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  • 99
    Millipore transwell inserts
    Evolution of mineralized deposits under porcine RPE in culture. Transmission electron microscopy ( A – D ), scanning electron microscopy ( E – H ), and microcomputed tomography for minerals ( I – L ). ( A , E , I ) Deposit-incapable, passaged cells at 6 weeks exhibited little or no mineralization. N, nucleus; Ins, culture dish insert. ( B , F , J ) Deposit-capable primary cells at 6 weeks produced a thin electron-dense sub-RPE deposit that was continuous with material in pores ( arrow ) and exhibited some mineralization. Due to the sectioning plane, the pore does not cross the <t>Transwell.</t> ( C , G , K ) At 12 weeks, deposit-capable cells developed a continuous layer of deposit with increased mineralization. ( D , H , L ) At 26.5 weeks, focal dome-shaped deposits were also present, with diffuse deposit, exhibited intense mineralization ( H , L ; arrowheads ). Diffuse and focal deposits had solid cores ( yellow asterisks ) with feathery surfaces ( red arrowhead ) ( C , D ). Cracks between cells ( G , H ) are due to brittleness conferred by mineralization. Scale bars denote the following: A – D , 2 μm; E – L , 100 μm.
    Transwell Inserts, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1587 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Corning Life Sciences transwell inserts
    Four conditions of bacterial <t>Transwell</t> culture. Bottom well contains bacterial sample, Transwell insert contains antibiotics and NO releasing biomimetic nanomatrix gel. (A) NO (-) antibiotics (-). (B) NO (+) antibiotics (-). (C) NO (-) antibiotics (+). (D) NO (+) antibiotics (+).
    Transwell Inserts, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 6883 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transwell inserts/product/Corning Life Sciences
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    92
    Becton Dickinson transwell inserts
    MELK knockdown reduces migration and invasion in vitro . (A) Wound healing assays revealed the reduced migratory potential of cells following knockdown of MELK. (B) <t>Transwell</t> assays revealed reduced migratory and invasive potential of MNNG/HOS cells following knockdown of MELK. (C) Migration (wound healing assay) and (D) migration and invasion (Transwell assays) were reduced in osteosarcoma cells following treatment with OTSSP167. **P
    Transwell Inserts, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 3514 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transwell inserts/product/Becton Dickinson
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    92
    Greiner Bio transwell inserts
    Xaliproden protects against the loss of tight junctions in the presence of paraquat. ARPE-19 cells were seeded in 24 <t>Transwell</t> plates and grown in low serum medium for 2 weeks. The cells were then treated with paraquat (300 µM), xaliproden (20 µM), or a combination of the two for 24 h before the transepithelial resistance (TEER) was measured in a volt/ohm meter. Bars represent the average electrical resistance from triplicates ± standard deviation (SD). *, p = 0.006 between paraquat, and paraquat + xaliproden, as determined with one-way ANOVA. Par = paraquat; Xali = xaliproden.
    Transwell Inserts, supplied by Greiner Bio, used in various techniques. Bioz Stars score: 92/100, based on 258 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 258 article reviews
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    90
    Becton Dickinson matrigel coated transwell inserts
    GBM-SKH cells were stably expressed without or with the control vector (pcDNA3.1) or with wild-type OPN (pDNA3.1/OPN). (A) MMP-2, OPN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression were analyzed by RT–PCR. (B) Cell lysates were harvested and immunoblotted with antibodies specific for OPN, MMP-2, and α-tubulin. These experiments were repeated, and the intensities of bands were quantitated and expressed as ratios to mock control cells. Results shown in the lower panel of (A) and (B) are the mean ± SE of three independent experiments. (C) Media were then collected for the zymographic assay of MMP-2 activity. In the lower panel, MMP-2 activity was quantitated by scanning the photographic negatives on a gel analysis system. (D) For the in vitro invasion assay, cells were seeded in equal amounts of experimental cells in the upper part of a <t>transwell</t> chamber separated by a <t>Matrigel-coated</t> membrane. Data represent the mean ± SE of three independent experiments. (E) Cells were seeded in culture plates in DMEM media supplemented with 10% heat-inactivated FCS. Viable cell numbers were counted at 24, 48, and 72 hours. Data represent the mean ± SE of three independent experiments. (F) Histopathological characteristics (H E) and tumor volumes of animals implanted with GBM/pcDNA or GBM/OPN were evaluated. Tumor sizes were measured through their largest diameter (upper panel), and the lower panel shows the mean ± SE of three independent experiments. * P
    Matrigel Coated Transwell Inserts, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 256 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 256 article reviews
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    90
    Corning Life Sciences polycarbonate transwell inserts
    Effect of the RBP-J deletion on the in vitro cultured EPC. (A) The expression of CXCR4 on the cultured EPC was accessed by FACS analysis. (B, C) In vitro migration assay. <t>Transwell</t> culture was set up, with EPC from the RBP-J-deleted and the control mice in the upper chamber, and SDF-1α in the lower chamber. A photograph of EPC in the lower chamber (B) was taken, and cells in the lower chamber (C) were counted 18 h after the starting of the culture (bars = mean±SD; n = 4; ** P
    Polycarbonate Transwell Inserts, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 90/100, based on 307 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polycarbonate transwell inserts/product/Corning Life Sciences
    Average 90 stars, based on 307 article reviews
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    92
    Becton Dickinson 24 well transwell inserts
    Effect of the RBP-J deletion on the in vitro cultured EPC. (A) The expression of CXCR4 on the cultured EPC was accessed by FACS analysis. (B, C) In vitro migration assay. <t>Transwell</t> culture was set up, with EPC from the RBP-J-deleted and the control mice in the upper chamber, and SDF-1α in the lower chamber. A photograph of EPC in the lower chamber (B) was taken, and cells in the lower chamber (C) were counted 18 h after the starting of the culture (bars = mean±SD; n = 4; ** P
    24 Well Transwell Inserts, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Corning Life Sciences transwell membrane inserts
    FOXK1 facilitates glioblastoma cell metastasis. (A) Following knockdown or ectopic expression of FOXK1 in LN18 cells, the protein or mRNA levels of FOXK1 were detected by western blotting or reverse transcription-quantitative polymerase chain reaction, respectively. (B and C) <t>Transwell</t> migration assay of LN18 cells after (B) ectopic expression and (C) knockdown of FOXK1 (48 h post-transfection). (D) Anchorage-independent cell growth assay. LN18 cells were transfected with FOXK1 siRNA and then placed on soft agar; the average number of colonies formed was determined. All experiments were performed at least three times. *P
    Transwell Membrane Inserts, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 91/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transwell membrane inserts/product/Corning Life Sciences
    Average 91 stars, based on 149 article reviews
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    91
    Corning Life Sciences polycarbonate membrane transwell inserts
    GRAMD1B negates JAK/STAT signaling. ( a ) Effects of GRAMD1B inhibition on JAK/STAT signaling. Increased p-JAK2 and p-STAT3 levels are detected on Gramd1b . ( b ) Treatment with AG490 almost completely suppresses the morphological changes of cells induced by Gramd1b knockdown. (Scale bar = 20 μm). ( c -c’) <t>Transwell</t> migration assay reveals that AG490 co-treatment efficiently suppresses the enhanced cell migration caused by GRAMD1B inhibition. (Scale bar = 100 μm). Data is represented as mean ± SEM of n = 3. ( d ) qRT-PCR analysis shows that AG490 treatment efficiently blocks the induction of Rac1 and RhoA in cells transfected with si-Gramd1b . Data is represented as mean ± SEM of n = 3. * P
    Polycarbonate Membrane Transwell Inserts, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 91/100, based on 268 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polycarbonate membrane transwell inserts/product/Corning Life Sciences
    Average 91 stars, based on 268 article reviews
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    89
    Corning Life Sciences collagen coated transwell inserts
    Primary CF epithelial cells with LPRs containing either 50 nM or 100 nM siRNA and peptide Y. ( A ) Percentage of expression of the α-ENaC mRNA after siRNA treatment in primary CF cells in submerged culture. The expression of α-ENaC was 40.5% at 50 nM and 27.2% at 100 nM, respectively. The difference was statistically significant (*** P ≤ 0.001) at 50 nM, whereas the significance (**P ≤ 0.01), was lower at 100 nM. The results show the means ± S.E.M. of triplicate experiments. ( B ) Viability (percentage) of primary CF cells following transfections with LPRs formulated with peptide Y compared to untransfected cells. The α-ENaC-siRNA treated cells showed a viability of 72% at 50 nM siRNA concentration and 57% at 100 nM. Cell survival was assessed using an LDH assay measuring the light emitted at 610 nm. Results represent mean values of triplicate repeats ± S.E.M. ( C ) Percentage of remaining expression of the α-ENaC gene transcript in primary CFBE cells grown on ALI in <t>transwell</t> plates. The expression of the transcript of the α-ENaC gene was 71.8% at 30 nM and 56.7% at 50 nM. The difference was statistically significant only at 50 nM (*P ≤ 0.05). qRT-PCR analysis was performed in triplicate on individual samples. The mRNA levels of the α-ENaC gene are expressed as percentage of irrelevant/non-targeted control siRNA.
    Collagen Coated Transwell Inserts, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 89/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Corning Life Sciences 24 well transwell insert
    Sepin-1 inhibits cell migration of triple-negative breast cancer cells. MD-MB-231 or MD-MB-468 cells were seeded on the top of the filter membrane in a <t>Transwell</t> ® insert, and medium with or without Sepin-1 was added to the bottom of the lower chamber in a <t>24-well</t> plate. After 24 h of incubation, the cells that had not migrated were removed. The cells on the other side of the membrane on Transwell ® insert were fixed and stained with crystal violet. The migrated cells (in purple) were counted. Representative images of migrated cells are shown in A. Percentage of cells migrated through the membrane is shown in B. The assay was performed in triplicate (n=3 ± SD). *** indicates p
    24 Well Transwell Insert, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 89/100, based on 144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/24 well transwell insert/product/Corning Life Sciences
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    89
    Becton Dickinson transwell precoated matrigel membrane filter inserts
    Effect of depleting or enforcing the expression of FoxQ1 on cell proliferation, migration and invasiveness of lung carcinoma cells A) qRT-PCR and B) western blot were used to select the most effective silencing siRNA targeting human FOXQ1. C) Two stably transfected cell lines silenced for FoxQ1 were established by G418 screening. G418-resistant clones were examined by qRT-PCR. D) Two stably transfected cell lines overexpressing FoxQ1 were identified by G418 screening, and qRT-PCR was used to confirm G418-resistant clones. E) The proliferation ability of the four experimental cell lines was examined using CCK-8 at 450 nm. Specifically, 5 × 10 3 cells were seeded in 100 μL of medium per well into 96-well plates (three wells per each group). Then, 10 μL of CCK8 solution was added to the culture medium in each well after 24h, 48h, 72h and 96h. Then cells were incubated for 3 h another. The absorbance was determined at 450 nm wavelength. F) Migration and G) invasion ability were presented as total number of cells that migrated to the bottom chamber without or with the <t>transwell-precoated</t> <t>matrigel,</t> respectively, as calculated in at least six random fields (total magnification ×200) per filter. (*P
    Transwell Precoated Matrigel Membrane Filter Inserts, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 89/100, based on 295 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transwell precoated matrigel membrane filter inserts/product/Becton Dickinson
    Average 89 stars, based on 295 article reviews
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    Image Search Results


    Evolution of mineralized deposits under porcine RPE in culture. Transmission electron microscopy ( A – D ), scanning electron microscopy ( E – H ), and microcomputed tomography for minerals ( I – L ). ( A , E , I ) Deposit-incapable, passaged cells at 6 weeks exhibited little or no mineralization. N, nucleus; Ins, culture dish insert. ( B , F , J ) Deposit-capable primary cells at 6 weeks produced a thin electron-dense sub-RPE deposit that was continuous with material in pores ( arrow ) and exhibited some mineralization. Due to the sectioning plane, the pore does not cross the Transwell. ( C , G , K ) At 12 weeks, deposit-capable cells developed a continuous layer of deposit with increased mineralization. ( D , H , L ) At 26.5 weeks, focal dome-shaped deposits were also present, with diffuse deposit, exhibited intense mineralization ( H , L ; arrowheads ). Diffuse and focal deposits had solid cores ( yellow asterisks ) with feathery surfaces ( red arrowhead ) ( C , D ). Cracks between cells ( G , H ) are due to brittleness conferred by mineralization. Scale bars denote the following: A – D , 2 μm; E – L , 100 μm.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model

    doi: 10.1167/iovs.16-21060

    Figure Lengend Snippet: Evolution of mineralized deposits under porcine RPE in culture. Transmission electron microscopy ( A – D ), scanning electron microscopy ( E – H ), and microcomputed tomography for minerals ( I – L ). ( A , E , I ) Deposit-incapable, passaged cells at 6 weeks exhibited little or no mineralization. N, nucleus; Ins, culture dish insert. ( B , F , J ) Deposit-capable primary cells at 6 weeks produced a thin electron-dense sub-RPE deposit that was continuous with material in pores ( arrow ) and exhibited some mineralization. Due to the sectioning plane, the pore does not cross the Transwell. ( C , G , K ) At 12 weeks, deposit-capable cells developed a continuous layer of deposit with increased mineralization. ( D , H , L ) At 26.5 weeks, focal dome-shaped deposits were also present, with diffuse deposit, exhibited intense mineralization ( H , L ; arrowheads ). Diffuse and focal deposits had solid cores ( yellow asterisks ) with feathery surfaces ( red arrowhead ) ( C , D ). Cracks between cells ( G , H ) are due to brittleness conferred by mineralization. Scale bars denote the following: A – D , 2 μm; E – L , 100 μm.

    Article Snippet: For our experiments, cells at passage 2 were cultured either on 10-μm-thick Transwell inserts with a pore diameter of 0.4 μm (Corning; Thermo Fisher Scientific) coated with Geltrex extracellular matrix (Thermo Fisher Scientific) or on 100-μm-thick Transwell inserts with a pore diameter of 0.45 μm (Millipore; Thermo Fisher Scientific) coated with laminin (Sigma-Aldrich Corp., St. Louis, MO, USA), in MEM-α modifications as previously described.

    Techniques: Transmission Assay, Electron Microscopy, Produced

    Lipids and calcium localize to sub-RPE deposits. ( A ) Primary porcine RPE cells cultured for 12.0 weeks are confluent. They exhibit a brush-border apically ( arrow ) and overlie a diffuse deposit ( arrowheads ). One-micrometer epoxy section, toluidine blue stain. ( B ) Deposit is a continuous and electron dense monolayer without evidence of thickened basal lamina; transmission electron microscopy. ( C ) Deposits contain neutral lipid, as do pores crossing the Transwell insert. Oil red O stain. ( D ) Cells form a continuous layer over HAP ( magenta ) positive deposits. Cryo-section; cells, autofluorescence; HAP deposit, bone-tag680RD (Li-Cor) fluorescent stain. ( E ) Deposits under nucleated cells ( blue ) contain HAP ( magenta ). Cryo-section, bone-tag680RD for HAP and 4′,6-diamidino-2-phenylindole (DAPI) for nucleic acids ( blue ). Scale bar denotes 20 μm and applies to all panels. Images in A and C were matched in magnification to images obtained with higher-resolution imaging technologies in B , D , and E and thus exhibit empty magnification.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model

    doi: 10.1167/iovs.16-21060

    Figure Lengend Snippet: Lipids and calcium localize to sub-RPE deposits. ( A ) Primary porcine RPE cells cultured for 12.0 weeks are confluent. They exhibit a brush-border apically ( arrow ) and overlie a diffuse deposit ( arrowheads ). One-micrometer epoxy section, toluidine blue stain. ( B ) Deposit is a continuous and electron dense monolayer without evidence of thickened basal lamina; transmission electron microscopy. ( C ) Deposits contain neutral lipid, as do pores crossing the Transwell insert. Oil red O stain. ( D ) Cells form a continuous layer over HAP ( magenta ) positive deposits. Cryo-section; cells, autofluorescence; HAP deposit, bone-tag680RD (Li-Cor) fluorescent stain. ( E ) Deposits under nucleated cells ( blue ) contain HAP ( magenta ). Cryo-section, bone-tag680RD for HAP and 4′,6-diamidino-2-phenylindole (DAPI) for nucleic acids ( blue ). Scale bar denotes 20 μm and applies to all panels. Images in A and C were matched in magnification to images obtained with higher-resolution imaging technologies in B , D , and E and thus exhibit empty magnification.

    Article Snippet: For our experiments, cells at passage 2 were cultured either on 10-μm-thick Transwell inserts with a pore diameter of 0.4 μm (Corning; Thermo Fisher Scientific) coated with Geltrex extracellular matrix (Thermo Fisher Scientific) or on 100-μm-thick Transwell inserts with a pore diameter of 0.45 μm (Millipore; Thermo Fisher Scientific) coated with laminin (Sigma-Aldrich Corp., St. Louis, MO, USA), in MEM-α modifications as previously described.

    Techniques: Cell Culture, Staining, Transmission Assay, Electron Microscopy, Imaging

    Retinal pigment epithelium–secreted materials in Transwell insert pores (0.4 μm diameter). Electron microscopy revealed cellular processes and material of varying electron density within the pores of the cell culture insert. ( A ) Membranous material in a pore under deposit-incapable cells at 6.0 weeks, indicating that these cells release material in culture, although not in sufficient quantity to accumulate. Cells in panels B through F are deposit-capable. ( B ) Spherical particles (26.5 weeks) demonstrating continuity of sub-RPE deposit into pores. ( C ) Cellular processes can enter Transwell pores alongside heterogeneous material (26.5 weeks). ( D – F ) Accumulation of material with varying electron density accumulates in pores in apparent continuity with sub-RPE deposits.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model

    doi: 10.1167/iovs.16-21060

    Figure Lengend Snippet: Retinal pigment epithelium–secreted materials in Transwell insert pores (0.4 μm diameter). Electron microscopy revealed cellular processes and material of varying electron density within the pores of the cell culture insert. ( A ) Membranous material in a pore under deposit-incapable cells at 6.0 weeks, indicating that these cells release material in culture, although not in sufficient quantity to accumulate. Cells in panels B through F are deposit-capable. ( B ) Spherical particles (26.5 weeks) demonstrating continuity of sub-RPE deposit into pores. ( C ) Cellular processes can enter Transwell pores alongside heterogeneous material (26.5 weeks). ( D – F ) Accumulation of material with varying electron density accumulates in pores in apparent continuity with sub-RPE deposits.

    Article Snippet: For our experiments, cells at passage 2 were cultured either on 10-μm-thick Transwell inserts with a pore diameter of 0.4 μm (Corning; Thermo Fisher Scientific) coated with Geltrex extracellular matrix (Thermo Fisher Scientific) or on 100-μm-thick Transwell inserts with a pore diameter of 0.45 μm (Millipore; Thermo Fisher Scientific) coated with laminin (Sigma-Aldrich Corp., St. Louis, MO, USA), in MEM-α modifications as previously described.

    Techniques: Electron Microscopy, Cell Culture

    Accumulation of ApoE in sub-RPE deposits in primary porcine and human fetal RPE cell culture models. Immunohistochemistry was performed on porcine RPE cells cultured for 26.5 weeks ( A – C ) or on human fetal RPE cells cultured for 6 weeks ( D , E ). Transwell membranes were either polyester ( A – D ) or composed of mixed cellulose ( E ). ( A ) Punctate ( arrowheads ) and diffuse ApoE immunofluorescence was present within focal deposits, as well as on the surface of the polyester insert when cells were cultured for 26.5 weeks ( arrows ). ( B ) Apolipoprotein E–immunofluorescent deposits accumulated along the membrane in a nonuniform manner ( arrows ). Note the intense labelling within the RPE cells bodies and less intense labeling within pores of the Transwell membranes on A and B . ( C ) Punctate ApoE labeling ( arrows ) is found within or under HAP sub-RPE deposits ( magenta ). ( D ) Human fetal primary RPE cells grown for 8 weeks on polyester membrane inserts show ApoE ( green ) accumulation on the surface of the membrane, similar to that for porcine cells. Unlike porcine cells, human RPE cell bodies stain lightly for ApoE. Red immunofluorescence corresponds to ZO1 staining associated with junctions, indicating that the cells are well differentiated. ( E ) Human cells grown on membranes of mixed cellulose esters showed the dispersed accumulation of ApoE ( green ) particles, identical to those published by Johnson et al., 9 in the membrane cavities. Red arrows between C and E indicate the surface of the membrane inserts. Scale bars denote 10 μm in A – E .

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model

    doi: 10.1167/iovs.16-21060

    Figure Lengend Snippet: Accumulation of ApoE in sub-RPE deposits in primary porcine and human fetal RPE cell culture models. Immunohistochemistry was performed on porcine RPE cells cultured for 26.5 weeks ( A – C ) or on human fetal RPE cells cultured for 6 weeks ( D , E ). Transwell membranes were either polyester ( A – D ) or composed of mixed cellulose ( E ). ( A ) Punctate ( arrowheads ) and diffuse ApoE immunofluorescence was present within focal deposits, as well as on the surface of the polyester insert when cells were cultured for 26.5 weeks ( arrows ). ( B ) Apolipoprotein E–immunofluorescent deposits accumulated along the membrane in a nonuniform manner ( arrows ). Note the intense labelling within the RPE cells bodies and less intense labeling within pores of the Transwell membranes on A and B . ( C ) Punctate ApoE labeling ( arrows ) is found within or under HAP sub-RPE deposits ( magenta ). ( D ) Human fetal primary RPE cells grown for 8 weeks on polyester membrane inserts show ApoE ( green ) accumulation on the surface of the membrane, similar to that for porcine cells. Unlike porcine cells, human RPE cell bodies stain lightly for ApoE. Red immunofluorescence corresponds to ZO1 staining associated with junctions, indicating that the cells are well differentiated. ( E ) Human cells grown on membranes of mixed cellulose esters showed the dispersed accumulation of ApoE ( green ) particles, identical to those published by Johnson et al., 9 in the membrane cavities. Red arrows between C and E indicate the surface of the membrane inserts. Scale bars denote 10 μm in A – E .

    Article Snippet: For our experiments, cells at passage 2 were cultured either on 10-μm-thick Transwell inserts with a pore diameter of 0.4 μm (Corning; Thermo Fisher Scientific) coated with Geltrex extracellular matrix (Thermo Fisher Scientific) or on 100-μm-thick Transwell inserts with a pore diameter of 0.45 μm (Millipore; Thermo Fisher Scientific) coated with laminin (Sigma-Aldrich Corp., St. Louis, MO, USA), in MEM-α modifications as previously described.

    Techniques: Cell Culture, Immunohistochemistry, Immunofluorescence, Labeling, Staining

    Distribution of the molecular ions identified by SIMS. An optical image of the focal deposit analyzed by SIMS analysis ( A ). Molecular ion mapping of calcium (Ca + ) ( B ) and calcium phosphate (Ca 2 PO 4 + ) ( C ) confirmed the presence of HAP within sub-RPE deposits. Lipid content of deposits was confirmed by molecular ion mapping of the phosphatidylcholine (PC) head group fragment ion ( D ). Mapping of amino acid signatures including glycine ( E ), alanine ( F ), proline ( G ), and leucine ( H ) confirmed the presence of protein within deposits. Thus, HAP, amino acids, and PC are all present within deposits. It should be noted that the culture insert is absent in the figure, during processing the cells and the sub-RPE deposits detached from the Transwell insert surface. Scale bars denote 40 μm.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model

    doi: 10.1167/iovs.16-21060

    Figure Lengend Snippet: Distribution of the molecular ions identified by SIMS. An optical image of the focal deposit analyzed by SIMS analysis ( A ). Molecular ion mapping of calcium (Ca + ) ( B ) and calcium phosphate (Ca 2 PO 4 + ) ( C ) confirmed the presence of HAP within sub-RPE deposits. Lipid content of deposits was confirmed by molecular ion mapping of the phosphatidylcholine (PC) head group fragment ion ( D ). Mapping of amino acid signatures including glycine ( E ), alanine ( F ), proline ( G ), and leucine ( H ) confirmed the presence of protein within deposits. Thus, HAP, amino acids, and PC are all present within deposits. It should be noted that the culture insert is absent in the figure, during processing the cells and the sub-RPE deposits detached from the Transwell insert surface. Scale bars denote 40 μm.

    Article Snippet: For our experiments, cells at passage 2 were cultured either on 10-μm-thick Transwell inserts with a pore diameter of 0.4 μm (Corning; Thermo Fisher Scientific) coated with Geltrex extracellular matrix (Thermo Fisher Scientific) or on 100-μm-thick Transwell inserts with a pore diameter of 0.45 μm (Millipore; Thermo Fisher Scientific) coated with laminin (Sigma-Aldrich Corp., St. Louis, MO, USA), in MEM-α modifications as previously described.

    Techniques:

    Diffuse and focal refractile deposits in a Transwell culture dish. Primary porcine RPE cells cultured ≥12 weeks overlie refractile focal ( red arrowheads ) and diffuse ( blue arrowheads ) deposits. The majority of cells are not strongly pigmented. Some cells lack melanosomes completely ( green asterisk ) as is common in RPE cell cultures. Pores through the Transwell insert are visible in places where cells are not present ( yellow arrows ).

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model

    doi: 10.1167/iovs.16-21060

    Figure Lengend Snippet: Diffuse and focal refractile deposits in a Transwell culture dish. Primary porcine RPE cells cultured ≥12 weeks overlie refractile focal ( red arrowheads ) and diffuse ( blue arrowheads ) deposits. The majority of cells are not strongly pigmented. Some cells lack melanosomes completely ( green asterisk ) as is common in RPE cell cultures. Pores through the Transwell insert are visible in places where cells are not present ( yellow arrows ).

    Article Snippet: For our experiments, cells at passage 2 were cultured either on 10-μm-thick Transwell inserts with a pore diameter of 0.4 μm (Corning; Thermo Fisher Scientific) coated with Geltrex extracellular matrix (Thermo Fisher Scientific) or on 100-μm-thick Transwell inserts with a pore diameter of 0.45 μm (Millipore; Thermo Fisher Scientific) coated with laminin (Sigma-Aldrich Corp., St. Louis, MO, USA), in MEM-α modifications as previously described.

    Techniques: Cell Culture

    LIPUS set-up experiment. Upper panel: LIPUS transducer device. Bottom panel: characteristic of the ultrasound signal: 200 μs burst of 1.5 MHz sine waves repeated at 1 kHz ( c ). MDA-MB-231 cells were seeded in 12-well plates employing four wells/plate. Two wells were placed on swich-on transducer receiving LIPUS stimuli to obtain LCM the other two were placed on swich-off transducer and used as untreated group to collect CM. LIPUS treatment was performed for 20 min/day for 10 days ( a ). MDA-MB-231 cells were seeded in 6-well plates using four wells/plate. After 24 h, a transwell insert was inserted into each well and Raw264.7 cells were seeded in the upper compartment. Two wells were placed on swich-on transducer receiving LIPUS stimuli to obtain LIPUS_CC group, the other two were placed on swich-off transducer and used as untreated to obtain Untreated_CC group. LIPUS treatment was performed for 20 min/day for 10 days ( b )

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Inhibitory effects of low intensity pulsed ultrasound on osteoclastogenesis induced in vitro by breast cancer cells

    doi: 10.1186/s13046-018-0868-2

    Figure Lengend Snippet: LIPUS set-up experiment. Upper panel: LIPUS transducer device. Bottom panel: characteristic of the ultrasound signal: 200 μs burst of 1.5 MHz sine waves repeated at 1 kHz ( c ). MDA-MB-231 cells were seeded in 12-well plates employing four wells/plate. Two wells were placed on swich-on transducer receiving LIPUS stimuli to obtain LCM the other two were placed on swich-off transducer and used as untreated group to collect CM. LIPUS treatment was performed for 20 min/day for 10 days ( a ). MDA-MB-231 cells were seeded in 6-well plates using four wells/plate. After 24 h, a transwell insert was inserted into each well and Raw264.7 cells were seeded in the upper compartment. Two wells were placed on swich-on transducer receiving LIPUS stimuli to obtain LIPUS_CC group, the other two were placed on swich-off transducer and used as untreated to obtain Untreated_CC group. LIPUS treatment was performed for 20 min/day for 10 days ( b )

    Article Snippet: After 24 h, a transwell insert (pore size 0.4 μm) (Millipore, Cork, Ireland) was inserted into each well and Raw264.7 cells were seeded in the upper compartment at a density of 15,000 cells/well.

    Techniques: Multiple Displacement Amplification, Laser Capture Microdissection

    Four conditions of bacterial Transwell culture. Bottom well contains bacterial sample, Transwell insert contains antibiotics and NO releasing biomimetic nanomatrix gel. (A) NO (-) antibiotics (-). (B) NO (+) antibiotics (-). (C) NO (-) antibiotics (+). (D) NO (+) antibiotics (+).

    Journal: PLoS ONE

    Article Title: Effects of the nitric oxide releasing biomimetic nanomatrix gel on pulp-dentin regeneration: Pilot study

    doi: 10.1371/journal.pone.0205534

    Figure Lengend Snippet: Four conditions of bacterial Transwell culture. Bottom well contains bacterial sample, Transwell insert contains antibiotics and NO releasing biomimetic nanomatrix gel. (A) NO (-) antibiotics (-). (B) NO (+) antibiotics (-). (C) NO (-) antibiotics (+). (D) NO (+) antibiotics (+).

    Article Snippet: The four different conditions ( ) of prepared antibiotics and NO releasing biomimetic nanomatrix gel were placed in Transwell inserts, with the sub-cultured bacteria and media distributed to the wells as described above.

    Techniques:

    Effects of miR-339-5p on breast cancer cells . A . Regulation of breast cancer cell growth by miR-339-5p. MCF-7 cells were grown and transiently transfected with miR-339-5p antisense oligonucleotide or scrambled sequence oligonucleotide as negative control for 4 days, and cell viability was analyzed using a kit. The experiments were in triplicate and repeated twice. B . Wound-healing assay. MCF-7 cells were grown and transiently transfected with miR-339-5p antisense oligonucleotides or scrambled sequence oligonucleotide as negative control for 3 days and subjected to the wound-healing assay. Magnification: ×100. C . Transwell migration and Matrigel invasion assays. MCF-7 cells were grown and transiently transfected with miR-339-5p antisense oligonucleotides or scrambled sequence oligonucleotide as negative control for 2 days and subjected to migration and invasion assays. Representative photographs (upper) and quantification (lower) are shown. Magnification: ×200.

    Journal: BMC Cancer

    Article Title: MiR-339-5p inhibits breast cancer cell migration and invasion in vitro and may be a potential biomarker for breast cancer prognosis

    doi: 10.1186/1471-2407-10-542

    Figure Lengend Snippet: Effects of miR-339-5p on breast cancer cells . A . Regulation of breast cancer cell growth by miR-339-5p. MCF-7 cells were grown and transiently transfected with miR-339-5p antisense oligonucleotide or scrambled sequence oligonucleotide as negative control for 4 days, and cell viability was analyzed using a kit. The experiments were in triplicate and repeated twice. B . Wound-healing assay. MCF-7 cells were grown and transiently transfected with miR-339-5p antisense oligonucleotides or scrambled sequence oligonucleotide as negative control for 3 days and subjected to the wound-healing assay. Magnification: ×100. C . Transwell migration and Matrigel invasion assays. MCF-7 cells were grown and transiently transfected with miR-339-5p antisense oligonucleotides or scrambled sequence oligonucleotide as negative control for 2 days and subjected to migration and invasion assays. Representative photographs (upper) and quantification (lower) are shown. Magnification: ×200.

    Article Snippet: Cell migration and invasion assay We used a Transwell insert (24-well insert, pore size 8 μm; Corning, Inc., Corning, NY) to determine the effect of miR-339-5p on breast cancer cell migration and invasion in vitro .

    Techniques: Transfection, Sequencing, Negative Control, Wound Healing Assay, Migration

    MiR-652-3p promotes the migration and invasion of NSCLC cells Twenty-four hours after miRNA transfection, serum was removed and cells were cultured in serum-free medium for 12 hours. Cells were plated into the upper chamber of 24-well transwell inserts uncoated or coated with Matrigel for migration or invasion assay for 12–24 hours. The cells on the lower side of the chamber without Matrigel ( A ) or coated with Matrigel ( C ) were fixed, stained and counted in three different areas at 100-fold magnification. The cell numbers of migration ( B ) and invasion ( D ) were compared between goups. Column, mean of three independent experiments, bar, S.E.M, each experiment performed in triplicates, t test was uesd. ** p

    Journal: Oncotarget

    Article Title: MiR-652-3p is upregulated in non-small cell lung cancer and promotes proliferation and metastasis by directly targeting Lgl1

    doi: 10.18632/oncotarget.7697

    Figure Lengend Snippet: MiR-652-3p promotes the migration and invasion of NSCLC cells Twenty-four hours after miRNA transfection, serum was removed and cells were cultured in serum-free medium for 12 hours. Cells were plated into the upper chamber of 24-well transwell inserts uncoated or coated with Matrigel for migration or invasion assay for 12–24 hours. The cells on the lower side of the chamber without Matrigel ( A ) or coated with Matrigel ( C ) were fixed, stained and counted in three different areas at 100-fold magnification. The cell numbers of migration ( B ) and invasion ( D ) were compared between goups. Column, mean of three independent experiments, bar, S.E.M, each experiment performed in triplicates, t test was uesd. ** p

    Article Snippet: Cells (5 × 104 or 1 × 105 ) in serum-free RPMI 1640 medium were plated into the upper chamber of 24-well transwell inserts (Corning, 8.0 μm pores) that were either uncoated or coated with Matrigel (BD Biosciences) for migration or invasion assay, the cells were then allowed to translocate toward medium containing 20% FBS for 12–24 hours.

    Techniques: Migration, Transfection, Cell Culture, Invasion Assay, Staining

    LCN2 induces cell migration of monocytic cells. Migration of murine monocyte/macrophage-like J774A.1 cells in response to LCN2 (0.5 μg/mL) was carried out in transwell cell culture inserts for 24 hours. Medium with 10% FCS was used as positive control. Migration is depicted as induction over unstimulated control. *P

    Journal: PLoS ONE

    Article Title: Lipocalin (LCN) 2 Mediates Pro-Atherosclerotic Processes and Is Elevated in Patients with Coronary Artery Disease

    doi: 10.1371/journal.pone.0137924

    Figure Lengend Snippet: LCN2 induces cell migration of monocytic cells. Migration of murine monocyte/macrophage-like J774A.1 cells in response to LCN2 (0.5 μg/mL) was carried out in transwell cell culture inserts for 24 hours. Medium with 10% FCS was used as positive control. Migration is depicted as induction over unstimulated control. *P

    Article Snippet: Migration Evaluation of cell migration of J774A.1 cells was performed using transwell cell culture inserts (8 μm pore size; Corning Life Sciences, Amsterdam, The Netherlands).

    Techniques: Migration, Cell Culture, Positive Control

    MEOX2 alters ECFC migration. (A) Representative images of transwell migration assay with transduced ECFCs (320 × magnification). Scale bar represents 30 μm. MEOX2 was overexpressed in ECFCs from control pregnancies using lentiviral techniques and cells were subjected to a transwell migration assay. Cells were stained with 1% crystal violet. (B) Quantitation of transwell migration assay with transduced ECFCs, n = 5 transductions, * P

    Journal: Journal of cellular physiology

    Article Title: Mesenchyme Homeobox 2 Enhances Migration of Endothelial Colony Forming Cells Exposed to Intrauterine Diabetes Mellitus

    doi: 10.1002/jcp.25734

    Figure Lengend Snippet: MEOX2 alters ECFC migration. (A) Representative images of transwell migration assay with transduced ECFCs (320 × magnification). Scale bar represents 30 μm. MEOX2 was overexpressed in ECFCs from control pregnancies using lentiviral techniques and cells were subjected to a transwell migration assay. Cells were stained with 1% crystal violet. (B) Quantitation of transwell migration assay with transduced ECFCs, n = 5 transductions, * P

    Article Snippet: Cells were plated on type I collagen-coated 8.0 mm pore size transwell inserts (Corning).

    Techniques: Migration, Transwell Migration Assay, Staining, Quantitation Assay

    Effects of miR-203 expression on proliferation, migration and invasion of renal cancer cells. A . Over-expression of miR-203 by transfection with miR-203 mimics in 786-O cells. B . Down-regulation of miR-203 by transfection with miR-203 inhibitor in 786-O cells. C,D . CCK-8 assay was performed to analyze the effect of miR-203 on cell proliferation of 786-O cells. E-H . Transwell migration and invasion assays were utilized to analyze the effect of miR-203 on cell migration (E,F) and invasion (G,H) of 786-O cells.* P

    Journal: Diagnostic Pathology

    Article Title: miR-203 inhibition of renal cancer cell proliferation, migration and invasion by targeting of FGF2

    doi: 10.1186/s13000-015-0255-7

    Figure Lengend Snippet: Effects of miR-203 expression on proliferation, migration and invasion of renal cancer cells. A . Over-expression of miR-203 by transfection with miR-203 mimics in 786-O cells. B . Down-regulation of miR-203 by transfection with miR-203 inhibitor in 786-O cells. C,D . CCK-8 assay was performed to analyze the effect of miR-203 on cell proliferation of 786-O cells. E-H . Transwell migration and invasion assays were utilized to analyze the effect of miR-203 on cell migration (E,F) and invasion (G,H) of 786-O cells.* P

    Article Snippet: Transwell migration and invasion assays The migration and invasion assays were carried out using Transwell insert chambers (Corning).

    Techniques: Expressing, Migration, Over Expression, Transfection, CCK-8 Assay

    Effect of TxB on the subcellular distribution of NHE3. Confluent LLC-PK 1 cells stably expressing NHE3′ 38HA3 were either left untreated (control), or treated with TxB. (A and B) Top (x/y axis) view of cells grown on coverslips, immunostained for surface NHE3 (exofacial epitope in intact cells). (A′ and B′) perpendicular (z axis) sections of control (A′) and toxin-treated (B′) cells grown on permeable Transwell filters and stained for surface NHE3 as in A and B. (D and E) Top (x/y axis) view of cells grown on coverslips, immunostained for total NHE3 (permeabilized cells). (D′ and E′) Perpendicular (z axis) sections of control (D′) and toxin-treated (E′) cells grown on permeable Transwell filters and stained for total NHE3 as in D and E. Representative of 4–8 similar experiments of each type. Bar, 15 μm. (C) Quantification of surface NHE3 in control and TxB-treated cells. The density of surface exchangers was measured by radiolabeling using anti-HA primary and 125 I-labeled secondary antibodies, as detailed in materials and methods , and is expressed relative to controls. Data are means ± SE of six determinations from two experiments, each with duplicate determinations. (F) Detection of total NHE3 content by immunoblotting. The cells were treated with or without TxB, as above and whole-cell lysates were solubilized in Laemmli sample buffer and used for SDS-PAGE and immunoblotting using anti-HA antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence and the radiogram shown is representative of three similar experiments.

    Journal: The Journal of General Physiology

    Article Title: Inhibition and Redistribution of NHE3, the Apical Na+/H+ Exchanger, by Clostridium difficile Toxin B

    doi: 10.1085/jgp.200308979

    Figure Lengend Snippet: Effect of TxB on the subcellular distribution of NHE3. Confluent LLC-PK 1 cells stably expressing NHE3′ 38HA3 were either left untreated (control), or treated with TxB. (A and B) Top (x/y axis) view of cells grown on coverslips, immunostained for surface NHE3 (exofacial epitope in intact cells). (A′ and B′) perpendicular (z axis) sections of control (A′) and toxin-treated (B′) cells grown on permeable Transwell filters and stained for surface NHE3 as in A and B. (D and E) Top (x/y axis) view of cells grown on coverslips, immunostained for total NHE3 (permeabilized cells). (D′ and E′) Perpendicular (z axis) sections of control (D′) and toxin-treated (E′) cells grown on permeable Transwell filters and stained for total NHE3 as in D and E. Representative of 4–8 similar experiments of each type. Bar, 15 μm. (C) Quantification of surface NHE3 in control and TxB-treated cells. The density of surface exchangers was measured by radiolabeling using anti-HA primary and 125 I-labeled secondary antibodies, as detailed in materials and methods , and is expressed relative to controls. Data are means ± SE of six determinations from two experiments, each with duplicate determinations. (F) Detection of total NHE3 content by immunoblotting. The cells were treated with or without TxB, as above and whole-cell lysates were solubilized in Laemmli sample buffer and used for SDS-PAGE and immunoblotting using anti-HA antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence and the radiogram shown is representative of three similar experiments.

    Article Snippet: Where indicated, LLC-PK1 cells were grown on Costar Transwell inserts (0.4 μm pore size; Corning, Inc.) in 12-well culture plates.

    Techniques: Stable Transfection, Expressing, Staining, Radioactivity, Labeling, SDS Page

    LEF1 is a facilitator of OCT4 in regulating the invasion and migration of Eca109 cells (scale bar = 50 μm). A and B, In the transwell assay, the p‐LEF1 group showed a higher invasion ability. Quantified data are presented as the number of invaded cells per HPF (n = 3 for each cell group), two‐tailed ANOVA was used, and LSD test was significant between shOCT4 and p‐LEF1 groups. A and C, In the transwell assay, the p‐LEF1 group showed a higher migration ability. Quantified data are presented as the number of migrated cells per HPF (n = 3 for each cell group), two‐tailed ANOVA was used, and LSD test was significant between shOCT4 and p‐LEF1 groups. D, Wound‐healing assays showed greater migration ability in the p‐LEF1 group. E and F, Quantified data are presented as the wound area and the area covered by migrating cells per HPF (n = 3 for each cell group), two‐tailed ANOVA was used, and LSD test was significant between shOCT4 and p‐LEF1 groups. All experiments were performed in triplicate

    Journal: Cancer Medicine

    Article Title: Prognostic value of association of OCT4 with LEF1 expression in esophageal squamous cell carcinoma and their impact on epithelial‐mesenchymal transition, invasion, and migration, et al. Prognostic value of association of OCT4 with LEF1 expression in esophageal squamous cell carcinoma and their impact on epithelial‐mesenchymal transition, invasion, and migration

    doi: 10.1002/cam4.1641

    Figure Lengend Snippet: LEF1 is a facilitator of OCT4 in regulating the invasion and migration of Eca109 cells (scale bar = 50 μm). A and B, In the transwell assay, the p‐LEF1 group showed a higher invasion ability. Quantified data are presented as the number of invaded cells per HPF (n = 3 for each cell group), two‐tailed ANOVA was used, and LSD test was significant between shOCT4 and p‐LEF1 groups. A and C, In the transwell assay, the p‐LEF1 group showed a higher migration ability. Quantified data are presented as the number of migrated cells per HPF (n = 3 for each cell group), two‐tailed ANOVA was used, and LSD test was significant between shOCT4 and p‐LEF1 groups. D, Wound‐healing assays showed greater migration ability in the p‐LEF1 group. E and F, Quantified data are presented as the wound area and the area covered by migrating cells per HPF (n = 3 for each cell group), two‐tailed ANOVA was used, and LSD test was significant between shOCT4 and p‐LEF1 groups. All experiments were performed in triplicate

    Article Snippet: The transwell filter inserts were coated with Matrigel (Corning).

    Techniques: Migration, Transwell Assay, Two Tailed Test

    MiR-363 reduces the migratory ability of HPV-negative SCCHN cells a JHU028 cells transfected with premiR-363 exhibited decreased Transwell migration at 1, 3, and 5 h as compared to cells transfected with a negative premiR control. b Quantification of cell migration data from A. * p

    Journal: BMC Cancer

    Article Title: MicroRNA-363 targets myosin 1B to reduce cellular migration in head and neck cancer

    doi: 10.1186/s12885-015-1888-3

    Figure Lengend Snippet: MiR-363 reduces the migratory ability of HPV-negative SCCHN cells a JHU028 cells transfected with premiR-363 exhibited decreased Transwell migration at 1, 3, and 5 h as compared to cells transfected with a negative premiR control. b Quantification of cell migration data from A. * p

    Article Snippet: Forty-eight hours after transfection, cells were harvested and reseeded into 24-well 8 μM pore Transwell inserts (Corning) in serum-free media.

    Techniques: Transfection, Migration

    MELK knockdown reduces migration and invasion in vitro . (A) Wound healing assays revealed the reduced migratory potential of cells following knockdown of MELK. (B) Transwell assays revealed reduced migratory and invasive potential of MNNG/HOS cells following knockdown of MELK. (C) Migration (wound healing assay) and (D) migration and invasion (Transwell assays) were reduced in osteosarcoma cells following treatment with OTSSP167. **P

    Journal: Oncology Reports

    Article Title: Maternal embryonic leucine zipper kinase serves as a poor prognosis marker and therapeutic target in osteosarcoma

    doi: 10.3892/or.2020.7686

    Figure Lengend Snippet: MELK knockdown reduces migration and invasion in vitro . (A) Wound healing assays revealed the reduced migratory potential of cells following knockdown of MELK. (B) Transwell assays revealed reduced migratory and invasive potential of MNNG/HOS cells following knockdown of MELK. (C) Migration (wound healing assay) and (D) migration and invasion (Transwell assays) were reduced in osteosarcoma cells following treatment with OTSSP167. **P

    Article Snippet: Osteosarcoma cells (1-2×105 cells) were seeded in the upper chambers of the Transwell inserts (24-wells, 8 µm pores; BD Falcon) with 200 µl serum-free media.

    Techniques: Migration, In Vitro, Wound Healing Assay

    A fixed number of cells were plated onto the upper part of the matrigel-coated Transwell chamber. After incubation for 24 h, invasive cells were counted at the lower part of Transwell

    Journal: Advanced Biomedical Research

    Article Title: Differential Immune Reactivity Pattern of SW48 and SW1116 Colorectal Cancer Cell Lines with Colorectal Cancer Patients Sera

    doi: 10.4103/2277-9175.199264

    Figure Lengend Snippet: A fixed number of cells were plated onto the upper part of the matrigel-coated Transwell chamber. After incubation for 24 h, invasive cells were counted at the lower part of Transwell

    Article Snippet: Invasion assays Invasion assay was carried out using a 24 well Transwell insert (8 µm pore filters, BD Bioscience, Bedford, MA, USA) with the matrigel-coated membrane.

    Techniques: Incubation

    Requirement of NRP1 and p130 Cas for HGF, PDGF, and VEGF stimulation of chemotactic migration of U87MG cells and HUVECs. (A) U87MG cells were transfected with 25 nM siNRP1, 25 nM sip130 Cas , or 25 nM siScr for 48 h. Cell migration in response to 25 ng/ml HGF or 50 ng/ml PDGF-BB was then determined in a Transwell migration assay as described in Materials and Methods. (B) HUVECs were transfected with 200 nM siNRP1, 200 nM sip130 Cas , or 200 nM siScr for 48 h, and cell migration in response to 25 ng/ml VEGF-A was determined as described for panel A. Values ( n ≥ 3) are means ± SEM, expressed as the number of cells migrating per field; *, P

    Journal: Molecular and Cellular Biology

    Article Title: Neuropilin-1 Signaling through p130Cas Tyrosine Phosphorylation Is Essential for Growth Factor-Dependent Migration of Glioma and Endothelial Cells ▿

    doi: 10.1128/MCB.00903-10

    Figure Lengend Snippet: Requirement of NRP1 and p130 Cas for HGF, PDGF, and VEGF stimulation of chemotactic migration of U87MG cells and HUVECs. (A) U87MG cells were transfected with 25 nM siNRP1, 25 nM sip130 Cas , or 25 nM siScr for 48 h. Cell migration in response to 25 ng/ml HGF or 50 ng/ml PDGF-BB was then determined in a Transwell migration assay as described in Materials and Methods. (B) HUVECs were transfected with 200 nM siNRP1, 200 nM sip130 Cas , or 200 nM siScr for 48 h, and cell migration in response to 25 ng/ml VEGF-A was determined as described for panel A. Values ( n ≥ 3) are means ± SEM, expressed as the number of cells migrating per field; *, P

    Article Snippet: Transwell cell culture inserts made of transparent, low-pore-density polyethylene terephthalate with an 8-μm pore size (Falcon; BD Biosciences, Oxford, United Kingdom) were inserted into a 24-well plate.

    Techniques: Migration, Transfection, Transwell Migration Assay

    Xaliproden protects against the loss of tight junctions in the presence of paraquat. ARPE-19 cells were seeded in 24 Transwell plates and grown in low serum medium for 2 weeks. The cells were then treated with paraquat (300 µM), xaliproden (20 µM), or a combination of the two for 24 h before the transepithelial resistance (TEER) was measured in a volt/ohm meter. Bars represent the average electrical resistance from triplicates ± standard deviation (SD). *, p = 0.006 between paraquat, and paraquat + xaliproden, as determined with one-way ANOVA. Par = paraquat; Xali = xaliproden.

    Journal: Molecular Vision

    Article Title: Repurposing an orally available drug for the treatment of geographic atrophy

    doi:

    Figure Lengend Snippet: Xaliproden protects against the loss of tight junctions in the presence of paraquat. ARPE-19 cells were seeded in 24 Transwell plates and grown in low serum medium for 2 weeks. The cells were then treated with paraquat (300 µM), xaliproden (20 µM), or a combination of the two for 24 h before the transepithelial resistance (TEER) was measured in a volt/ohm meter. Bars represent the average electrical resistance from triplicates ± standard deviation (SD). *, p = 0.006 between paraquat, and paraquat + xaliproden, as determined with one-way ANOVA. Par = paraquat; Xali = xaliproden.

    Article Snippet: Measurement of transepithelial electrical resistance We plated the ARPE-19 cells on Transwell inserts (33.6 mm2 , pore size 0.4 µm; Greiner Bio-One, Monroe, NC) in DMEM/F12 including 1% FBS and allowed the cells to grow in 24-well plates for 4 weeks.

    Techniques: Standard Deviation

    Xaliproden protects against the loss of tight junctions in the presence of TNF-α. ARPE-19 cells were seeded in 24 Transwell plates and grown in low serum medium for 4 weeks. The cells were then treated with tumor necrosis factor-α (TNF-α; 10 ng/ml), xaliproden (20 M), or a combination of the two or left untreated for 48 h before the transepithelial electrical resistance (TEER) was measured using a volt/ohm meter. Bars represent the average electrical resistance from triplicates ± standard deviation (SD). *, p = 0.005, as determined with one-way ANOVA. xal = xaliproden.

    Journal: Molecular Vision

    Article Title: Repurposing an orally available drug for the treatment of geographic atrophy

    doi:

    Figure Lengend Snippet: Xaliproden protects against the loss of tight junctions in the presence of TNF-α. ARPE-19 cells were seeded in 24 Transwell plates and grown in low serum medium for 4 weeks. The cells were then treated with tumor necrosis factor-α (TNF-α; 10 ng/ml), xaliproden (20 M), or a combination of the two or left untreated for 48 h before the transepithelial electrical resistance (TEER) was measured using a volt/ohm meter. Bars represent the average electrical resistance from triplicates ± standard deviation (SD). *, p = 0.005, as determined with one-way ANOVA. xal = xaliproden.

    Article Snippet: Measurement of transepithelial electrical resistance We plated the ARPE-19 cells on Transwell inserts (33.6 mm2 , pore size 0.4 µm; Greiner Bio-One, Monroe, NC) in DMEM/F12 including 1% FBS and allowed the cells to grow in 24-well plates for 4 weeks.

    Techniques: Standard Deviation

    GBM-SKH cells were stably expressed without or with the control vector (pcDNA3.1) or with wild-type OPN (pDNA3.1/OPN). (A) MMP-2, OPN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression were analyzed by RT–PCR. (B) Cell lysates were harvested and immunoblotted with antibodies specific for OPN, MMP-2, and α-tubulin. These experiments were repeated, and the intensities of bands were quantitated and expressed as ratios to mock control cells. Results shown in the lower panel of (A) and (B) are the mean ± SE of three independent experiments. (C) Media were then collected for the zymographic assay of MMP-2 activity. In the lower panel, MMP-2 activity was quantitated by scanning the photographic negatives on a gel analysis system. (D) For the in vitro invasion assay, cells were seeded in equal amounts of experimental cells in the upper part of a transwell chamber separated by a Matrigel-coated membrane. Data represent the mean ± SE of three independent experiments. (E) Cells were seeded in culture plates in DMEM media supplemented with 10% heat-inactivated FCS. Viable cell numbers were counted at 24, 48, and 72 hours. Data represent the mean ± SE of three independent experiments. (F) Histopathological characteristics (H E) and tumor volumes of animals implanted with GBM/pcDNA or GBM/OPN were evaluated. Tumor sizes were measured through their largest diameter (upper panel), and the lower panel shows the mean ± SE of three independent experiments. * P

    Journal: Neuro-Oncology

    Article Title: Osteopontin regulates human glioma cell invasiveness and tumor growth in mice

    doi: 10.1093/neuonc/nop013

    Figure Lengend Snippet: GBM-SKH cells were stably expressed without or with the control vector (pcDNA3.1) or with wild-type OPN (pDNA3.1/OPN). (A) MMP-2, OPN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression were analyzed by RT–PCR. (B) Cell lysates were harvested and immunoblotted with antibodies specific for OPN, MMP-2, and α-tubulin. These experiments were repeated, and the intensities of bands were quantitated and expressed as ratios to mock control cells. Results shown in the lower panel of (A) and (B) are the mean ± SE of three independent experiments. (C) Media were then collected for the zymographic assay of MMP-2 activity. In the lower panel, MMP-2 activity was quantitated by scanning the photographic negatives on a gel analysis system. (D) For the in vitro invasion assay, cells were seeded in equal amounts of experimental cells in the upper part of a transwell chamber separated by a Matrigel-coated membrane. Data represent the mean ± SE of three independent experiments. (E) Cells were seeded in culture plates in DMEM media supplemented with 10% heat-inactivated FCS. Viable cell numbers were counted at 24, 48, and 72 hours. Data represent the mean ± SE of three independent experiments. (F) Histopathological characteristics (H E) and tumor volumes of animals implanted with GBM/pcDNA or GBM/OPN were evaluated. Tumor sizes were measured through their largest diameter (upper panel), and the lower panel shows the mean ± SE of three independent experiments. * P

    Article Snippet: In Vitro Matrigel Invasion Assay (Boyden Chamber) In vitro invasion of glioma cells was measured using Matrigel-coated transwell inserts (BD Biosciences, Mansfield, Massachusetts).

    Techniques: Stable Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Activity Assay, In Vitro, Invasion Assay

    U87MG cells were either stably transfected with an OPN shRNA plasmid or with a plKo shRNA vector as mock control. (A) MMP-2, OPN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expressions were analyzed by RT–PCR. (B) Cell lysates were harvested and immunoblotted with antibodies specific for OPN, MMP-2, and α-tubulin. These experiments were repeated, and the intensities of bands were quantitated and expressed as ratios to mock control cells. Results shown in the lower panel of (A) and (B) are the mean ± SE of three independent experiments. (C) Media were collected for the zymographic assay of MMP-2 activity. In the lower panel, MMP-2 activity was quantitated by scanning the photographic negatives on a gel analysis system. (D) For the in vitro invasion assay, cells were seeded in equal amounts cells in the upper part of transwell chamber separated by a Matrigel-coated membrane. Migrated cells on the bottom of the membrane were counted. Data represent the mean ± SE of three independent experiments. (E) Cells were seeded in culture plates in DMEM media supplemented with 10% heat-inactivated FCS. Viable cell numbers were counted. Data represent the mean ± SE of three independent experiments. (F) Histopathological characteristics (H E) and tumor volumes of animals implanted with U87/plKo or U87MG/shOPN. Tumor sizes were measured through their largest diameter (upper panel), and the lower panel shows the mean ± SE of three independent experiments. * P

    Journal: Neuro-Oncology

    Article Title: Osteopontin regulates human glioma cell invasiveness and tumor growth in mice

    doi: 10.1093/neuonc/nop013

    Figure Lengend Snippet: U87MG cells were either stably transfected with an OPN shRNA plasmid or with a plKo shRNA vector as mock control. (A) MMP-2, OPN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expressions were analyzed by RT–PCR. (B) Cell lysates were harvested and immunoblotted with antibodies specific for OPN, MMP-2, and α-tubulin. These experiments were repeated, and the intensities of bands were quantitated and expressed as ratios to mock control cells. Results shown in the lower panel of (A) and (B) are the mean ± SE of three independent experiments. (C) Media were collected for the zymographic assay of MMP-2 activity. In the lower panel, MMP-2 activity was quantitated by scanning the photographic negatives on a gel analysis system. (D) For the in vitro invasion assay, cells were seeded in equal amounts cells in the upper part of transwell chamber separated by a Matrigel-coated membrane. Migrated cells on the bottom of the membrane were counted. Data represent the mean ± SE of three independent experiments. (E) Cells were seeded in culture plates in DMEM media supplemented with 10% heat-inactivated FCS. Viable cell numbers were counted. Data represent the mean ± SE of three independent experiments. (F) Histopathological characteristics (H E) and tumor volumes of animals implanted with U87/plKo or U87MG/shOPN. Tumor sizes were measured through their largest diameter (upper panel), and the lower panel shows the mean ± SE of three independent experiments. * P

    Article Snippet: In Vitro Matrigel Invasion Assay (Boyden Chamber) In vitro invasion of glioma cells was measured using Matrigel-coated transwell inserts (BD Biosciences, Mansfield, Massachusetts).

    Techniques: Stable Transfection, Transfection, shRNA, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Activity Assay, In Vitro, Invasion Assay

    Cell lines derived from human gliomas exhibit different growth rates, invasiveness, and intravasation. (A) For the in vitro invasion assay, equal numbers of glioma cells were seeded in the upper part of a transwell coated with Matrigel. After 24 hours, cells on the bottom side of filter were fixed, stained, and counted. Data represent the mean ± SE of three independent experiments. (B) Viable cell numbers were counted at 24, 48, and 72 hours. Data represent the mean ± SE of three independent experiments. (C) A chicken embryo CAM model was used for the in vivo intravasation assay. Intravasation was demonstrated by amplification of the human Alu sequence from DNA extracted from the isolated CAMs. P, positive control; N, negative control. (D) Medium was collected and MMP-2 gelatinolytic activities were analyzed by zymography. In the lower panel, the MMP-2 activity was quantitated by scanning the photographic negatives on a gel analysis system. (E) MMP-2, OPN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expressions were analyzed by RT–PCR. (F) Cell lysates were harvested and immunoblotted with antibodies specific for MMP-2, OPN, and α-tubulin. The upper panels of (E) and (F) are representative data, these experiments were repeated, and the intensities of bands were quantitated and expressed as ratios to equal loading controls. Results shown in the lower panels are the mean ± SE of three independent experiments. * P

    Journal: Neuro-Oncology

    Article Title: Osteopontin regulates human glioma cell invasiveness and tumor growth in mice

    doi: 10.1093/neuonc/nop013

    Figure Lengend Snippet: Cell lines derived from human gliomas exhibit different growth rates, invasiveness, and intravasation. (A) For the in vitro invasion assay, equal numbers of glioma cells were seeded in the upper part of a transwell coated with Matrigel. After 24 hours, cells on the bottom side of filter were fixed, stained, and counted. Data represent the mean ± SE of three independent experiments. (B) Viable cell numbers were counted at 24, 48, and 72 hours. Data represent the mean ± SE of three independent experiments. (C) A chicken embryo CAM model was used for the in vivo intravasation assay. Intravasation was demonstrated by amplification of the human Alu sequence from DNA extracted from the isolated CAMs. P, positive control; N, negative control. (D) Medium was collected and MMP-2 gelatinolytic activities were analyzed by zymography. In the lower panel, the MMP-2 activity was quantitated by scanning the photographic negatives on a gel analysis system. (E) MMP-2, OPN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expressions were analyzed by RT–PCR. (F) Cell lysates were harvested and immunoblotted with antibodies specific for MMP-2, OPN, and α-tubulin. The upper panels of (E) and (F) are representative data, these experiments were repeated, and the intensities of bands were quantitated and expressed as ratios to equal loading controls. Results shown in the lower panels are the mean ± SE of three independent experiments. * P

    Article Snippet: In Vitro Matrigel Invasion Assay (Boyden Chamber) In vitro invasion of glioma cells was measured using Matrigel-coated transwell inserts (BD Biosciences, Mansfield, Massachusetts).

    Techniques: Derivative Assay, In Vitro, Invasion Assay, Staining, Chick Chorioallantoic Membrane Assay, In Vivo, Amplification, Sequencing, Isolation, Positive Control, Negative Control, Zymography, Activity Assay, Reverse Transcription Polymerase Chain Reaction

    Human glioma cell lines were treated with different concentrations of 5-aza-dC. (A) Media were then collected for a zymographic assay for MMP-2 activity. In the lower panel, effects of 5-aza-dC on MMP-2 secretion in various cells were quantitated by scanning the photographic negatives on a gel analysis system and are expressed as a percent of basal level for each cell line. (B) Cell viability was analyzed by the MTT assay after incubation of U87MG cells with 5-aza-dC for 24 hours. (C) Cell lysates were harvested and immunoblotted with antibodies specific for OPN, MMP-2, GFAP, vimentin, and α-tubulin. (D) For the in vitro invasion assay, equal numbers of glioma cells were seeded in the upper part of a transwell coated with Matrigel in the presence of different concentrations of 5-aza-dC. After 24 hours, cells on the bottom side of the filter were fixed, stained, and counted. Data represent the mean ± SE of three independent experiments. (E) An intravasation assay was carried out. PCR amplification for Alu sequence is shown. P, positive control; N, negative control. (F) A trypan blue exclusion assay was carried out after treating cells with different concentrations of 5-aza-dC in 5% FBS medium for different time periods. Data represent the mean ± SE of three independent experiments. (G) Measurement of implanted tumor sizes in mice without or with 5-aza-dC administration. Tumor sizes were measured through their largest diameter (upper panel), and the lower panel shows the mean ± SE of three independent experiments. * P

    Journal: Neuro-Oncology

    Article Title: Osteopontin regulates human glioma cell invasiveness and tumor growth in mice

    doi: 10.1093/neuonc/nop013

    Figure Lengend Snippet: Human glioma cell lines were treated with different concentrations of 5-aza-dC. (A) Media were then collected for a zymographic assay for MMP-2 activity. In the lower panel, effects of 5-aza-dC on MMP-2 secretion in various cells were quantitated by scanning the photographic negatives on a gel analysis system and are expressed as a percent of basal level for each cell line. (B) Cell viability was analyzed by the MTT assay after incubation of U87MG cells with 5-aza-dC for 24 hours. (C) Cell lysates were harvested and immunoblotted with antibodies specific for OPN, MMP-2, GFAP, vimentin, and α-tubulin. (D) For the in vitro invasion assay, equal numbers of glioma cells were seeded in the upper part of a transwell coated with Matrigel in the presence of different concentrations of 5-aza-dC. After 24 hours, cells on the bottom side of the filter were fixed, stained, and counted. Data represent the mean ± SE of three independent experiments. (E) An intravasation assay was carried out. PCR amplification for Alu sequence is shown. P, positive control; N, negative control. (F) A trypan blue exclusion assay was carried out after treating cells with different concentrations of 5-aza-dC in 5% FBS medium for different time periods. Data represent the mean ± SE of three independent experiments. (G) Measurement of implanted tumor sizes in mice without or with 5-aza-dC administration. Tumor sizes were measured through their largest diameter (upper panel), and the lower panel shows the mean ± SE of three independent experiments. * P

    Article Snippet: In Vitro Matrigel Invasion Assay (Boyden Chamber) In vitro invasion of glioma cells was measured using Matrigel-coated transwell inserts (BD Biosciences, Mansfield, Massachusetts).

    Techniques: Activity Assay, MTT Assay, Incubation, In Vitro, Invasion Assay, Staining, Polymerase Chain Reaction, Amplification, Sequencing, Positive Control, Negative Control, Trypan Blue Exclusion Assay, Mouse Assay

    Effect of the RBP-J deletion on the in vitro cultured EPC. (A) The expression of CXCR4 on the cultured EPC was accessed by FACS analysis. (B, C) In vitro migration assay. Transwell culture was set up, with EPC from the RBP-J-deleted and the control mice in the upper chamber, and SDF-1α in the lower chamber. A photograph of EPC in the lower chamber (B) was taken, and cells in the lower chamber (C) were counted 18 h after the starting of the culture (bars = mean±SD; n = 4; ** P

    Journal: PLoS ONE

    Article Title: Notch-RBP-J Signaling Regulates the Mobilization and Function of Endothelial Progenitor Cells by Dynamic Modulation of CXCR4 Expression in Mice

    doi: 10.1371/journal.pone.0007572

    Figure Lengend Snippet: Effect of the RBP-J deletion on the in vitro cultured EPC. (A) The expression of CXCR4 on the cultured EPC was accessed by FACS analysis. (B, C) In vitro migration assay. Transwell culture was set up, with EPC from the RBP-J-deleted and the control mice in the upper chamber, and SDF-1α in the lower chamber. A photograph of EPC in the lower chamber (B) was taken, and cells in the lower chamber (C) were counted 18 h after the starting of the culture (bars = mean±SD; n = 4; ** P

    Article Snippet: Migration assay Chemotaxis experiments were performed in polycarbonate transwell inserts (5-µm pore, Corning Costar Corp.).

    Techniques: In Vitro, Cell Culture, Expressing, FACS, Migration, Mouse Assay

    FOXK1 facilitates glioblastoma cell metastasis. (A) Following knockdown or ectopic expression of FOXK1 in LN18 cells, the protein or mRNA levels of FOXK1 were detected by western blotting or reverse transcription-quantitative polymerase chain reaction, respectively. (B and C) Transwell migration assay of LN18 cells after (B) ectopic expression and (C) knockdown of FOXK1 (48 h post-transfection). (D) Anchorage-independent cell growth assay. LN18 cells were transfected with FOXK1 siRNA and then placed on soft agar; the average number of colonies formed was determined. All experiments were performed at least three times. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: FOXK1 promotes glioblastoma proliferation and metastasis through activation of Snail transcription

    doi: 10.3892/etm.2018.5732

    Figure Lengend Snippet: FOXK1 facilitates glioblastoma cell metastasis. (A) Following knockdown or ectopic expression of FOXK1 in LN18 cells, the protein or mRNA levels of FOXK1 were detected by western blotting or reverse transcription-quantitative polymerase chain reaction, respectively. (B and C) Transwell migration assay of LN18 cells after (B) ectopic expression and (C) knockdown of FOXK1 (48 h post-transfection). (D) Anchorage-independent cell growth assay. LN18 cells were transfected with FOXK1 siRNA and then placed on soft agar; the average number of colonies formed was determined. All experiments were performed at least three times. *P

    Article Snippet: LN18 cells with knockdown or ectopic expression of FOXK1 (transfection time, 48 h) were seeded onto Transwell membrane inserts (Corning, Inc., Corning, NY, USA) at 5×103 cells per well in DMEM without serum.

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Transwell Migration Assay, Transfection, Growth Assay

    GRAMD1B negates JAK/STAT signaling. ( a ) Effects of GRAMD1B inhibition on JAK/STAT signaling. Increased p-JAK2 and p-STAT3 levels are detected on Gramd1b . ( b ) Treatment with AG490 almost completely suppresses the morphological changes of cells induced by Gramd1b knockdown. (Scale bar = 20 μm). ( c -c’) Transwell migration assay reveals that AG490 co-treatment efficiently suppresses the enhanced cell migration caused by GRAMD1B inhibition. (Scale bar = 100 μm). Data is represented as mean ± SEM of n = 3. ( d ) qRT-PCR analysis shows that AG490 treatment efficiently blocks the induction of Rac1 and RhoA in cells transfected with si-Gramd1b . Data is represented as mean ± SEM of n = 3. * P

    Journal: Scientific Reports

    Article Title: GRAMD1B regulates cell migration in breast cancer cells through JAK/STAT and Akt signaling

    doi: 10.1038/s41598-018-27864-6

    Figure Lengend Snippet: GRAMD1B negates JAK/STAT signaling. ( a ) Effects of GRAMD1B inhibition on JAK/STAT signaling. Increased p-JAK2 and p-STAT3 levels are detected on Gramd1b . ( b ) Treatment with AG490 almost completely suppresses the morphological changes of cells induced by Gramd1b knockdown. (Scale bar = 20 μm). ( c -c’) Transwell migration assay reveals that AG490 co-treatment efficiently suppresses the enhanced cell migration caused by GRAMD1B inhibition. (Scale bar = 100 μm). Data is represented as mean ± SEM of n = 3. ( d ) qRT-PCR analysis shows that AG490 treatment efficiently blocks the induction of Rac1 and RhoA in cells transfected with si-Gramd1b . Data is represented as mean ± SEM of n = 3. * P

    Article Snippet: Following the treatment, cells were re-suspended in serum free RPMI-1640 medium and seeded into polycarbonate membrane transwell inserts (Corning Inc., USA).

    Techniques: Inhibition, Transwell Migration Assay, Migration, Quantitative RT-PCR, Transfection

    GRAMD1B inhibition promotes cell migration in breast cancer cells. ( a -a’) Transwell migration assay shows an increase in cell migration on Gramd1b knockdown. (Scale bar = 100 μm). Data is represented as mean ± SEM of n = 3. ( b -b’) Wound healing assay confirms the inhibitory effects of GRAMD1B on cell migration. Data is represented as mean ± SEM of n = 3. ( c , d ) qRT-PCR and Western blot analyses show a significant induction of the Rho GTPase Rac1, RhoA and Cdc42 at both mRNA and protein levels. Data is represented as mean ± SEM of n = 3. * P

    Journal: Scientific Reports

    Article Title: GRAMD1B regulates cell migration in breast cancer cells through JAK/STAT and Akt signaling

    doi: 10.1038/s41598-018-27864-6

    Figure Lengend Snippet: GRAMD1B inhibition promotes cell migration in breast cancer cells. ( a -a’) Transwell migration assay shows an increase in cell migration on Gramd1b knockdown. (Scale bar = 100 μm). Data is represented as mean ± SEM of n = 3. ( b -b’) Wound healing assay confirms the inhibitory effects of GRAMD1B on cell migration. Data is represented as mean ± SEM of n = 3. ( c , d ) qRT-PCR and Western blot analyses show a significant induction of the Rho GTPase Rac1, RhoA and Cdc42 at both mRNA and protein levels. Data is represented as mean ± SEM of n = 3. * P

    Article Snippet: Following the treatment, cells were re-suspended in serum free RPMI-1640 medium and seeded into polycarbonate membrane transwell inserts (Corning Inc., USA).

    Techniques: Inhibition, Migration, Transwell Migration Assay, Wound Healing Assay, Quantitative RT-PCR, Western Blot

    FYN knockdown in glioma decrease MDSC migration potential and immune suppressive activity within the tumor microenvironment. (A) Diagram of experimental design of MDSC transwell migration assay. MDSCs derived from bone marrow and induced with IL6 and GM-CSF were seeded on the top of the Transwell and incubated for 15 hours in NT and shFYN conditioned media. The migrated cells were analyzed using CellTiter-Glo ® . (B) MDSC migration assay results. Data is expressed as percentage of migrating cells relative to the control (plain media). Error bars represent ±SEM. Experiment was performed 3 times with 3 replicates per treatment. Statistical significance was determined using One-way ANOVA test, followed by Duncan multiple comparisons. ns: Non-significant, *p

    Journal: bioRxiv

    Article Title: FYN tyrosine kinase, a downstream target of receptor tyrosine kinases, modulates anti-glioma immune responses

    doi: 10.1101/608505

    Figure Lengend Snippet: FYN knockdown in glioma decrease MDSC migration potential and immune suppressive activity within the tumor microenvironment. (A) Diagram of experimental design of MDSC transwell migration assay. MDSCs derived from bone marrow and induced with IL6 and GM-CSF were seeded on the top of the Transwell and incubated for 15 hours in NT and shFYN conditioned media. The migrated cells were analyzed using CellTiter-Glo ® . (B) MDSC migration assay results. Data is expressed as percentage of migrating cells relative to the control (plain media). Error bars represent ±SEM. Experiment was performed 3 times with 3 replicates per treatment. Statistical significance was determined using One-way ANOVA test, followed by Duncan multiple comparisons. ns: Non-significant, *p

    Article Snippet: We analyzed in vitro MDSC (M-MDSC and PMN-MDSC) migration using Transwell® polycarbonate membrane inserts (Corning Inc.) of 6.5 mm diameter and 8 um pore size.

    Techniques: Migration, Activity Assay, Transwell Migration Assay, Derivative Assay, Incubation

    Primary CF epithelial cells with LPRs containing either 50 nM or 100 nM siRNA and peptide Y. ( A ) Percentage of expression of the α-ENaC mRNA after siRNA treatment in primary CF cells in submerged culture. The expression of α-ENaC was 40.5% at 50 nM and 27.2% at 100 nM, respectively. The difference was statistically significant (*** P ≤ 0.001) at 50 nM, whereas the significance (**P ≤ 0.01), was lower at 100 nM. The results show the means ± S.E.M. of triplicate experiments. ( B ) Viability (percentage) of primary CF cells following transfections with LPRs formulated with peptide Y compared to untransfected cells. The α-ENaC-siRNA treated cells showed a viability of 72% at 50 nM siRNA concentration and 57% at 100 nM. Cell survival was assessed using an LDH assay measuring the light emitted at 610 nm. Results represent mean values of triplicate repeats ± S.E.M. ( C ) Percentage of remaining expression of the α-ENaC gene transcript in primary CFBE cells grown on ALI in transwell plates. The expression of the transcript of the α-ENaC gene was 71.8% at 30 nM and 56.7% at 50 nM. The difference was statistically significant only at 50 nM (*P ≤ 0.05). qRT-PCR analysis was performed in triplicate on individual samples. The mRNA levels of the α-ENaC gene are expressed as percentage of irrelevant/non-targeted control siRNA.

    Journal: Scientific Reports

    Article Title: Delivery of ENaC siRNA to epithelial cells mediated by a targeted nanocomplex: a therapeutic strategy for cystic fibrosis

    doi: 10.1038/s41598-017-00662-2

    Figure Lengend Snippet: Primary CF epithelial cells with LPRs containing either 50 nM or 100 nM siRNA and peptide Y. ( A ) Percentage of expression of the α-ENaC mRNA after siRNA treatment in primary CF cells in submerged culture. The expression of α-ENaC was 40.5% at 50 nM and 27.2% at 100 nM, respectively. The difference was statistically significant (*** P ≤ 0.001) at 50 nM, whereas the significance (**P ≤ 0.01), was lower at 100 nM. The results show the means ± S.E.M. of triplicate experiments. ( B ) Viability (percentage) of primary CF cells following transfections with LPRs formulated with peptide Y compared to untransfected cells. The α-ENaC-siRNA treated cells showed a viability of 72% at 50 nM siRNA concentration and 57% at 100 nM. Cell survival was assessed using an LDH assay measuring the light emitted at 610 nm. Results represent mean values of triplicate repeats ± S.E.M. ( C ) Percentage of remaining expression of the α-ENaC gene transcript in primary CFBE cells grown on ALI in transwell plates. The expression of the transcript of the α-ENaC gene was 71.8% at 30 nM and 56.7% at 50 nM. The difference was statistically significant only at 50 nM (*P ≤ 0.05). qRT-PCR analysis was performed in triplicate on individual samples. The mRNA levels of the α-ENaC gene are expressed as percentage of irrelevant/non-targeted control siRNA.

    Article Snippet: For ALI culture, primary cells were grown in 12 mm collagen-coated transwell inserts (Polyester (PET) Membrane Transwell-Clear Inserts, Corning, Corning Inc. Life Sciences, Tewksbury, MA, USA) at a seeding density of 1.5 × 105 or 1 × 106 viable cells per insert and fed with BEGM ALI medium (1:1 DMEM-Hi glucose: BEGM containing supplements) supplemented with 100 nM retinoic acid (Sigma).

    Techniques: Expressing, Transfection, Concentration Assay, Lactate Dehydrogenase Assay, Quantitative RT-PCR

    Sepin-1 inhibits cell migration of triple-negative breast cancer cells. MD-MB-231 or MD-MB-468 cells were seeded on the top of the filter membrane in a Transwell ® insert, and medium with or without Sepin-1 was added to the bottom of the lower chamber in a 24-well plate. After 24 h of incubation, the cells that had not migrated were removed. The cells on the other side of the membrane on Transwell ® insert were fixed and stained with crystal violet. The migrated cells (in purple) were counted. Representative images of migrated cells are shown in A. Percentage of cells migrated through the membrane is shown in B. The assay was performed in triplicate (n=3 ± SD). *** indicates p

    Journal: Journal of cancer science & therapy

    Article Title: Separase Inhibitor Sepin-1 Inhibits Foxm1 Expression and Breast Cancer Cell Growth

    doi: 10.4172/1948-5956.1000517

    Figure Lengend Snippet: Sepin-1 inhibits cell migration of triple-negative breast cancer cells. MD-MB-231 or MD-MB-468 cells were seeded on the top of the filter membrane in a Transwell ® insert, and medium with or without Sepin-1 was added to the bottom of the lower chamber in a 24-well plate. After 24 h of incubation, the cells that had not migrated were removed. The cells on the other side of the membrane on Transwell ® insert were fixed and stained with crystal violet. The migrated cells (in purple) were counted. Representative images of migrated cells are shown in A. Percentage of cells migrated through the membrane is shown in B. The assay was performed in triplicate (n=3 ± SD). *** indicates p

    Article Snippet: One hundred μl of the cell solution was plated on the top of the filter membrane in a 24-well Transwell® insert (Corning) and incubated for 10 minutes at 37°C to allow the cells to settle down.

    Techniques: Migration, Incubation, Staining

    Effect of depleting or enforcing the expression of FoxQ1 on cell proliferation, migration and invasiveness of lung carcinoma cells A) qRT-PCR and B) western blot were used to select the most effective silencing siRNA targeting human FOXQ1. C) Two stably transfected cell lines silenced for FoxQ1 were established by G418 screening. G418-resistant clones were examined by qRT-PCR. D) Two stably transfected cell lines overexpressing FoxQ1 were identified by G418 screening, and qRT-PCR was used to confirm G418-resistant clones. E) The proliferation ability of the four experimental cell lines was examined using CCK-8 at 450 nm. Specifically, 5 × 10 3 cells were seeded in 100 μL of medium per well into 96-well plates (three wells per each group). Then, 10 μL of CCK8 solution was added to the culture medium in each well after 24h, 48h, 72h and 96h. Then cells were incubated for 3 h another. The absorbance was determined at 450 nm wavelength. F) Migration and G) invasion ability were presented as total number of cells that migrated to the bottom chamber without or with the transwell-precoated matrigel, respectively, as calculated in at least six random fields (total magnification ×200) per filter. (*P

    Journal: Oncotarget

    Article Title: Involvement of FoxQ1 in NSCLC through regulating EMT and increasing chemosensitivity

    doi:

    Figure Lengend Snippet: Effect of depleting or enforcing the expression of FoxQ1 on cell proliferation, migration and invasiveness of lung carcinoma cells A) qRT-PCR and B) western blot were used to select the most effective silencing siRNA targeting human FOXQ1. C) Two stably transfected cell lines silenced for FoxQ1 were established by G418 screening. G418-resistant clones were examined by qRT-PCR. D) Two stably transfected cell lines overexpressing FoxQ1 were identified by G418 screening, and qRT-PCR was used to confirm G418-resistant clones. E) The proliferation ability of the four experimental cell lines was examined using CCK-8 at 450 nm. Specifically, 5 × 10 3 cells were seeded in 100 μL of medium per well into 96-well plates (three wells per each group). Then, 10 μL of CCK8 solution was added to the culture medium in each well after 24h, 48h, 72h and 96h. Then cells were incubated for 3 h another. The absorbance was determined at 450 nm wavelength. F) Migration and G) invasion ability were presented as total number of cells that migrated to the bottom chamber without or with the transwell-precoated matrigel, respectively, as calculated in at least six random fields (total magnification ×200) per filter. (*P

    Article Snippet: Transwell Migration and Invasion Assays For cell invasion assays, modified Boyden Chambers consisting of Transwell-precoated Matrigel membrane filter inserts with 8 μm pores were used in 24-well tissue culture plates (BD Biosciences, Bedford, MA).

    Techniques: Expressing, Migration, Quantitative RT-PCR, Western Blot, Stable Transfection, Transfection, Clone Assay, CCK-8 Assay, Incubation