca 2+ ]i fluorescence of HUVECs/si-NC are shown in the (A) absence and (B) presence of LPS, at the following time points: (a) 1 sec; (b) 17 sec; (c) 35 sec; (d) 70 sec; (e) 99 sec; (f) 122 sec; (g) 158 sec; (h) 199 sec; (i) 212 sec; (j) 232 sec; (k) 268 sec; and (l) 334 sec. In HUVECs/si-NC without LPS treatment, a characteristic biphasic [Ca 2+ ]i response was observed following the addition of ACh (10 µM) at 10 sec, with an initial rapid increase in [Ca 2+ ]i, reaching a maximum level at a different time-points for every cell, followed by a sustained plateau in [Ca 2+ ]i that declined slowly toward the baseline. In addition, a [Ca 2+ ]i oscillation phenomenon was observed with peak and plateau levels occurring regularly. In the HUVECs/si-NC group with LPS treatment, the addition of ACh (10 µM) at 10 sec resulted in a smaller peak and plateau compared with those observed in the group without LPS treatment, with certain cells not presenting any response. [Ca 2+ ]i, intracellular Ca 2+ ; LPS, lipopolysaccharide; ACh, acetylcholine; HUVECs, human umbilical vein endothelial cells; Cav-1, caveolin-1; si, small interfering RNA; NC, negative control.

    Article Snippet: Small interfering RNA (siRNA) duplex oligonucleotides that were specific for Cav-1 were synthesized by Suzhou GenePharma Co., Ltd. (Suzhou; China).

    Techniques: Size-exclusion Chromatography, Fluorescence, Small Interfering RNA, Negative Control

    Production of NO with or without ACh exposure in HUVECs/si-NC and HUVECs/siCav-1 with or without LPS pretreatment. ACh stimulated the production of NO in HUVECs/si-NC and HUVECs/siCav-1. The NO level was lower when cells were treated with LPS compared with the levels observed without LPS treatment. ACh stimulated the LPS-induced HUVECs/siCav-1 to produce significantly higher NO levels compared with those produced by LPS-induced HUVECs/si-NC. *P≤0.05, **P≤0.01, ***P≤0.001. NO, nitric oxide; HUVECs, human umbilical vein endothelial cells; LPS, lipopolysaccharide; ACh, acetylcholine; Cav-1, caveolin-1; si, small interfering RNA; NC, negative control; IOD, integrated optical density.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Downregulated caveolin-1 expression serves a potential role in coronary artery spasm by inducing nitric oxide production in vitro

    doi: 10.3892/etm.2018.6646

    Figure Lengend Snippet: Production of NO with or without ACh exposure in HUVECs/si-NC and HUVECs/siCav-1 with or without LPS pretreatment. ACh stimulated the production of NO in HUVECs/si-NC and HUVECs/siCav-1. The NO level was lower when cells were treated with LPS compared with the levels observed without LPS treatment. ACh stimulated the LPS-induced HUVECs/siCav-1 to produce significantly higher NO levels compared with those produced by LPS-induced HUVECs/si-NC. *P≤0.05, **P≤0.01, ***P≤0.001. NO, nitric oxide; HUVECs, human umbilical vein endothelial cells; LPS, lipopolysaccharide; ACh, acetylcholine; Cav-1, caveolin-1; si, small interfering RNA; NC, negative control; IOD, integrated optical density.

    Article Snippet: Small interfering RNA (siRNA) duplex oligonucleotides that were specific for Cav-1 were synthesized by Suzhou GenePharma Co., Ltd. (Suzhou; China).

    Techniques: Produced, Small Interfering RNA, Negative Control

    Effects of treatment with LPS (10–100 µg/ml) for 24 h on the viability of HUVECs/si-NC and HUVECs/siCav-1. Treatment with 75 and 100 µg/ml LPS induced a significant decrease in the viability of HUVECs/si-NC and HUVECs/siCav-1. There was no significant difference in the viability of HUVECs/si-NC and HUVECs/siCav-1 treated with LPS at 75 or 100 µg/ml. *P≤0.001 vs. absence of LPS treatment. HUVECs, human umbilical vein endothelial cells; LPS, lipopolysaccharide; Cav-1, caveolin-1; si, small interfering RNA; NC, negative control.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Downregulated caveolin-1 expression serves a potential role in coronary artery spasm by inducing nitric oxide production in vitro

    doi: 10.3892/etm.2018.6646

    Figure Lengend Snippet: Effects of treatment with LPS (10–100 µg/ml) for 24 h on the viability of HUVECs/si-NC and HUVECs/siCav-1. Treatment with 75 and 100 µg/ml LPS induced a significant decrease in the viability of HUVECs/si-NC and HUVECs/siCav-1. There was no significant difference in the viability of HUVECs/si-NC and HUVECs/siCav-1 treated with LPS at 75 or 100 µg/ml. *P≤0.001 vs. absence of LPS treatment. HUVECs, human umbilical vein endothelial cells; LPS, lipopolysaccharide; Cav-1, caveolin-1; si, small interfering RNA; NC, negative control.

    Article Snippet: Small interfering RNA (siRNA) duplex oligonucleotides that were specific for Cav-1 were synthesized by Suzhou GenePharma Co., Ltd. (Suzhou; China).

    Techniques: Small Interfering RNA, Negative Control

    CFL2 inhibits the proliferation and migration of MGC803 cells. ( A ) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and ( B ) Western blotting were used to detect CFL2 mRNA and protein expression in MGC803 cells after transfecting with CFL2 -siRNA or NC-siRNA. The effect of CFL2 knockdown on the proliferation and migration of MGC803 cells was examined by ( C ) CCK8, ( D ) transwell, and ( E ) wound healing assays, respectively. ( D , E ) left, magnifcation, ×100. Data are from three independent experiments. * p

    Journal: International Journal of Molecular Sciences

    Article Title: miR-3189-3p Mimics Enhance the Effects of S100A4 siRNA on the Inhibition of Proliferation and Migration of Gastric Cancer Cells by Targeting CFL2

    doi: 10.3390/ijms19010236

    Figure Lengend Snippet: CFL2 inhibits the proliferation and migration of MGC803 cells. ( A ) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and ( B ) Western blotting were used to detect CFL2 mRNA and protein expression in MGC803 cells after transfecting with CFL2 -siRNA or NC-siRNA. The effect of CFL2 knockdown on the proliferation and migration of MGC803 cells was examined by ( C ) CCK8, ( D ) transwell, and ( E ) wound healing assays, respectively. ( D , E ) left, magnifcation, ×100. Data are from three independent experiments. * p

    Article Snippet: Cell Transfection The duplex siRNA oligonucleotides specific for human S100A4 or CFL2 , and oligonucleotides specific for hsa-miR-3189-3p were synthesized by GenePharma (Shanghai, China), and are listed in .

    Techniques: Migration, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing

    S100A4 knockdown leads to decreased expression of miR-3189-3p in MGC803 cells. MGC803 cells were transfected with either S100A4 -siRNA (small interfering RNA) or NC-siRNA, mRNA was extracted for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of ( A ) S100A4, ( B ) GDF15 , ( C ) pri-miR-3189, and ( D ) miR-3189-3p at 48 h after transfection. Data represent the mean of three independent experiments. GAPDH was used for the internal control. * p

    Journal: International Journal of Molecular Sciences

    Article Title: miR-3189-3p Mimics Enhance the Effects of S100A4 siRNA on the Inhibition of Proliferation and Migration of Gastric Cancer Cells by Targeting CFL2

    doi: 10.3390/ijms19010236

    Figure Lengend Snippet: S100A4 knockdown leads to decreased expression of miR-3189-3p in MGC803 cells. MGC803 cells were transfected with either S100A4 -siRNA (small interfering RNA) or NC-siRNA, mRNA was extracted for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of ( A ) S100A4, ( B ) GDF15 , ( C ) pri-miR-3189, and ( D ) miR-3189-3p at 48 h after transfection. Data represent the mean of three independent experiments. GAPDH was used for the internal control. * p

    Article Snippet: Cell Transfection The duplex siRNA oligonucleotides specific for human S100A4 or CFL2 , and oligonucleotides specific for hsa-miR-3189-3p were synthesized by GenePharma (Shanghai, China), and are listed in .

    Techniques: Expressing, Transfection, Small Interfering RNA, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    miR-3189-3p mimics enhanced the effects of S100A4 small interfering RNA (siRNA) on the inhibition of the proliferation and migration of MGC803 cells. MGC803 cells were co-transfected with S100A4 -siRNA and miR-3189-3p mimics or miR-3189-3p NC. The effect on proliferation or migration was analyzed by ( A ) CCK8, ( B ) transwell, and ( C ) wound healing assays. ( B , C ) left, magnifcation, ×100. All the results were obtained from three independent experiments. * p

    Journal: International Journal of Molecular Sciences

    Article Title: miR-3189-3p Mimics Enhance the Effects of S100A4 siRNA on the Inhibition of Proliferation and Migration of Gastric Cancer Cells by Targeting CFL2

    doi: 10.3390/ijms19010236

    Figure Lengend Snippet: miR-3189-3p mimics enhanced the effects of S100A4 small interfering RNA (siRNA) on the inhibition of the proliferation and migration of MGC803 cells. MGC803 cells were co-transfected with S100A4 -siRNA and miR-3189-3p mimics or miR-3189-3p NC. The effect on proliferation or migration was analyzed by ( A ) CCK8, ( B ) transwell, and ( C ) wound healing assays. ( B , C ) left, magnifcation, ×100. All the results were obtained from three independent experiments. * p

    Article Snippet: Cell Transfection The duplex siRNA oligonucleotides specific for human S100A4 or CFL2 , and oligonucleotides specific for hsa-miR-3189-3p were synthesized by GenePharma (Shanghai, China), and are listed in .

    Techniques: Small Interfering RNA, Inhibition, Migration, Transfection

    (A) Schematic illustration of the sequence motifs in the helicase core of DEAD box RNA helicase and the p68 LGLD and HLIGR mutants. (B) Coomassie blue staining of SDS-PAGE of recombinant p68 wt (lane 1), LGLD mutant (lane 2), or HILGR mutant (lane 3) that were expressed and purified from bacterial E. coli. (C) Cross-links of p68 wt (lane 1), LGLD mutant (lane 2), or HILGR mutant (lane 3) to [α- 32 P]ATP were analyzed by SDS-PAGE followed by autoradiography. (D) RNA unwinding by p68 wt (lane 3), LGLD mutant (lane 4), or HILGR mutant (lane 5) was analyzed by SDS-PAGE followed by autoradiography. Lane 1 is duplex RNA denatured by heating to 95°C. Lane 2 is the duplex RNA. (E) ATPase activities of p68 wt/mutant (indicated) were measured by colorimetric assay. The ATPase activity ( y axis) is expressed as μM P i /mg of p68 or mutants.

    Journal: Molecular and Cellular Biology

    Article Title: ATPase/Helicase Activities of p68 RNA Helicase Are Required for Pre-mRNA Splicing but Not for Assembly of the Spliceosome

    doi: 10.1128/MCB.25.17.7484-7493.2005

    Figure Lengend Snippet: (A) Schematic illustration of the sequence motifs in the helicase core of DEAD box RNA helicase and the p68 LGLD and HLIGR mutants. (B) Coomassie blue staining of SDS-PAGE of recombinant p68 wt (lane 1), LGLD mutant (lane 2), or HILGR mutant (lane 3) that were expressed and purified from bacterial E. coli. (C) Cross-links of p68 wt (lane 1), LGLD mutant (lane 2), or HILGR mutant (lane 3) to [α- 32 P]ATP were analyzed by SDS-PAGE followed by autoradiography. (D) RNA unwinding by p68 wt (lane 3), LGLD mutant (lane 4), or HILGR mutant (lane 5) was analyzed by SDS-PAGE followed by autoradiography. Lane 1 is duplex RNA denatured by heating to 95°C. Lane 2 is the duplex RNA. (E) ATPase activities of p68 wt/mutant (indicated) were measured by colorimetric assay. The ATPase activity ( y axis) is expressed as μM P i /mg of p68 or mutants.

    Article Snippet: The duplex RNA oligonucleotides for targeting p68 RNA helicase were purchased from Dharmacon siGENOME and SMARTpool.

    Techniques: Sequencing, Staining, SDS Page, Recombinant, Mutagenesis, Purification, Autoradiography, Colorimetric Assay, Activity Assay

    Flotillin upregulation stimulates AXL endocytosis towards non-degradative late endosomes and promotes its stabilization. A) AXL knock-down in MCF10AF1F2 cells decreases ZEB1 and N-cadherin expression . MCF10AmCh and MCF10AF1F2 cells were transfected with control siRNA (CTL) or against AXL and probed by western blotting with antibodies for the indicated proteins. The quantification results are the mean ± SEM of 4 independent experiment, and are expressed as fold-increase compared with control (MCF10AmCh). B) ZEB1 nuclear localization analyzed by immunocytochemistry and co-staining with Hoechst (Hst) and C) E-cadherin distribution analyzed by immunocytochemistry in MCF10AmCh and MCF10AF1F2 cells transfected with control (CTL) or anti-AXL siRNAs. Results shown are representative of 4 independent experiments. Scale bar: 10 µm. C) Flotillin-oligomerization induced by a CRY2-CIBN-based optogenetic approach promotes co-clustering of AXL . Still images from a TIRF-microscopy movie of a MCF10A cell that co-expresses CRY2-mCitrin, flotillin 2-CIBN-mCherry, and AXL-Halo labeled with Halo-Tag-Janelia Fluor 646. Representative image of a whole cell 15s after starting the 488 nm illumination to perform CRY2 binding to flotillin 2-CIBN-mCh and subsequent oligomerization (see also supplementary Fig. S6 for the details regarding the validation of the constructs and the optogenetic approach). Magnified images from the boxed area taken before and after 488 nm illumination. Arrowheads indicate clusters of CRY2-mCitrin and flotillin 2-CIBN-mCherry where AXL-Halo accumulates (see also video 4). Scale bars: 10 µm for the main image and 2 µm for the images from the boxed area. D) Co-accumulation of AXL and flotillin 2 at the PM prior to their co-endocytosis . Still images of a time-lapse series of one MCF10AF1F2 cell that expresses AXL-GFP to show flotillin 2/AXL co-localization at the PM and their co-endocytosis at PM sites (showed by arrowheads). Kymographs show the temporal variation of the GFP and mCherry signals along two line-scans (blue at the PM endocytic site, and yellow close to the PM endocytic site). Graphs show the intensity variation of both signals along the dotted white lines on the kymographs. The left kymograph shows the accumulation of AXL-GFP and flotillin 2-mCherry at the PM followed by a sudden concomitant drop of both signals (around 83s, indicated by a star). The right kymograph shows that in the meantime, both AXL-GFP and flotillin 2-mCherry signals remained stable at the PM outside and close to this endocytic site (See also video 5). E) Flotillin 1 co-precipitates with AXL. AXL was immunoprecipitated from MCF10AF1F2 cell lysates, and flotillin 1 co-immunoprecipitation was assessed by immunoblotting. Control immunoprecipitation was performed with non-relevant immunoglobulins (Ig). F) Flotillin upregulation accelerates AXL endocytosis. Kinetics of AXL internalization in MCF10AmCh and MCF10AF1F2 cells (left), and in MDA-MB-231shLuci and MDA-MB-231shFlot2 cells (right). Surface proteins were labeled with biotin at 4°C, and cells were incubated at 37°C for the indicated times to allow endocytosis. AXL presence in the internalized biotinylated fraction was probed by western blotting using relevant antibodies and quantified as the percentage of the maximum level of internalized protein (reached at 12 and 8 min, respectively, in MCF10AmCh and MCF10AF1F2 cells). Results are expressed as the mean ± SEM of 4 independent experiments. The AXL internalization rates (dashed lines) were 8.48 ± 1.28 and 12.43 ± 0.48 %/min in MCF10AmCh and MCF10AF1F2 cells, respectively, revealing a significant 1.45-fold increase (p=0.0088) upon flotillin upregulation. Comparison of the AXL internalization rates (dashed lines) in MDA-MB-231shLuci and MDA-MB-231shFlot2 cells showed a significant 3.66-fold decrease (p=0.0009) upon flotillin downregulation. Results are expressed as the percentage of the maximum level of surface labeled AXL at T0 and are the mean ± SEM of 4 independent experiments. G) Flotillin upregulation promotes AXL protein level increase . Cell lysates from MCF10AmCh and MCF10AF1F2 cells were probed by western blotting with antibodies against AXL phosphorylated on Y702, AXL and tubulin. Results are expressed as fold-increase compared with MCF10AmCh cells and are the mean ± SEM of 6 independent experiments. H) Flotillin upregulation does not increase AXL mRNA level. RT-qPCR analysis of AXL expression in MCF10AmCh and MCF10AF1F2 cells. Results are expressed relative to the level in MCF10AmCh cells and are the mean ± SEM of 4 independent experiments. I) Flotillin upregulation stabilizes AXL. MCF10A and MCF10AF1F2 cells were incubated with cycloheximide (CHX, 100µg/ml) and cell lysates collected at the indicated time points. AXL and tubulin levels were assessed by western blotting. The graph shows the normalized AXL levels in each cell line during CHX incubation. CHX treatment was stopped after 9 hours because MCF10Amch cells did not survive longer treatment. *P

    Journal: bioRxiv

    Article Title: Flotillin-upregulation acts as an epithelial-mesenchymal transition driver by promoting sphingosine kinase 2-dependent AXL stabilization

    doi: 10.1101/2020.05.12.090571

    Figure Lengend Snippet: Flotillin upregulation stimulates AXL endocytosis towards non-degradative late endosomes and promotes its stabilization. A) AXL knock-down in MCF10AF1F2 cells decreases ZEB1 and N-cadherin expression . MCF10AmCh and MCF10AF1F2 cells were transfected with control siRNA (CTL) or against AXL and probed by western blotting with antibodies for the indicated proteins. The quantification results are the mean ± SEM of 4 independent experiment, and are expressed as fold-increase compared with control (MCF10AmCh). B) ZEB1 nuclear localization analyzed by immunocytochemistry and co-staining with Hoechst (Hst) and C) E-cadherin distribution analyzed by immunocytochemistry in MCF10AmCh and MCF10AF1F2 cells transfected with control (CTL) or anti-AXL siRNAs. Results shown are representative of 4 independent experiments. Scale bar: 10 µm. C) Flotillin-oligomerization induced by a CRY2-CIBN-based optogenetic approach promotes co-clustering of AXL . Still images from a TIRF-microscopy movie of a MCF10A cell that co-expresses CRY2-mCitrin, flotillin 2-CIBN-mCherry, and AXL-Halo labeled with Halo-Tag-Janelia Fluor 646. Representative image of a whole cell 15s after starting the 488 nm illumination to perform CRY2 binding to flotillin 2-CIBN-mCh and subsequent oligomerization (see also supplementary Fig. S6 for the details regarding the validation of the constructs and the optogenetic approach). Magnified images from the boxed area taken before and after 488 nm illumination. Arrowheads indicate clusters of CRY2-mCitrin and flotillin 2-CIBN-mCherry where AXL-Halo accumulates (see also video 4). Scale bars: 10 µm for the main image and 2 µm for the images from the boxed area. D) Co-accumulation of AXL and flotillin 2 at the PM prior to their co-endocytosis . Still images of a time-lapse series of one MCF10AF1F2 cell that expresses AXL-GFP to show flotillin 2/AXL co-localization at the PM and their co-endocytosis at PM sites (showed by arrowheads). Kymographs show the temporal variation of the GFP and mCherry signals along two line-scans (blue at the PM endocytic site, and yellow close to the PM endocytic site). Graphs show the intensity variation of both signals along the dotted white lines on the kymographs. The left kymograph shows the accumulation of AXL-GFP and flotillin 2-mCherry at the PM followed by a sudden concomitant drop of both signals (around 83s, indicated by a star). The right kymograph shows that in the meantime, both AXL-GFP and flotillin 2-mCherry signals remained stable at the PM outside and close to this endocytic site (See also video 5). E) Flotillin 1 co-precipitates with AXL. AXL was immunoprecipitated from MCF10AF1F2 cell lysates, and flotillin 1 co-immunoprecipitation was assessed by immunoblotting. Control immunoprecipitation was performed with non-relevant immunoglobulins (Ig). F) Flotillin upregulation accelerates AXL endocytosis. Kinetics of AXL internalization in MCF10AmCh and MCF10AF1F2 cells (left), and in MDA-MB-231shLuci and MDA-MB-231shFlot2 cells (right). Surface proteins were labeled with biotin at 4°C, and cells were incubated at 37°C for the indicated times to allow endocytosis. AXL presence in the internalized biotinylated fraction was probed by western blotting using relevant antibodies and quantified as the percentage of the maximum level of internalized protein (reached at 12 and 8 min, respectively, in MCF10AmCh and MCF10AF1F2 cells). Results are expressed as the mean ± SEM of 4 independent experiments. The AXL internalization rates (dashed lines) were 8.48 ± 1.28 and 12.43 ± 0.48 %/min in MCF10AmCh and MCF10AF1F2 cells, respectively, revealing a significant 1.45-fold increase (p=0.0088) upon flotillin upregulation. Comparison of the AXL internalization rates (dashed lines) in MDA-MB-231shLuci and MDA-MB-231shFlot2 cells showed a significant 3.66-fold decrease (p=0.0009) upon flotillin downregulation. Results are expressed as the percentage of the maximum level of surface labeled AXL at T0 and are the mean ± SEM of 4 independent experiments. G) Flotillin upregulation promotes AXL protein level increase . Cell lysates from MCF10AmCh and MCF10AF1F2 cells were probed by western blotting with antibodies against AXL phosphorylated on Y702, AXL and tubulin. Results are expressed as fold-increase compared with MCF10AmCh cells and are the mean ± SEM of 6 independent experiments. H) Flotillin upregulation does not increase AXL mRNA level. RT-qPCR analysis of AXL expression in MCF10AmCh and MCF10AF1F2 cells. Results are expressed relative to the level in MCF10AmCh cells and are the mean ± SEM of 4 independent experiments. I) Flotillin upregulation stabilizes AXL. MCF10A and MCF10AF1F2 cells were incubated with cycloheximide (CHX, 100µg/ml) and cell lysates collected at the indicated time points. AXL and tubulin levels were assessed by western blotting. The graph shows the normalized AXL levels in each cell line during CHX incubation. CHX treatment was stopped after 9 hours because MCF10Amch cells did not survive longer treatment. *P

    Article Snippet: For RNA-mediated interference experiments, cells were transfected using Interferin (Polyplus Transfection) with siRNA oligonucleotide duplexes (Eurogentec, Belgium).

    Techniques: Expressing, Transfection, Western Blot, Immunocytochemistry, Staining, Microscopy, Labeling, Binding Assay, Construct, Immunoprecipitation, Multiple Displacement Amplification, Incubation, Quantitative RT-PCR

    Sphingosine kinase 2 is required for flotillin-induced AXL stabilization A, B, D) SphK2 is enriched in flotillin-positive late endosomes and co-accumulates at the PM with flotillins prior to endocytosis. MCF10AF1F2 cells that expressing SphK2-GFP (A,B) and MDA-MB-231 cells that co-express flotillin 1-mCherry or flotillin 2-mCherry and SphK2-GFP (A,B,D) were imaged by spinning disk confocal microscopy (See also videos 6, 7, 8, 9). ( A,B ) SphK2-GFP, but not SphK1-GFP is abundantly present in flotillin 2-mCherry-positive vesicles (Fig. S9 A). Quantification of flotillin 2-mCherry-positive vesicles in which SphK2-GFP or SphK1-GFP is detected (B). Results are expressed as the percentage of vesicles and are the mean ± SEM (n=300 vesicles in 10 MCF10AF1F2 cells, and n=100 vesicles in 10 MDA-MB-231 cells that co-express flotillin 2-mCherry and SPK2 or SPK1-GFP). (D) Fast co-endocytosis of flotillin 2-mCherry and SphK2-GFP following their co-accumulation at the plasma membrane (indicated by the arrows) in MCF10AF1F2 cells (upper panel) and MDA-MB-231 cells (lower panel). Shown are still images from spinning disk confocal videomicroscopy. A) C) AXL is present in flotillin- and SphK2-positive vesicles. Still image from spinning disk confocal microscopy of live MCF10AF1F2 cells that co-expressing SphK2-GFP and AXL-Halo labeled with Halo-tag-Janelia Fluor 646. Arrows show flotillin-positive vesicles that contain SphK2-GFP and AXL-Halo (See also video 7). B) E) SphK2 inhibition decreases AXL and ZEB1 levels in MCF10AF1F2 cells. MCF10AmCh and MCF10AF1F2 cells were incubated with opaganib (50 µM) for the indicated times. AXL, ZEB1, flotillin 1 and 2 were analyzed by western blotting in whole cell lysates. Results correspond to the level of each protein normalized to the actin signal, expressed as the fold increase compared to control (MCF10AmCh cells) and are the mean ± SEM of 4 independent experiments. C) F) SphK2 downregulation decreases AXL and ZEB1 levels in MCF10AF1F2 cells. MCF10AF1F2 cells were transfected with siRNAs against luciferase (CTL) or SphK2, and siRNA efficacy was tested by western blotting with antibodies against SphK2 and also AXL, ZEB1 and tubulin. Histograms show the level of each protein normalized to the tubulin signal, expressed as fold increase compared with CTL and are the mean ± SEM of 4 independent experiments. D) G) SphK2 inhibition decreases AXL and ZEB1 levels in MDA-MB-231 cells. MDA-MB-231shLuci (control) and MDA-MB-231shFlot2 cells were incubated with opaganib (50 µM) for the indicated times. AXL, ZEB1 and actin were analyzed by western blotting in whole cell lysates. The histograms show the level of each protein normalized to the actin signal, expressed as fold increase compared with control and are the mean ± SEM of 5 independent experiments. E) H) AXL decrease upon SphK2 inhibition is not due to protein synthesis inhibition. MCF10AF1F2 and MDA-MB-231shLuci cells were incubated or not with opaganib for 10 hours. When indicated, cycloheximide (CHX) was added 4 hours after the beginning of opaganib incubation. AXL was analyzed by western blotting in whole cell lysates. The histograms show the level of AXL normalized to the tubulin signal, expressed as fold increase compared with control (no treatment) and are the mean ± SEM of 4 independent experiments. I,J) SphK2 inhibition increases AXL level at the cell surface in flotillin-upregulated cells. Fixed non-permeabilized cells were immunostained for AXL using the monoclonal D4 antibody against AXL extracellular domain and an Alexa488-conjugated anti-human secondary antibody. Images of control- and opaganib-treated cells (50µM, 4h) were acquired in the same conditions. Representative images are shown (for MCF10AF1F2 cells, the insets show flotillin 2-mCherry signal). The mean fluorescence intensity was quantified in the area defined by the outline of each individual cell. Results are expressed as arbitrary units and are the mean ± SEM of 23 measurements for MCF10AF1F2 cells (I), and 22 measurements for MDA-MB-231shLuci cells (J) for each condition (control and opaganib). Scale bars: 10 µm for the main images and 2 µm in the magnified images from the boxed area. *P

    Journal: bioRxiv

    Article Title: Flotillin-upregulation acts as an epithelial-mesenchymal transition driver by promoting sphingosine kinase 2-dependent AXL stabilization

    doi: 10.1101/2020.05.12.090571

    Figure Lengend Snippet: Sphingosine kinase 2 is required for flotillin-induced AXL stabilization A, B, D) SphK2 is enriched in flotillin-positive late endosomes and co-accumulates at the PM with flotillins prior to endocytosis. MCF10AF1F2 cells that expressing SphK2-GFP (A,B) and MDA-MB-231 cells that co-express flotillin 1-mCherry or flotillin 2-mCherry and SphK2-GFP (A,B,D) were imaged by spinning disk confocal microscopy (See also videos 6, 7, 8, 9). ( A,B ) SphK2-GFP, but not SphK1-GFP is abundantly present in flotillin 2-mCherry-positive vesicles (Fig. S9 A). Quantification of flotillin 2-mCherry-positive vesicles in which SphK2-GFP or SphK1-GFP is detected (B). Results are expressed as the percentage of vesicles and are the mean ± SEM (n=300 vesicles in 10 MCF10AF1F2 cells, and n=100 vesicles in 10 MDA-MB-231 cells that co-express flotillin 2-mCherry and SPK2 or SPK1-GFP). (D) Fast co-endocytosis of flotillin 2-mCherry and SphK2-GFP following their co-accumulation at the plasma membrane (indicated by the arrows) in MCF10AF1F2 cells (upper panel) and MDA-MB-231 cells (lower panel). Shown are still images from spinning disk confocal videomicroscopy. A) C) AXL is present in flotillin- and SphK2-positive vesicles. Still image from spinning disk confocal microscopy of live MCF10AF1F2 cells that co-expressing SphK2-GFP and AXL-Halo labeled with Halo-tag-Janelia Fluor 646. Arrows show flotillin-positive vesicles that contain SphK2-GFP and AXL-Halo (See also video 7). B) E) SphK2 inhibition decreases AXL and ZEB1 levels in MCF10AF1F2 cells. MCF10AmCh and MCF10AF1F2 cells were incubated with opaganib (50 µM) for the indicated times. AXL, ZEB1, flotillin 1 and 2 were analyzed by western blotting in whole cell lysates. Results correspond to the level of each protein normalized to the actin signal, expressed as the fold increase compared to control (MCF10AmCh cells) and are the mean ± SEM of 4 independent experiments. C) F) SphK2 downregulation decreases AXL and ZEB1 levels in MCF10AF1F2 cells. MCF10AF1F2 cells were transfected with siRNAs against luciferase (CTL) or SphK2, and siRNA efficacy was tested by western blotting with antibodies against SphK2 and also AXL, ZEB1 and tubulin. Histograms show the level of each protein normalized to the tubulin signal, expressed as fold increase compared with CTL and are the mean ± SEM of 4 independent experiments. D) G) SphK2 inhibition decreases AXL and ZEB1 levels in MDA-MB-231 cells. MDA-MB-231shLuci (control) and MDA-MB-231shFlot2 cells were incubated with opaganib (50 µM) for the indicated times. AXL, ZEB1 and actin were analyzed by western blotting in whole cell lysates. The histograms show the level of each protein normalized to the actin signal, expressed as fold increase compared with control and are the mean ± SEM of 5 independent experiments. E) H) AXL decrease upon SphK2 inhibition is not due to protein synthesis inhibition. MCF10AF1F2 and MDA-MB-231shLuci cells were incubated or not with opaganib for 10 hours. When indicated, cycloheximide (CHX) was added 4 hours after the beginning of opaganib incubation. AXL was analyzed by western blotting in whole cell lysates. The histograms show the level of AXL normalized to the tubulin signal, expressed as fold increase compared with control (no treatment) and are the mean ± SEM of 4 independent experiments. I,J) SphK2 inhibition increases AXL level at the cell surface in flotillin-upregulated cells. Fixed non-permeabilized cells were immunostained for AXL using the monoclonal D4 antibody against AXL extracellular domain and an Alexa488-conjugated anti-human secondary antibody. Images of control- and opaganib-treated cells (50µM, 4h) were acquired in the same conditions. Representative images are shown (for MCF10AF1F2 cells, the insets show flotillin 2-mCherry signal). The mean fluorescence intensity was quantified in the area defined by the outline of each individual cell. Results are expressed as arbitrary units and are the mean ± SEM of 23 measurements for MCF10AF1F2 cells (I), and 22 measurements for MDA-MB-231shLuci cells (J) for each condition (control and opaganib). Scale bars: 10 µm for the main images and 2 µm in the magnified images from the boxed area. *P

    Article Snippet: For RNA-mediated interference experiments, cells were transfected using Interferin (Polyplus Transfection) with siRNA oligonucleotide duplexes (Eurogentec, Belgium).

    Techniques: Expressing, Multiple Displacement Amplification, Confocal Microscopy, Labeling, Inhibition, Incubation, Western Blot, Transfection, Luciferase, Fluorescence