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Corning Life Sciences sirna duplexes
Drug specificity and final phenotypes. A: Drug specificity tests. <t>HeLa</t> H2B-GFP cells were transfected with <t>siRNA</t> pools as indicated, or “Mock” transfected in the absence of siRNA. 24 hours after transfection, medium containing either 150 nM of taxol or 300 nM of nocadazole was added to all of the samples. Cells were then incubated for an additional 24 hours and imaged for phenotypic changes. The pools were scored visually for cells in mitotic arrest, and this value was quantified for the pool by counting > 100 cells. (B) Phenotypic categorization of hits on the basis of data in Figure 7 and 8A . Category I) failure to enter mitosis, II) brief mitotic arrest without cytokinesis, and III) brief mitotic arrest with cytokinesis (true suppressors). Shown in bold are 5 expected genes, 4 known SAC genes and KifC1, which is a known true suppressor of loss of Kinesin-5 function [28] .
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1) Product Images from "An Intermittent Live Cell Imaging Screen for siRNA Enhancers and Suppressors of a Kinesin-5 Inhibitor"

Article Title: An Intermittent Live Cell Imaging Screen for siRNA Enhancers and Suppressors of a Kinesin-5 Inhibitor

Journal: PLoS ONE

doi: 10.1371/journal.pone.0007339

Drug specificity and final phenotypes. A: Drug specificity tests. HeLa H2B-GFP cells were transfected with siRNA pools as indicated, or “Mock” transfected in the absence of siRNA. 24 hours after transfection, medium containing either 150 nM of taxol or 300 nM of nocadazole was added to all of the samples. Cells were then incubated for an additional 24 hours and imaged for phenotypic changes. The pools were scored visually for cells in mitotic arrest, and this value was quantified for the pool by counting > 100 cells. (B) Phenotypic categorization of hits on the basis of data in Figure 7 and 8A . Category I) failure to enter mitosis, II) brief mitotic arrest without cytokinesis, and III) brief mitotic arrest with cytokinesis (true suppressors). Shown in bold are 5 expected genes, 4 known SAC genes and KifC1, which is a known true suppressor of loss of Kinesin-5 function [28] .
Figure Legend Snippet: Drug specificity and final phenotypes. A: Drug specificity tests. HeLa H2B-GFP cells were transfected with siRNA pools as indicated, or “Mock” transfected in the absence of siRNA. 24 hours after transfection, medium containing either 150 nM of taxol or 300 nM of nocadazole was added to all of the samples. Cells were then incubated for an additional 24 hours and imaged for phenotypic changes. The pools were scored visually for cells in mitotic arrest, and this value was quantified for the pool by counting > 100 cells. (B) Phenotypic categorization of hits on the basis of data in Figure 7 and 8A . Category I) failure to enter mitosis, II) brief mitotic arrest without cytokinesis, and III) brief mitotic arrest with cytokinesis (true suppressors). Shown in bold are 5 expected genes, 4 known SAC genes and KifC1, which is a known true suppressor of loss of Kinesin-5 function [28] .

Techniques Used: Transfection, Incubation

Sample strong hit phenotypes from the K5I Suppressor screen. Nuclei from each time point are color-coded to illustrate their CellProfiler classifications: yellow = interphase, red = monopolar, blue = unclassified objects. As illustrated in Fig. 2 , transfection occurred at T = 0. (A) untreated HeLa H2B-GFP cells, (B) untransfected cells treated with 1 uM K5I, and (C) mock transfected cells treated with HiPerFect transfection reagent and no K5I. In panels D–F, 1 uM K5I was added to cells 24 hours after transfection with (D) non-targeting siRNA, or siRNAs directed against (E) TTK or (F) Bub3.
Figure Legend Snippet: Sample strong hit phenotypes from the K5I Suppressor screen. Nuclei from each time point are color-coded to illustrate their CellProfiler classifications: yellow = interphase, red = monopolar, blue = unclassified objects. As illustrated in Fig. 2 , transfection occurred at T = 0. (A) untreated HeLa H2B-GFP cells, (B) untransfected cells treated with 1 uM K5I, and (C) mock transfected cells treated with HiPerFect transfection reagent and no K5I. In panels D–F, 1 uM K5I was added to cells 24 hours after transfection with (D) non-targeting siRNA, or siRNAs directed against (E) TTK or (F) Bub3.

Techniques Used: Transfection

Results of time-lapse experiments to examine duration of mitotic arrest and percent of cells entering cytokinesis after treatment with K5I suppressor siRNAs. HeLa H2B-GFP cells were transfected with siRNA pools and individual siRNA duplexes directed against the indicated genes or with a non-targeting control siRNA. “Mock” cells were treated with HiPerFect transfection agent in the absence of siRNA. 24 hours after transfection, K5I was added to all of the samples, so that the final inhibitor concentration was 1 uM. Cells were imaged at 10 minute intervals for 48 hours on an automated inverted fluorescence microscope and automated focus. 50–100 cells from each condition were scored by visual inspection for (A) the average duration of mitosis in the population, and (B) the percent of cells going through cytokinesis upon exit from mitosis. For each of the genes shown, both the siRNA pool and the individual siRNA duplexes were observed to cause the same phenotypes, but only data for the siRNA pools is graphed here.
Figure Legend Snippet: Results of time-lapse experiments to examine duration of mitotic arrest and percent of cells entering cytokinesis after treatment with K5I suppressor siRNAs. HeLa H2B-GFP cells were transfected with siRNA pools and individual siRNA duplexes directed against the indicated genes or with a non-targeting control siRNA. “Mock” cells were treated with HiPerFect transfection agent in the absence of siRNA. 24 hours after transfection, K5I was added to all of the samples, so that the final inhibitor concentration was 1 uM. Cells were imaged at 10 minute intervals for 48 hours on an automated inverted fluorescence microscope and automated focus. 50–100 cells from each condition were scored by visual inspection for (A) the average duration of mitosis in the population, and (B) the percent of cells going through cytokinesis upon exit from mitosis. For each of the genes shown, both the siRNA pool and the individual siRNA duplexes were observed to cause the same phenotypes, but only data for the siRNA pools is graphed here.

Techniques Used: Transfection, Concentration Assay, Fluorescence, Microscopy

Sample strong hit phenotypes from the K5I Enhancer screen. The nuclei from each time point are color-coded to illustrate their CellProfiler classifications: yellow = interphase, red = monopolar, blue = unclassified objects. As illustrated in Fig. 2 , transfection occurred at T = 0. (A) untreated HeLa H2B-GFP cells, (B) untransfected cells treated with 60 nM K5I, (C) mock transfected cells treated with HiPerFect transfection reagent and no K5I. In panels D–F, 60 nM K5I was added to cells 24 hours after transfection with (D) non-targeting siRNA, or siRNAs directed against (E) PLK1 or (F) AURKA.
Figure Legend Snippet: Sample strong hit phenotypes from the K5I Enhancer screen. The nuclei from each time point are color-coded to illustrate their CellProfiler classifications: yellow = interphase, red = monopolar, blue = unclassified objects. As illustrated in Fig. 2 , transfection occurred at T = 0. (A) untreated HeLa H2B-GFP cells, (B) untransfected cells treated with 60 nM K5I, (C) mock transfected cells treated with HiPerFect transfection reagent and no K5I. In panels D–F, 60 nM K5I was added to cells 24 hours after transfection with (D) non-targeting siRNA, or siRNAs directed against (E) PLK1 or (F) AURKA.

Techniques Used: Transfection

Examples of chromosomal phenotypes in the screening assay. All cells were grown in 384-well plates and treated according to the primary screen assay protocol outlined in Figure 2 and described in Materials and Methods . These samples were all transfected with a non-targeting siRNA duplexes. (A) Interphase cells and (B) Mitotic cells selected from control wells undergoing normal cell cycle progression; no Kinesin 5 inhibitor (K5I) added. (C) Monopolar spindles formed in the presence of 1 uM K5I. (D) Analysis of cells treated with 1 uM K5I after 48 hours.
Figure Legend Snippet: Examples of chromosomal phenotypes in the screening assay. All cells were grown in 384-well plates and treated according to the primary screen assay protocol outlined in Figure 2 and described in Materials and Methods . These samples were all transfected with a non-targeting siRNA duplexes. (A) Interphase cells and (B) Mitotic cells selected from control wells undergoing normal cell cycle progression; no Kinesin 5 inhibitor (K5I) added. (C) Monopolar spindles formed in the presence of 1 uM K5I. (D) Analysis of cells treated with 1 uM K5I after 48 hours.

Techniques Used: Screening Assay, Transfection

Related Articles

Concentration Assay:

Article Title: An Intermittent Live Cell Imaging Screen for siRNA Enhancers and Suppressors of a Kinesin-5 Inhibitor
Article Snippet: .. Taxol and Nocodazole AssaysHeLa H2B-GFP cells were reverse transfected by hand with individual siRNA duplexes and pools (both at 10 nM final concentration) in 384 well plates (Corning Costar #3712). .. 24 hours after transfection, cells were imaged to determine initial cell density.

Transfection:

Article Title: An Intermittent Live Cell Imaging Screen for siRNA Enhancers and Suppressors of a Kinesin-5 Inhibitor
Article Snippet: .. Taxol and Nocodazole AssaysHeLa H2B-GFP cells were reverse transfected by hand with individual siRNA duplexes and pools (both at 10 nM final concentration) in 384 well plates (Corning Costar #3712). .. 24 hours after transfection, cells were imaged to determine initial cell density.

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    Corning Life Sciences sirnas
    MPCs regulated cellular NADH level. ( A ) Knocking down <t>MPC1</t> or MPC2 increased the lactate/pyruvate ratio in MEFs. Mouse MEFs transfected with <t>siRNAs</t> against MPC1 or MPC2 increased lactate/pyruvate ratios. ( B ) Western blot assay for flag-tagged human MPC1 and MPC2 overexpression in MEFs cells. The relative protein expression levels were quantified and are shown in the bar diagram. ( C ) Transfection of MPC1 and MPC2 to MEFs cells decreased lactate/pyruvate ratio. * Indicates P
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    Corning Life Sciences rhcxcl5
    CXCL5 enhances blood vessel growth in Matrigel plugs in vivo. a Representative photographs of the Matrigel plug sections stained with H E. Number of cells manifest the ability of angiogenesis in different groups. Arrows on the lower columns indicate areas of blood cells. Scale bars, 50 μm (magnification ×200). b Representative photographs of the Matrigel plug sections stained with Masson’s trichrome. Number of cells manifest the ability of angiogenesis in different groups. Arrows on the lower columns indicate areas of blood cells. Scale bars, 50 μm (magnification ×200). c Cells migrating to form microvessels in Matrigel containing rhCXLC5 (10 ng) were more than PBS control. d Hemoglobin content of the <t>rhCXCL5-</t> (10 ng) treated group was significantly great compared with that of the PBS control. Matrigel containing bFGF (100 ng) served as a positive control. Data represent the mean ± SD, * P
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    Corning Life Sciences transwell migration chambers
    Impact of cystic fibrosis transmembrane conductance regulator (CFTR) on the invasion of A549 cells in vitro . A549 cells were infected with Ad/BgLII (BgLII), Ad/CFTR (CFTR), and Ad/CFTRi (CFTRi) for 24 hours; the capability of cell invasion was assessed by a <t>transwell</t> analysis in the absence or presence of nicotine. A, Representative images of transwell assay for A549 cells cultured in indicated conditions. B, Relevant quantification of the numbers of invasive cells in (A). An overexpression of CFTR showed an ability to significantly suppress cell invasion in A549 cells. In contrast, an exposure of nicotine or knockdown of CFTR by short hairpin RNA (shRNA) dramatically enhanced cell invasion in A549 cells. Compared between indicated groups (B), ** P
    Transwell Migration Chambers, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corning Life Sciences transwells
    Rapamycin, WYE-354 and Torin-1 differently affect cancer properties of distinct CoCSC subpopulations (A) Flow cytometry dot plots ( left ) showing gates used for sorting Tu12 cells. Cells were stained with CD326 PerCP-Cy5.5, CD24 FITC, CD49f PE, CD29 APC, and Sytox Blue or CD326 PerCP-Cy5.5, CD166 PE, CD44 SAv-APC, and Sytox Blue. CD326 + /CD24 + /CD49f + /CD29 + and CD326 + /CD44 + /CD166 + subpopulations were sorted directly into 96-well plates in medium containing vehicle, 1μM Rapamycin, WYE-354 or Torin-1 (see the table for cell densities, middle panel ). Medium was replaced every 72h up to 12 days, when wells containing colonies were scored and CFU frequencies (±SE) estimated using the L-Calc™ software (StemCell Technologies) ( upper right panel ). Optical imaging of CV-stained wells ( lower right panel ) showing confluence rates of CD326 + /CD44 + /CD166 + cells exposed to different drugs (pictures are from wells originally plated with the highest number of cells; white arrows indicate colonies). Scale bar, 200μm. (B) Optical imaging of unstained or CV-stained CD326 + /CD44 + /CD166 + cells treated as in (A) and migrating across the gap up to 72h. Scale bar, 200μm. Thirty thousand cells were seeded into each well of a culture insert and grown overnight. After removal of the insert, a 500μm cell-free gap was created. Cells were therefore treated, and migration monitored at different time points. (C) Optical imaging of CV-stained <t>transwells</t> ( left ) showing invasive potential of CD326 + /CD24 + /CD49f + /CD29 + and CD326 + /CD44 + /CD166 + Tu12 cells treated as in (A). Scale bar, 100μm. Fifteen thousand cells were sorted directly into growth factor reduced Matrigel (2mg/ml)-coated transwells and allowed to invade for 72h. Drugs were added to bottom chambers. Bar graph ( middle ) shows the mean (±SD) invasion area (pixels) from one representative experiment of two in triplicate, with similar results (statistical analysis was performed with respect to control samples; *p
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    MPCs regulated cellular NADH level. ( A ) Knocking down MPC1 or MPC2 increased the lactate/pyruvate ratio in MEFs. Mouse MEFs transfected with siRNAs against MPC1 or MPC2 increased lactate/pyruvate ratios. ( B ) Western blot assay for flag-tagged human MPC1 and MPC2 overexpression in MEFs cells. The relative protein expression levels were quantified and are shown in the bar diagram. ( C ) Transfection of MPC1 and MPC2 to MEFs cells decreased lactate/pyruvate ratio. * Indicates P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: C-Terminal Binding Protein 1 Modulates Cellular Redox via Feedback Regulation of MPC1 and MPC2 in Melanoma Cells

    doi: 10.12659/MSM.912735

    Figure Lengend Snippet: MPCs regulated cellular NADH level. ( A ) Knocking down MPC1 or MPC2 increased the lactate/pyruvate ratio in MEFs. Mouse MEFs transfected with siRNAs against MPC1 or MPC2 increased lactate/pyruvate ratios. ( B ) Western blot assay for flag-tagged human MPC1 and MPC2 overexpression in MEFs cells. The relative protein expression levels were quantified and are shown in the bar diagram. ( C ) Transfection of MPC1 and MPC2 to MEFs cells decreased lactate/pyruvate ratio. * Indicates P

    Article Snippet: Invasion assay A375 or SK-MEL-28 cells were transfected with scramble or empty vector, or PCDNA3 vector containing MPC1 and MPC2, or siRNAs against to MPC1 and MPC2 for 48 h. Then, the transfected 1×105 cells seeded on the upper side of a Matrigel-coated 6.5 mm polycarbonic Transwell chamber (Corning Costar, USA) with serum-free DMEM culture medium, and the lower side of the Transwell chamber was filled with DMEM culture medium with 10% FBS.

    Techniques: Transfection, Western Blot, Over Expression, Expressing

    MPC1 and MPC2 repress melanoma cells proliferation and migration. ( A ) Western blot analysis showed overexpression or knockdown of MPC1 and MPC2 in A375 and SK-MEL-28 cells. The relative protein expression levels were quantified and are shown in the bar diagram. ( B ) Growth curves of A375 and SK-MEL-28 cells, which were transfected with MPC1 and MPC2, siRNAs against MPC1 and MPC2, scramble or treated with 10 mM 2-DG, respectively. * Indicates P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: C-Terminal Binding Protein 1 Modulates Cellular Redox via Feedback Regulation of MPC1 and MPC2 in Melanoma Cells

    doi: 10.12659/MSM.912735

    Figure Lengend Snippet: MPC1 and MPC2 repress melanoma cells proliferation and migration. ( A ) Western blot analysis showed overexpression or knockdown of MPC1 and MPC2 in A375 and SK-MEL-28 cells. The relative protein expression levels were quantified and are shown in the bar diagram. ( B ) Growth curves of A375 and SK-MEL-28 cells, which were transfected with MPC1 and MPC2, siRNAs against MPC1 and MPC2, scramble or treated with 10 mM 2-DG, respectively. * Indicates P

    Article Snippet: Invasion assay A375 or SK-MEL-28 cells were transfected with scramble or empty vector, or PCDNA3 vector containing MPC1 and MPC2, or siRNAs against to MPC1 and MPC2 for 48 h. Then, the transfected 1×105 cells seeded on the upper side of a Matrigel-coated 6.5 mm polycarbonic Transwell chamber (Corning Costar, USA) with serum-free DMEM culture medium, and the lower side of the Transwell chamber was filled with DMEM culture medium with 10% FBS.

    Techniques: Migration, Western Blot, Over Expression, Expressing, Transfection

    CXCL5 enhances blood vessel growth in Matrigel plugs in vivo. a Representative photographs of the Matrigel plug sections stained with H E. Number of cells manifest the ability of angiogenesis in different groups. Arrows on the lower columns indicate areas of blood cells. Scale bars, 50 μm (magnification ×200). b Representative photographs of the Matrigel plug sections stained with Masson’s trichrome. Number of cells manifest the ability of angiogenesis in different groups. Arrows on the lower columns indicate areas of blood cells. Scale bars, 50 μm (magnification ×200). c Cells migrating to form microvessels in Matrigel containing rhCXLC5 (10 ng) were more than PBS control. d Hemoglobin content of the rhCXCL5- (10 ng) treated group was significantly great compared with that of the PBS control. Matrigel containing bFGF (100 ng) served as a positive control. Data represent the mean ± SD, * P

    Journal: Cell Death & Disease

    Article Title: CXCL5 induces tumor angiogenesis via enhancing the expression of FOXD1 mediated by the AKT/NF-κB pathway in colorectal cancer

    doi: 10.1038/s41419-019-1431-6

    Figure Lengend Snippet: CXCL5 enhances blood vessel growth in Matrigel plugs in vivo. a Representative photographs of the Matrigel plug sections stained with H E. Number of cells manifest the ability of angiogenesis in different groups. Arrows on the lower columns indicate areas of blood cells. Scale bars, 50 μm (magnification ×200). b Representative photographs of the Matrigel plug sections stained with Masson’s trichrome. Number of cells manifest the ability of angiogenesis in different groups. Arrows on the lower columns indicate areas of blood cells. Scale bars, 50 μm (magnification ×200). c Cells migrating to form microvessels in Matrigel containing rhCXLC5 (10 ng) were more than PBS control. d Hemoglobin content of the rhCXCL5- (10 ng) treated group was significantly great compared with that of the PBS control. Matrigel containing bFGF (100 ng) served as a positive control. Data represent the mean ± SD, * P

    Article Snippet: In total, 1 × 105 cells were added to serum-free DMEM medium with or without 10 ng/ml rhCXCL5 and plated in transwell chambers (8 μm for a 24-well plate; Corning Costar, NY, USA).

    Techniques: In Vivo, Staining, Positive Control

    rhCXCL5 promotes HUVEC tube formation, migration, and proliferation through the CXCR2 pathway. a Images of HUVEC tube formation assay in different groups. Formation of tube-like networks was stimulated by the addition of rhCXCL5. Number of tubes and total length of tubes demonstrate the ability of angiogenesis in each group. Scale bars, 200 μm (magnification ×40). b , c Number of tubes and total length of tubes significantly increase in the rhCXCL5 group and decrease in the CXCR2-shRNA group. d – f VEGF-A protein and mRNA expression were detected by ELISA, western blot, and qPCR in different groups. rhCXCL5 increases VEGF-A expression which is inhibited by CXCR2-shRNA. g Images of transwell assay in different groups. Scale bar, 100 μm (magnification ×100). h Migration cell numbers are increased in the rhCXCL5 group compared with rhCXCL5-stimulated HUVEC-shCXCR2 group and control group. i EdU assay results in different groups. Scale bar, 200 μm (magnification ×40). j Proportion of cells in the S phase markedly increase in the rhCXCL5 group compared with rhCXCL5-stimulated HUVEC-shCXCR2 group and control group. Data represent the mean ± SD, * P

    Journal: Cell Death & Disease

    Article Title: CXCL5 induces tumor angiogenesis via enhancing the expression of FOXD1 mediated by the AKT/NF-κB pathway in colorectal cancer

    doi: 10.1038/s41419-019-1431-6

    Figure Lengend Snippet: rhCXCL5 promotes HUVEC tube formation, migration, and proliferation through the CXCR2 pathway. a Images of HUVEC tube formation assay in different groups. Formation of tube-like networks was stimulated by the addition of rhCXCL5. Number of tubes and total length of tubes demonstrate the ability of angiogenesis in each group. Scale bars, 200 μm (magnification ×40). b , c Number of tubes and total length of tubes significantly increase in the rhCXCL5 group and decrease in the CXCR2-shRNA group. d – f VEGF-A protein and mRNA expression were detected by ELISA, western blot, and qPCR in different groups. rhCXCL5 increases VEGF-A expression which is inhibited by CXCR2-shRNA. g Images of transwell assay in different groups. Scale bar, 100 μm (magnification ×100). h Migration cell numbers are increased in the rhCXCL5 group compared with rhCXCL5-stimulated HUVEC-shCXCR2 group and control group. i EdU assay results in different groups. Scale bar, 200 μm (magnification ×40). j Proportion of cells in the S phase markedly increase in the rhCXCL5 group compared with rhCXCL5-stimulated HUVEC-shCXCR2 group and control group. Data represent the mean ± SD, * P

    Article Snippet: In total, 1 × 105 cells were added to serum-free DMEM medium with or without 10 ng/ml rhCXCL5 and plated in transwell chambers (8 μm for a 24-well plate; Corning Costar, NY, USA).

    Techniques: Migration, HUVEC Tube Formation Assay, shRNA, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Real-time Polymerase Chain Reaction, Transwell Assay, EdU Assay

    CXCL5/CXCR2 axis promotes FOXD1 activity through the AKT/NF-κB pathway. a Screening of CXCL5/CXCR2 downstream signaling pathway using western blot after different treatments in HUVECs. b Western blot shows that the AKT pathway rather than the ERK pathway regulates the expression of FOXD1 and VEGF-A using pathway inhibitors U0126 and LY294002. c Western blot analyses of p-P65, FOXD1, and VEGF-A in indicated groups after using the NF-κB inhibitor PDTC. d Images of HUVEC tube formation in indicated groups. Scale bars, 200 μm (magnification ×40). e , f Number of tubes and total length of tubes in different groups. Inhibitor of LY294002 and PDTC obviously inhibited the ability of HUVEC tube formation. g A reporter plasmid for FOXD1 (pGL3-FOXD1) was generated by cloning the FOXD1 promoter region (WT) or its NF-κB binding site mutants (MT) into the pGL3 basic vector. rhCXCL5 significantly increased the luciferase activity of the FOXD1 promoter region (WT), while the activity was significantly decreased when transfected with MT sequence. Meanwhile, FOXD1 luciferase activity can be inhibited by PDTC. h CCK8 assay in different groups. Inhibitor of LY294002 and PDTC noticeably reduced HUVEC proliferation ability. i Images of transwell assay in different groups. Scale bars, 100 μm (magnification ×100). j The migration ability of HUVECs was suppressed by the inhibitor of LY294002 and PDTC. Data represent the mean ± SD, * P

    Journal: Cell Death & Disease

    Article Title: CXCL5 induces tumor angiogenesis via enhancing the expression of FOXD1 mediated by the AKT/NF-κB pathway in colorectal cancer

    doi: 10.1038/s41419-019-1431-6

    Figure Lengend Snippet: CXCL5/CXCR2 axis promotes FOXD1 activity through the AKT/NF-κB pathway. a Screening of CXCL5/CXCR2 downstream signaling pathway using western blot after different treatments in HUVECs. b Western blot shows that the AKT pathway rather than the ERK pathway regulates the expression of FOXD1 and VEGF-A using pathway inhibitors U0126 and LY294002. c Western blot analyses of p-P65, FOXD1, and VEGF-A in indicated groups after using the NF-κB inhibitor PDTC. d Images of HUVEC tube formation in indicated groups. Scale bars, 200 μm (magnification ×40). e , f Number of tubes and total length of tubes in different groups. Inhibitor of LY294002 and PDTC obviously inhibited the ability of HUVEC tube formation. g A reporter plasmid for FOXD1 (pGL3-FOXD1) was generated by cloning the FOXD1 promoter region (WT) or its NF-κB binding site mutants (MT) into the pGL3 basic vector. rhCXCL5 significantly increased the luciferase activity of the FOXD1 promoter region (WT), while the activity was significantly decreased when transfected with MT sequence. Meanwhile, FOXD1 luciferase activity can be inhibited by PDTC. h CCK8 assay in different groups. Inhibitor of LY294002 and PDTC noticeably reduced HUVEC proliferation ability. i Images of transwell assay in different groups. Scale bars, 100 μm (magnification ×100). j The migration ability of HUVECs was suppressed by the inhibitor of LY294002 and PDTC. Data represent the mean ± SD, * P

    Article Snippet: In total, 1 × 105 cells were added to serum-free DMEM medium with or without 10 ng/ml rhCXCL5 and plated in transwell chambers (8 μm for a 24-well plate; Corning Costar, NY, USA).

    Techniques: Activity Assay, Western Blot, Expressing, Plasmid Preparation, Generated, Clone Assay, Binding Assay, Luciferase, Transfection, Sequencing, CCK-8 Assay, Transwell Assay, Migration

    The CXCL5/CXCR2 induces the expression of VEGF-A dependent on FOXD1. a Western blot analyses of HIF-1α, C-JUN, and FOXD1 expression in the different groups. b A heat map displays some FOX protein expression level after being treated with or without rhCXCL5 in HUVECs. c Fold changes of the relative mRNA level of VEGF-A-related FOX gene after being treated with or without rhCXCL5 in HUVECs. d Images of HUVEC tube formation assay in each group. Scale bar, 200 μm (magnification ×40). e , f Number of tubes and total length of tubes in different groups, FOXD1 silencing in HUVECs significantly decreases HUVEC tube formation. g , h VEGF-A protein expression is examined by western blot and ELISA in different groups. i ChIP-qPCR assay using Flag antibody or control IgG in HUVECs transfected with a FOXD1 (Flag-tagged) plasmid shows the binding of FOXD1 on the VEGF-A promoter. j A reporter plasmid for VEGF-A (pGL3-VEGF-A) was generated by cloning the VEGF-A promoter region (WT) or its FOXD1 binding site mutants (MT) into the pGL3 basic vector. rhCXCL5 significantly increased the luciferase activity of the VEGF-A promoter region (WT), while the activity was significantly decreased when transfected with MT sequence. Meanwhile, VEGF-A luciferase activity was inhibited when HUVECs were transfected with the shFOXD1 plasmid. k Images of transwell assay in each group. Scale bars, 100 μm (magnification ×100). l Migration cell numbers were reduced by knocking down FOXD1 in HUVECs. m Images of the EdU assay in each group. Scale bars, 200 μm (magnification ×40). n Proportion of cells in the S phase were reduced by knocking down FOXD1 in HUVECs. Data represent the mean ± SD, * P

    Journal: Cell Death & Disease

    Article Title: CXCL5 induces tumor angiogenesis via enhancing the expression of FOXD1 mediated by the AKT/NF-κB pathway in colorectal cancer

    doi: 10.1038/s41419-019-1431-6

    Figure Lengend Snippet: The CXCL5/CXCR2 induces the expression of VEGF-A dependent on FOXD1. a Western blot analyses of HIF-1α, C-JUN, and FOXD1 expression in the different groups. b A heat map displays some FOX protein expression level after being treated with or without rhCXCL5 in HUVECs. c Fold changes of the relative mRNA level of VEGF-A-related FOX gene after being treated with or without rhCXCL5 in HUVECs. d Images of HUVEC tube formation assay in each group. Scale bar, 200 μm (magnification ×40). e , f Number of tubes and total length of tubes in different groups, FOXD1 silencing in HUVECs significantly decreases HUVEC tube formation. g , h VEGF-A protein expression is examined by western blot and ELISA in different groups. i ChIP-qPCR assay using Flag antibody or control IgG in HUVECs transfected with a FOXD1 (Flag-tagged) plasmid shows the binding of FOXD1 on the VEGF-A promoter. j A reporter plasmid for VEGF-A (pGL3-VEGF-A) was generated by cloning the VEGF-A promoter region (WT) or its FOXD1 binding site mutants (MT) into the pGL3 basic vector. rhCXCL5 significantly increased the luciferase activity of the VEGF-A promoter region (WT), while the activity was significantly decreased when transfected with MT sequence. Meanwhile, VEGF-A luciferase activity was inhibited when HUVECs were transfected with the shFOXD1 plasmid. k Images of transwell assay in each group. Scale bars, 100 μm (magnification ×100). l Migration cell numbers were reduced by knocking down FOXD1 in HUVECs. m Images of the EdU assay in each group. Scale bars, 200 μm (magnification ×40). n Proportion of cells in the S phase were reduced by knocking down FOXD1 in HUVECs. Data represent the mean ± SD, * P

    Article Snippet: In total, 1 × 105 cells were added to serum-free DMEM medium with or without 10 ng/ml rhCXCL5 and plated in transwell chambers (8 μm for a 24-well plate; Corning Costar, NY, USA).

    Techniques: Expressing, Western Blot, HUVEC Tube Formation Assay, Enzyme-linked Immunosorbent Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Binding Assay, Generated, Clone Assay, Luciferase, Activity Assay, Sequencing, Transwell Assay, Migration, EdU Assay

    Impact of cystic fibrosis transmembrane conductance regulator (CFTR) on the invasion of A549 cells in vitro . A549 cells were infected with Ad/BgLII (BgLII), Ad/CFTR (CFTR), and Ad/CFTRi (CFTRi) for 24 hours; the capability of cell invasion was assessed by a transwell analysis in the absence or presence of nicotine. A, Representative images of transwell assay for A549 cells cultured in indicated conditions. B, Relevant quantification of the numbers of invasive cells in (A). An overexpression of CFTR showed an ability to significantly suppress cell invasion in A549 cells. In contrast, an exposure of nicotine or knockdown of CFTR by short hairpin RNA (shRNA) dramatically enhanced cell invasion in A549 cells. Compared between indicated groups (B), ** P

    Journal: Technology in Cancer Research & Treatment

    Article Title: Nicotine Induces Progressive Properties of Lung Adenocarcinoma A549 Cells by Inhibiting Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Expression and Plasma Membrane Localization

    doi: 10.1177/1533033818809984

    Figure Lengend Snippet: Impact of cystic fibrosis transmembrane conductance regulator (CFTR) on the invasion of A549 cells in vitro . A549 cells were infected with Ad/BgLII (BgLII), Ad/CFTR (CFTR), and Ad/CFTRi (CFTRi) for 24 hours; the capability of cell invasion was assessed by a transwell analysis in the absence or presence of nicotine. A, Representative images of transwell assay for A549 cells cultured in indicated conditions. B, Relevant quantification of the numbers of invasive cells in (A). An overexpression of CFTR showed an ability to significantly suppress cell invasion in A549 cells. In contrast, an exposure of nicotine or knockdown of CFTR by short hairpin RNA (shRNA) dramatically enhanced cell invasion in A549 cells. Compared between indicated groups (B), ** P

    Article Snippet: Transwell Assay The invasive capacity of A549 infected with different adenoviral vectors was ascertained by a transwell assay using transwell migration chambers (Cat#3413, 8.0 μm pore; Corning Incorporated, Corning, New York).

    Techniques: In Vitro, Infection, Transwell Assay, Cell Culture, Over Expression, shRNA

    Rapamycin, WYE-354 and Torin-1 differently affect cancer properties of distinct CoCSC subpopulations (A) Flow cytometry dot plots ( left ) showing gates used for sorting Tu12 cells. Cells were stained with CD326 PerCP-Cy5.5, CD24 FITC, CD49f PE, CD29 APC, and Sytox Blue or CD326 PerCP-Cy5.5, CD166 PE, CD44 SAv-APC, and Sytox Blue. CD326 + /CD24 + /CD49f + /CD29 + and CD326 + /CD44 + /CD166 + subpopulations were sorted directly into 96-well plates in medium containing vehicle, 1μM Rapamycin, WYE-354 or Torin-1 (see the table for cell densities, middle panel ). Medium was replaced every 72h up to 12 days, when wells containing colonies were scored and CFU frequencies (±SE) estimated using the L-Calc™ software (StemCell Technologies) ( upper right panel ). Optical imaging of CV-stained wells ( lower right panel ) showing confluence rates of CD326 + /CD44 + /CD166 + cells exposed to different drugs (pictures are from wells originally plated with the highest number of cells; white arrows indicate colonies). Scale bar, 200μm. (B) Optical imaging of unstained or CV-stained CD326 + /CD44 + /CD166 + cells treated as in (A) and migrating across the gap up to 72h. Scale bar, 200μm. Thirty thousand cells were seeded into each well of a culture insert and grown overnight. After removal of the insert, a 500μm cell-free gap was created. Cells were therefore treated, and migration monitored at different time points. (C) Optical imaging of CV-stained transwells ( left ) showing invasive potential of CD326 + /CD24 + /CD49f + /CD29 + and CD326 + /CD44 + /CD166 + Tu12 cells treated as in (A). Scale bar, 100μm. Fifteen thousand cells were sorted directly into growth factor reduced Matrigel (2mg/ml)-coated transwells and allowed to invade for 72h. Drugs were added to bottom chambers. Bar graph ( middle ) shows the mean (±SD) invasion area (pixels) from one representative experiment of two in triplicate, with similar results (statistical analysis was performed with respect to control samples; *p

    Journal: Oncotarget

    Article Title: Selective targeting of human colon cancer stem-like cells by the mTOR inhibitor Torin-1

    doi:

    Figure Lengend Snippet: Rapamycin, WYE-354 and Torin-1 differently affect cancer properties of distinct CoCSC subpopulations (A) Flow cytometry dot plots ( left ) showing gates used for sorting Tu12 cells. Cells were stained with CD326 PerCP-Cy5.5, CD24 FITC, CD49f PE, CD29 APC, and Sytox Blue or CD326 PerCP-Cy5.5, CD166 PE, CD44 SAv-APC, and Sytox Blue. CD326 + /CD24 + /CD49f + /CD29 + and CD326 + /CD44 + /CD166 + subpopulations were sorted directly into 96-well plates in medium containing vehicle, 1μM Rapamycin, WYE-354 or Torin-1 (see the table for cell densities, middle panel ). Medium was replaced every 72h up to 12 days, when wells containing colonies were scored and CFU frequencies (±SE) estimated using the L-Calc™ software (StemCell Technologies) ( upper right panel ). Optical imaging of CV-stained wells ( lower right panel ) showing confluence rates of CD326 + /CD44 + /CD166 + cells exposed to different drugs (pictures are from wells originally plated with the highest number of cells; white arrows indicate colonies). Scale bar, 200μm. (B) Optical imaging of unstained or CV-stained CD326 + /CD44 + /CD166 + cells treated as in (A) and migrating across the gap up to 72h. Scale bar, 200μm. Thirty thousand cells were seeded into each well of a culture insert and grown overnight. After removal of the insert, a 500μm cell-free gap was created. Cells were therefore treated, and migration monitored at different time points. (C) Optical imaging of CV-stained transwells ( left ) showing invasive potential of CD326 + /CD24 + /CD49f + /CD29 + and CD326 + /CD44 + /CD166 + Tu12 cells treated as in (A). Scale bar, 100μm. Fifteen thousand cells were sorted directly into growth factor reduced Matrigel (2mg/ml)-coated transwells and allowed to invade for 72h. Drugs were added to bottom chambers. Bar graph ( middle ) shows the mean (±SD) invasion area (pixels) from one representative experiment of two in triplicate, with similar results (statistical analysis was performed with respect to control samples; *p

    Article Snippet: Transwells (07-200-150) were purchased from Corning.

    Techniques: Flow Cytometry, Cytometry, Staining, Software, Optical Imaging, Migration

    SGK1 knockdown reduces CoCSC growth, invasive ability and chemoresistance Tu12, Tu21 and Tu22 were infected with copGFP control, control shRNA or SGK1 shRNA lentiviral particles (MOI=6.5). 72h post-transfection, cells were subjected to (A) light microscopy, and (B, left ) RT-PCR analysis for SGK1 or 18S housekeeping gene as internal control. Scale bars, 100μm. After additional 5 days of puromycin selection (1.5-2μg/mL), cells were analyzed by immunofluorescence for SGK1 Ser422 (B, right ). Scale bar, 100μm. (C) Decreased CoCSC clonogenicity following SGK1 knockdown. One hundred thousand cells infected with control or SGK1 shRNA lentiviral particles were plated in each well of a 6-well plate. 2 weeks later, colonies were fixed and stained with 0.2% crystal violet (CV) in 10% ethanol. (D) Bar graph ( left ) and optical imaging ( right ) of CV-stained transwells showing reduced invasive potential of SGK1-silenced Tu12 cells. Scale bar, 100μm. Fifteen thousand cells infected as in (C) were seeded on growth factor reduced Matrigel (2mg/ml)-coated transwells and allowed to invade for 48h. Data are presented as relative ratio of invasion from one experiment representative of two independent experiments carried out in triplicate (*** p

    Journal: Oncotarget

    Article Title: Selective targeting of human colon cancer stem-like cells by the mTOR inhibitor Torin-1

    doi:

    Figure Lengend Snippet: SGK1 knockdown reduces CoCSC growth, invasive ability and chemoresistance Tu12, Tu21 and Tu22 were infected with copGFP control, control shRNA or SGK1 shRNA lentiviral particles (MOI=6.5). 72h post-transfection, cells were subjected to (A) light microscopy, and (B, left ) RT-PCR analysis for SGK1 or 18S housekeeping gene as internal control. Scale bars, 100μm. After additional 5 days of puromycin selection (1.5-2μg/mL), cells were analyzed by immunofluorescence for SGK1 Ser422 (B, right ). Scale bar, 100μm. (C) Decreased CoCSC clonogenicity following SGK1 knockdown. One hundred thousand cells infected with control or SGK1 shRNA lentiviral particles were plated in each well of a 6-well plate. 2 weeks later, colonies were fixed and stained with 0.2% crystal violet (CV) in 10% ethanol. (D) Bar graph ( left ) and optical imaging ( right ) of CV-stained transwells showing reduced invasive potential of SGK1-silenced Tu12 cells. Scale bar, 100μm. Fifteen thousand cells infected as in (C) were seeded on growth factor reduced Matrigel (2mg/ml)-coated transwells and allowed to invade for 48h. Data are presented as relative ratio of invasion from one experiment representative of two independent experiments carried out in triplicate (*** p

    Article Snippet: Transwells (07-200-150) were purchased from Corning.

    Techniques: Infection, shRNA, Transfection, Light Microscopy, Reverse Transcription Polymerase Chain Reaction, Selection, Immunofluorescence, Staining, Optical Imaging