Structured Review

Thermo Fisher fbs
CXCR4 surface expression in PANC-1 adherent cells and spheres CXCR4 cell surface expression in PANC-1 adherently growing cells ( A ; 6.4%) and spheres ( B ; 19.4%) using <t>FACS</t> analysis. All results are expressed as means of three independent experiments. ( C ) Transwell migration assay of PANC-1 spheres compared with adherently growing cells. Average percentage of cells (normalized to DMEM serum-free medium as the control) that migrated after 48 h from the upper to the lower chamber is shown. The medium in the lower chamber contained the following chemoattractants: DMEM serum-free medium (as the control, ctrl); complete CSM (CSC); HGF at 200 ng/ml; DMEM serum-free medium supplemented with SDF-1 (200 ng/ml; SDF) and DMEM supplemented with 10% <t>FBS</t> (10% FBS). Statistically significant (Student's t test; * P
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1) Product Images from "Pancreatic cancer spheres are more than just aggregates of stem marker-positive cells"

Article Title: Pancreatic cancer spheres are more than just aggregates of stem marker-positive cells

Journal: Bioscience Reports

doi: 10.1042/BSR20100018

CXCR4 surface expression in PANC-1 adherent cells and spheres CXCR4 cell surface expression in PANC-1 adherently growing cells ( A ; 6.4%) and spheres ( B ; 19.4%) using FACS analysis. All results are expressed as means of three independent experiments. ( C ) Transwell migration assay of PANC-1 spheres compared with adherently growing cells. Average percentage of cells (normalized to DMEM serum-free medium as the control) that migrated after 48 h from the upper to the lower chamber is shown. The medium in the lower chamber contained the following chemoattractants: DMEM serum-free medium (as the control, ctrl); complete CSM (CSC); HGF at 200 ng/ml; DMEM serum-free medium supplemented with SDF-1 (200 ng/ml; SDF) and DMEM supplemented with 10% FBS (10% FBS). Statistically significant (Student's t test; * P
Figure Legend Snippet: CXCR4 surface expression in PANC-1 adherent cells and spheres CXCR4 cell surface expression in PANC-1 adherently growing cells ( A ; 6.4%) and spheres ( B ; 19.4%) using FACS analysis. All results are expressed as means of three independent experiments. ( C ) Transwell migration assay of PANC-1 spheres compared with adherently growing cells. Average percentage of cells (normalized to DMEM serum-free medium as the control) that migrated after 48 h from the upper to the lower chamber is shown. The medium in the lower chamber contained the following chemoattractants: DMEM serum-free medium (as the control, ctrl); complete CSM (CSC); HGF at 200 ng/ml; DMEM serum-free medium supplemented with SDF-1 (200 ng/ml; SDF) and DMEM supplemented with 10% FBS (10% FBS). Statistically significant (Student's t test; * P

Techniques Used: Expressing, FACS, Transwell Migration Assay

2) Product Images from "A murine–human chimeric IgG antibody against vascular endothelial growth factor receptor 2 inhibits angiogenesis in vitro"

Article Title: A murine–human chimeric IgG antibody against vascular endothelial growth factor receptor 2 inhibits angiogenesis in vitro

Journal: Cytotechnology

doi: 10.1007/s10616-013-9587-x

Stability and affinity determination of the 2F12 antibody. a Cells of clone 2F12 were cultured in 10 %FBS/OPTI-MEM I medium without l -histidinol or MPA. After 30 passages, the total RNA from cells of the 30th passage was extracted, and the V H
Figure Legend Snippet: Stability and affinity determination of the 2F12 antibody. a Cells of clone 2F12 were cultured in 10 %FBS/OPTI-MEM I medium without l -histidinol or MPA. After 30 passages, the total RNA from cells of the 30th passage was extracted, and the V H

Techniques Used: Cell Culture

3) Product Images from "MitosRNAs and extreme anoxia tolerance in embryos of the annual killifish Austrofundulus limnaeus"

Article Title: MitosRNAs and extreme anoxia tolerance in embryos of the annual killifish Austrofundulus limnaeus

Journal: Scientific Reports

doi: 10.1038/s41598-019-56231-2

MitosRNA-tRNA-cys sequences are present and induced in response to anoxia in the A . limnaeus WS40NE cell line. ( a ) Heat map of mitosRNAs differentially expressed in response to anoxia in WS40NE cells. Log 2 fold change is calculated relative to normoxic control with fetal bovine serum (N + FBS). For heat map details see Fig. 1 caption. ( b ) MitosRNAs-tRNA-cys 3′ variants northern blot of normoxic and 24 h anoxic WS40NE cells shows increased expression of a variety of mitosRNAs derived from tRNA-cys during anoxia. 20 µg RNA was loaded for normoxic and anoxic cells and the blot was hybridized with 2 nM of mitosRNA-tRNA-cys probe. Note: smear of RNA at top of blot is due to need to load high amount of RNA in order to get a signal for the mitosRNAs. This is likely due to relatively low expression of mitosRNAs in the cells. ( c ) Line graphs of the 6 most highly differentially expressed mitosRNAs in response to anoxia. Note appearance of 3 variants of mitosRNAs annotating to mitochondrial tRNA-cys.
Figure Legend Snippet: MitosRNA-tRNA-cys sequences are present and induced in response to anoxia in the A . limnaeus WS40NE cell line. ( a ) Heat map of mitosRNAs differentially expressed in response to anoxia in WS40NE cells. Log 2 fold change is calculated relative to normoxic control with fetal bovine serum (N + FBS). For heat map details see Fig. 1 caption. ( b ) MitosRNAs-tRNA-cys 3′ variants northern blot of normoxic and 24 h anoxic WS40NE cells shows increased expression of a variety of mitosRNAs derived from tRNA-cys during anoxia. 20 µg RNA was loaded for normoxic and anoxic cells and the blot was hybridized with 2 nM of mitosRNA-tRNA-cys probe. Note: smear of RNA at top of blot is due to need to load high amount of RNA in order to get a signal for the mitosRNAs. This is likely due to relatively low expression of mitosRNAs in the cells. ( c ) Line graphs of the 6 most highly differentially expressed mitosRNAs in response to anoxia. Note appearance of 3 variants of mitosRNAs annotating to mitochondrial tRNA-cys.

Techniques Used: Northern Blot, Expressing, Derivative Assay

4) Product Images from "Aggregation of Human Eyelid Adipose-derived Stem Cells by Human Body Fluids"

Article Title: Aggregation of Human Eyelid Adipose-derived Stem Cells by Human Body Fluids

Journal: Development & Reproduction/Balsaeng'gwa saengsig

doi: 10.12717/DR.2012.16.4.339

Chemotactic aggregation of HEACs during culture in different conditions. HEACs were cultivated in DMEM-LG containing 10% HS, 10% FBS, 5% HS plus 5% FBS, or 0.4 BSA.
Figure Legend Snippet: Chemotactic aggregation of HEACs during culture in different conditions. HEACs were cultivated in DMEM-LG containing 10% HS, 10% FBS, 5% HS plus 5% FBS, or 0.4 BSA.

Techniques Used:

5) Product Images from "Cbl-transforming Variants Trigger a Cascade of Molecular Alterations That Lead to Epithelial Mesenchymal Conversion"

Article Title: Cbl-transforming Variants Trigger a Cascade of Molecular Alterations That Lead to Epithelial Mesenchymal Conversion

Journal: Molecular Biology of the Cell

doi:

Overexpression of 70z-Cbl promotes the loss of cell–cell contacts. (A) Parental MDCK cells, unstimulated or stimulated with 5 U/ml HGF for 24 h, as well as stable cell lines expressing c-Cbl (clone 8) and 70z-Cbl (clone 20), were grown on glass coverslips in DMEM containing 10% FBS. Cells were treated for 10 min with CSK buffer, fixed in 3.7% formaldehyde, and then labeled with anti-E-cadherin antibody followed by Cy3-conjugated anti-mouse antiserum (a, c, e, and g). Matching phase-contrast images are shown (b, d, f, and h). Photographs were taken at a magnification of ×63. (B) Whole cell lysates were separated on an 8% SDS-PAGE gel and immunoblotted with anti-E-cadherin antibody.
Figure Legend Snippet: Overexpression of 70z-Cbl promotes the loss of cell–cell contacts. (A) Parental MDCK cells, unstimulated or stimulated with 5 U/ml HGF for 24 h, as well as stable cell lines expressing c-Cbl (clone 8) and 70z-Cbl (clone 20), were grown on glass coverslips in DMEM containing 10% FBS. Cells were treated for 10 min with CSK buffer, fixed in 3.7% formaldehyde, and then labeled with anti-E-cadherin antibody followed by Cy3-conjugated anti-mouse antiserum (a, c, e, and g). Matching phase-contrast images are shown (b, d, f, and h). Photographs were taken at a magnification of ×63. (B) Whole cell lysates were separated on an 8% SDS-PAGE gel and immunoblotted with anti-E-cadherin antibody.

Techniques Used: Over Expression, Stable Transfection, Expressing, Labeling, SDS Page

6) Product Images from "Improved Approach for Chondrogenic Differentiation of Human Induced Pluripotent Stem Cells"

Article Title: Improved Approach for Chondrogenic Differentiation of Human Induced Pluripotent Stem Cells

Journal: Stem Cell Reviews

doi: 10.1007/s12015-014-9581-5

Chondrogenic differentiation of hiPSC. ( a ) Classical chondrogenic differentiation of hiPSCs via formation of embryoid bodies, outgrowth of endodermal ( green ), ectodermal ( yellow ) and mesodermal ( red ) cell lineages, selection of mesodermal cells, and induction of MSC and induction of chondrocytes. In this method hiPS cells were detached from matrigel coated dish and moved to ultra low attachment culture dish for 5 days to induce the EB formation, then EBs moved to plastic culture dish to select the hMSCs by collecting the outgrowing cells from EB (from day 5 to day 14) after collecting the attached fibroblast-like cells. These cells were cultured for 3 weeks in media containing FBS to prepare the hiPSC-MSCs (day 35 of differentiation). Then, hiPSC-MSCs were differentiated in a pellet culture system using serum free chondrogenic media for 3 weeks. ( b ) Embryoid body free method of direct differentiation of hiPSCs into hiPSC-MSCs, followed by chondrogenic differentiation. In embryoid body free method hiPSCs were cultured in matrigel coated dish and media was changed to hMSC media (DMEM supplemented with FBS) for 5 days to induce the hMSC differentiation (Day 5). Then, cells were detached and moved to a plastic culture dish for 4 passages to prepare the hiPSC-MSCs (Day 28). To differentiate the hiPSC-MSCs to chondrocytes, cells were used in pellet culture system using serum free chondrogenic media for 3 weeks
Figure Legend Snippet: Chondrogenic differentiation of hiPSC. ( a ) Classical chondrogenic differentiation of hiPSCs via formation of embryoid bodies, outgrowth of endodermal ( green ), ectodermal ( yellow ) and mesodermal ( red ) cell lineages, selection of mesodermal cells, and induction of MSC and induction of chondrocytes. In this method hiPS cells were detached from matrigel coated dish and moved to ultra low attachment culture dish for 5 days to induce the EB formation, then EBs moved to plastic culture dish to select the hMSCs by collecting the outgrowing cells from EB (from day 5 to day 14) after collecting the attached fibroblast-like cells. These cells were cultured for 3 weeks in media containing FBS to prepare the hiPSC-MSCs (day 35 of differentiation). Then, hiPSC-MSCs were differentiated in a pellet culture system using serum free chondrogenic media for 3 weeks. ( b ) Embryoid body free method of direct differentiation of hiPSCs into hiPSC-MSCs, followed by chondrogenic differentiation. In embryoid body free method hiPSCs were cultured in matrigel coated dish and media was changed to hMSC media (DMEM supplemented with FBS) for 5 days to induce the hMSC differentiation (Day 5). Then, cells were detached and moved to a plastic culture dish for 4 passages to prepare the hiPSC-MSCs (Day 28). To differentiate the hiPSC-MSCs to chondrocytes, cells were used in pellet culture system using serum free chondrogenic media for 3 weeks

Techniques Used: Selection, Cell Culture

7) Product Images from "Ex vivo Expansion of Bovine Corneal Endothelial Cells in Xeno-Free Medium Supplemented with Platelet Releasate"

Article Title: Ex vivo Expansion of Bovine Corneal Endothelial Cells in Xeno-Free Medium Supplemented with Platelet Releasate

Journal: PLoS ONE

doi: 10.1371/journal.pone.0099145

Western blot analysis of membrane markers ZO-1, N-cadherin (N-CAD), Na-K ATPase (ATPase), connexin 43 (CX43), in cells isolated and expanded in DMEM + F12 medium supplemented with 10% heat-treated platelet releasate (HPR) or complete SHEM medium (FBS). Molecular weight (kDa) of membrane markers is shown in parenthesis. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase experimental marker control.
Figure Legend Snippet: Western blot analysis of membrane markers ZO-1, N-cadherin (N-CAD), Na-K ATPase (ATPase), connexin 43 (CX43), in cells isolated and expanded in DMEM + F12 medium supplemented with 10% heat-treated platelet releasate (HPR) or complete SHEM medium (FBS). Molecular weight (kDa) of membrane markers is shown in parenthesis. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase experimental marker control.

Techniques Used: Western Blot, Isolation, Molecular Weight, Marker

8) Product Images from "Neural crest–derived SEMA3C activates endothelial NRP1 for cardiac outflow tract septation"

Article Title: Neural crest–derived SEMA3C activates endothelial NRP1 for cardiac outflow tract septation

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI79668

SEMA3C signals via NRP1 to induce endoMT of OFT endothelium. OFT sections from E10.5 Wnt1-Cre Rosa Yfp ( A ) and Tie2-Cre Rosa Yfp mice at E10.5 and E12.5 ( B and C ) were immunolabeled for YFP and PECAM. Arrows indicate YFP-positive cells within the endocardial cushions; arrowheads indicate YFP-positive OFT endothelium. Curved arrows highlight YFP-positive cells that have undergone endoMT; the open curved arrow shows a lack of PECAM expression in these cells. The wavy arrow labels PECAM-positive capillaries. ( D and E ) E10.5 OFT explants of the indicated genotypes were labeled for F-actin and YFP ( D ) or PECAM ( E ) and counterstained with DAPI after 72 hours of culture. Curved arrows indicate YFP-positive outgrowth cells, demonstrating their origin by endoMT, while open curved arrows show that outgrowth cells lack PECAM after endoMT. ( F and G ) E10.5 Wnt1-Cre Sema3c fl/fl ( n = 6) ( F ) and Nrp1 -null ( n = 5) ( G ) OFTs with their WT littermates ( n = 14 and n = 4, respectively) were labeled for F-actin and DAPI after 72 hours of culture and the number of F-actin–positive emigrated cells quantitated. ( H and I ) E10.5 WT and Nrp1 –/– OFTs ( n = 3 each) were cultured for 72 hours in 1% FBS with or without 400 ng/ml SEMA3C and labeled for F-actin and DAPI. The number of F-actin–positive emigrated cells was quantitated. Mean ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, 2-tailed, unpaired Student’s t test. Scale bars: 100 μm ( A and B ); 200 μm ( C – I ).
Figure Legend Snippet: SEMA3C signals via NRP1 to induce endoMT of OFT endothelium. OFT sections from E10.5 Wnt1-Cre Rosa Yfp ( A ) and Tie2-Cre Rosa Yfp mice at E10.5 and E12.5 ( B and C ) were immunolabeled for YFP and PECAM. Arrows indicate YFP-positive cells within the endocardial cushions; arrowheads indicate YFP-positive OFT endothelium. Curved arrows highlight YFP-positive cells that have undergone endoMT; the open curved arrow shows a lack of PECAM expression in these cells. The wavy arrow labels PECAM-positive capillaries. ( D and E ) E10.5 OFT explants of the indicated genotypes were labeled for F-actin and YFP ( D ) or PECAM ( E ) and counterstained with DAPI after 72 hours of culture. Curved arrows indicate YFP-positive outgrowth cells, demonstrating their origin by endoMT, while open curved arrows show that outgrowth cells lack PECAM after endoMT. ( F and G ) E10.5 Wnt1-Cre Sema3c fl/fl ( n = 6) ( F ) and Nrp1 -null ( n = 5) ( G ) OFTs with their WT littermates ( n = 14 and n = 4, respectively) were labeled for F-actin and DAPI after 72 hours of culture and the number of F-actin–positive emigrated cells quantitated. ( H and I ) E10.5 WT and Nrp1 –/– OFTs ( n = 3 each) were cultured for 72 hours in 1% FBS with or without 400 ng/ml SEMA3C and labeled for F-actin and DAPI. The number of F-actin–positive emigrated cells was quantitated. Mean ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, 2-tailed, unpaired Student’s t test. Scale bars: 100 μm ( A and B ); 200 μm ( C – I ).

Techniques Used: Mouse Assay, Immunolabeling, Expressing, Labeling, Cell Culture

9) Product Images from "High-Discrimination Factor Nanosensor Based on Tetrahedral DNA Nanostructures and Gold Nanoparticles for Detection of MiRNA-21 in Live Cells"

Article Title: High-Discrimination Factor Nanosensor Based on Tetrahedral DNA Nanostructures and Gold Nanoparticles for Detection of MiRNA-21 in Live Cells

Journal: Theranostics

doi: 10.7150/thno.23852

(A, B) Fluorescence kinetic curves of mutation base in the middle, distal or proximal site of the target and target detection by ExP and OrP, respectively. (C) Relative fluorescence intensity of the ExP and OrP in detecting single-base mutation target and wild-type target. (D) Corresponding discrimination factors (DFs) of ExP and OrP. (E) UV-vis characterization of the stability of Au-TDNNs in SSC, PBS and DMEM (10% FBS). (F) Zeta potential analysis of the step-by-step assembly of the Au-TDNNs. RFI: relative fluorescence intensity. DF = ∆F target /∆F target analogue (∆F = F-F 0 , F 0 : background signal.). * P
Figure Legend Snippet: (A, B) Fluorescence kinetic curves of mutation base in the middle, distal or proximal site of the target and target detection by ExP and OrP, respectively. (C) Relative fluorescence intensity of the ExP and OrP in detecting single-base mutation target and wild-type target. (D) Corresponding discrimination factors (DFs) of ExP and OrP. (E) UV-vis characterization of the stability of Au-TDNNs in SSC, PBS and DMEM (10% FBS). (F) Zeta potential analysis of the step-by-step assembly of the Au-TDNNs. RFI: relative fluorescence intensity. DF = ∆F target /∆F target analogue (∆F = F-F 0 , F 0 : background signal.). * P

Techniques Used: Fluorescence, Mutagenesis

10) Product Images from "Dicer1/miR-29/HMGCR axis contributes to hepatic free cholesterol accumulation in mouse non-alcoholic steatohepatitis"

Article Title: Dicer1/miR-29/HMGCR axis contributes to hepatic free cholesterol accumulation in mouse non-alcoholic steatohepatitis

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2016.158

miR-29a levels and HMGCR levels change inversely in two steatosis hepatic cell models. (A – F) SMMC-7721 and HL-7702 cells were incubated with or without cholesterol/25-hydroxycholesterol (10 μg·mL -1 /1 μg·mL -1 ) for 24 h, 48 h and 72 h. (G – L) SMMC-7721 and HL-7702 cells were exposed to 10% FBS-RPMI-1640 medium or 50% FBS-RPMI-1640 medium for 24 h, 48 h and 72 h. The miR-29a levels (A, D, G and J) and HMGCR mRNA levels (B, E, H, K) were determined by qRT-PCR, and the HMGCR protein levels (C, F, I, L) were determined by Western blot, which were normalized to control ones at the same time points. GAPDH and RNU6-2 were used as internal controls for protein and miRNA, respectively. (C and F) – , without cholesterol/25-hydroxycholesterol (chol); +, with cholesterol/25-hydroxycholesterol. (I and L) –, 10% FBS; +, 50% FBS. Data are the mean±SD of at least three independent experiments. * P
Figure Legend Snippet: miR-29a levels and HMGCR levels change inversely in two steatosis hepatic cell models. (A – F) SMMC-7721 and HL-7702 cells were incubated with or without cholesterol/25-hydroxycholesterol (10 μg·mL -1 /1 μg·mL -1 ) for 24 h, 48 h and 72 h. (G – L) SMMC-7721 and HL-7702 cells were exposed to 10% FBS-RPMI-1640 medium or 50% FBS-RPMI-1640 medium for 24 h, 48 h and 72 h. The miR-29a levels (A, D, G and J) and HMGCR mRNA levels (B, E, H, K) were determined by qRT-PCR, and the HMGCR protein levels (C, F, I, L) were determined by Western blot, which were normalized to control ones at the same time points. GAPDH and RNU6-2 were used as internal controls for protein and miRNA, respectively. (C and F) – , without cholesterol/25-hydroxycholesterol (chol); +, with cholesterol/25-hydroxycholesterol. (I and L) –, 10% FBS; +, 50% FBS. Data are the mean±SD of at least three independent experiments. * P

Techniques Used: Incubation, Quantitative RT-PCR, Western Blot

11) Product Images from "K-Ras mutation-mediated IGF-1-induced feedback ERK activation contributes to the rapalog resistance in pancreatic ductal adenocarcinomas"

Article Title: K-Ras mutation-mediated IGF-1-induced feedback ERK activation contributes to the rapalog resistance in pancreatic ductal adenocarcinomas

Journal: Cancer Letters

doi: 10.1016/j.canlet.2012.02.005

K- Ras mutations contribute to the everolimus-induced ERK signal. (A) The K- Ras mt PANC-1 cells were subjected to western blotting after treated first with everolimus and then exposed to 10% FBS, EGF, FGF2, HGF and IGF-1, as indicated on the top of the panel. (B) The experiment was repeated with using NVP-AEW541. (C) The empty vector and K- Ras shRNA expressing PANC-1 cells were examined by western blotting after treated first with everolimus first and then with IGF-1. (D) The empty vector and K- Ras shRNA expressing PANC-1 cells, treated as described above in B, were examined by by BrdU labeling assay. The experiment was repeated three times and the data was presented as mean ± SD. **, p
Figure Legend Snippet: K- Ras mutations contribute to the everolimus-induced ERK signal. (A) The K- Ras mt PANC-1 cells were subjected to western blotting after treated first with everolimus and then exposed to 10% FBS, EGF, FGF2, HGF and IGF-1, as indicated on the top of the panel. (B) The experiment was repeated with using NVP-AEW541. (C) The empty vector and K- Ras shRNA expressing PANC-1 cells were examined by western blotting after treated first with everolimus first and then with IGF-1. (D) The empty vector and K- Ras shRNA expressing PANC-1 cells, treated as described above in B, were examined by by BrdU labeling assay. The experiment was repeated three times and the data was presented as mean ± SD. **, p

Techniques Used: Western Blot, Plasmid Preparation, shRNA, Expressing, Labeling

12) Product Images from "Cortactin promotes colorectal cancer cell proliferation by activating the EGFR-MAPK pathway"

Article Title: Cortactin promotes colorectal cancer cell proliferation by activating the EGFR-MAPK pathway

Journal: Oncotarget

doi: 10.18632/oncotarget.13652

CTTN attenuates EGF-induced EGFR ubiquitination by interaction with c-Cbl A. SW1116 cells were pretreated with 10 μM of cycloheximide for 1 h followed by stimulation with 10 ng/mL EGF at the indicated time. The cell lysates were immunoprecipitated with EGFR and followed with anti-Ubiquitin, EGFR(Tyr), EGFR (Tyr 1045), c-Cbl and EGFR. B. SW1116 cells were cultured conventionally with 10% FBS, cell lysates were immunoprecipitated with CTTN, and then immunobloted with anti-EGFR, c-Cbl and CTTN. C. The SW1116-sh182 cells with CTTN rescue expression were lysed and immunoprecipitated with HA, and then immunobloted with anti-EGFR, c-Cbl. D. Serum starved SW1116 control or CTTN-sh182 cells were pretreated with 10 μM of cycloheximide and 100 μM Bafilomycin A1 (Baf-A1) for 1 h, followed by stimulation with 10 ng/mL EGF at different times. Cell lysates were analyzed with antibodies as indicated by Western Blot.
Figure Legend Snippet: CTTN attenuates EGF-induced EGFR ubiquitination by interaction with c-Cbl A. SW1116 cells were pretreated with 10 μM of cycloheximide for 1 h followed by stimulation with 10 ng/mL EGF at the indicated time. The cell lysates were immunoprecipitated with EGFR and followed with anti-Ubiquitin, EGFR(Tyr), EGFR (Tyr 1045), c-Cbl and EGFR. B. SW1116 cells were cultured conventionally with 10% FBS, cell lysates were immunoprecipitated with CTTN, and then immunobloted with anti-EGFR, c-Cbl and CTTN. C. The SW1116-sh182 cells with CTTN rescue expression were lysed and immunoprecipitated with HA, and then immunobloted with anti-EGFR, c-Cbl. D. Serum starved SW1116 control or CTTN-sh182 cells were pretreated with 10 μM of cycloheximide and 100 μM Bafilomycin A1 (Baf-A1) for 1 h, followed by stimulation with 10 ng/mL EGF at different times. Cell lysates were analyzed with antibodies as indicated by Western Blot.

Techniques Used: Immunoprecipitation, Cell Culture, Expressing, Western Blot

The effect of Cortactin overexpression on cell proliferation and colony formation is enhanced by EGF A, B, C, D. The knockdown of CTTN attenuates EGF-induced proliferation and colony formation of HT-29 and SW1116 cells. E, F. The rescue expression of CTTN restores the EGF-dependent proliferation and colony formation in SW1116 cells. G, H. CTTN overexpression enhances EGF-induced proliferation and colony formation in SW480 cells. All assays were performed in plates containing 1% FBS and 10 ng/mL EGF. Representative images from three independent experiments (* P
Figure Legend Snippet: The effect of Cortactin overexpression on cell proliferation and colony formation is enhanced by EGF A, B, C, D. The knockdown of CTTN attenuates EGF-induced proliferation and colony formation of HT-29 and SW1116 cells. E, F. The rescue expression of CTTN restores the EGF-dependent proliferation and colony formation in SW1116 cells. G, H. CTTN overexpression enhances EGF-induced proliferation and colony formation in SW480 cells. All assays were performed in plates containing 1% FBS and 10 ng/mL EGF. Representative images from three independent experiments (* P

Techniques Used: Over Expression, Expressing

13) Product Images from "Cortactin promotes colorectal cancer cell proliferation by activating the EGFR-MAPK pathway"

Article Title: Cortactin promotes colorectal cancer cell proliferation by activating the EGFR-MAPK pathway

Journal: Oncotarget

doi: 10.18632/oncotarget.13652

CTTN attenuates EGF-induced EGFR ubiquitination by interaction with c-Cbl A. SW1116 cells were pretreated with 10 μM of cycloheximide for 1 h followed by stimulation with 10 ng/mL EGF at the indicated time. The cell lysates were immunoprecipitated with EGFR and followed with anti-Ubiquitin, EGFR(Tyr), EGFR (Tyr 1045), c-Cbl and EGFR. B. SW1116 cells were cultured conventionally with 10% FBS, cell lysates were immunoprecipitated with CTTN, and then immunobloted with anti-EGFR, c-Cbl and CTTN. C. The SW1116-sh182 cells with CTTN rescue expression were lysed and immunoprecipitated with HA, and then immunobloted with anti-EGFR, c-Cbl. D. Serum starved SW1116 control or CTTN-sh182 cells were pretreated with 10 μM of cycloheximide and 100 μM Bafilomycin A1 (Baf-A1) for 1 h, followed by stimulation with 10 ng/mL EGF at different times. Cell lysates were analyzed with antibodies as indicated by Western Blot.
Figure Legend Snippet: CTTN attenuates EGF-induced EGFR ubiquitination by interaction with c-Cbl A. SW1116 cells were pretreated with 10 μM of cycloheximide for 1 h followed by stimulation with 10 ng/mL EGF at the indicated time. The cell lysates were immunoprecipitated with EGFR and followed with anti-Ubiquitin, EGFR(Tyr), EGFR (Tyr 1045), c-Cbl and EGFR. B. SW1116 cells were cultured conventionally with 10% FBS, cell lysates were immunoprecipitated with CTTN, and then immunobloted with anti-EGFR, c-Cbl and CTTN. C. The SW1116-sh182 cells with CTTN rescue expression were lysed and immunoprecipitated with HA, and then immunobloted with anti-EGFR, c-Cbl. D. Serum starved SW1116 control or CTTN-sh182 cells were pretreated with 10 μM of cycloheximide and 100 μM Bafilomycin A1 (Baf-A1) for 1 h, followed by stimulation with 10 ng/mL EGF at different times. Cell lysates were analyzed with antibodies as indicated by Western Blot.

Techniques Used: Immunoprecipitation, Cell Culture, Expressing, Western Blot

The effect of Cortactin overexpression on cell proliferation and colony formation is enhanced by EGF A, B, C, D. The knockdown of CTTN attenuates EGF-induced proliferation and colony formation of HT-29 and SW1116 cells. E, F. The rescue expression of CTTN restores the EGF-dependent proliferation and colony formation in SW1116 cells. G, H. CTTN overexpression enhances EGF-induced proliferation and colony formation in SW480 cells. All assays were performed in plates containing 1% FBS and 10 ng/mL EGF. Representative images from three independent experiments (* P
Figure Legend Snippet: The effect of Cortactin overexpression on cell proliferation and colony formation is enhanced by EGF A, B, C, D. The knockdown of CTTN attenuates EGF-induced proliferation and colony formation of HT-29 and SW1116 cells. E, F. The rescue expression of CTTN restores the EGF-dependent proliferation and colony formation in SW1116 cells. G, H. CTTN overexpression enhances EGF-induced proliferation and colony formation in SW480 cells. All assays were performed in plates containing 1% FBS and 10 ng/mL EGF. Representative images from three independent experiments (* P

Techniques Used: Over Expression, Expressing

14) Product Images from "Rosuvastatin Attenuates Myocardial Ischemia-Reperfusion Injury via Upregulating miR-17-3p-Mediated Autophagy"

Article Title: Rosuvastatin Attenuates Myocardial Ischemia-Reperfusion Injury via Upregulating miR-17-3p-Mediated Autophagy

Journal: Cellular Reprogramming

doi: 10.1089/cell.2018.0053

OGD/R decreases H9C2 cell viability and increases the expression of miR-17-3p. OGD/R was performed by means of free FBS and no-glucose Dulbecco's modified Eagle's medium (DMEM) in 5% CO 2 , 1% O 2 , and 37°C for 6 hours, then higher-glucose DMEM, 10% FBS and 1% penicillin-streptomycin in 5% CO 2 , 95% air, and 37°C for 1 hour. H9C2 cells were seeded in 96-well plate at 4 × 10 3 cells/hole for 24 hours. Then, H9C2 cells were treated with OGD/R for 24 hours or 48 hours. (A) Cell viability was detected by MTT assay. (B) The expression level of miR-17-3p was analyzed via qRT-PCR assays. Values were presented by mean ± SD, and the relationship between the two groups was analyzed by ANOVA with Tukey's test (* vs. Control group, * p
Figure Legend Snippet: OGD/R decreases H9C2 cell viability and increases the expression of miR-17-3p. OGD/R was performed by means of free FBS and no-glucose Dulbecco's modified Eagle's medium (DMEM) in 5% CO 2 , 1% O 2 , and 37°C for 6 hours, then higher-glucose DMEM, 10% FBS and 1% penicillin-streptomycin in 5% CO 2 , 95% air, and 37°C for 1 hour. H9C2 cells were seeded in 96-well plate at 4 × 10 3 cells/hole for 24 hours. Then, H9C2 cells were treated with OGD/R for 24 hours or 48 hours. (A) Cell viability was detected by MTT assay. (B) The expression level of miR-17-3p was analyzed via qRT-PCR assays. Values were presented by mean ± SD, and the relationship between the two groups was analyzed by ANOVA with Tukey's test (* vs. Control group, * p

Techniques Used: Expressing, Modification, MTT Assay, Quantitative RT-PCR

15) Product Images from "Measurement and analysis of traction force dynamics in response to vasoactive agonists"

Article Title: Measurement and analysis of traction force dynamics in response to vasoactive agonists

Journal: Integrative biology : quantitative biosciences from nano to macro

doi: 10.1039/c0ib00156b

Effects of vasoactive agonists on actin organization and myosin activity (a) Representative images of cells stimulated with BSA (10 μg/mL), histamine (3 μM), LPA (10 μg/mL), S1P (0.5 μM), serum (20% v/v FBS), thrombin (1 U/mL), or VEGF (50 ng/mL) for 10 min. F-actin (green) and ppMLC (magenta) are labeled. Scale bars are 25 μm. (b) Fold change in ppMLC immunofluorescence in cells stimulated with BSA (10 μg/mL), histamine (His; 3 μM), LPA (10 μg/mL), S1P (0.5 μM), serum (20% v/v FBS), thrombin (Thr; 1 U/mL), or VEGF (50 ng/mL) for 10 (white bars) and 30 (black bars) min. ppMLC is normalized to the BSA treatment condition for each stimulation duration. Data for each treatment condition is the average of n = 20 cells from one experiment. Error bars indicate SEM. * p
Figure Legend Snippet: Effects of vasoactive agonists on actin organization and myosin activity (a) Representative images of cells stimulated with BSA (10 μg/mL), histamine (3 μM), LPA (10 μg/mL), S1P (0.5 μM), serum (20% v/v FBS), thrombin (1 U/mL), or VEGF (50 ng/mL) for 10 min. F-actin (green) and ppMLC (magenta) are labeled. Scale bars are 25 μm. (b) Fold change in ppMLC immunofluorescence in cells stimulated with BSA (10 μg/mL), histamine (His; 3 μM), LPA (10 μg/mL), S1P (0.5 μM), serum (20% v/v FBS), thrombin (Thr; 1 U/mL), or VEGF (50 ng/mL) for 10 (white bars) and 30 (black bars) min. ppMLC is normalized to the BSA treatment condition for each stimulation duration. Data for each treatment condition is the average of n = 20 cells from one experiment. Error bars indicate SEM. * p

Techniques Used: Activity Assay, Labeling, Immunofluorescence

16) Product Images from "Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo"

Article Title: Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo

Journal: Nature biotechnology

doi: 10.1038/nbt.3081

DNA sequence specificity of Cas9-mediated endogenous gene cleavage in cultured human cells by plasmid transfection or by cationic lipid-mediated protein:sgRNA delivery using 1.6 µL RNAiMAX complexed with 100 nM Cas9 and 100 nM sgRNA. ( a ) T7EI assay was performed for on-target modification of endogenous CLTA , EMX , and VEGF genes in HEK293T cells. ( b–d ) On-target:off-target DNA modification ratio resulting from Cas9:sgRNA for plasmid transfection or cationic lipid-mediated protein:sgRNA delivery. The conditions for each treatment were adjusted to result in ~10% on-target cleavage, enabling a comparison of DNA cleavage specificity between the two delivery methods under conditions in which on-target gene modification efficiencies are similar. P values for a single biological replicate are listed in Supplementary Table 2 . Each on- and off-target sample was sequenced once with > 10,000 sequences analyzed per on-target sample and an average of > 111,000 sequences analyzed per off-target sample ( Supplementary Table 2 ). All protein:sgRNA deliveries and plasmid transfections were performed in 24-well format using 1.6 µL RNAiMAX in 550 µL DMEM-FBS without antibiotics. Error bars reflect s.d. from three biological replicates performed on different days.
Figure Legend Snippet: DNA sequence specificity of Cas9-mediated endogenous gene cleavage in cultured human cells by plasmid transfection or by cationic lipid-mediated protein:sgRNA delivery using 1.6 µL RNAiMAX complexed with 100 nM Cas9 and 100 nM sgRNA. ( a ) T7EI assay was performed for on-target modification of endogenous CLTA , EMX , and VEGF genes in HEK293T cells. ( b–d ) On-target:off-target DNA modification ratio resulting from Cas9:sgRNA for plasmid transfection or cationic lipid-mediated protein:sgRNA delivery. The conditions for each treatment were adjusted to result in ~10% on-target cleavage, enabling a comparison of DNA cleavage specificity between the two delivery methods under conditions in which on-target gene modification efficiencies are similar. P values for a single biological replicate are listed in Supplementary Table 2 . Each on- and off-target sample was sequenced once with > 10,000 sequences analyzed per on-target sample and an average of > 111,000 sequences analyzed per off-target sample ( Supplementary Table 2 ). All protein:sgRNA deliveries and plasmid transfections were performed in 24-well format using 1.6 µL RNAiMAX in 550 µL DMEM-FBS without antibiotics. Error bars reflect s.d. from three biological replicates performed on different days.

Techniques Used: Sequencing, Cell Culture, Plasmid Preparation, Transfection, T7EI Assay, Modification

Delivery of Cre recombinase to cultured human cells. ( a ) Fusion of either highly cationic (+36)GFP or highly anionic (−30)GFP to Cre recombinase. We used a HeLa reporter cell line that expresses DsRed upon Cre-mediated recombination to evaluate Cre delivery efficiency. ( b ) HeLa dsRed cells treated with 10 nM (−30)GFP-Cre and 1.5 µL of the cationic lipid formulation RNAiMAX. Cells were visualized after incubation for 48 hours in media containing 10% fetal bovine serum (FBS). ( c ) Delivery of (+36)GFP-Cre in 10% FBS media or in serum-free media, and (−30)GFP-Cre with or without the cationic lipid RNAiMAX (0.8 µL) in full-serum media. ( d ) Effect of cationic lipid dose on functional (−30)GFP-Cre delivery efficacy after 48 hours. ( e ) Comparison of several commercially available cationic lipids and polymers for functional delivery efficacy of (−30)dGFP-Cre. ( f ) RNAiMAX-mediated delivery of multiple anionic peptide or protein sequences fused to Cre. The net theoretical charge of the VP64 activation domain and the 3xFLAG tag is −22 and −7, respectively. All experiments were performed with 25 nM protein in 48-well plate format using 275 µL DMEM with 10% FBS and no antibiotics. Error bars reflect s.d. from three biological replicates performed on different days.
Figure Legend Snippet: Delivery of Cre recombinase to cultured human cells. ( a ) Fusion of either highly cationic (+36)GFP or highly anionic (−30)GFP to Cre recombinase. We used a HeLa reporter cell line that expresses DsRed upon Cre-mediated recombination to evaluate Cre delivery efficiency. ( b ) HeLa dsRed cells treated with 10 nM (−30)GFP-Cre and 1.5 µL of the cationic lipid formulation RNAiMAX. Cells were visualized after incubation for 48 hours in media containing 10% fetal bovine serum (FBS). ( c ) Delivery of (+36)GFP-Cre in 10% FBS media or in serum-free media, and (−30)GFP-Cre with or without the cationic lipid RNAiMAX (0.8 µL) in full-serum media. ( d ) Effect of cationic lipid dose on functional (−30)GFP-Cre delivery efficacy after 48 hours. ( e ) Comparison of several commercially available cationic lipids and polymers for functional delivery efficacy of (−30)dGFP-Cre. ( f ) RNAiMAX-mediated delivery of multiple anionic peptide or protein sequences fused to Cre. The net theoretical charge of the VP64 activation domain and the 3xFLAG tag is −22 and −7, respectively. All experiments were performed with 25 nM protein in 48-well plate format using 275 µL DMEM with 10% FBS and no antibiotics. Error bars reflect s.d. from three biological replicates performed on different days.

Techniques Used: Cell Culture, Incubation, Functional Assay, Activation Assay

Delivery of TALE transcriptional activators into cultured human cells. ( a ) Design of an 18.5-repeat TALE activator fused C-terminally to a VP64 activation domain and N-terminally to (−30)GFP and an NLS. The overall net theoretical charge of the fusion is −43. ( b ) Activation of NTF3 transcription by traditional transfection of plasmids encoding TALE-VP64 activators that target sites in the NTF3 gene, or by RNAiMAX cationic lipid-mediated delivery of the corresponding NTF3 -targeting (−30)GFP-TALE-VP64 proteins. For protein delivery experiments, 25 nM VEGF TALE, 25 nM NTF3 TALE 1, or 25 nM NTF3 TALEs 1–5 (5 nM each) were delivered with 1.5 µL RNAiMAX in 275 µL DMEM-FBS without antibiotics for 4 hours before being harvested. For plasmid transfections, a total of 300 ng of one or all five NTF3 TALE expression plasmids (60 ng each) were transfected with 0.8 µL Lipofectamine 2000 in 275 µL DMEM-FBS without antibiotics and harvested 48 hours later. Gene expression levels of harvested cells were measured by qRT-PCR and are normalized to GAPDH expression levels. Incubation times for TALE activators by plasmid transfection and protein delivery were those found to give maximal increases in NTF3 mRNA levels. ( c ) Time course of TALE activation for protein delivery and plasmid transfection by measuring NTF3 mRNA levels and then normalizing each method to the highest activation value achieved over any time point for that method. Optimal protein (25–50 nM) and lipid dosage (1.5 µL RNAiMAX) was used for each delivery technique. Error bars reflect s.d. from three biological replicates performed on different days.
Figure Legend Snippet: Delivery of TALE transcriptional activators into cultured human cells. ( a ) Design of an 18.5-repeat TALE activator fused C-terminally to a VP64 activation domain and N-terminally to (−30)GFP and an NLS. The overall net theoretical charge of the fusion is −43. ( b ) Activation of NTF3 transcription by traditional transfection of plasmids encoding TALE-VP64 activators that target sites in the NTF3 gene, or by RNAiMAX cationic lipid-mediated delivery of the corresponding NTF3 -targeting (−30)GFP-TALE-VP64 proteins. For protein delivery experiments, 25 nM VEGF TALE, 25 nM NTF3 TALE 1, or 25 nM NTF3 TALEs 1–5 (5 nM each) were delivered with 1.5 µL RNAiMAX in 275 µL DMEM-FBS without antibiotics for 4 hours before being harvested. For plasmid transfections, a total of 300 ng of one or all five NTF3 TALE expression plasmids (60 ng each) were transfected with 0.8 µL Lipofectamine 2000 in 275 µL DMEM-FBS without antibiotics and harvested 48 hours later. Gene expression levels of harvested cells were measured by qRT-PCR and are normalized to GAPDH expression levels. Incubation times for TALE activators by plasmid transfection and protein delivery were those found to give maximal increases in NTF3 mRNA levels. ( c ) Time course of TALE activation for protein delivery and plasmid transfection by measuring NTF3 mRNA levels and then normalizing each method to the highest activation value achieved over any time point for that method. Optimal protein (25–50 nM) and lipid dosage (1.5 µL RNAiMAX) was used for each delivery technique. Error bars reflect s.d. from three biological replicates performed on different days.

Techniques Used: Cell Culture, Activation Assay, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Incubation

17) Product Images from "Transplantation of a bone marrow mesenchymal stem cell line increases neuronal progenitor cell migration in a cerebral ischemia animal model"

Article Title: Transplantation of a bone marrow mesenchymal stem cell line increases neuronal progenitor cell migration in a cerebral ischemia animal model

Journal: Scientific Reports

doi: 10.1038/s41598-018-33030-9

Effects of B10 cells on neuronal cell migration in culture. ( A ) To assess the effects of B10-secreted molecules on neuronal migration, B10 cells were cultured to confluency in complete medium. Then the medium was changed to 0.5% FBS containing DMEM, cultured for further 24 h and the culture supernatant was used for A1 migration assay. DMEM-only, DMEM containing 0.5% FBS (medium) or A1 culture supernatant was used as control. * p
Figure Legend Snippet: Effects of B10 cells on neuronal cell migration in culture. ( A ) To assess the effects of B10-secreted molecules on neuronal migration, B10 cells were cultured to confluency in complete medium. Then the medium was changed to 0.5% FBS containing DMEM, cultured for further 24 h and the culture supernatant was used for A1 migration assay. DMEM-only, DMEM containing 0.5% FBS (medium) or A1 culture supernatant was used as control. * p

Techniques Used: Migration, Cell Culture

18) Product Images from "The prognostic significance of Cdc6 and Cdt1 in breast cancer"

Article Title: The prognostic significance of Cdc6 and Cdt1 in breast cancer

Journal: Scientific Reports

doi: 10.1038/s41598-017-00998-9

The expression of Cdc6 and Cdt1 in breast cell lines. Histograms showing ( A ) Cdc6 and ( B ) Cdt1 mRNA expression as measured by qPCR in normal breast epithelial cell line MCF10A, and two breast cancer cell lines, MCF7 and MDA MB231 in medium without Fetal Bovine Serum (FBS). ( C ) Western blot analysis of Cdc6, Cdt1 and β-actin in MCF10A, MCF7 and MDA MB231 cells in normal culture media (left panel) and in media without Fetal Bovine Serum (FBS; right panel).
Figure Legend Snippet: The expression of Cdc6 and Cdt1 in breast cell lines. Histograms showing ( A ) Cdc6 and ( B ) Cdt1 mRNA expression as measured by qPCR in normal breast epithelial cell line MCF10A, and two breast cancer cell lines, MCF7 and MDA MB231 in medium without Fetal Bovine Serum (FBS). ( C ) Western blot analysis of Cdc6, Cdt1 and β-actin in MCF10A, MCF7 and MDA MB231 cells in normal culture media (left panel) and in media without Fetal Bovine Serum (FBS; right panel).

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Western Blot

19) Product Images from "Changes in expression of cartilaginous genes during chondrogenesis of Wharton’s jelly mesenchymal stem cells on three-dimensional biodegradable poly(L-lactide-co-glycolide) scaffolds"

Article Title: Changes in expression of cartilaginous genes during chondrogenesis of Wharton’s jelly mesenchymal stem cells on three-dimensional biodegradable poly(L-lactide-co-glycolide) scaffolds

Journal: Cellular & Molecular Biology Letters

doi: 10.1186/s11658-016-0012-2

Transcriptional activity of collagen type II, aggrecan, collagen type I, and collagen type III genes expressed as fold change in relation to control (expression = 1) in WJ-MSCs cultured for 21 days on: a 2D – monolayer, b 3D PLGA scaffold. Control culture was carried out in growth medium in: a 2D – monolayer, b PLGA scaffold. Growth medium contained inter alia α-MEM medium with 20 % FBS. Chondrogenic medium contained inter alia differentiation factors such as dexamethasone and TGFβ3, ascorbic acid 2-phosphate, ITS + Premix, sodium pyruvate and 2 % FBS. * p
Figure Legend Snippet: Transcriptional activity of collagen type II, aggrecan, collagen type I, and collagen type III genes expressed as fold change in relation to control (expression = 1) in WJ-MSCs cultured for 21 days on: a 2D – monolayer, b 3D PLGA scaffold. Control culture was carried out in growth medium in: a 2D – monolayer, b PLGA scaffold. Growth medium contained inter alia α-MEM medium with 20 % FBS. Chondrogenic medium contained inter alia differentiation factors such as dexamethasone and TGFβ3, ascorbic acid 2-phosphate, ITS + Premix, sodium pyruvate and 2 % FBS. * p

Techniques Used: Activity Assay, Expressing, Cell Culture

Transcriptional activity of collagen type II, aggrecan, collagen type I, and collagen type III genes expressed as fold change in relation to control (expression = 1) in chondrocytes cultured for 21 days on: a 2D – monolayer, b 3D – PLGA scaffold. Control culture was carried out in growth medium in: a 2D – monolayer, b PLGA scaffold. Growth medium contained inter alia α-MEM medium with 10 % FBS. Chondrogenic medium contained inter alia differentiation factors such as dexamethasone and TGFβ3, ascorbic acid 2-phosphate, ITS + Premix, sodium pyruvate and 2 % FBS. * p
Figure Legend Snippet: Transcriptional activity of collagen type II, aggrecan, collagen type I, and collagen type III genes expressed as fold change in relation to control (expression = 1) in chondrocytes cultured for 21 days on: a 2D – monolayer, b 3D – PLGA scaffold. Control culture was carried out in growth medium in: a 2D – monolayer, b PLGA scaffold. Growth medium contained inter alia α-MEM medium with 10 % FBS. Chondrogenic medium contained inter alia differentiation factors such as dexamethasone and TGFβ3, ascorbic acid 2-phosphate, ITS + Premix, sodium pyruvate and 2 % FBS. * p

Techniques Used: Activity Assay, Expressing, Cell Culture

20) Product Images from "Photodynamic Inactivation Potentiates the Susceptibility of Antifungal Agents against the Planktonic and Biofilm Cells of Candida albicans"

Article Title: Photodynamic Inactivation Potentiates the Susceptibility of Antifungal Agents against the Planktonic and Biofilm Cells of Candida albicans

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19020434

The degree of cell damage post-PDI in C. albicans biofilms. ( A ) The growth ability of biofilm cells was measured at different time points post PDI (2.5 mM TBO plus 50 J/cm 2 ). Cells obtained from PDI-treated biofilms were suspended in YPD medium and subjected to a plate count. Each point is the mean of results obtained from three independent experiments and shown as mean ± SD. ( B ) Effect of PDI on hyphal formation in C. albicans biofilms. Cells obtained from PDI-treated biofilms were incubated in YPD medium supplemented with 10% FBS for 3 h. The ability of hyphal formation was observed and counted under microscope to determine the ratio of hyphal formation. The scale bar in the left panel is 20 µm. ☐: non-PDI. ■: PDI. Each value is obtained from three independent experiments and shown as the mean ± SD. *** p
Figure Legend Snippet: The degree of cell damage post-PDI in C. albicans biofilms. ( A ) The growth ability of biofilm cells was measured at different time points post PDI (2.5 mM TBO plus 50 J/cm 2 ). Cells obtained from PDI-treated biofilms were suspended in YPD medium and subjected to a plate count. Each point is the mean of results obtained from three independent experiments and shown as mean ± SD. ( B ) Effect of PDI on hyphal formation in C. albicans biofilms. Cells obtained from PDI-treated biofilms were incubated in YPD medium supplemented with 10% FBS for 3 h. The ability of hyphal formation was observed and counted under microscope to determine the ratio of hyphal formation. The scale bar in the left panel is 20 µm. ☐: non-PDI. ■: PDI. Each value is obtained from three independent experiments and shown as the mean ± SD. *** p

Techniques Used: Incubation, Microscopy

21) Product Images from "Identification of valid reference genes for mRNA and microRNA normalisation in prostate cancer cell lines"

Article Title: Identification of valid reference genes for mRNA and microRNA normalisation in prostate cancer cell lines

Journal: Scientific Reports

doi: 10.1038/s41598-018-19458-z

Expression stability and ranking of the six npcRNA reference genes as calculated by geNorm in various DHT treatments and all samples sets. Cells were cultured using RPMI 1640 medium containing 10% charcoal-stripped FBS for 24 hours prior to treatment with different DHT concentrations. cDNA for the qPCR experiments was prepared from these cells. The mean expression stability (M) was calculated by geNorm program. The least stable genes are presented on the left and the most stable on the right side of the plot.
Figure Legend Snippet: Expression stability and ranking of the six npcRNA reference genes as calculated by geNorm in various DHT treatments and all samples sets. Cells were cultured using RPMI 1640 medium containing 10% charcoal-stripped FBS for 24 hours prior to treatment with different DHT concentrations. cDNA for the qPCR experiments was prepared from these cells. The mean expression stability (M) was calculated by geNorm program. The least stable genes are presented on the left and the most stable on the right side of the plot.

Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

Box plots of absolute Cq values for each mRNA reference gene ( A ) and each npcRNA reference gene ( B ). Cells were cultured using RPMI 1640 medium containing 10% charcoal-stripped FBS for 24 hours, and then treated with different DHT concentrations. cDNA was prepared from these cells for the qPCR experiments. Expression of selected mRNA genes displayed as Cq across all prostate cell lines treated with various concentrations of DHT. The median is indicated by a line in each box, which in turn represents the 25 th and 75 th percentile. Whiskers indicate the 10/90 percentile ranges, and circles represent potential outliers.
Figure Legend Snippet: Box plots of absolute Cq values for each mRNA reference gene ( A ) and each npcRNA reference gene ( B ). Cells were cultured using RPMI 1640 medium containing 10% charcoal-stripped FBS for 24 hours, and then treated with different DHT concentrations. cDNA was prepared from these cells for the qPCR experiments. Expression of selected mRNA genes displayed as Cq across all prostate cell lines treated with various concentrations of DHT. The median is indicated by a line in each box, which in turn represents the 25 th and 75 th percentile. Whiskers indicate the 10/90 percentile ranges, and circles represent potential outliers.

Techniques Used: Cell Culture, Real-time Polymerase Chain Reaction, Expressing

Relative quantification of miR-141 expression depends on different npcRNA reference genes. Cells were cultured using RPMI 1640 medium containing 10% charcoal-stripping FBS for 24 hours prior to treatment with different DHT concentrations. The cDNA was prepared from these cells for the amplification of miR-141 and npcRNA reference genes by qPCR. Relative expression of miR-141 across all cell lines was normalised with the best reference genes combination ( miR-16-miR-1228-3p ) ( A ), by the most stable single gene miR-16 ( B ) or miR-1228-3p ( C ), by the least stable single gene miR-130b ( D ), or RNU43 ( E ), or RNU6-2 ( F ). Y-axis indicates the fold change of relative expression of miR-141 in RWPE-1 cells set to 1. Error bars indicate the standard error (± SE) evaluated from three biological replicates. * and **Indicate P
Figure Legend Snippet: Relative quantification of miR-141 expression depends on different npcRNA reference genes. Cells were cultured using RPMI 1640 medium containing 10% charcoal-stripping FBS for 24 hours prior to treatment with different DHT concentrations. The cDNA was prepared from these cells for the amplification of miR-141 and npcRNA reference genes by qPCR. Relative expression of miR-141 across all cell lines was normalised with the best reference genes combination ( miR-16-miR-1228-3p ) ( A ), by the most stable single gene miR-16 ( B ) or miR-1228-3p ( C ), by the least stable single gene miR-130b ( D ), or RNU43 ( E ), or RNU6-2 ( F ). Y-axis indicates the fold change of relative expression of miR-141 in RWPE-1 cells set to 1. Error bars indicate the standard error (± SE) evaluated from three biological replicates. * and **Indicate P

Techniques Used: Expressing, Cell Culture, Stripping Membranes, Amplification, Real-time Polymerase Chain Reaction

Expression stability and ranking of the ten reference genes as calculated by geNorm in various DHT treatments and all samples sets. Cells were cultured using RPMI 1640 medium containing 10% charcoal-stripped FBS for 24 hours prior to treatment with different DHT concentrations. cDNA was prepared from these cells and then used to perform qPCR experiments. The mean expression stability (M) was calculated by stepwise exclusion of the least stable gene by geNorm program. A lower M value indicates more stable expression. The least stable genes are presented on the left and the most stable on the right side of the plot.
Figure Legend Snippet: Expression stability and ranking of the ten reference genes as calculated by geNorm in various DHT treatments and all samples sets. Cells were cultured using RPMI 1640 medium containing 10% charcoal-stripped FBS for 24 hours prior to treatment with different DHT concentrations. cDNA was prepared from these cells and then used to perform qPCR experiments. The mean expression stability (M) was calculated by stepwise exclusion of the least stable gene by geNorm program. A lower M value indicates more stable expression. The least stable genes are presented on the left and the most stable on the right side of the plot.

Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

Determination of the optimal number of reference genes for normalisation. Cells were cultured using RPMI 1640 medium containing 10% charcoal-stripped FBS for 24 hours prior to treatment with different DHT concentrations. The obtained Cq values were analysed and a pair-wise variation value (Vn/n + 1) generated by geNorm analysis across all prostate cancer cell lines with different DHT concentrations for mRNA reference genes ( A ), and for miRNA reference genes ( B ).
Figure Legend Snippet: Determination of the optimal number of reference genes for normalisation. Cells were cultured using RPMI 1640 medium containing 10% charcoal-stripped FBS for 24 hours prior to treatment with different DHT concentrations. The obtained Cq values were analysed and a pair-wise variation value (Vn/n + 1) generated by geNorm analysis across all prostate cancer cell lines with different DHT concentrations for mRNA reference genes ( A ), and for miRNA reference genes ( B ).

Techniques Used: Cell Culture, Generated

Validation of DHT treatments by Western blot. AR+ cells (LNCaP and 22RV1), AR − cells (DU145 and PC-3), and normal prostate epithelial cell (RWPE-1) were cultured using RPMI 1640 medium containing 10% charcoal-stripped FBS for 24 hours, and then treated with different DHT treatments. Validation of DHT treatments were performed through Western blot experiments using PSA as a marker, and GAPDH and Actin as control proteins. The grouping of blots were cropped from different parts of the same gel, or from different gels. Full-length unadjusted Western blot images for this figure as shown in Supplementary Fig. S3 .
Figure Legend Snippet: Validation of DHT treatments by Western blot. AR+ cells (LNCaP and 22RV1), AR − cells (DU145 and PC-3), and normal prostate epithelial cell (RWPE-1) were cultured using RPMI 1640 medium containing 10% charcoal-stripped FBS for 24 hours, and then treated with different DHT treatments. Validation of DHT treatments were performed through Western blot experiments using PSA as a marker, and GAPDH and Actin as control proteins. The grouping of blots were cropped from different parts of the same gel, or from different gels. Full-length unadjusted Western blot images for this figure as shown in Supplementary Fig. S3 .

Techniques Used: Western Blot, Cell Culture, Marker

Relative quantification of PCA3 expression depends on mRNA reference gene. Cells were cultured using RPMI 1640 medium containing 10% charcoal-stripped FBS for 24 hours prior to treatment with different DHT concentrations. The cDNA was prepared from these cells for the amplification of PCA3 and mRNA reference genes by qPCR. Relative expression of PCA3 across all cell lines was normalised by the best reference genes combination ( ACTB-GAPDH ) ( A ), by the most stable single gene ACTB ( B ) or GAPDH ( C ), by the least stable single gene 18 S rRNA ( D ), or K-ALPHA-1 ( E ), or ALAS1 ( F ). Y-axis indicates the fold change of relative expression of PCA3 in RWPE-1 cells set to 1. Error bars indicate the standard error (±SE) evaluated from three biological replicates. * and **Indicate P
Figure Legend Snippet: Relative quantification of PCA3 expression depends on mRNA reference gene. Cells were cultured using RPMI 1640 medium containing 10% charcoal-stripped FBS for 24 hours prior to treatment with different DHT concentrations. The cDNA was prepared from these cells for the amplification of PCA3 and mRNA reference genes by qPCR. Relative expression of PCA3 across all cell lines was normalised by the best reference genes combination ( ACTB-GAPDH ) ( A ), by the most stable single gene ACTB ( B ) or GAPDH ( C ), by the least stable single gene 18 S rRNA ( D ), or K-ALPHA-1 ( E ), or ALAS1 ( F ). Y-axis indicates the fold change of relative expression of PCA3 in RWPE-1 cells set to 1. Error bars indicate the standard error (±SE) evaluated from three biological replicates. * and **Indicate P

Techniques Used: Expressing, Cell Culture, Amplification, Real-time Polymerase Chain Reaction

22) Product Images from "Aqueous Humor Rapidly Stimulates Myocilin Secretion from Human Trabecular Meshwork Cells"

Article Title: Aqueous Humor Rapidly Stimulates Myocilin Secretion from Human Trabecular Meshwork Cells

Journal: Experimental eye research

doi: 10.1016/j.exer.2010.09.017

HCE cell conditioned media and myocilin secretion from NTM cells Conditioned media collected from HCE cells maintained for 72 hours in FBS (10%), human serum (HS, 0.2%), porcine aqueous humor (pAq, 50%) or BSA (0.1%) was incubated with NTM cells for 24 hours. Myocilin accumulation in HCE conditioned media following incubation with NTM cells was determined by Western blot. UnCM=unconditioned media, CM=conditioned media.
Figure Legend Snippet: HCE cell conditioned media and myocilin secretion from NTM cells Conditioned media collected from HCE cells maintained for 72 hours in FBS (10%), human serum (HS, 0.2%), porcine aqueous humor (pAq, 50%) or BSA (0.1%) was incubated with NTM cells for 24 hours. Myocilin accumulation in HCE conditioned media following incubation with NTM cells was determined by Western blot. UnCM=unconditioned media, CM=conditioned media.

Techniques Used: Incubation, Western Blot

Human aqueous humor stimulation of myocilin secretion from trabecular meshwork cells Human NTM cells (mean ± SEM, n=3) were treated with 50% hAq or 10% FBS for up to 48 hours and myocilin accumulation in conditioned media was quantified by Western blot (representative image shown under bar graph). Myocilin in 50% hAq conditioned media was normalized to myocilin levels in unconditioned media (UnCM) containing 50% hAq. Asterisk (*) represents p
Figure Legend Snippet: Human aqueous humor stimulation of myocilin secretion from trabecular meshwork cells Human NTM cells (mean ± SEM, n=3) were treated with 50% hAq or 10% FBS for up to 48 hours and myocilin accumulation in conditioned media was quantified by Western blot (representative image shown under bar graph). Myocilin in 50% hAq conditioned media was normalized to myocilin levels in unconditioned media (UnCM) containing 50% hAq. Asterisk (*) represents p

Techniques Used: Western Blot

23) Product Images from "Cellularity and structure of fresh human coronary thrombectomy specimens; presence of cells with markers of progenitor cells"

Article Title: Cellularity and structure of fresh human coronary thrombectomy specimens; presence of cells with markers of progenitor cells

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2012.01629.x

Immunohistochemistry of cells grown in M199 20% FBS: pancytokeratin (negative), CD31, CD34, vWF (A–D).
Figure Legend Snippet: Immunohistochemistry of cells grown in M199 20% FBS: pancytokeratin (negative), CD31, CD34, vWF (A–D).

Techniques Used: Immunohistochemistry

Growth pattern of cultured cells grown in DMEM 20% FBS (A–C); shapes of cultured cells, dividing cells, multinucleated cells (D–F), spindle-shaped cells reminiscent of mesenchymal cells/fibroblasts (G); cells grown in M199 20% FBS (EC medium) grew in small clusters (H) and then became confluent in a cobblestone pattern (I).
Figure Legend Snippet: Growth pattern of cultured cells grown in DMEM 20% FBS (A–C); shapes of cultured cells, dividing cells, multinucleated cells (D–F), spindle-shaped cells reminiscent of mesenchymal cells/fibroblasts (G); cells grown in M199 20% FBS (EC medium) grew in small clusters (H) and then became confluent in a cobblestone pattern (I).

Techniques Used: Cell Culture

24) Product Images from "Peroxidasin contributes to lung host defense by direct binding and killing of gram-negative bacteria"

Article Title: Peroxidasin contributes to lung host defense by direct binding and killing of gram-negative bacteria

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007026

PXDN is expressed in the lung epithelium and contributes to bacterial killing. (A) Detection of PXDN in human bronchoalveolar lavage fluid (BALF). Right middle lobe BALF was collected and processed within 2 h. BALF samples were then centrifuged at 400 x g for 10 min at 4°C. BALF were concentrated by Centricon (~10x). Samples of BALF (1), positive control of human plasma containing ~100 ng of PXDN (2), and purified MPO (50 ng) (3) were subjected to immunoblotting using anti-PXDN or anti-MPO antibodies. (B) IHC of the lung. IHC of lung sections from WT and PXDN-deficient mice was performed using anti-PXDN antibody (1:600). Images were taken using BZ-X710 All-in-One Fluorescence Microscope. PXDN: dark brown. Magnification: 400x. (C) PXDN expresses in mouse primary lung type II alveolar epithelial cells (AECs). Upper panel showed immunoblot analysis of AECs and fibroblasts while lower panel showed phase contrast microscopy of AECs (left) and fibroblasts (right); magnification 100x. Cell lysates were subjected to conventional immunoblotting by using anti-PXDN antibody. β-actin was used as loading control. (D) LPS induces PXDN expression. AECs were induced by LPS as indicated. The cell lysates were subject to immunoblot analysis as in ( C ). (E) Mouse primary type II AECs mediate P . aeruginosa killing. AECs were grown in 12-well plate in DMEM with 10% FBS without antibiotics until 70% confluence. Cells were serum-starved for 16 h prior to bactericidal assays; cells were stimulated with/without TGF-β and hematin/NaBu. Cells and the overlying medium (supernatant) were separated and utilized for evaluation of PXDN-mediated bactericidal killing. 1 mL of fresh DMEM containing 10 4 P . aeruginosa strain K and 10 μM H 2 O 2 was added to AECs or supernatant. The mixture was incubated at 37°C for 1 h, and then plated on LB agar plates prior to overnight incubation at 37°C. The control group contained untreated AECs and P . aeruginosa . The relative survival rate was calculated as CFUs in the experimental group divided by those in the control group; * P
Figure Legend Snippet: PXDN is expressed in the lung epithelium and contributes to bacterial killing. (A) Detection of PXDN in human bronchoalveolar lavage fluid (BALF). Right middle lobe BALF was collected and processed within 2 h. BALF samples were then centrifuged at 400 x g for 10 min at 4°C. BALF were concentrated by Centricon (~10x). Samples of BALF (1), positive control of human plasma containing ~100 ng of PXDN (2), and purified MPO (50 ng) (3) were subjected to immunoblotting using anti-PXDN or anti-MPO antibodies. (B) IHC of the lung. IHC of lung sections from WT and PXDN-deficient mice was performed using anti-PXDN antibody (1:600). Images were taken using BZ-X710 All-in-One Fluorescence Microscope. PXDN: dark brown. Magnification: 400x. (C) PXDN expresses in mouse primary lung type II alveolar epithelial cells (AECs). Upper panel showed immunoblot analysis of AECs and fibroblasts while lower panel showed phase contrast microscopy of AECs (left) and fibroblasts (right); magnification 100x. Cell lysates were subjected to conventional immunoblotting by using anti-PXDN antibody. β-actin was used as loading control. (D) LPS induces PXDN expression. AECs were induced by LPS as indicated. The cell lysates were subject to immunoblot analysis as in ( C ). (E) Mouse primary type II AECs mediate P . aeruginosa killing. AECs were grown in 12-well plate in DMEM with 10% FBS without antibiotics until 70% confluence. Cells were serum-starved for 16 h prior to bactericidal assays; cells were stimulated with/without TGF-β and hematin/NaBu. Cells and the overlying medium (supernatant) were separated and utilized for evaluation of PXDN-mediated bactericidal killing. 1 mL of fresh DMEM containing 10 4 P . aeruginosa strain K and 10 μM H 2 O 2 was added to AECs or supernatant. The mixture was incubated at 37°C for 1 h, and then plated on LB agar plates prior to overnight incubation at 37°C. The control group contained untreated AECs and P . aeruginosa . The relative survival rate was calculated as CFUs in the experimental group divided by those in the control group; * P

Techniques Used: Positive Control, Purification, Immunohistochemistry, Mouse Assay, Fluorescence, Microscopy, Expressing, Incubation

25) Product Images from "DAB2IP loss confers the resistance of prostate cancer to androgen deprivation therapy through activating STAT3 and inhibiting apoptosis"

Article Title: DAB2IP loss confers the resistance of prostate cancer to androgen deprivation therapy through activating STAT3 and inhibiting apoptosis

Journal: Cell Death & Disease

doi: 10.1038/cddis.2015.289

STAT3/survivin signaling pathway mediates the resistance to ADT-induced apoptosis in DAB2IP-deficient PCa. C4-2 or LAPC-4 KD cells were pretreated with specific STAT3 inhibitor Stattic (10 or 20 μ M) for 1 h or transfected with siRNA oligonucleotides specific to STAT3 or survivin for 24 h, and then cultured in Phenol Red-free RPMI-1640+5% CS-FBS for another 48 h. ( a and b ) Cell lysates after treatment were subjected to western blotting for detecting p-STAT3 (Y705), t-STAT3, survivin, cleaved PARP and Caspase-3. Actin was used as a loading control ( c ) C4-2 cells after treatment were cultured in Phenol Red-free RPMI-1640+5% CS-FBS for another 48 h and cell growth was determined by MTT assay. ( d ) C4-2 cells after treatment were cultured in Phenol Red-free RPMI-1640+5% CS-FBS for another 48 h, then subjected to Annexin V/FITC staining and flow cytometric analysis. ( e ) C4-2 cells after treatment were cultured in Phenol Red-free RPMI-1640+5% CS-FBS for another 48 h, then subjected to JC-1 staining and flow cytometric analysis. All data (means±S.E.M.) shown in c , d and e were obtained from three independent experiments. * P
Figure Legend Snippet: STAT3/survivin signaling pathway mediates the resistance to ADT-induced apoptosis in DAB2IP-deficient PCa. C4-2 or LAPC-4 KD cells were pretreated with specific STAT3 inhibitor Stattic (10 or 20 μ M) for 1 h or transfected with siRNA oligonucleotides specific to STAT3 or survivin for 24 h, and then cultured in Phenol Red-free RPMI-1640+5% CS-FBS for another 48 h. ( a and b ) Cell lysates after treatment were subjected to western blotting for detecting p-STAT3 (Y705), t-STAT3, survivin, cleaved PARP and Caspase-3. Actin was used as a loading control ( c ) C4-2 cells after treatment were cultured in Phenol Red-free RPMI-1640+5% CS-FBS for another 48 h and cell growth was determined by MTT assay. ( d ) C4-2 cells after treatment were cultured in Phenol Red-free RPMI-1640+5% CS-FBS for another 48 h, then subjected to Annexin V/FITC staining and flow cytometric analysis. ( e ) C4-2 cells after treatment were cultured in Phenol Red-free RPMI-1640+5% CS-FBS for another 48 h, then subjected to JC-1 staining and flow cytometric analysis. All data (means±S.E.M.) shown in c , d and e were obtained from three independent experiments. * P

Techniques Used: Transfection, Cell Culture, Western Blot, MTT Assay, Staining, Flow Cytometry

DAB2IP controls the intrinsic apoptosis of PCa cells following ADT. PCa sublines were cultured in Phenol Red-free RPMI-1640+5% CS-FBS for 3 days. ( a ) C4-2 (left) and LAPC-4 (right) sublines were then collected for Annexin V/FITC staining and flow cytometric analysis, and data (means±S.E.M.) were obtained from three independent experiments; * P
Figure Legend Snippet: DAB2IP controls the intrinsic apoptosis of PCa cells following ADT. PCa sublines were cultured in Phenol Red-free RPMI-1640+5% CS-FBS for 3 days. ( a ) C4-2 (left) and LAPC-4 (right) sublines were then collected for Annexin V/FITC staining and flow cytometric analysis, and data (means±S.E.M.) were obtained from three independent experiments; * P

Techniques Used: Cell Culture, Staining, Flow Cytometry

DAB2IP modulates the cell survival of PCa cells under androgen-depleted condition. ( a – c ) C4-2 and LAPC-4 sublines were cultured in Phenol Red-free RPMI-1640+5% CS-FBS and cell growth was determined by MTT or colony formation assay; data (means±S.E.M.) were obtained from three independent experiments; * P
Figure Legend Snippet: DAB2IP modulates the cell survival of PCa cells under androgen-depleted condition. ( a – c ) C4-2 and LAPC-4 sublines were cultured in Phenol Red-free RPMI-1640+5% CS-FBS and cell growth was determined by MTT or colony formation assay; data (means±S.E.M.) were obtained from three independent experiments; * P

Techniques Used: Cell Culture, MTT Assay, Colony Assay

26) Product Images from "STAT3 Undergoes Acetylation-dependent Mitochondrial Translocation to Regulate Pyruvate Metabolism"

Article Title: STAT3 Undergoes Acetylation-dependent Mitochondrial Translocation to Regulate Pyruvate Metabolism

Journal: Scientific Reports

doi: 10.1038/srep39517

Insulin triggers conversion of glucose to fatty acids. ( a ) Cellular glucose levels were measured in control or STAT3−/− MEFs receiving insulin (5 μg/ml) treatment for 30 min or no treatment. ( b ) Seahorse extracellular acidification rates (ECAR) were measured in quadruplicate wells containing equal numbers of wild-type or STAT3−/− MEFs treated with insulin (5 μg/ml). ( c ) The Seahorse oxygen consumption rate (OCR) was measured in quadruplicate wells containing equal numbers of wild-type or STAT3−/− MEFs treated with insulin (5 μg/ml). ( d ) α-ketoglutaric acid concentrations were measured in control or STAT−/− MEFs receiving insulin (5 μg/ml) treatment for 30 min or no treatment. ( e ) Cytoplasmic and mitochondrial Acetyl-CoA levels were measured in MEFs treated with insulin for various times as indicated. ( f ) Oil red O staining of control MEFs and STAT3−/− MEFs, treated with or without insulin (5 μg/ml, 30 min). Lipid droplet accumulation was visualized with a microscope. ( g ) The IOD (integrated optical density) of the lipid droplets from ( c ) was measured with IPP software ( d ). ( h ) The IOD (integrated optical density) of lipid droplets was measured in STAT3−/− MEFs stained with Oil red O after SSR (90% DMEM and 10% FBS, 30 min), insulin (5 μg/ml, 30 min) and NAM (100 μM, 6 hrs) treatment.
Figure Legend Snippet: Insulin triggers conversion of glucose to fatty acids. ( a ) Cellular glucose levels were measured in control or STAT3−/− MEFs receiving insulin (5 μg/ml) treatment for 30 min or no treatment. ( b ) Seahorse extracellular acidification rates (ECAR) were measured in quadruplicate wells containing equal numbers of wild-type or STAT3−/− MEFs treated with insulin (5 μg/ml). ( c ) The Seahorse oxygen consumption rate (OCR) was measured in quadruplicate wells containing equal numbers of wild-type or STAT3−/− MEFs treated with insulin (5 μg/ml). ( d ) α-ketoglutaric acid concentrations were measured in control or STAT−/− MEFs receiving insulin (5 μg/ml) treatment for 30 min or no treatment. ( e ) Cytoplasmic and mitochondrial Acetyl-CoA levels were measured in MEFs treated with insulin for various times as indicated. ( f ) Oil red O staining of control MEFs and STAT3−/− MEFs, treated with or without insulin (5 μg/ml, 30 min). Lipid droplet accumulation was visualized with a microscope. ( g ) The IOD (integrated optical density) of the lipid droplets from ( c ) was measured with IPP software ( d ). ( h ) The IOD (integrated optical density) of lipid droplets was measured in STAT3−/− MEFs stained with Oil red O after SSR (90% DMEM and 10% FBS, 30 min), insulin (5 μg/ml, 30 min) and NAM (100 μM, 6 hrs) treatment.

Techniques Used: Staining, Microscopy, Software

27) Product Images from "Modeling and antitumor studies of a modified L–penetratin peptide targeting E2F in lung cancer and prostate cancer"

Article Title: Modeling and antitumor studies of a modified L–penetratin peptide targeting E2F in lung cancer and prostate cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.26064

D- Arg Peptide is more effective than the L- Arg Peptide in (A) DU145 cells and (B) H196 SCLC cells at 24 hours of treatment In this assay, 5000 cells per well were plated for the 24-hour time point in a 96 well plate on day zero in RPMI media containing 10% FBS. After 24 hours either the D- Arg peptide or the L- Arg Peptide was added in serial dilutions across the plate. Cell viability was assessed at 24 hours of treatment by measuring the absorption at 490nm using the MTS tetrazolium Promega CellTitre 96 ® Aqueous One Solution Cell proliferation assay according to the manufacturer’s instructions. Each time point was done in triplicate and values are represented by mean with standard mean deviation.
Figure Legend Snippet: D- Arg Peptide is more effective than the L- Arg Peptide in (A) DU145 cells and (B) H196 SCLC cells at 24 hours of treatment In this assay, 5000 cells per well were plated for the 24-hour time point in a 96 well plate on day zero in RPMI media containing 10% FBS. After 24 hours either the D- Arg peptide or the L- Arg Peptide was added in serial dilutions across the plate. Cell viability was assessed at 24 hours of treatment by measuring the absorption at 490nm using the MTS tetrazolium Promega CellTitre 96 ® Aqueous One Solution Cell proliferation assay according to the manufacturer’s instructions. Each time point was done in triplicate and values are represented by mean with standard mean deviation.

Techniques Used: Proliferation Assay

A. D- Arg Peptide incubated in 10% FBS RPMI media at 37°C for 24 hours is resistant to proteolysis; it is as effective as the non-incubated D- Arg Peptide in DU145 cells. D- Arg Peptide was incubated in RPMI media containing 10% FBS at 37°C for 24 hours. DU145 cells were seeded in a 96 well plate at 5000 cells in each well. After 24 hours either the incubated D- Arg Peptide or the non-incubated D- Arg Peptide was added to the cells. B. D- Arg and L-Arg Peptide incubated in 10% FBS RPMI media at 37°C for 24 hours. DU145 cells were seeded in a 96 well plate at 5000 cells in each well. After 24 hours either the incubated D- Arg Peptide or the L- Arg Peptide was added to the cells. The cell viability in both experiments were assessed after another 24 hours by measuring the absorption at 490nm using the MTS tetrazolium Promega CellTitre 96 ® Aqueous One Solution Cell proliferation assay according to the manufacturer’s instructions. Each time point was done in triplicate and values are represented by mean with standard mean deviation.
Figure Legend Snippet: A. D- Arg Peptide incubated in 10% FBS RPMI media at 37°C for 24 hours is resistant to proteolysis; it is as effective as the non-incubated D- Arg Peptide in DU145 cells. D- Arg Peptide was incubated in RPMI media containing 10% FBS at 37°C for 24 hours. DU145 cells were seeded in a 96 well plate at 5000 cells in each well. After 24 hours either the incubated D- Arg Peptide or the non-incubated D- Arg Peptide was added to the cells. B. D- Arg and L-Arg Peptide incubated in 10% FBS RPMI media at 37°C for 24 hours. DU145 cells were seeded in a 96 well plate at 5000 cells in each well. After 24 hours either the incubated D- Arg Peptide or the L- Arg Peptide was added to the cells. The cell viability in both experiments were assessed after another 24 hours by measuring the absorption at 490nm using the MTS tetrazolium Promega CellTitre 96 ® Aqueous One Solution Cell proliferation assay according to the manufacturer’s instructions. Each time point was done in triplicate and values are represented by mean with standard mean deviation.

Techniques Used: Incubation, Proliferation Assay

28) Product Images from "Three-dimensional retinal organoids from mouse pluripotent stem cells mimic in vivo development with enhanced stratification and rod photoreceptor differentiation"

Article Title: Three-dimensional retinal organoids from mouse pluripotent stem cells mimic in vivo development with enhanced stratification and rod photoreceptor differentiation

Journal: Molecular Vision

doi:

Generation of retinal organoids from Nrl -GFP mouse ESCs and iPSCs. A : Schematic representation of the High Efficiency Hypoxia Induced Generation of Photoreceptors in Retinal Organoids (HIPRO) protocol for differentiation of mouse stem cells into the three-dimensional (3D) neural retina. GMEM, Glasgow minimum essential medium; KSR, knockout serum replacement; poly-HEMA, polyhydroxyethylmethacrylate; RMM, retinal maturation medium constituted of DMEM/F12 with GlutaMAX, 1X penicillin-streptomycin, and 1X N 2 supplement; RA, retinoic acid; FBS, fetal bovine serum; N2, N2 supplement; B27, B27 supplement. B : Morphology of organoids at different time points using normoxic and hypoxic conditions. C : Percentage of organoids that form optic vesicles at day (D)7 and optic cups at D10. Optic vesicles are defined as protrusions of neuroepithelia from the organoids. Optic cups are characterized by a hinge region due to inward folding of the neuroepithelia of the optic vesicles. The data were obtained from ten independent experiments (n = 10, each had a total of 144 organoids) and represented as mean ± standard error of the mean (SEM); * p
Figure Legend Snippet: Generation of retinal organoids from Nrl -GFP mouse ESCs and iPSCs. A : Schematic representation of the High Efficiency Hypoxia Induced Generation of Photoreceptors in Retinal Organoids (HIPRO) protocol for differentiation of mouse stem cells into the three-dimensional (3D) neural retina. GMEM, Glasgow minimum essential medium; KSR, knockout serum replacement; poly-HEMA, polyhydroxyethylmethacrylate; RMM, retinal maturation medium constituted of DMEM/F12 with GlutaMAX, 1X penicillin-streptomycin, and 1X N 2 supplement; RA, retinoic acid; FBS, fetal bovine serum; N2, N2 supplement; B27, B27 supplement. B : Morphology of organoids at different time points using normoxic and hypoxic conditions. C : Percentage of organoids that form optic vesicles at day (D)7 and optic cups at D10. Optic vesicles are defined as protrusions of neuroepithelia from the organoids. Optic cups are characterized by a hinge region due to inward folding of the neuroepithelia of the optic vesicles. The data were obtained from ten independent experiments (n = 10, each had a total of 144 organoids) and represented as mean ± standard error of the mean (SEM); * p

Techniques Used: Knock-Out

29) Product Images from "Adaptive response to l‐serine deficiency is mediated by p38 MAPK activation via 1‐deoxysphinganine in normal fibroblasts"

Article Title: Adaptive response to l‐serine deficiency is mediated by p38 MAPK activation via 1‐deoxysphinganine in normal fibroblasts

Journal: FEBS Open Bio

doi: 10.1002/2211-5463.12038

External l ‐Ser is necessary for proliferation of KO ‐ MEF s. (A) The indicated cell lines were cultured in DMEM containing 10% FBS for 24 h, and Phgdh expression was examined by western blot analysis. (B–D) Live cell numbers of WT ‐, HT ‐, and KO ‐ MEF s were determined after incubation in complete DMEM medium containing 10% FBS for 0, 24, 48, and 72 h (B); EMEM containing 10% FBS for 0, 48, 96, and 144 h (Dunnett's post hoc test, †† P
Figure Legend Snippet: External l ‐Ser is necessary for proliferation of KO ‐ MEF s. (A) The indicated cell lines were cultured in DMEM containing 10% FBS for 24 h, and Phgdh expression was examined by western blot analysis. (B–D) Live cell numbers of WT ‐, HT ‐, and KO ‐ MEF s were determined after incubation in complete DMEM medium containing 10% FBS for 0, 24, 48, and 72 h (B); EMEM containing 10% FBS for 0, 48, 96, and 144 h (Dunnett's post hoc test, †† P

Techniques Used: Cell Culture, Expressing, Western Blot, Incubation

30) Product Images from "Loss of STK11 expression is an early event in prostate carcinogenesis and predicts therapeutic response to targeted therapy against MAPK/p38"

Article Title: Loss of STK11 expression is an early event in prostate carcinogenesis and predicts therapeutic response to targeted therapy against MAPK/p38

Journal: Autophagy

doi: 10.1080/15548627.2015.1091910

Role of STK11 as a predictive marker of MAPK14/11i-based therapy response in PCa irrespective of AR status. ( A, B ) LNCaP cells were cultured in phenol red–free RPMI containing 10% charcoal stripped-FBS (CS-FBS) or RPMI containing 10% FBS for 30 h
Figure Legend Snippet: Role of STK11 as a predictive marker of MAPK14/11i-based therapy response in PCa irrespective of AR status. ( A, B ) LNCaP cells were cultured in phenol red–free RPMI containing 10% charcoal stripped-FBS (CS-FBS) or RPMI containing 10% FBS for 30 h

Techniques Used: Marker, Cell Culture

31) Product Images from "Latexin inhibits the proliferation of CD133+ miapaca-2 pancreatic cancer stem-like cells"

Article Title: Latexin inhibits the proliferation of CD133+ miapaca-2 pancreatic cancer stem-like cells

Journal: World Journal of Surgical Oncology

doi: 10.1186/1477-7819-12-404

Proliferation ability of the CD133+ and CD133- cells in serum-free medium. CD133- cells showed slow growth and good adherence; and almost all cells died within 2 weeks (A) . CD133+ cells formed floating spheres (B) . When the floating spheres were cultured in DMEM supplemented with 10% FBS, the cells were adherent and proliferated similarly to unsorted miapaca-2 cells (C) . The expression of CD133 mRNA in CD133+ cells was dramatically higher than unsorted miapaca-2 cells and re-adherence CD133+ cells (D) . a P
Figure Legend Snippet: Proliferation ability of the CD133+ and CD133- cells in serum-free medium. CD133- cells showed slow growth and good adherence; and almost all cells died within 2 weeks (A) . CD133+ cells formed floating spheres (B) . When the floating spheres were cultured in DMEM supplemented with 10% FBS, the cells were adherent and proliferated similarly to unsorted miapaca-2 cells (C) . The expression of CD133 mRNA in CD133+ cells was dramatically higher than unsorted miapaca-2 cells and re-adherence CD133+ cells (D) . a P

Techniques Used: Cell Culture, Expressing

32) Product Images from "Neuroprotective effects of blockers for T-type calcium channels"

Article Title: Neuroprotective effects of blockers for T-type calcium channels

Journal: Molecular Neurodegeneration

doi: 10.1186/1750-1326-4-44

Neuroprotection by Nimodipine in long-term culture . Hippocampal neurons from E18 C57BL/6J mice were cultured for 7-8 days in neurobasal medium containing 2% FBS. (A) On the 8 DIV, the medium was replenished and the neurons treated with either 0 or 1 μM nimodipine (control, n = 6; nimodipine, n = 6). Cell death was measured using LDH assay on 15 DIV. Mean values were expressed as % of control ± SEM. *p ≤ 0.05 and **p ≤ 0.01 compared with the control condition. (B) Cortical neurons were treated with either 0 or 1 μM nimodipine (control, n = 6; nimodipine, n = 6). Cell death was measured using LDH assay on 15 DIV. Mean values were expressed as % of control. *p ≤ 0.05 and **p ≤ 0.01 compared with the control condition using student's t test.
Figure Legend Snippet: Neuroprotection by Nimodipine in long-term culture . Hippocampal neurons from E18 C57BL/6J mice were cultured for 7-8 days in neurobasal medium containing 2% FBS. (A) On the 8 DIV, the medium was replenished and the neurons treated with either 0 or 1 μM nimodipine (control, n = 6; nimodipine, n = 6). Cell death was measured using LDH assay on 15 DIV. Mean values were expressed as % of control ± SEM. *p ≤ 0.05 and **p ≤ 0.01 compared with the control condition. (B) Cortical neurons were treated with either 0 or 1 μM nimodipine (control, n = 6; nimodipine, n = 6). Cell death was measured using LDH assay on 15 DIV. Mean values were expressed as % of control. *p ≤ 0.05 and **p ≤ 0.01 compared with the control condition using student's t test.

Techniques Used: Mouse Assay, Cell Culture, Lactate Dehydrogenase Assay

Neuronal protection by trimethadione . (A) E18 hippocampal neurons cultured in neurobasal medium with 2% FBS for 7-8 days. Medium was replenished at the 8 th day of culture and neurons treated with either 0 mM, 0.3 mM, 0.6 mM, or 0.9 mM (control, n = 16; treatment groups, n = 8 each). Cell death was performed with LDH assay 10 DIV (24 h later); mean LDH value expressed as % of control. *p ≤ 0.05 and **p ≤ 0.01 compared with the control condition. Raw data was used for one-way ANOVA. (B) E18 cortical neurons cultured in neurobasal medium with 2% FBS for 7-8 days. Medium was replenished on the 8 th day and neurons treated with either 0 mM, 0.3 mM, 0.6 mM, or 0.9 mM (n = 12 each). Cell viability was performed with LDH assay on 10 DIV. Mean LDH value expressed as % of control. *p ≤ 0.05 compared with the control using student's t test. Raw data was used for one-way ANOVA.
Figure Legend Snippet: Neuronal protection by trimethadione . (A) E18 hippocampal neurons cultured in neurobasal medium with 2% FBS for 7-8 days. Medium was replenished at the 8 th day of culture and neurons treated with either 0 mM, 0.3 mM, 0.6 mM, or 0.9 mM (control, n = 16; treatment groups, n = 8 each). Cell death was performed with LDH assay 10 DIV (24 h later); mean LDH value expressed as % of control. *p ≤ 0.05 and **p ≤ 0.01 compared with the control condition. Raw data was used for one-way ANOVA. (B) E18 cortical neurons cultured in neurobasal medium with 2% FBS for 7-8 days. Medium was replenished on the 8 th day and neurons treated with either 0 mM, 0.3 mM, 0.6 mM, or 0.9 mM (n = 12 each). Cell viability was performed with LDH assay on 10 DIV. Mean LDH value expressed as % of control. *p ≤ 0.05 compared with the control using student's t test. Raw data was used for one-way ANOVA.

Techniques Used: Cell Culture, Lactate Dehydrogenase Assay

Neuronal protection by Mibefradil . Using cells from the hippocampi of E18 mice, neurons were cultured for 7-8 days with neurobasal medium with 2% FBS. On the 8 th day of culture, medium was replaced and cells treated with mibefradil in concentrations of 0 μM, 0.5 μM, and 1 μM (all groups, n = 12). Cell death was quantified using LDH assay 48 hours after treatment. Raw data was used for one-way ANOVA. When the data was expressed as % of control. *p ≤ 0.05 compared to the control was significant using student's t test.
Figure Legend Snippet: Neuronal protection by Mibefradil . Using cells from the hippocampi of E18 mice, neurons were cultured for 7-8 days with neurobasal medium with 2% FBS. On the 8 th day of culture, medium was replaced and cells treated with mibefradil in concentrations of 0 μM, 0.5 μM, and 1 μM (all groups, n = 12). Cell death was quantified using LDH assay 48 hours after treatment. Raw data was used for one-way ANOVA. When the data was expressed as % of control. *p ≤ 0.05 compared to the control was significant using student's t test.

Techniques Used: Mouse Assay, Cell Culture, Lactate Dehydrogenase Assay

Neuronal protection by nimodipine . Hippocampal neurons from E18 C57BL/6J mice and cultured for 7-8 days in neurobasal medium with 2% FBS. Fresh medium was placed in wells and neurons were treated with either 0 or 1 μM nimodipine (n = 12 each). Neurons were subjected to LDH assay to quantify cell death 48 h later (10 DIV); nimodipine remained in the cultures throughout this time. Mean LDH value expressed as % of control. *p ≤ 0.05 compared with control condition.
Figure Legend Snippet: Neuronal protection by nimodipine . Hippocampal neurons from E18 C57BL/6J mice and cultured for 7-8 days in neurobasal medium with 2% FBS. Fresh medium was placed in wells and neurons were treated with either 0 or 1 μM nimodipine (n = 12 each). Neurons were subjected to LDH assay to quantify cell death 48 h later (10 DIV); nimodipine remained in the cultures throughout this time. Mean LDH value expressed as % of control. *p ≤ 0.05 compared with control condition.

Techniques Used: Mouse Assay, Cell Culture, Lactate Dehydrogenase Assay

33) Product Images from "Periodic acid-Schiff staining method for function detection of liver cells is affected by 2% horse serum in induction medium"

Article Title: Periodic acid-Schiff staining method for function detection of liver cells is affected by 2% horse serum in induction medium

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2017.7587

Replacement of 2% HS with 10% FBS ahead of PAS staining detection reflects the glycogen storage and accumulation function of induced HP14.5d cells. HP14.5d cells were treated with DMEM containing 2% HS + 0.1 µmol/l Dex + 10 ng/ml HGF + 20 ng/ml FGF4 for 12 days, then the induction medium was replaced with complete DMEM with 10% FBS + 0.1 µmol/l Dex + 10 ng/ml HGF + 20 ng/ml FGF4 for different time periods. (A) PAS staining and (B) ICG uptake were assessed at 12, 24, 48 and 72 h after the induction medium was changed. (a) Uninduced HP14.5d cells as control; (b) HP14.5d cells induced for 12 days with no media shift; (c) HP14.5d cells induced for 12 days and induction medium changed for 12 h; (d) HP14.5d cells induced for 12 days and induction medium changed for 24 h; (e) HP14.5d cells induced for 12 days and induction medium changed for 48 h; (f) HP14.5d cells induced for 12 days and induction medium changed for 72 h. Photomicrographs were captured under ×100 magnification. Representative images and data from ≥3 independent experiments are presented. *P
Figure Legend Snippet: Replacement of 2% HS with 10% FBS ahead of PAS staining detection reflects the glycogen storage and accumulation function of induced HP14.5d cells. HP14.5d cells were treated with DMEM containing 2% HS + 0.1 µmol/l Dex + 10 ng/ml HGF + 20 ng/ml FGF4 for 12 days, then the induction medium was replaced with complete DMEM with 10% FBS + 0.1 µmol/l Dex + 10 ng/ml HGF + 20 ng/ml FGF4 for different time periods. (A) PAS staining and (B) ICG uptake were assessed at 12, 24, 48 and 72 h after the induction medium was changed. (a) Uninduced HP14.5d cells as control; (b) HP14.5d cells induced for 12 days with no media shift; (c) HP14.5d cells induced for 12 days and induction medium changed for 12 h; (d) HP14.5d cells induced for 12 days and induction medium changed for 24 h; (e) HP14.5d cells induced for 12 days and induction medium changed for 48 h; (f) HP14.5d cells induced for 12 days and induction medium changed for 72 h. Photomicrographs were captured under ×100 magnification. Representative images and data from ≥3 independent experiments are presented. *P

Techniques Used: Staining

Replacement of 2% HS with 10% FBS following 12 days of induction does not influence the induced differentiation of HP14.5d cells. HP14.5d cells were treated with DMEM containing 2% HS + 0.1 µmol/l Dex + 10 ng/ml HGF + 20 ng/ml FGF4 for 12 days, then the induction medium was replaced with complete DMEM with 10% FBS + 0.1 µmol/l Dex + 10 ng/ml HGF + 20 ng/ml FGF4 for different time periods: (a) Uninduced HP14.5d cells as control; (b) HP14.5d cells induced for 12 days with no media shift; (c) HP14.5d cells induced for 12 days and induction medium changed for 12 h; (d) HP14.5d cells induced for 12 days and induction medium changed for 24 h; (e) HP14.5d cells induced for 12 days and induction medium changed for 48 h; (f) HP14.5d cells induced for 12 days and induction medium changed for 72 h. (A) HP14.5d cell morphologies. Photomicrographs were captured under ×200 magnification. (B) ALB-GLuc activity in HP14.5d cells. *P
Figure Legend Snippet: Replacement of 2% HS with 10% FBS following 12 days of induction does not influence the induced differentiation of HP14.5d cells. HP14.5d cells were treated with DMEM containing 2% HS + 0.1 µmol/l Dex + 10 ng/ml HGF + 20 ng/ml FGF4 for 12 days, then the induction medium was replaced with complete DMEM with 10% FBS + 0.1 µmol/l Dex + 10 ng/ml HGF + 20 ng/ml FGF4 for different time periods: (a) Uninduced HP14.5d cells as control; (b) HP14.5d cells induced for 12 days with no media shift; (c) HP14.5d cells induced for 12 days and induction medium changed for 12 h; (d) HP14.5d cells induced for 12 days and induction medium changed for 24 h; (e) HP14.5d cells induced for 12 days and induction medium changed for 48 h; (f) HP14.5d cells induced for 12 days and induction medium changed for 72 h. (A) HP14.5d cell morphologies. Photomicrographs were captured under ×200 magnification. (B) ALB-GLuc activity in HP14.5d cells. *P

Techniques Used: Activity Assay

HS influences PAS staining in induced HP14.5d cells. (A) PAS staining and ICG uptake demonstrated the glycogen storage and transport-metabolism function, respectively, of HP14.5d cells induced for 12 days. Uninduced HP14.5d cells were set up as a control. Photomicrographs were captured under ×200 magnification. (B) PAS staining of HP14.5d cells following induction for 12 days in: (a) complete DMEM containing 10% FBS (uninduced); (b) induction medium (DMEM containing 0.1 µmol/l Dex, 10 ng/ml HGF, 20 ng/ml FGF4 and 2% HS); (c) induction medium without Dex; (d) induction medium without HGF; (e) induction medium without FGF4; (f) induction medium with 2% HS replaced with 10% FBS. Photomicrographs were captured under ×100 magnification. Representative images of ≥3 independent experiments are presented. HS, horse serum; PAS, periodic acid-Schiff; ICG, indocyanine green; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; Dex, dexamethasone; HGF, hepatic growth factor; FGF4, fibroblast growth factor 4.
Figure Legend Snippet: HS influences PAS staining in induced HP14.5d cells. (A) PAS staining and ICG uptake demonstrated the glycogen storage and transport-metabolism function, respectively, of HP14.5d cells induced for 12 days. Uninduced HP14.5d cells were set up as a control. Photomicrographs were captured under ×200 magnification. (B) PAS staining of HP14.5d cells following induction for 12 days in: (a) complete DMEM containing 10% FBS (uninduced); (b) induction medium (DMEM containing 0.1 µmol/l Dex, 10 ng/ml HGF, 20 ng/ml FGF4 and 2% HS); (c) induction medium without Dex; (d) induction medium without HGF; (e) induction medium without FGF4; (f) induction medium with 2% HS replaced with 10% FBS. Photomicrographs were captured under ×100 magnification. Representative images of ≥3 independent experiments are presented. HS, horse serum; PAS, periodic acid-Schiff; ICG, indocyanine green; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; Dex, dexamethasone; HGF, hepatic growth factor; FGF4, fibroblast growth factor 4.

Techniques Used: Staining, Modification

34) Product Images from "Platelet Lysates Produced from Expired Platelet Concentrates Support Growth and Osteogenic Differentiation of Mesenchymal Stem Cells"

Article Title: Platelet Lysates Produced from Expired Platelet Concentrates Support Growth and Osteogenic Differentiation of Mesenchymal Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0068984

Cumulative population doublings of MSC after culture in FBS, HPLF or HPLO. MSC were cultured in culture media supplemented with 10% of FBS, platelet lysate from fresh platelet concentrates (HPLF) or lysate from expired platelet concentrates (HPLO). Population doubling assay was performed at the end of every passage for a total of six passages (P1–P6, n = 3). MSC cultured in either HPLF or HPLO consistently had higher numbers of population doublings at the end of every passage compared to MSC cultured in FBS. By the end of the sixth passage cumulative population doublings were 13.77±0.77 CPD for HPLO, 12.77±0.12 CPD for HPLF and 9.50±1.14 CPD for FBS supplemented cultures, respectively.
Figure Legend Snippet: Cumulative population doublings of MSC after culture in FBS, HPLF or HPLO. MSC were cultured in culture media supplemented with 10% of FBS, platelet lysate from fresh platelet concentrates (HPLF) or lysate from expired platelet concentrates (HPLO). Population doubling assay was performed at the end of every passage for a total of six passages (P1–P6, n = 3). MSC cultured in either HPLF or HPLO consistently had higher numbers of population doublings at the end of every passage compared to MSC cultured in FBS. By the end of the sixth passage cumulative population doublings were 13.77±0.77 CPD for HPLO, 12.77±0.12 CPD for HPLF and 9.50±1.14 CPD for FBS supplemented cultures, respectively.

Techniques Used: Cell Culture

Mesenchymal stem cell morphology during expansion and differentiation. MSC were stained with crystal violet after expansion in media containing 10% of FBS, HPLF or HPLO for three passages (A–C). MSC cultured in HPLF or HPLO developed more spindle shaped morphology than cells cultured in FBS, proliferated faster and left circular areas containing no cells between them. Differentiation towards osteoblasts, adipocytes and chondrocytes was initiated after expansion in the three supplements. After 28 days of osteogenic differentiation mineralization in the culture could be visualized with a Von Kossa staining (D–F). Successful adipogenic differentiation was confirmed with Oil red O staining (G–I) and after 28 days of chondrogenic pellet cultures, the pellets were sectioned and stained with toludine blue staining (J–L). Pictures representative of three experiments.
Figure Legend Snippet: Mesenchymal stem cell morphology during expansion and differentiation. MSC were stained with crystal violet after expansion in media containing 10% of FBS, HPLF or HPLO for three passages (A–C). MSC cultured in HPLF or HPLO developed more spindle shaped morphology than cells cultured in FBS, proliferated faster and left circular areas containing no cells between them. Differentiation towards osteoblasts, adipocytes and chondrocytes was initiated after expansion in the three supplements. After 28 days of osteogenic differentiation mineralization in the culture could be visualized with a Von Kossa staining (D–F). Successful adipogenic differentiation was confirmed with Oil red O staining (G–I) and after 28 days of chondrogenic pellet cultures, the pellets were sectioned and stained with toludine blue staining (J–L). Pictures representative of three experiments.

Techniques Used: Staining, Cell Culture

Increased enzymatic activity of alkaline phosphatase during osteogenic differentiation. MSC that had previously been cultured in media containing 10% of FBS, HPLF or HPLO underwent osteogenic differentiation. ALP activity was evaluated after 7 and 14 days of differentiation. ALP activity increased significantly from day 7 to day 14 independent of the type of culture conditions the MSC had been exposed to prior to osteogenic differentiation (p≤0.05, n = 3). * = p≤0.05.
Figure Legend Snippet: Increased enzymatic activity of alkaline phosphatase during osteogenic differentiation. MSC that had previously been cultured in media containing 10% of FBS, HPLF or HPLO underwent osteogenic differentiation. ALP activity was evaluated after 7 and 14 days of differentiation. ALP activity increased significantly from day 7 to day 14 independent of the type of culture conditions the MSC had been exposed to prior to osteogenic differentiation (p≤0.05, n = 3). * = p≤0.05.

Techniques Used: Activity Assay, Cell Culture, ALP Assay

Expression of osteogenic marker genes during osteogenic differentiation of MSC. Osteogenic differentiation was initiated after MSC expansion in 10% of FBS, HPLF or HPLO. Expression of osteogenic marker genes was evaluated after 7 and 21 days of osteogenic differentiation and is represented with graphs for each gene (A–C, n = 3) and a heat-map (D) where green shades represent lower levels of expression and red shades higher levels of expression. ALP was evenly expressed throughout the differentiation (A,D) while the expression of SPP1 increased from day 7 to day 21 (B,D), irrespective of the culture supplements the MSC had previously been exposed to (p≤0.01). Expression of RUNX2 decreased from day 7 to day 21 for all cultures (C–D), still expression of RUNX2 in cultures originating from HPLO treated MSC was higher at both time-points compared to FBS and also at day 21 for HPLF compared to FBS (p≤0.05). * = p≤0.05, ** = p≤0.01.
Figure Legend Snippet: Expression of osteogenic marker genes during osteogenic differentiation of MSC. Osteogenic differentiation was initiated after MSC expansion in 10% of FBS, HPLF or HPLO. Expression of osteogenic marker genes was evaluated after 7 and 21 days of osteogenic differentiation and is represented with graphs for each gene (A–C, n = 3) and a heat-map (D) where green shades represent lower levels of expression and red shades higher levels of expression. ALP was evenly expressed throughout the differentiation (A,D) while the expression of SPP1 increased from day 7 to day 21 (B,D), irrespective of the culture supplements the MSC had previously been exposed to (p≤0.01). Expression of RUNX2 decreased from day 7 to day 21 for all cultures (C–D), still expression of RUNX2 in cultures originating from HPLO treated MSC was higher at both time-points compared to FBS and also at day 21 for HPLF compared to FBS (p≤0.05). * = p≤0.05, ** = p≤0.01.

Techniques Used: Expressing, Marker, ALP Assay

Reduction of mononuclear cell proliferation during co-culture with MSC. Mononuclear cells (MNC) were stimulated with phytohemaglutinin (PHA) and then cultured with or without MSC that had been previously expanded in media containing 10% of FBS, HPLF or HPLO. MNC proliferation was significantly less when co-cultured with MSC than if cultured on their own (p≤0.05, n = 3). The reduced proliferation was seen irrespective of the culture conditions the MSC had previously been exposed to. * = p≤0.05.
Figure Legend Snippet: Reduction of mononuclear cell proliferation during co-culture with MSC. Mononuclear cells (MNC) were stimulated with phytohemaglutinin (PHA) and then cultured with or without MSC that had been previously expanded in media containing 10% of FBS, HPLF or HPLO. MNC proliferation was significantly less when co-cultured with MSC than if cultured on their own (p≤0.05, n = 3). The reduced proliferation was seen irrespective of the culture conditions the MSC had previously been exposed to. * = p≤0.05.

Techniques Used: Co-Culture Assay, Cell Culture

35) Product Images from "Impact of Serum Proteins on MRI Contrast Agents: Cellular Binding and T2 relaxation"

Article Title: Impact of Serum Proteins on MRI Contrast Agents: Cellular Binding and T2 relaxation

Journal: RSC advances

doi: 10.1039/C4RA04246H

Flow cytometry was used to measure the cellular binding of CMD-PS NPs (60 pM) to CHO cells in the absence or presence of FBS (10%) and BSA (3.5 mg/mL). The mean fluorescence intensity is normalized against the cellular binding of CMD-PS NPs in the absence
Figure Legend Snippet: Flow cytometry was used to measure the cellular binding of CMD-PS NPs (60 pM) to CHO cells in the absence or presence of FBS (10%) and BSA (3.5 mg/mL). The mean fluorescence intensity is normalized against the cellular binding of CMD-PS NPs in the absence

Techniques Used: Flow Cytometry, Cytometry, Binding Assay, Fluorescence

Competition of SPIONs with the mixture of FBS proteins and isolated BSA for binding to CHO cells was quantified with flow cytometry. (A) The formation of a FBS corona on the SPION inhibits binding to CHO cells. In comparison, a corona formed using isolated
Figure Legend Snippet: Competition of SPIONs with the mixture of FBS proteins and isolated BSA for binding to CHO cells was quantified with flow cytometry. (A) The formation of a FBS corona on the SPION inhibits binding to CHO cells. In comparison, a corona formed using isolated

Techniques Used: Isolation, Binding Assay, Flow Cytometry, Cytometry

36) Product Images from "Fatty acid binding protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells"

Article Title: Fatty acid binding protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells

Journal: The FASEB Journal

doi: 10.1096/fj.09-134882

FABP4 expression is induced by VEGF and bFGF. A, C ) HUVECs were starved in ECGF-free M199 with 2% FBS for 8 h, then stimulated with 50 ng/ml VEGFA 165 ( A ) or 10 ng/ml bFGF ( C ). Cells were harvested at the indicated time points, and FABP4 mRNA expression was analyzed by real-time PCR. * P
Figure Legend Snippet: FABP4 expression is induced by VEGF and bFGF. A, C ) HUVECs were starved in ECGF-free M199 with 2% FBS for 8 h, then stimulated with 50 ng/ml VEGFA 165 ( A ) or 10 ng/ml bFGF ( C ). Cells were harvested at the indicated time points, and FABP4 mRNA expression was analyzed by real-time PCR. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

37) Product Images from "Salinity-Induced Anti-Angiogenesis Activities and Structural Changes of the Polysaccharides from Cultured Cordyceps Militaris"

Article Title: Salinity-Induced Anti-Angiogenesis Activities and Structural Changes of the Polysaccharides from Cultured Cordyceps Militaris

Journal: PLoS ONE

doi: 10.1371/journal.pone.0103880

P5 reduced HUVEC cell migration and VEGF expression in HUVEC cells. P5 reduced HUVEC cell migration. The transwell assay was performed by using a 24-well chamber as the outer chambers and polycarbonate filters (8 µm pores) as the inner chambers as described in Methods . After incubation for 18 hrs at 37°C, the migrated cells on the lower surface of the filter were fixed in 90% ethanol and stained with 0.1% crystal violet. Images of the migrated cells were taken using a microscope (Olympus, CKX41, Japan) ( a ). The migrated cells were quantified at 595 nm after extraction with 10% acetic acid ( b ). Migrated cells in the outer chambers were also quantified independently by incubating the out chambers in F12 media with 10% FBS for 8 days followed by resazurin quantification ( b ). The untreated cells (control) were assigned values of 100 and the results were presented as mean ± S.D. (n = 3). Significance: *P
Figure Legend Snippet: P5 reduced HUVEC cell migration and VEGF expression in HUVEC cells. P5 reduced HUVEC cell migration. The transwell assay was performed by using a 24-well chamber as the outer chambers and polycarbonate filters (8 µm pores) as the inner chambers as described in Methods . After incubation for 18 hrs at 37°C, the migrated cells on the lower surface of the filter were fixed in 90% ethanol and stained with 0.1% crystal violet. Images of the migrated cells were taken using a microscope (Olympus, CKX41, Japan) ( a ). The migrated cells were quantified at 595 nm after extraction with 10% acetic acid ( b ). Migrated cells in the outer chambers were also quantified independently by incubating the out chambers in F12 media with 10% FBS for 8 days followed by resazurin quantification ( b ). The untreated cells (control) were assigned values of 100 and the results were presented as mean ± S.D. (n = 3). Significance: *P

Techniques Used: Migration, Expressing, Transwell Assay, Incubation, Staining, Microscopy

38) Product Images from "Treatment of autosomal dominant hearing loss by invivo delivery of genome editing agents"

Article Title: Treatment of autosomal dominant hearing loss by invivo delivery of genome editing agents

Journal: Nature

doi: 10.1038/nature25164

Allele-selective editing of wild-type or Bth mutant Tmc1 in cleavage assays in vitro and in lipid-mediated delivery into primary fibroblasts. ( a ) In vitro Cas9:sgRNA-mediated Tmc1 DNA cleavage. 100 nM of a 995-bp DNA fragment containing wild-type Tmc1 (lanes 1-5) or Bth mutant Tmc1 (lanes 6-10) was incubated with 300 nM of each of the four Cas9:sgRNAs shown for 15 min at 37 °C. Expected cleavage products are 774-778 bp and 217-221 bp. M = 100-bp ladder; the lower two heavy bands are 500 and 1,000 bp. ( b ) Quantification of DNA cleavage in (a) by densitometry using imageJ. ( c ) Transfection efficiency comparison of HEK293T cells and wild-type primary fibroblasts. 50 ng GFP plasmid, 10 nM Cas9:FitC-Tmc1-mut3 sgRNA RNP, or 10 nM Cas9:CrRNA-Tmc1-mut3:atto-550-TracrRNA RNP were delivered into HEK293T cells or wild-type primary fibroblasts using 3 μL Lipofectamine 2000. For samples with GFP plasmid, the fraction of GFP-positive cells was measured by flow cytometry 24 h after delivery. For samples with Cas9:FitC-Tmc1-mut3 RNP, or Cas9:CrRNA-Tmc1-mut3:atto-550-TracrRNA RNP, media was removed 6 h after delivery. The cells were trypsinized, washed three times with 500 μL PBS containing 20 U/mL heparin, and subjected to flow cytometry. ( d ) Wild-type or Bth mutant Tmc1 allele editing in primary fibroblasts derived from wild-type or Bth/Bth mice as a function of the dose of Cas9:Tmc1-mut3:lipid complex. 12.5, 25, 50, 100, 200, or 400 nM of Cas9:Tmc1-mut3 were delivered into the primary fibroblasts using Lipofectamine 2000 in DMEM-FBS. ( e ) Lipid-mediated delivery of Cas9:sgRNA complexes into primary fibroblasts derived from wild-type or Bth/Bth mice. 100 nM of purified Cas9 protein and each wild-type Tmc1 -targeting sgRNA (Tmc1-wt1, Tmc1-wt2, or Tmc1-wt3) or Bth mutant-targeting sgRNA (Tmc1-mut1, Tmc1-mut2, or Tmc1-mut3) were delivered into wild-type fibroblasts (red) and Bth/Bth fibroblasts (blue) using Lipofectamine 2000 in DMEM-FBS. Primary fibroblast cells were harvested 96 h after treatment. Genomic DNA was extracted and indels were detected by HTS. Values and error bars reflect the mean ± standard deviation of three or more biological replicates.
Figure Legend Snippet: Allele-selective editing of wild-type or Bth mutant Tmc1 in cleavage assays in vitro and in lipid-mediated delivery into primary fibroblasts. ( a ) In vitro Cas9:sgRNA-mediated Tmc1 DNA cleavage. 100 nM of a 995-bp DNA fragment containing wild-type Tmc1 (lanes 1-5) or Bth mutant Tmc1 (lanes 6-10) was incubated with 300 nM of each of the four Cas9:sgRNAs shown for 15 min at 37 °C. Expected cleavage products are 774-778 bp and 217-221 bp. M = 100-bp ladder; the lower two heavy bands are 500 and 1,000 bp. ( b ) Quantification of DNA cleavage in (a) by densitometry using imageJ. ( c ) Transfection efficiency comparison of HEK293T cells and wild-type primary fibroblasts. 50 ng GFP plasmid, 10 nM Cas9:FitC-Tmc1-mut3 sgRNA RNP, or 10 nM Cas9:CrRNA-Tmc1-mut3:atto-550-TracrRNA RNP were delivered into HEK293T cells or wild-type primary fibroblasts using 3 μL Lipofectamine 2000. For samples with GFP plasmid, the fraction of GFP-positive cells was measured by flow cytometry 24 h after delivery. For samples with Cas9:FitC-Tmc1-mut3 RNP, or Cas9:CrRNA-Tmc1-mut3:atto-550-TracrRNA RNP, media was removed 6 h after delivery. The cells were trypsinized, washed three times with 500 μL PBS containing 20 U/mL heparin, and subjected to flow cytometry. ( d ) Wild-type or Bth mutant Tmc1 allele editing in primary fibroblasts derived from wild-type or Bth/Bth mice as a function of the dose of Cas9:Tmc1-mut3:lipid complex. 12.5, 25, 50, 100, 200, or 400 nM of Cas9:Tmc1-mut3 were delivered into the primary fibroblasts using Lipofectamine 2000 in DMEM-FBS. ( e ) Lipid-mediated delivery of Cas9:sgRNA complexes into primary fibroblasts derived from wild-type or Bth/Bth mice. 100 nM of purified Cas9 protein and each wild-type Tmc1 -targeting sgRNA (Tmc1-wt1, Tmc1-wt2, or Tmc1-wt3) or Bth mutant-targeting sgRNA (Tmc1-mut1, Tmc1-mut2, or Tmc1-mut3) were delivered into wild-type fibroblasts (red) and Bth/Bth fibroblasts (blue) using Lipofectamine 2000 in DMEM-FBS. Primary fibroblast cells were harvested 96 h after treatment. Genomic DNA was extracted and indels were detected by HTS. Values and error bars reflect the mean ± standard deviation of three or more biological replicates.

Techniques Used: Mutagenesis, In Vitro, Incubation, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry, Derivative Assay, Mouse Assay, Purification, Standard Deviation

Delivery of Cas9:Tmc1-mut3 sgRNA complexes into primary fibroblasts derived from wild-type or homozygous Bth/Bth mice using ( a ) seven commercially available lipids. LPF2000 = Lipofectamine 2000; RNAiMAX = Lipofectamine® RNAiMAX; LPF3000 = Lipofectamine 3000; CRISPRMAX = Lipofectamine CRISPRMAX; LTX = Lipofectamine LTX, or ( b ) ten biodegradable, bioreducible lipids. Lipid 1 = 75-O14B; Lipid 2 = 76-O14B; Lipid 3 = 80-O18B; Lipid 4 = 87-O16B; Lipid 5 = 113-O18B; Lipid 6 = 306-O12B; Lipid 7 = 306-O16B; Lipid 8 = 306-O18B; Lipid 9 = 400-O12B; Lipid 10 = 400-O16B. 100 nM purified Cas9:Tmc1-mut3 RNP was delivered using 3 μL of the cationic lipid shown in DMEM-FBS. Fibroblast cells were harvested 96 h after treatment, genomic DNA was extracted, and indels were detected by HTS. (c ) Synthetic route and chemical structure of lipids. ( d ) Commercially available amine head groups used in lipid synthesis. Lipids were synthesized as previously described 26 . Values and error bars reflect the mean ± standard deviation of three or more biological replicates.
Figure Legend Snippet: Delivery of Cas9:Tmc1-mut3 sgRNA complexes into primary fibroblasts derived from wild-type or homozygous Bth/Bth mice using ( a ) seven commercially available lipids. LPF2000 = Lipofectamine 2000; RNAiMAX = Lipofectamine® RNAiMAX; LPF3000 = Lipofectamine 3000; CRISPRMAX = Lipofectamine CRISPRMAX; LTX = Lipofectamine LTX, or ( b ) ten biodegradable, bioreducible lipids. Lipid 1 = 75-O14B; Lipid 2 = 76-O14B; Lipid 3 = 80-O18B; Lipid 4 = 87-O16B; Lipid 5 = 113-O18B; Lipid 6 = 306-O12B; Lipid 7 = 306-O16B; Lipid 8 = 306-O18B; Lipid 9 = 400-O12B; Lipid 10 = 400-O16B. 100 nM purified Cas9:Tmc1-mut3 RNP was delivered using 3 μL of the cationic lipid shown in DMEM-FBS. Fibroblast cells were harvested 96 h after treatment, genomic DNA was extracted, and indels were detected by HTS. (c ) Synthetic route and chemical structure of lipids. ( d ) Commercially available amine head groups used in lipid synthesis. Lipids were synthesized as previously described 26 . Values and error bars reflect the mean ± standard deviation of three or more biological replicates.

Techniques Used: Derivative Assay, Mouse Assay, Purification, Synthesized, Standard Deviation

39) Product Images from "Optimization of a Method to Isolate and Culture Adult Porcine, Rats and Mice Müller Glia in Order to Study Retinal Diseases"

Article Title: Optimization of a Method to Isolate and Culture Adult Porcine, Rats and Mice Müller Glia in Order to Study Retinal Diseases

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2020.00007

Analysis of pig Müller cells when cultured in different media: DMEM + 10% FBS, DMEM-F12, and NBA/B27 + 10% FBS. The purity and survival of the cells maintained in DMEM + 10% FBS (A) , DMEM/F12 (B) or NBA B27 + 10% FBS (C) was analyzed. Neurons (RGCs) were labeled with an antibody against βIII-tubulin (red), Müller cells with an antibody against vimentin (green) and the nuclei were stained with DAPI. Note that the cultures maintained in NBA/B27 + 10% FBS were not pure (C) . In DMEM + 10% FBS, Müller cells reached confluency more rapidly (A) and there were fewer Müller cells in the cultures grown in NBA + 10% FBS and DMEM-F12 at both time points, as seen in the histogram (D) : ∗ p -value
Figure Legend Snippet: Analysis of pig Müller cells when cultured in different media: DMEM + 10% FBS, DMEM-F12, and NBA/B27 + 10% FBS. The purity and survival of the cells maintained in DMEM + 10% FBS (A) , DMEM/F12 (B) or NBA B27 + 10% FBS (C) was analyzed. Neurons (RGCs) were labeled with an antibody against βIII-tubulin (red), Müller cells with an antibody against vimentin (green) and the nuclei were stained with DAPI. Note that the cultures maintained in NBA/B27 + 10% FBS were not pure (C) . In DMEM + 10% FBS, Müller cells reached confluency more rapidly (A) and there were fewer Müller cells in the cultures grown in NBA + 10% FBS and DMEM-F12 at both time points, as seen in the histogram (D) : ∗ p -value

Techniques Used: Cell Culture, Labeling, Staining

Scheme summarizing the main steps of the protocol to establish adult Müller cell cultures. The culture method for pig Müller cells from adult animals uses papain as the enzyme to digest the retina, poly- L -Lysine and laminin as the substrate and DMEM + 10% FBS as the culture medium.
Figure Legend Snippet: Scheme summarizing the main steps of the protocol to establish adult Müller cell cultures. The culture method for pig Müller cells from adult animals uses papain as the enzyme to digest the retina, poly- L -Lysine and laminin as the substrate and DMEM + 10% FBS as the culture medium.

Techniques Used:

40) Product Images from "Regeneration of kidney tissue using in vitro cultured fetal kidney cells"

Article Title: Regeneration of kidney tissue using in vitro cultured fetal kidney cells

Journal: Experimental & Molecular Medicine

doi: 10.3858/emm.2008.40.4.361

Cellular appearance of cultured fetal kidney cells. (A) Fetal kidney cells cultured in DMEM with 10% (v/v) FBS show spindle-shaped and polygonal morphology, whereas (B) fetal kidney cells cultured in KSFM with 2% (v/v) FBS and ITS show cobblestone-like morphology. The scale bars indicate 20 µm.
Figure Legend Snippet: Cellular appearance of cultured fetal kidney cells. (A) Fetal kidney cells cultured in DMEM with 10% (v/v) FBS show spindle-shaped and polygonal morphology, whereas (B) fetal kidney cells cultured in KSFM with 2% (v/v) FBS and ITS show cobblestone-like morphology. The scale bars indicate 20 µm.

Techniques Used: Cell Culture

41) Product Images from "The intrinsically kinase-inactive EPHB6 receptor predisposes cancer cells to DR5-induced apoptosis by promoting mitochondrial fragmentation"

Article Title: The intrinsically kinase-inactive EPHB6 receptor predisposes cancer cells to DR5-induced apoptosis by promoting mitochondrial fragmentation

Journal: Oncotarget

doi: 10.18632/oncotarget.12838

The EPHB6 receptor enhances DR5-induced cell death and CASPASE-3 activation in triple-negative breast cancer cells A. Western blot analysis of EPHB6 expression in triple-negative breast cancer (TNBC) cells, MDA-MB-231, stably transfected with the pcDNA3 expression vector encoding myc-tagged EPHB6 (MDA-B6-M) or mock-transfected with the empty vector (MDA-pc3). Western blotting with anti-tubulin was used as a loading control. B. Anti-DR5 activating antibody or matching non-specific IgG (both at 10 μg/mL) were immobilised overnight at 4°C on 96-well plates. MDA-B6-M and MDA-pc3 cells were loaded onto the pre-coated plates (5 × 10 4 cells/well) in DMEM containing 0.5% FBS, and incubated at 37°C for 24 h or 36 h. To assess cell survival, cells were stained with resazurin and the fluorescence was measured using a SpectraMax M5 plate reader. Each graph represents the analysis of quadruplicates. The graphs present cell viability in DR5-treated cells as percentages relative to matching non-specific IgG controls. C. TNBC cells, BT-20, were transduced with EPHB6-targeting shRNA (BT20-B6-shRNA) or non-silencing shRNA (BT20-NS). EPHB6 expression was analysed by Western blotting as in (A). D. BT20-NS and BT20-B6-shRNA cells were stimulated in 96-well plates (1.5 × 10 4 cells/well) for 36 h with anti-DR5 pre-immobilised at 20 μg/mL, as in (B). Matching non-specific IgG was used as a control. Cell survival was monitored by the MTT assay. A Spectramax 340PC plate reader was used to measure absorbance. Each graph represents the analysis of five replicates. The graph shows cell survival as a percentage relative to matching IgG controls. E. The indicated cells (1 × 10 6 cells/well) were incubated in 6-well plates pre-coated with anti-DR5 or matching control IgG (both at 10 μg/mL) for 5 h and CASPASE-3 activity was measured using the EnzChek® CASPASE-3 Assay Kit #1 (Abcam) according to the manufacturer's instructions. Each graph represents the analysis of triplicates. The graphs present CASPASE-3 activity as percentages relative to IgG controls. All experiments were performed at least three times. *, P
Figure Legend Snippet: The EPHB6 receptor enhances DR5-induced cell death and CASPASE-3 activation in triple-negative breast cancer cells A. Western blot analysis of EPHB6 expression in triple-negative breast cancer (TNBC) cells, MDA-MB-231, stably transfected with the pcDNA3 expression vector encoding myc-tagged EPHB6 (MDA-B6-M) or mock-transfected with the empty vector (MDA-pc3). Western blotting with anti-tubulin was used as a loading control. B. Anti-DR5 activating antibody or matching non-specific IgG (both at 10 μg/mL) were immobilised overnight at 4°C on 96-well plates. MDA-B6-M and MDA-pc3 cells were loaded onto the pre-coated plates (5 × 10 4 cells/well) in DMEM containing 0.5% FBS, and incubated at 37°C for 24 h or 36 h. To assess cell survival, cells were stained with resazurin and the fluorescence was measured using a SpectraMax M5 plate reader. Each graph represents the analysis of quadruplicates. The graphs present cell viability in DR5-treated cells as percentages relative to matching non-specific IgG controls. C. TNBC cells, BT-20, were transduced with EPHB6-targeting shRNA (BT20-B6-shRNA) or non-silencing shRNA (BT20-NS). EPHB6 expression was analysed by Western blotting as in (A). D. BT20-NS and BT20-B6-shRNA cells were stimulated in 96-well plates (1.5 × 10 4 cells/well) for 36 h with anti-DR5 pre-immobilised at 20 μg/mL, as in (B). Matching non-specific IgG was used as a control. Cell survival was monitored by the MTT assay. A Spectramax 340PC plate reader was used to measure absorbance. Each graph represents the analysis of five replicates. The graph shows cell survival as a percentage relative to matching IgG controls. E. The indicated cells (1 × 10 6 cells/well) were incubated in 6-well plates pre-coated with anti-DR5 or matching control IgG (both at 10 μg/mL) for 5 h and CASPASE-3 activity was measured using the EnzChek® CASPASE-3 Assay Kit #1 (Abcam) according to the manufacturer's instructions. Each graph represents the analysis of triplicates. The graphs present CASPASE-3 activity as percentages relative to IgG controls. All experiments were performed at least three times. *, P

Techniques Used: Activation Assay, Western Blot, Expressing, Multiple Displacement Amplification, Stable Transfection, Transfection, Plasmid Preparation, Incubation, Staining, Fluorescence, Transduction, shRNA, MTT Assay, Activity Assay, Caspase-3 Assay

42) Product Images from "Comparative analysis of differentially secreted proteins in serum-free and serum-containing media by using BONCAT and pulsed SILAC"

Article Title: Comparative analysis of differentially secreted proteins in serum-free and serum-containing media by using BONCAT and pulsed SILAC

Journal: Scientific Reports

doi: 10.1038/s41598-019-39650-z

Analysis of hWJ-MSC secretome from serum-containing medium (SCM) and serum-free medium (SFM). ( a ) Schematic workflow. ( b ) The number of identified proteins in the secretome. ( c ) The distribution of differentially secreted proteins between SFM and SCM. The H/M ratios are log2-transformed after normalization by the difference in growth rate. ( d , e ) Secretion pathways and subcellular localization of the differentially secreted proteins. ( f ) MMP14 was measured by western blot analysis. Note that 10% FBS was added to SFM just before SDS-PAGE in order to view any background effect stemming from FBS itself. Equal loading was confirmed by Commassie staining of the membrane. Full-length western blot images are shown in Supplementary Fig. S5b .
Figure Legend Snippet: Analysis of hWJ-MSC secretome from serum-containing medium (SCM) and serum-free medium (SFM). ( a ) Schematic workflow. ( b ) The number of identified proteins in the secretome. ( c ) The distribution of differentially secreted proteins between SFM and SCM. The H/M ratios are log2-transformed after normalization by the difference in growth rate. ( d , e ) Secretion pathways and subcellular localization of the differentially secreted proteins. ( f ) MMP14 was measured by western blot analysis. Note that 10% FBS was added to SFM just before SDS-PAGE in order to view any background effect stemming from FBS itself. Equal loading was confirmed by Commassie staining of the membrane. Full-length western blot images are shown in Supplementary Fig. S5b .

Techniques Used: Transformation Assay, Western Blot, SDS Page, Staining

43) Product Images from "cGMP-dependent protein kinase and the regulation of vascular smooth muscle cell gene expression: possible involvement of Elk-1 sumoylation"

Article Title: cGMP-dependent protein kinase and the regulation of vascular smooth muscle cell gene expression: possible involvement of Elk-1 sumoylation

Journal: American Journal of Physiology - Heart and Circulatory Physiology

doi: 10.1152/ajpheart.00677.2010

Detection of higher molecular species of phospho-E26-like protein-1 (pElk-1) in SMCs. A : low-passaged rat aortic SMCs were cultured in 10% FBS in DMEM until grown to confluence, incubated in serum-deprived medium (1 mg/ml of BSA in DMEM) for 3 days, and then stimulated with or without PDGF-BB (10 ng/ml) for 30 min. B , top : rat aortic SMCs were treated with PDGF-BB or 8-( p -chlorophenylthio)-cGMP (1 or 20 μM) for 30 min after culturing in serum-deprived medium for 3 days. B , bottom : pooled data from 3 experiments quantifying higher molecular mass species of pElk-1 (arrow). # P
Figure Legend Snippet: Detection of higher molecular species of phospho-E26-like protein-1 (pElk-1) in SMCs. A : low-passaged rat aortic SMCs were cultured in 10% FBS in DMEM until grown to confluence, incubated in serum-deprived medium (1 mg/ml of BSA in DMEM) for 3 days, and then stimulated with or without PDGF-BB (10 ng/ml) for 30 min. B , top : rat aortic SMCs were treated with PDGF-BB or 8-( p -chlorophenylthio)-cGMP (1 or 20 μM) for 30 min after culturing in serum-deprived medium for 3 days. B , bottom : pooled data from 3 experiments quantifying higher molecular mass species of pElk-1 (arrow). # P

Techniques Used: Cell Culture, Incubation

44) Product Images from "Impact of E1 and Cre on Adenovirus Vector Amplification: Developing MDCK CAV-2-E1 and E1-Cre Transcomplementing Cell Lines"

Article Title: Impact of E1 and Cre on Adenovirus Vector Amplification: Developing MDCK CAV-2-E1 and E1-Cre Transcomplementing Cell Lines

Journal: PLoS ONE

doi: 10.1371/journal.pone.0060342

Cell specific productivity and corresponding E1 gene expression of MDCK-E1 cell clones. a. Cell specific infectious viral titer produced in MDCK-E1 clones using DMEM 10% (v/v) FBS and 1% (v/v) NEAA. Error bars represent a 25% inter-assay variability error. b and c. Levels of mRNA E1A ( b ) and E1B expression ( c ) obtained for the different MDCK-E1 clones. The gene expression was determined by Real Time reverse transcriptase PCR and normalized to the housekeeping gene (GAPDH). Error bars represent a 10% variability error associated with the method. d. Cell concentration obtained for the different MDCK-E1 clones at virus harvest time. Cells were infected at the same cell concentration. Error bars represent the standard deviation of three independent experiments (n = 3).
Figure Legend Snippet: Cell specific productivity and corresponding E1 gene expression of MDCK-E1 cell clones. a. Cell specific infectious viral titer produced in MDCK-E1 clones using DMEM 10% (v/v) FBS and 1% (v/v) NEAA. Error bars represent a 25% inter-assay variability error. b and c. Levels of mRNA E1A ( b ) and E1B expression ( c ) obtained for the different MDCK-E1 clones. The gene expression was determined by Real Time reverse transcriptase PCR and normalized to the housekeeping gene (GAPDH). Error bars represent a 10% variability error associated with the method. d. Cell concentration obtained for the different MDCK-E1 clones at virus harvest time. Cells were infected at the same cell concentration. Error bars represent the standard deviation of three independent experiments (n = 3).

Techniques Used: Expressing, Clone Assay, Produced, Inter Assay, Polymerase Chain Reaction, Concentration Assay, Infection, Standard Deviation

45) Product Images from "Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies"

Article Title: Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies

Journal: Molecular Biotechnology

doi: 10.1007/s12033-010-9367-1

Binding studies of Z mab25 or protein G with fetal bovine serum and species selective affinity recovery of two monoclonal antibodies. a 10% FBS was flowed over a Z mab25 ( gray solid trace ) or protein G C2–C3 fragment (SPG) ( black dashed trace ) BIAcore biosensor surface. For comparison, a sample containing 10% FBS spiked with mouse IgG 1 mAb3 was also flowed over the Z mab25 surface ( black solid trace ). b : SDS-PAGE analysis (reducing conditions) of samples, flow-through (FT), and eluate (E) fractions from affinity chromatography experiments using either a protein G (SPG) column or a Z mab25 column, and samples of 10% FBS only ( lanes 1 – 6 ) or 10% FBS with in-spiked mouse IgG 1 mAb3 ( lanes 7 – 12 ) or 10% FBS with in-spiked mouse IgG 1 8F11 ( lanes 13 – 15 ). M LMW-SDS molecular weight marker; lane 1 : 10% FBS sample; lane 2 : SPG column, FT; lane 3 : SPG column, E; lane 4 : 10% FBS sample; lane 5 : Z mab25 column, FT; lane 6 : Z mab25 column, E; lane 7 : 10% FBS + mAb3 sample; lane 8 : SPG column, FT; lane 9 : SPG column, E; lane 10 : 10% FBS + mAb3 sample; lane 11 : Z mab25 column, FT; lane 12 : Z mab25 column, E; lane 13 : 10% FBS + mAb 8F11 sample; lane 14 : Z mab25 column, FT; lane 15 : Z mab25 column, E. The black arrows to the right indicate nominal molecular weights of antibody heavy and light chains, respectively. The numbers to the left indicate molecular weights in kilo Dalton (kDa). c Results from a biosensor analysis of pH neutralized eluates from the SPG and Z mab25 columns, originating from the use of the 10% FBS sample containing in-spiked mAb3 ( 1 – 4 ) or mAb 8F11 ( 5 , 6 ). On separate flow cell surfaces, anti-cow IgG and anti-mouse Ig antibodies were immobilized, and samples were injected. (1) black solid trace SPG column eluate, anti-cow IgG surface; (2) black dashed trace SPG column eluate, anti-mouse Ig surface; (3) gray solid trace Z mab25 column eluate, anti-cow IgG surface; (4) gray dashed trace Z mab25 column eluate, anti-mouse Ig surface; (5) black dotted trace Z mab25 column eluate, anti-cow IgG surface; (6) gray dotted trace Z mab25 column eluate, anti-mouse Ig surface
Figure Legend Snippet: Binding studies of Z mab25 or protein G with fetal bovine serum and species selective affinity recovery of two monoclonal antibodies. a 10% FBS was flowed over a Z mab25 ( gray solid trace ) or protein G C2–C3 fragment (SPG) ( black dashed trace ) BIAcore biosensor surface. For comparison, a sample containing 10% FBS spiked with mouse IgG 1 mAb3 was also flowed over the Z mab25 surface ( black solid trace ). b : SDS-PAGE analysis (reducing conditions) of samples, flow-through (FT), and eluate (E) fractions from affinity chromatography experiments using either a protein G (SPG) column or a Z mab25 column, and samples of 10% FBS only ( lanes 1 – 6 ) or 10% FBS with in-spiked mouse IgG 1 mAb3 ( lanes 7 – 12 ) or 10% FBS with in-spiked mouse IgG 1 8F11 ( lanes 13 – 15 ). M LMW-SDS molecular weight marker; lane 1 : 10% FBS sample; lane 2 : SPG column, FT; lane 3 : SPG column, E; lane 4 : 10% FBS sample; lane 5 : Z mab25 column, FT; lane 6 : Z mab25 column, E; lane 7 : 10% FBS + mAb3 sample; lane 8 : SPG column, FT; lane 9 : SPG column, E; lane 10 : 10% FBS + mAb3 sample; lane 11 : Z mab25 column, FT; lane 12 : Z mab25 column, E; lane 13 : 10% FBS + mAb 8F11 sample; lane 14 : Z mab25 column, FT; lane 15 : Z mab25 column, E. The black arrows to the right indicate nominal molecular weights of antibody heavy and light chains, respectively. The numbers to the left indicate molecular weights in kilo Dalton (kDa). c Results from a biosensor analysis of pH neutralized eluates from the SPG and Z mab25 columns, originating from the use of the 10% FBS sample containing in-spiked mAb3 ( 1 – 4 ) or mAb 8F11 ( 5 , 6 ). On separate flow cell surfaces, anti-cow IgG and anti-mouse Ig antibodies were immobilized, and samples were injected. (1) black solid trace SPG column eluate, anti-cow IgG surface; (2) black dashed trace SPG column eluate, anti-mouse Ig surface; (3) gray solid trace Z mab25 column eluate, anti-cow IgG surface; (4) gray dashed trace Z mab25 column eluate, anti-mouse Ig surface; (5) black dotted trace Z mab25 column eluate, anti-cow IgG surface; (6) gray dotted trace Z mab25 column eluate, anti-mouse Ig surface

Techniques Used: Binding Assay, SDS Page, Flow Cytometry, Affinity Chromatography, Molecular Weight, Marker, Injection

46) Product Images from "Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages"

Article Title: Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages

Journal: Infection and Immunity

doi: 10.1128/IAI.00033-15

Differentiation of monocyte-derived human macrophages in fetal bovine serum versus human serum affects the outcome of Salmonella Typhimurium infection. Cells were cultured in media containing FBS or HuS for 7 days as described in the text and then infected with Salmonella WT strains SL1344 and14028. (A) Phase-contrast images of uninfected cells. Bar, 100 μm. (B) Gentamicin protection assay. Shown is the fold change in CFU (at 18 and 2 h p.i.). Results are means ± SD from 4 independent experiments. *, P
Figure Legend Snippet: Differentiation of monocyte-derived human macrophages in fetal bovine serum versus human serum affects the outcome of Salmonella Typhimurium infection. Cells were cultured in media containing FBS or HuS for 7 days as described in the text and then infected with Salmonella WT strains SL1344 and14028. (A) Phase-contrast images of uninfected cells. Bar, 100 μm. (B) Gentamicin protection assay. Shown is the fold change in CFU (at 18 and 2 h p.i.). Results are means ± SD from 4 independent experiments. *, P

Techniques Used: Derivative Assay, Infection, Cell Culture

47) Product Images from "Aberrant over-expression of TRPM7 ion channels in pancreatic cancer: required for cancer cell invasion and implicated in tumor growth and metastasis"

Article Title: Aberrant over-expression of TRPM7 ion channels in pancreatic cancer: required for cancer cell invasion and implicated in tumor growth and metastasis

Journal: Biology Open

doi: 10.1242/bio.20137088

ShRNA-mediated silencing of TRPM7 impaired invasion in pancreatic adenocarcinoma cells. (A) Non-transfected BxPC-3 cells assayed for invasion using the trans-well assay in the presence of 3% FBS or no FBS. (B,C) BxPC-3 cells transfected with non-targeting control (NC) shRNA or anti- TRPM7 shRNA analyzed for cell invasion in the presence of either 3% FBS (B) or 10% FBS (C). A representative image of the invaded cells stained with crystal violet is shown for each experimental group. Cell invasion is expressed as % control, and each column represents the mean ± standard error.
Figure Legend Snippet: ShRNA-mediated silencing of TRPM7 impaired invasion in pancreatic adenocarcinoma cells. (A) Non-transfected BxPC-3 cells assayed for invasion using the trans-well assay in the presence of 3% FBS or no FBS. (B,C) BxPC-3 cells transfected with non-targeting control (NC) shRNA or anti- TRPM7 shRNA analyzed for cell invasion in the presence of either 3% FBS (B) or 10% FBS (C). A representative image of the invaded cells stained with crystal violet is shown for each experimental group. Cell invasion is expressed as % control, and each column represents the mean ± standard error.

Techniques Used: shRNA, Transfection, Staining

48) Product Images from "Bone marrow-derived progenitor cells prevent thrombin-induced increase in lung vascular permeability"

Article Title: Bone marrow-derived progenitor cells prevent thrombin-induced increase in lung vascular permeability

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

doi: 10.1152/ajplung.00064.2009

Bone marrow-derived progenitor cells (BMPCs) exhibit the characteristics of progenitor cells. A – C : colony-forming units (CFU-C) assays demonstrating the progenitor potential of BMPCs. BMPCs or mouse lung endothelial cells (ECs) were plated at a density of 10 3 cells/well with MethodCult RH4100 mixed with EBM-2MV medium (1:1) containing 5% FBS. Following 18 days in culture, the colonies formed in collagen gels were counted. Data are expressed as means ± SD ( n = 3 experiments). * P
Figure Legend Snippet: Bone marrow-derived progenitor cells (BMPCs) exhibit the characteristics of progenitor cells. A – C : colony-forming units (CFU-C) assays demonstrating the progenitor potential of BMPCs. BMPCs or mouse lung endothelial cells (ECs) were plated at a density of 10 3 cells/well with MethodCult RH4100 mixed with EBM-2MV medium (1:1) containing 5% FBS. Following 18 days in culture, the colonies formed in collagen gels were counted. Data are expressed as means ± SD ( n = 3 experiments). * P

Techniques Used: Derivative Assay

49) Product Images from "The oncogenic transcription factor c-Jun regulates glutaminase expression and sensitizes cells to glutaminase-targeted therapy"

Article Title: The oncogenic transcription factor c-Jun regulates glutaminase expression and sensitizes cells to glutaminase-targeted therapy

Journal: Nature Communications

doi: 10.1038/ncomms11321

c-Jun correlates strongly with GLS levels in human breast cancer cell lines. Cells were collected at ∼60% confluency from RPMI growth medium supplemented with 10% FBS, and whole-cell lysates prepared and analysed by western blot. Samples were ordered according to glutamine dependence, increasing from left to right ( Supplementary Fig. 3 ). ( a ) Correlation between c-Jun/phospho-c-Jun and GLS levels. Quantification of GLS and c-Jun band intensities allowed a Pearson correlation coefficient of 0.85 to be determined ( Supplementary Fig. 4a ). ( b ) Other Jun-family members do not correlate strongly with GLS levels. ( c ) Under 10% FBS culture conditions, p46 JNK (lower band) is active in all of the breast cancer cell lines. Neither JNK nor phospho-JNK correlate with GLS levels. ( d ) Other reported regulators of GLS expression do not strongly correlate with GLS levels.
Figure Legend Snippet: c-Jun correlates strongly with GLS levels in human breast cancer cell lines. Cells were collected at ∼60% confluency from RPMI growth medium supplemented with 10% FBS, and whole-cell lysates prepared and analysed by western blot. Samples were ordered according to glutamine dependence, increasing from left to right ( Supplementary Fig. 3 ). ( a ) Correlation between c-Jun/phospho-c-Jun and GLS levels. Quantification of GLS and c-Jun band intensities allowed a Pearson correlation coefficient of 0.85 to be determined ( Supplementary Fig. 4a ). ( b ) Other Jun-family members do not correlate strongly with GLS levels. ( c ) Under 10% FBS culture conditions, p46 JNK (lower band) is active in all of the breast cancer cell lines. Neither JNK nor phospho-JNK correlate with GLS levels. ( d ) Other reported regulators of GLS expression do not strongly correlate with GLS levels.

Techniques Used: Western Blot, Expressing

50) Product Images from "Imaging flow cytometry facilitates multiparametric characterization of extracellular vesicles in malignant brain tumours"

Article Title: Imaging flow cytometry facilitates multiparametric characterization of extracellular vesicles in malignant brain tumours

Journal: Journal of Extracellular Vesicles

doi: 10.1080/20013078.2019.1588555

Cellular and vesicular tetraspanin responses to various cell stimuli. (a) Three glioblastoma cell lines (GS-8 (triangle), GS-57 (circle), BT112 (square)) were cultured under hypoxic conditions (0.1% O 2 ) or differentiating condition (medium containing EV-depleted FBS). Cellular CD81, CD9 and CD63 expression was measured by qPCR, and transcript levels were normalized to 18S. Expression values of pooled GS-8, GS-57 and BT112 cells are shown as means + SD and were normalized to cells under normoxia. (b) Hypoxic cell adaptation was confirmed by induction HK2 and GLUT1, and differentiation was confirmed by elevated expression of GFAP as well as downregulation of OLIG2, PAX6, POU5F1 and SOX2. Expression values of pooled GS-8, GS-57 and BT112 are shown as means + SD. (c) Corresponding EVs were analysed for CD81, CD9 and CD63 by IFCM, and single (left) as well as double (right) positive EVs are shown and calculated as fraction of positive EVs of CD9, CD63 or CD81 positive EVs. EV profiles of GS-8, GS-57 and BT112 were pooled and values are shown as mean + SD normalized to cells under normoxia. Results are representative of at least three biological repeat experiments. ANOVA, posthoc Bonferroni was performed for multiple group comparisons and student’s t -test was performed to compare two individual groups (**** = p
Figure Legend Snippet: Cellular and vesicular tetraspanin responses to various cell stimuli. (a) Three glioblastoma cell lines (GS-8 (triangle), GS-57 (circle), BT112 (square)) were cultured under hypoxic conditions (0.1% O 2 ) or differentiating condition (medium containing EV-depleted FBS). Cellular CD81, CD9 and CD63 expression was measured by qPCR, and transcript levels were normalized to 18S. Expression values of pooled GS-8, GS-57 and BT112 cells are shown as means + SD and were normalized to cells under normoxia. (b) Hypoxic cell adaptation was confirmed by induction HK2 and GLUT1, and differentiation was confirmed by elevated expression of GFAP as well as downregulation of OLIG2, PAX6, POU5F1 and SOX2. Expression values of pooled GS-8, GS-57 and BT112 are shown as means + SD. (c) Corresponding EVs were analysed for CD81, CD9 and CD63 by IFCM, and single (left) as well as double (right) positive EVs are shown and calculated as fraction of positive EVs of CD9, CD63 or CD81 positive EVs. EV profiles of GS-8, GS-57 and BT112 were pooled and values are shown as mean + SD normalized to cells under normoxia. Results are representative of at least three biological repeat experiments. ANOVA, posthoc Bonferroni was performed for multiple group comparisons and student’s t -test was performed to compare two individual groups (**** = p

Techniques Used: Cell Culture, Expressing, Real-time Polymerase Chain Reaction

51) Product Images from "Diesel and biodiesel exhaust particle effects on rat alveolar macrophages with in vitro exposure"

Article Title: Diesel and biodiesel exhaust particle effects on rat alveolar macrophages with in vitro exposure

Journal: Chemosphere

doi: 10.1016/j.chemosphere.2013.10.080

B20 and PDEP binding of hot ( 3 H-PGE 2 ) and cold (PGE 2 ) in a cell free system. Particles were suspended in RPMI media with 2.5% FBS for 24h, particles were centrifuged and supernatant was collected for standard scintillation counting. B20 at both 100µg/mL
Figure Legend Snippet: B20 and PDEP binding of hot ( 3 H-PGE 2 ) and cold (PGE 2 ) in a cell free system. Particles were suspended in RPMI media with 2.5% FBS for 24h, particles were centrifuged and supernatant was collected for standard scintillation counting. B20 at both 100µg/mL

Techniques Used: Binding Assay

52) Product Images from "Pentabromopseudilin: a myosin V inhibitor suppresses TGF- β activity by recruiting the type II TGF- β receptor to lysosomal degradation "

Article Title: Pentabromopseudilin: a myosin V inhibitor suppresses TGF- β activity by recruiting the type II TGF- β receptor to lysosomal degradation

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

doi: 10.1080/14756366.2018.1465416

PBrP delays TGF- β -induced cell migration. Confluent monolayers of A549 cells in a 24-well cluster tissue culture plate were scratched and incubated at 37 °C for 20 h in DMEM containing 0.2% of FBS (Control; top). Cell motility was measured using a fully automatic microscope at 10× phase objective. Cell migration was observed by performing time-lapse microscopy and images of all four experimental conditions were captured simultaneously every 20 min. The red lines indicate the starting point of cell migration. Representative micrographs from three experiments are shown in panel (A). Right graphs illustrate of quantitative analyses of cell covering area (mean ± SD) from three independent experiments are shown (B) (% of TGF- β treatment alone); ** p
Figure Legend Snippet: PBrP delays TGF- β -induced cell migration. Confluent monolayers of A549 cells in a 24-well cluster tissue culture plate were scratched and incubated at 37 °C for 20 h in DMEM containing 0.2% of FBS (Control; top). Cell motility was measured using a fully automatic microscope at 10× phase objective. Cell migration was observed by performing time-lapse microscopy and images of all four experimental conditions were captured simultaneously every 20 min. The red lines indicate the starting point of cell migration. Representative micrographs from three experiments are shown in panel (A). Right graphs illustrate of quantitative analyses of cell covering area (mean ± SD) from three independent experiments are shown (B) (% of TGF- β treatment alone); ** p

Techniques Used: Migration, Incubation, Microscopy, Time-lapse Microscopy

PBrP induces T β RII turnover through the late endosome–lysosome pathway and is impaired by lysosome inhibitors. Mv1Lu cells grown in 0.2% of FBS-containing DMEM were incubated with PBrP (0.5 μ M) with chloroquine (100 μ M) or NH 4 Cl (20 mM) (A) for 1, 3 and 6 h or MG132 (20 μ M) and carfilzomib (0.5 μ M) (B). Subsequently, the cell lysates were subjected to SDS-PAGE, and T β RII expression was analysed through Western blotting and quantified through densitometry. (C) T β RII was enriched in the Rab11-positive endosomal compartment in PBrP-treated cells. Mv1Lu cells were treated with 0.5 μ M of PBrP for 1 h in low-serum DMEM. Endogenous T β RII and Rab11 were visualised through immunofluorescence staining using Alexa Fluor 488- and 594-conjugated secondary antibodies, respectively. (D) T β RII was internalised and directed into lysosomes in PBrP-treated cells. HEK293 cells transiently co-expressing T β RII-flag and Lamp-1-GFP were treated with 0.5 μ M of PBrP for 2.5 h in low-serum DMEM. After fixation and permeabilisation, T β RII-flag was visualised through immunofluorescence staining using anti-flag antibodies and an Alexa Fluor 594-conjugated secondary antibody. PBrP reduced membrane-associated T β RII-flag (red) and increased co-localisation with the lysosome marker Lamp-1 (green). Bar: 10 μ m.
Figure Legend Snippet: PBrP induces T β RII turnover through the late endosome–lysosome pathway and is impaired by lysosome inhibitors. Mv1Lu cells grown in 0.2% of FBS-containing DMEM were incubated with PBrP (0.5 μ M) with chloroquine (100 μ M) or NH 4 Cl (20 mM) (A) for 1, 3 and 6 h or MG132 (20 μ M) and carfilzomib (0.5 μ M) (B). Subsequently, the cell lysates were subjected to SDS-PAGE, and T β RII expression was analysed through Western blotting and quantified through densitometry. (C) T β RII was enriched in the Rab11-positive endosomal compartment in PBrP-treated cells. Mv1Lu cells were treated with 0.5 μ M of PBrP for 1 h in low-serum DMEM. Endogenous T β RII and Rab11 were visualised through immunofluorescence staining using Alexa Fluor 488- and 594-conjugated secondary antibodies, respectively. (D) T β RII was internalised and directed into lysosomes in PBrP-treated cells. HEK293 cells transiently co-expressing T β RII-flag and Lamp-1-GFP were treated with 0.5 μ M of PBrP for 2.5 h in low-serum DMEM. After fixation and permeabilisation, T β RII-flag was visualised through immunofluorescence staining using anti-flag antibodies and an Alexa Fluor 594-conjugated secondary antibody. PBrP reduced membrane-associated T β RII-flag (red) and increased co-localisation with the lysosome marker Lamp-1 (green). Bar: 10 μ m.

Techniques Used: Incubation, SDS Page, Expressing, Western Blot, Immunofluorescence, Staining, Marker

53) Product Images from "6-Substituted Pyrrolo[2,3-d]pyrimidine Thienoyl Regioisomers as Targeted Antifolates for Folate Receptor α and the Proton-Coupled Folate Transporter in Human Tumors"

Article Title: 6-Substituted Pyrrolo[2,3-d]pyrimidine Thienoyl Regioisomers as Targeted Antifolates for Folate Receptor α and the Proton-Coupled Folate Transporter in Human Tumors

Journal: Journal of medicinal chemistry

doi: 10.1021/acs.jmedchem.5b00801

Growth inhibition of KB cells by 4 and 6 – 8 and protection by excess folic acid, nucleosides, or 5-aminoimidazole-4-carboxamide (AICA). KB cells were plated (4000 cells/well) in folate-free RPMI 1640 medium with 10% FBS, antibiotics, L-glutamine,
Figure Legend Snippet: Growth inhibition of KB cells by 4 and 6 – 8 and protection by excess folic acid, nucleosides, or 5-aminoimidazole-4-carboxamide (AICA). KB cells were plated (4000 cells/well) in folate-free RPMI 1640 medium with 10% FBS, antibiotics, L-glutamine,

Techniques Used: Inhibition

54) Product Images from "Grp94 in complexes with IgG is a soluble diagnostic marker of gastrointestinal tumors and displays immune-stimulating activity on peripheral blood immune cells"

Article Title: Grp94 in complexes with IgG is a soluble diagnostic marker of gastrointestinal tumors and displays immune-stimulating activity on peripheral blood immune cells

Journal: Oncotarget

doi: 10.18632/oncotarget.12141

Grp94 in complexes with IgG is the form in which the tumor antigen Grp94 circulates in plasma and is presented by plasma cells ( A ) WB for Grp94 and IgG in plasma of all patients. Patients are numbered following the order by which they were analyzed. Ten μg of proteins of each plasma sample was loaded on to 4–20% polyacrylamide gel in denaturing conditions of PAGE, without boiling and reducing treatment of samples. WB was performed as specified in Methods, probing the membrane with primary anti-Grp94 monoclonal Abs (clone 9G10, Santa Cruz Biotech., Santa Cruz, CA, USA) followed by anti-rat HRP-conjugated secondary Abs, and with primary anti-human IgG polyclonal Abs followed by anti-sheep HRP-conjugated secondary Abs. Cropped WB of samples 1–14 and samples 15–28 originate from two distinct membranes. Patient #16 was then excluded from the following analyses for having a tumor that secondarily involved the large bowel. Only the Grp94-positive bands in the membranes are shown since no other band was visualized. Grp94 at masses higher than 200 kDa indicates the formation of complexes with IgG, as proved by the intense co-positivity for human IgG of the Grp94-positve bands (see WB on right). Specificity of the immune reaction was also evaluated by probing the membranes with only secondary Abs. ( B ) In the upper panel, scatter plots are shown of ELISA determinations of Grp94-IgG complexes in plasma of 15 healthy subjects and 22 cancer patients (13 males, 9 females). Measurements were made by testing each plasma sample in duplicate at the dilution of 1:128, and absorbance was measured at 450 nm (OD, optical density). Horizontal lines indicate the mean value with SE. Minimum and maximum OD values were, respectively, 0.27 and 0.458 in healthy subjects, and 0.506 and 2.31 in patients. The mean ± SE of OD values in healthy subjects was 0.346 ± 0.013, and in patients 0.796 ± 0.0817. Lower and upper 95% CI of the mean was 0.318 and 0.375 in healthy subjects, 0.627 and 0.966 in patients. The difference between the two groups was statistically significant (Mann-Whitney test, two-tailed). The lower panel shows the ELISA measurements in the same patients grouped by type of tumor. The mean ± SE OD values did not differ significantly among groups. The patient with the highest value of OD (2.31) died after two months from the admission. ( C ) Cropped WB for Grp94 on lysates of PBMCs of a representative number of patients (eight with large bowel adenocarcinomas and two with gastric carcinomas). Patients #3, #7, #9, #10 and #13 had tumors at stage III/IV. PBMCs were incubated in absence (control) and presence of the B cell stimulatory agent Pokeweed mitogen (PWM) at 20 μg/ml (final concentration), and after 10 days of incubation cells were lysed and treated as specified in Methods. The protein concentration was previously determined by means of calibration SDS-PAGE (10% polyacrylamide gel) and the same protein quantity (without boiling and reducing treatments) was loaded for each sample on to a 4–20% polyacrylamide gel and then probed with anti-Grp94 rat monoclonal Abs. Specificity of the reaction was assessed by probing the membrane with secondary Abs only. Stimulation by PWM leads to the increased expression of Grp94 at molecular masses much higher than the expected mass of the protein, consistent with the association of Grp94 in big complexes with IgG (WB for human IgG in Supplementary Figure S2B ). Arrows mark the bands positive for Grp94 that in control PBMCs focus at molecular masses of both the monomer (100 kDa) and dimer (200 kDa), whereas in PWM-treated cells mostly appear at masses consistent with the formation of big complexes. Adjustments of the contrast and brightness were applied to each WB image. ( D ) Cropped WB for Grp94 and IgG on Grp94 in culture medium of PBMCs in absence (control, without incubation) and presence of 10% FBS (after incubation). Fifty μg of Grp94, in absence of reducing treatment, were loaded in each lane of a 4–20% polyacrylamide gel and probed with anti-Grp94 rat monoclonal Abs and anti-IgG sheep polyclonal Abs. Arrows mark the band of Grp94 that in absence of FBS appears in its free form, prevalently as monomer (100 kDa), whereas in the presence of FBS Grp94 focuses exclusively at high molecular masses in association with IgG. Blots for incubated Grp94 derive from a single membrane. No adjustments were made for the blots of Grp94, whereas slight adjustments for contrast and brightness were applied to the blot of IgG.
Figure Legend Snippet: Grp94 in complexes with IgG is the form in which the tumor antigen Grp94 circulates in plasma and is presented by plasma cells ( A ) WB for Grp94 and IgG in plasma of all patients. Patients are numbered following the order by which they were analyzed. Ten μg of proteins of each plasma sample was loaded on to 4–20% polyacrylamide gel in denaturing conditions of PAGE, without boiling and reducing treatment of samples. WB was performed as specified in Methods, probing the membrane with primary anti-Grp94 monoclonal Abs (clone 9G10, Santa Cruz Biotech., Santa Cruz, CA, USA) followed by anti-rat HRP-conjugated secondary Abs, and with primary anti-human IgG polyclonal Abs followed by anti-sheep HRP-conjugated secondary Abs. Cropped WB of samples 1–14 and samples 15–28 originate from two distinct membranes. Patient #16 was then excluded from the following analyses for having a tumor that secondarily involved the large bowel. Only the Grp94-positive bands in the membranes are shown since no other band was visualized. Grp94 at masses higher than 200 kDa indicates the formation of complexes with IgG, as proved by the intense co-positivity for human IgG of the Grp94-positve bands (see WB on right). Specificity of the immune reaction was also evaluated by probing the membranes with only secondary Abs. ( B ) In the upper panel, scatter plots are shown of ELISA determinations of Grp94-IgG complexes in plasma of 15 healthy subjects and 22 cancer patients (13 males, 9 females). Measurements were made by testing each plasma sample in duplicate at the dilution of 1:128, and absorbance was measured at 450 nm (OD, optical density). Horizontal lines indicate the mean value with SE. Minimum and maximum OD values were, respectively, 0.27 and 0.458 in healthy subjects, and 0.506 and 2.31 in patients. The mean ± SE of OD values in healthy subjects was 0.346 ± 0.013, and in patients 0.796 ± 0.0817. Lower and upper 95% CI of the mean was 0.318 and 0.375 in healthy subjects, 0.627 and 0.966 in patients. The difference between the two groups was statistically significant (Mann-Whitney test, two-tailed). The lower panel shows the ELISA measurements in the same patients grouped by type of tumor. The mean ± SE OD values did not differ significantly among groups. The patient with the highest value of OD (2.31) died after two months from the admission. ( C ) Cropped WB for Grp94 on lysates of PBMCs of a representative number of patients (eight with large bowel adenocarcinomas and two with gastric carcinomas). Patients #3, #7, #9, #10 and #13 had tumors at stage III/IV. PBMCs were incubated in absence (control) and presence of the B cell stimulatory agent Pokeweed mitogen (PWM) at 20 μg/ml (final concentration), and after 10 days of incubation cells were lysed and treated as specified in Methods. The protein concentration was previously determined by means of calibration SDS-PAGE (10% polyacrylamide gel) and the same protein quantity (without boiling and reducing treatments) was loaded for each sample on to a 4–20% polyacrylamide gel and then probed with anti-Grp94 rat monoclonal Abs. Specificity of the reaction was assessed by probing the membrane with secondary Abs only. Stimulation by PWM leads to the increased expression of Grp94 at molecular masses much higher than the expected mass of the protein, consistent with the association of Grp94 in big complexes with IgG (WB for human IgG in Supplementary Figure S2B ). Arrows mark the bands positive for Grp94 that in control PBMCs focus at molecular masses of both the monomer (100 kDa) and dimer (200 kDa), whereas in PWM-treated cells mostly appear at masses consistent with the formation of big complexes. Adjustments of the contrast and brightness were applied to each WB image. ( D ) Cropped WB for Grp94 and IgG on Grp94 in culture medium of PBMCs in absence (control, without incubation) and presence of 10% FBS (after incubation). Fifty μg of Grp94, in absence of reducing treatment, were loaded in each lane of a 4–20% polyacrylamide gel and probed with anti-Grp94 rat monoclonal Abs and anti-IgG sheep polyclonal Abs. Arrows mark the band of Grp94 that in absence of FBS appears in its free form, prevalently as monomer (100 kDa), whereas in the presence of FBS Grp94 focuses exclusively at high molecular masses in association with IgG. Blots for incubated Grp94 derive from a single membrane. No adjustments were made for the blots of Grp94, whereas slight adjustments for contrast and brightness were applied to the blot of IgG.

Techniques Used: Western Blot, Polyacrylamide Gel Electrophoresis, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Two Tailed Test, Incubation, Concentration Assay, Protein Concentration, SDS Page, Expressing

55) Product Images from "Transplantation of a bone marrow mesenchymal stem cell line increases neuronal progenitor cell migration in a cerebral ischemia animal model"

Article Title: Transplantation of a bone marrow mesenchymal stem cell line increases neuronal progenitor cell migration in a cerebral ischemia animal model

Journal: Scientific Reports

doi: 10.1038/s41598-018-33030-9

Effects of B10 cells on neuronal cell migration in culture. ( A ) To assess the effects of B10-secreted molecules on neuronal migration, B10 cells were cultured to confluency in complete medium. Then the medium was changed to 0.5% FBS containing DMEM, cultured for further 24 h and the culture supernatant was used for A1 migration assay. DMEM-only, DMEM containing 0.5% FBS (medium) or A1 culture supernatant was used as control. * p
Figure Legend Snippet: Effects of B10 cells on neuronal cell migration in culture. ( A ) To assess the effects of B10-secreted molecules on neuronal migration, B10 cells were cultured to confluency in complete medium. Then the medium was changed to 0.5% FBS containing DMEM, cultured for further 24 h and the culture supernatant was used for A1 migration assay. DMEM-only, DMEM containing 0.5% FBS (medium) or A1 culture supernatant was used as control. * p

Techniques Used: Migration, Cell Culture

56) Product Images from "Abi-1 forms an epidermal growth factor-inducible complex with Cbl: role in receptor endocytosis"

Article Title: Abi-1 forms an epidermal growth factor-inducible complex with Cbl: role in receptor endocytosis

Journal: Cellular signalling

doi: 10.1016/j.cellsig.2007.02.008

Endogenous Abi-1 forms a complex with Endogenous Cbl upon EGF-stimulation in COS-7 cells COS7 cells (70 % confluency) were placed in 0.1% FBS for 24 hours and subsequently treated with EGF for the times indicated. Abi-1 was immunoprecipitated with a combination of two polyclonal antibodies (6987/6988) (odd lanes). Normal rabbit serum (NRS) was employed as a negative control (even lanes). Twelve hours prior to serum-starvation, COS7 cells were plated at 2.5 ×10 6 cells/dish. Lanes 1,2: serum-starved cells. Lanes 3, 4: EGF 5 min. Lanes 5,6: EGF 15 min. Lanes 7,8: EGF 60 min. Lanes 9,10: cells in DMEM 10% FBS. Whole cell lysates showing the levels of Cbl and Abi-1 are shown in the lower panels. These data are representative of 5 independent experiments.
Figure Legend Snippet: Endogenous Abi-1 forms a complex with Endogenous Cbl upon EGF-stimulation in COS-7 cells COS7 cells (70 % confluency) were placed in 0.1% FBS for 24 hours and subsequently treated with EGF for the times indicated. Abi-1 was immunoprecipitated with a combination of two polyclonal antibodies (6987/6988) (odd lanes). Normal rabbit serum (NRS) was employed as a negative control (even lanes). Twelve hours prior to serum-starvation, COS7 cells were plated at 2.5 ×10 6 cells/dish. Lanes 1,2: serum-starved cells. Lanes 3, 4: EGF 5 min. Lanes 5,6: EGF 15 min. Lanes 7,8: EGF 60 min. Lanes 9,10: cells in DMEM 10% FBS. Whole cell lysates showing the levels of Cbl and Abi-1 are shown in the lower panels. These data are representative of 5 independent experiments.

Techniques Used: Immunoprecipitation, Negative Control

57) Product Images from "Cancer-derived immunoglobulin G promotes tumor cell growth and proliferation through inducing production of reactive oxygen species"

Article Title: Cancer-derived immunoglobulin G promotes tumor cell growth and proliferation through inducing production of reactive oxygen species

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.474

Downregulation of cancer-derived IgG-lowered cellular ROS level. ( a ) HeLa cells were treated with IGHG1 siRNA or scrambled siRNA for 72 h. Cells were then incubated with fluorescent probe DHE at a final concentration of 10 μ M in high-glucose DMEM without FBS for 20 min at 37 °C, washed three times with serum-free medium and analyzed using a flow cytometry. For each analysis, 10 000 events were recorded. Similarly treated cells were observed with a confocal microscope. The images are shown in the right panel. Scale bar, 50 μ m. ( b ) Similar experiments were performed for HEp-2 and PC3 cells. ( c ) HeLa cells were transfected with IGHG1 siRNA or scrambled siRNA for 72 h. Cells were then incubated with fluorescent probe DCFH-DA at a final concentration of 10 μ M in high-glucose DMEM without FBS for 20 min at 37 °C, washed three times with serum-free medium and analyzed with a flow cytometry. For each analysis, 10 000 events were recorded. Similarly treated cells were observed with a confocal microscope. The images are shown in the right panel. Scale bar, 50 μ m. ( d ) Intracellular ROS (H 2 O 2 ) level was monitored with flow cytometry with fluorescent probe DCFH-DA in HEp-2 and PC3 cells. HeLa cells were treated with IGHG1 siRNA or scrambled siRNA for 72 h. Total lysates ( e ) and culture medium ( f ) were used to detect cellular H 2 O 2 level respectively. Similar experiments were performed using HeLa cells stably expressing IGHG1 shRNA or control shRNA ( a , c , e , and f ). Data shown are the mean±S.D. of three independent experiments (* P
Figure Legend Snippet: Downregulation of cancer-derived IgG-lowered cellular ROS level. ( a ) HeLa cells were treated with IGHG1 siRNA or scrambled siRNA for 72 h. Cells were then incubated with fluorescent probe DHE at a final concentration of 10 μ M in high-glucose DMEM without FBS for 20 min at 37 °C, washed three times with serum-free medium and analyzed using a flow cytometry. For each analysis, 10 000 events were recorded. Similarly treated cells were observed with a confocal microscope. The images are shown in the right panel. Scale bar, 50 μ m. ( b ) Similar experiments were performed for HEp-2 and PC3 cells. ( c ) HeLa cells were transfected with IGHG1 siRNA or scrambled siRNA for 72 h. Cells were then incubated with fluorescent probe DCFH-DA at a final concentration of 10 μ M in high-glucose DMEM without FBS for 20 min at 37 °C, washed three times with serum-free medium and analyzed with a flow cytometry. For each analysis, 10 000 events were recorded. Similarly treated cells were observed with a confocal microscope. The images are shown in the right panel. Scale bar, 50 μ m. ( d ) Intracellular ROS (H 2 O 2 ) level was monitored with flow cytometry with fluorescent probe DCFH-DA in HEp-2 and PC3 cells. HeLa cells were treated with IGHG1 siRNA or scrambled siRNA for 72 h. Total lysates ( e ) and culture medium ( f ) were used to detect cellular H 2 O 2 level respectively. Similar experiments were performed using HeLa cells stably expressing IGHG1 shRNA or control shRNA ( a , c , e , and f ). Data shown are the mean±S.D. of three independent experiments (* P

Techniques Used: Derivative Assay, Incubation, Concentration Assay, Flow Cytometry, Cytometry, Microscopy, Transfection, Stable Transfection, Expressing, shRNA

58) Product Images from "1, 25-dihydroxy-vitamin D3 with tumor necrosis factor-alpha protects against rheumatoid arthritis by promoting p53 acetylation-mediated apoptosis via Sirt1 in synoviocytes"

Article Title: 1, 25-dihydroxy-vitamin D3 with tumor necrosis factor-alpha protects against rheumatoid arthritis by promoting p53 acetylation-mediated apoptosis via Sirt1 in synoviocytes

Journal: Cell Death & Disease

doi: 10.1038/cddis.2016.300

VD with TNF- α promoted apoptosis of rheumatoid FLSs. Human rheumatoid FLS-MH7A cells were treated with DMEM and 10% FBS (serum control), DMEM (serum-free control), DMEM and indicated concentrations of VD with or without TNF- α . ( a ) Flow cytometry of double-stained cells using annexin V (AV) and propidium iodide (PI). ( b ) AV-positive but PI-negative cells (AV+PI–), AV and PI double-positive cells (AV+PI+), and total AV-positive cells (AV+) were quantified. Values are mean±S.E.M. of six determinations per group. * P
Figure Legend Snippet: VD with TNF- α promoted apoptosis of rheumatoid FLSs. Human rheumatoid FLS-MH7A cells were treated with DMEM and 10% FBS (serum control), DMEM (serum-free control), DMEM and indicated concentrations of VD with or without TNF- α . ( a ) Flow cytometry of double-stained cells using annexin V (AV) and propidium iodide (PI). ( b ) AV-positive but PI-negative cells (AV+PI–), AV and PI double-positive cells (AV+PI+), and total AV-positive cells (AV+) were quantified. Values are mean±S.E.M. of six determinations per group. * P

Techniques Used: Flow Cytometry, Cytometry, Staining

VD with TNF- α induced apoptosis of rheumatoid FLSs through VDR and p53 pro-apoptotic signaling. Human rheumatoid FLS-MH7A cells were treated with DMEM and 10% FBS (serum control), DMEM (serum-free control), DMEM and 10 –7 M VD and VDR-negative control small interfering RNA (10 –7 M VD+NC siRNA), DMEM and 10 –7 M VD and VDR siRNA (10 –7 M VD+VDR siRNA), DMEM and 10 –7 M VD and 30 μ M pifithrin- α (PFT- α ) (10 –7 M VD+30 μ M PFT- α ), DMEM and 30 ng/ml TNF- α and NC siRNA (30 ng/ml TNF- α + NC siRNA), DMEM and 30 ng/ml TNF- α and 10 –7 M VD and NC siRNA (30 ng/ml TNF- α + 10 –7 M VD+NC siRNA), DMEM and 30 ng/ml TNF- α and 10 –7 M VD and 30 μ M PFT- α (30 ng/ml TNF- α + 10 –7 M VD+30 μ M PFT- α ). ( a ) Flow cytometry of cells double-stained for annexin V (AV) and propidium iodide (PI). ( b ) AV-positive but PI-negative cells (AV+PI–), AV and PI double-positive cells (AV+PI+) and total AV-positive cells (AV+) were quantified. Values are mean±S.E.M. of six determinations per group. * P
Figure Legend Snippet: VD with TNF- α induced apoptosis of rheumatoid FLSs through VDR and p53 pro-apoptotic signaling. Human rheumatoid FLS-MH7A cells were treated with DMEM and 10% FBS (serum control), DMEM (serum-free control), DMEM and 10 –7 M VD and VDR-negative control small interfering RNA (10 –7 M VD+NC siRNA), DMEM and 10 –7 M VD and VDR siRNA (10 –7 M VD+VDR siRNA), DMEM and 10 –7 M VD and 30 μ M pifithrin- α (PFT- α ) (10 –7 M VD+30 μ M PFT- α ), DMEM and 30 ng/ml TNF- α and NC siRNA (30 ng/ml TNF- α + NC siRNA), DMEM and 30 ng/ml TNF- α and 10 –7 M VD and NC siRNA (30 ng/ml TNF- α + 10 –7 M VD+NC siRNA), DMEM and 30 ng/ml TNF- α and 10 –7 M VD and 30 μ M PFT- α (30 ng/ml TNF- α + 10 –7 M VD+30 μ M PFT- α ). ( a ) Flow cytometry of cells double-stained for annexin V (AV) and propidium iodide (PI). ( b ) AV-positive but PI-negative cells (AV+PI–), AV and PI double-positive cells (AV+PI+) and total AV-positive cells (AV+) were quantified. Values are mean±S.E.M. of six determinations per group. * P

Techniques Used: Negative Control, Small Interfering RNA, Flow Cytometry, Cytometry, Staining

VD with TNF- α promoted p53 acetylation-mediated apoptosis in human rheumatoid FLSs. Human rheumatoid FLS-MH7A cells were treated with DMEM and 10% FBS (serum control), DMEM (serum-free control), DMEM and indicated concentrations of VD with or without TNF- α . ( a ) Western blots of cells by group for Caspase-3, Bax, Puma, Bcl-2, p53, p53 (acetyl K382), NF- κ B-p65 and NF- κ B-p65 (phospho Ser536). β -actin was the loading control. ( b - h ) Protein relative to β -actin was assessed by densitometry. Values are mean±S.E.M. of six determinations per group. * P
Figure Legend Snippet: VD with TNF- α promoted p53 acetylation-mediated apoptosis in human rheumatoid FLSs. Human rheumatoid FLS-MH7A cells were treated with DMEM and 10% FBS (serum control), DMEM (serum-free control), DMEM and indicated concentrations of VD with or without TNF- α . ( a ) Western blots of cells by group for Caspase-3, Bax, Puma, Bcl-2, p53, p53 (acetyl K382), NF- κ B-p65 and NF- κ B-p65 (phospho Ser536). β -actin was the loading control. ( b - h ) Protein relative to β -actin was assessed by densitometry. Values are mean±S.E.M. of six determinations per group. * P

Techniques Used: Western Blot

59) Product Images from "Field-deployable, quantitative, rapid identification of active Ebola virus infection in unprocessed blood quantitative, rapid identification of active Ebola virus infection in unprocessed blood †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc03281a"

Article Title: Field-deployable, quantitative, rapid identification of active Ebola virus infection in unprocessed blood quantitative, rapid identification of active Ebola virus infection in unprocessed blood †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc03281a

Journal: Chemical Science

doi: 10.1039/c7sc03281a

Optimised, controlled freeze–thawing of RNA viruses in probe-based qRT-PCR containing blood or serum. (a) Optimal freeze–thaw extraction of RNA from viruses within qRT-PCR master mixes in the same reaction vessel is achieved with 8–10 freeze–thaw cycles as determined through threshold cycle (Ct) detection of template amplification using the Trombley+ GP assay against 10 5 GE per reaction of AR14 templates. No blood was used in these reactions. (b) Increasing amounts of blood spiked into reactions containing 6.6 × 10 8 GE per reaction AR14 subjected to 10 automated freeze–thaw cycles results in a 2 Ct drop in GP Trombley+ assay performance (4×) against reactions containing no blood. The Ct call in the absence of blood in the reaction (0% v/v final blood concentration) is used as an optimal performance reference. (c) Amplification discrimination for the Trombley+ assay between no template control (NTC) reactions, and reactions containing 66 GE of AR14. Reactions were spiked with 5 microliters of human blood (8% v/v final blood concentration in the reaction) and viral genomes were extracted in the qRT-PCR blood-containing mix using 8 freeze–thaw cycles. (d) Ten-fold serial dilutions in tissue culture media of tissue cultured derived, 700 PFU per ml Ebola virus were tested in three independent reactions using the Trombley+ GP assay. Fetal bovine serum was spiked into these reactions as a fresh patient sample matrix surrogate, to an 8% v/v final concentration. (e) Calibration of Ebola virus sample PFUs against GE content, as independently determined on the QuRapID under BSL4 testing using AR14 pre-qualification runs, reveals strong linear concordance between two Ebola virus strains diluted in FBS and the ArmoredRNA surrogate template (99.96% probability of single curve fit, Akaike's Informative Criteria test; individual replicates and 95% confidence bands shown).
Figure Legend Snippet: Optimised, controlled freeze–thawing of RNA viruses in probe-based qRT-PCR containing blood or serum. (a) Optimal freeze–thaw extraction of RNA from viruses within qRT-PCR master mixes in the same reaction vessel is achieved with 8–10 freeze–thaw cycles as determined through threshold cycle (Ct) detection of template amplification using the Trombley+ GP assay against 10 5 GE per reaction of AR14 templates. No blood was used in these reactions. (b) Increasing amounts of blood spiked into reactions containing 6.6 × 10 8 GE per reaction AR14 subjected to 10 automated freeze–thaw cycles results in a 2 Ct drop in GP Trombley+ assay performance (4×) against reactions containing no blood. The Ct call in the absence of blood in the reaction (0% v/v final blood concentration) is used as an optimal performance reference. (c) Amplification discrimination for the Trombley+ assay between no template control (NTC) reactions, and reactions containing 66 GE of AR14. Reactions were spiked with 5 microliters of human blood (8% v/v final blood concentration in the reaction) and viral genomes were extracted in the qRT-PCR blood-containing mix using 8 freeze–thaw cycles. (d) Ten-fold serial dilutions in tissue culture media of tissue cultured derived, 700 PFU per ml Ebola virus were tested in three independent reactions using the Trombley+ GP assay. Fetal bovine serum was spiked into these reactions as a fresh patient sample matrix surrogate, to an 8% v/v final concentration. (e) Calibration of Ebola virus sample PFUs against GE content, as independently determined on the QuRapID under BSL4 testing using AR14 pre-qualification runs, reveals strong linear concordance between two Ebola virus strains diluted in FBS and the ArmoredRNA surrogate template (99.96% probability of single curve fit, Akaike's Informative Criteria test; individual replicates and 95% confidence bands shown).

Techniques Used: Quantitative RT-PCR, Amplification, Concentration Assay, Cell Culture, Derivative Assay

60) Product Images from "Primary Respiratory Chain Disease Causes Tissue-Specific Dysregulation of the Global Transcriptome and Nutrient-Sensing Signaling Network"

Article Title: Primary Respiratory Chain Disease Causes Tissue-Specific Dysregulation of the Global Transcriptome and Nutrient-Sensing Signaling Network

Journal: PLoS ONE

doi: 10.1371/journal.pone.0069282

Primary RC dysfunction transcriptionally and post-transcriptionally dysregulates the integrated nutrient-sensing signaling network. ( A ) Integrated overview modeling general interactions between central nutrient-sensing signaling pathways. Arrows and bars convey activating and inhibiting effects, respectively. TFs, physiologic signals, and drugs known to modulate this pathway are indicated in green, blue, and purple font, respectively. “P” indicates pathway components whose activity is modulated by phosphorylation. Red boxes detail physiologic effects. ( B ) Oligomycin-based pharmacologic RC inhibition in human FCLs alters mTORC1 and AMPK pathway activities. To confirm primary mitochondrial RC dysfunction was sufficient to alter mTORC1 signaling, FCLs from a healthy individual were treated in DMEM medium containing 20% fetal bovine serum for 24 hours and either low (1 g/L or 5 mMol) or high (4.5 g/L or 25 mMol) glucose, with either the complex V inhibitor, Oligomycin, (“O”, 5 uMol), an AMPK activator, AICAR (“A”, 2 mMol), or the mTORC1 inhibitor, rapamycin (“R”, 100 nMol). Regardless of glucose concentration, expression of a standard readout of mTORC1 pathway activity, phospho-S6 protein level, was reduced by oligomycin treatment, while increased phospho-AMPK expression was evident in high glucose media. ( C ) Oligomycin-based alteration of mTORC1 and AMPK pathway activities is dose- and nutrient-dependent. The same control FCLs as in (B) were treated for 24 hours in either low (1 g/L or 5 mMol) or high (4.5 g/L or 25 mMol) glucose media with oligomycin in doses ranging from 0.25 uMol to 5 uMol. A general trend is evident of overall reduced phospho-S6 and phospho-AMPK at increasing doses of oligomycin. See also Fig. S13 in File S3 . ( D ) Pharmacologic RC inhibition increases FOXO1 expression in high-glucose conditions. FCLs were treated for 24 hours in either low (1 g/L or 5 mMol) or high (4.5 g/L or 25 mMol) glucose media with a RC inhibitor targeting either complex I (rotenone, 0.1 uMol), III (antimycin A, 0.5 uMol), or V (oligomycin, 1 uMol). As expected, FOXO1 expression was inhibited by high-glucose media. All three RC inhibitors increased FOXO1 relative expression in high-glucose conditions, whereas FOXO1 expression was decreased by oligomycin in low-glucose media. ( E–F ) Integrated view of detailed gene and sub-gene level transcriptional alterations in RC disease among central nutrient-sensing signaling network modulators. Their major downstream targets and pathways were presented in separated plots. An overall inverse expression pattern is evident in RC disease between ( E ) skeletal muscle and ( F ) FCL.
Figure Legend Snippet: Primary RC dysfunction transcriptionally and post-transcriptionally dysregulates the integrated nutrient-sensing signaling network. ( A ) Integrated overview modeling general interactions between central nutrient-sensing signaling pathways. Arrows and bars convey activating and inhibiting effects, respectively. TFs, physiologic signals, and drugs known to modulate this pathway are indicated in green, blue, and purple font, respectively. “P” indicates pathway components whose activity is modulated by phosphorylation. Red boxes detail physiologic effects. ( B ) Oligomycin-based pharmacologic RC inhibition in human FCLs alters mTORC1 and AMPK pathway activities. To confirm primary mitochondrial RC dysfunction was sufficient to alter mTORC1 signaling, FCLs from a healthy individual were treated in DMEM medium containing 20% fetal bovine serum for 24 hours and either low (1 g/L or 5 mMol) or high (4.5 g/L or 25 mMol) glucose, with either the complex V inhibitor, Oligomycin, (“O”, 5 uMol), an AMPK activator, AICAR (“A”, 2 mMol), or the mTORC1 inhibitor, rapamycin (“R”, 100 nMol). Regardless of glucose concentration, expression of a standard readout of mTORC1 pathway activity, phospho-S6 protein level, was reduced by oligomycin treatment, while increased phospho-AMPK expression was evident in high glucose media. ( C ) Oligomycin-based alteration of mTORC1 and AMPK pathway activities is dose- and nutrient-dependent. The same control FCLs as in (B) were treated for 24 hours in either low (1 g/L or 5 mMol) or high (4.5 g/L or 25 mMol) glucose media with oligomycin in doses ranging from 0.25 uMol to 5 uMol. A general trend is evident of overall reduced phospho-S6 and phospho-AMPK at increasing doses of oligomycin. See also Fig. S13 in File S3 . ( D ) Pharmacologic RC inhibition increases FOXO1 expression in high-glucose conditions. FCLs were treated for 24 hours in either low (1 g/L or 5 mMol) or high (4.5 g/L or 25 mMol) glucose media with a RC inhibitor targeting either complex I (rotenone, 0.1 uMol), III (antimycin A, 0.5 uMol), or V (oligomycin, 1 uMol). As expected, FOXO1 expression was inhibited by high-glucose media. All three RC inhibitors increased FOXO1 relative expression in high-glucose conditions, whereas FOXO1 expression was decreased by oligomycin in low-glucose media. ( E–F ) Integrated view of detailed gene and sub-gene level transcriptional alterations in RC disease among central nutrient-sensing signaling network modulators. Their major downstream targets and pathways were presented in separated plots. An overall inverse expression pattern is evident in RC disease between ( E ) skeletal muscle and ( F ) FCL.

Techniques Used: Activity Assay, Inhibition, Concentration Assay, Expressing

61) Product Images from "Molecular Imaging of Intracellular Drug-Membrane Aggregate Formation"

Article Title: Molecular Imaging of Intracellular Drug-Membrane Aggregate Formation

Journal: Molecular pharmaceutics

doi: 10.1021/mp200101b

TEM images of control cells (A, vehicle-only with 5% FBS-DMEM after 24 hr treatment); and cells incubated with 10 μM clofazimine for 24 hr (B) or 87 hr (C). D, E, and F are zoom-in of boxed regions in A, B, and C, respectively. Cytoplasmic objects
Figure Legend Snippet: TEM images of control cells (A, vehicle-only with 5% FBS-DMEM after 24 hr treatment); and cells incubated with 10 μM clofazimine for 24 hr (B) or 87 hr (C). D, E, and F are zoom-in of boxed regions in A, B, and C, respectively. Cytoplasmic objects

Techniques Used: Transmission Electron Microscopy, Incubation

62) Product Images from "Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data"

Article Title: Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data

Journal: Stem Cell Research & Therapy

doi: 10.1186/scrt398

Gene expression profiles of UCX® cultured in HS (A, C) and hPL (B, C) versus FBS . Scatterplots for all genes represented on the HG-U133Plus2.0 array (A, B) or 132 selected MSC-relevant gene sets representing surface markers and immune suppression related genes (C, D) are shown. Mean Spearman pairwise correlation coefficients (ρ) are indicated. FBS, fetal bovine serum; hPL, human Platelet Lysate; HS, human serum.
Figure Legend Snippet: Gene expression profiles of UCX® cultured in HS (A, C) and hPL (B, C) versus FBS . Scatterplots for all genes represented on the HG-U133Plus2.0 array (A, B) or 132 selected MSC-relevant gene sets representing surface markers and immune suppression related genes (C, D) are shown. Mean Spearman pairwise correlation coefficients (ρ) are indicated. FBS, fetal bovine serum; hPL, human Platelet Lysate; HS, human serum.

Techniques Used: Expressing, Cell Culture

Isolation of UCX® cells . A standard protocol using a mixture of crude collagenase and porcine trypsin for cell isolation (ENZ(RG)), and fetal bovine serum (FBS) for cell culture was adapted by replacing the digestion enzymes with clinical grade enzymes (ENZ(CG)) and culturing the cells in human serum (HS) or human Platelet Lysate (hPL). The efficiency of the process was plotted taking into consideration the total cells at the end of passage 0 (P0) isolated from a standard cord (17.5 g, considering an average weight of 1 gram per centimeter of cord), and the days to confluence. Results are represented as mean ± SEM.
Figure Legend Snippet: Isolation of UCX® cells . A standard protocol using a mixture of crude collagenase and porcine trypsin for cell isolation (ENZ(RG)), and fetal bovine serum (FBS) for cell culture was adapted by replacing the digestion enzymes with clinical grade enzymes (ENZ(CG)) and culturing the cells in human serum (HS) or human Platelet Lysate (hPL). The efficiency of the process was plotted taking into consideration the total cells at the end of passage 0 (P0) isolated from a standard cord (17.5 g, considering an average weight of 1 gram per centimeter of cord), and the days to confluence. Results are represented as mean ± SEM.

Techniques Used: Isolation, Cell Isolation, Cell Culture

63) Product Images from "Serum deprivation elevates the levels of microvesicles with different size distributions and selectively enriched proteins in human myeloma cells in vitro"

Article Title: Serum deprivation elevates the levels of microvesicles with different size distributions and selectively enriched proteins in human myeloma cells in vitro

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2013.166

SD treatment resulted in significantly decreased proliferation and G 0 /G 1 state arrest without increased apoptosis in RPMI 8226 cells. RPMI 8226 cells were cultured with 10% FBS and 1% FBS (SD) for 24 h, and proliferation and apoptosis were analyzed using MTT and flow cytometry, respectively. (A) For the growth assays, MTT was used and the results were indicated. (B) Cell cycle was determined by FACS analysis of DNA fragmentation of propidium iodide-stained nuclei. SD resulted in increased RPMI 8226 cells at the G 0 /G 1 phase (50.75%±1.13% vs 62.23%±1.26%, P
Figure Legend Snippet: SD treatment resulted in significantly decreased proliferation and G 0 /G 1 state arrest without increased apoptosis in RPMI 8226 cells. RPMI 8226 cells were cultured with 10% FBS and 1% FBS (SD) for 24 h, and proliferation and apoptosis were analyzed using MTT and flow cytometry, respectively. (A) For the growth assays, MTT was used and the results were indicated. (B) Cell cycle was determined by FACS analysis of DNA fragmentation of propidium iodide-stained nuclei. SD resulted in increased RPMI 8226 cells at the G 0 /G 1 phase (50.75%±1.13% vs 62.23%±1.26%, P

Techniques Used: Cell Culture, MTT Assay, Flow Cytometry, Cytometry, FACS, Staining

64) Product Images from "Plexin-B2 Negatively Regulates Macrophage Motility, Rac, and Cdc42 Activation"

Article Title: Plexin-B2 Negatively Regulates Macrophage Motility, Rac, and Cdc42 Activation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0024795

Plxnb2 −/− cells demonstrate faster wound healing than wild type macrophages. A) F-actin distribution in sham and M-CSF treated macrophages. Wild type and Plxnb2 −/− macrophages were plated on glass coverslips, rested overnight, and fixed following sham (plain DMEM) treatment or treatment with 50 ng/ml M-CSF in DMEM for 2 or 10 minutes. Cells were imaged on an Olympus IX-70 (Center Valley, PA). Pictures are representatives of two different experiments with 10 pictures per treatment group, with cells from four mice per group. B) Migration of macrophages into extracellular matrix protein. Wild type and Plxnb2 −/− macrophages were seeded into the top chamber of a fibronectin coated transwell apparatus in plain DMEM. Cells were allowed to migrate towards plain DMEM, DMEM with 100 ng/ml M-CSF, and DMEM with 10% FBS for 20 hours and quantified by optical density. Graph is representative of two separate experiments performed with cells from four mice per group. C) Adhesion to extracellular matrix protein. Wild type and Plxnb2 −/− macrophages were plated on fibronectin (20 ug/ml) coated plates and allowed to adhere for 20 minutes. Plates were washed and adherent cells were quantified by ToxiLight Bioassay Kit (Lonza, Rockland, ME). Graph is representative of two separate experiments with cells from four mice per group. D) Wound healing assay. Wild type and Plxnb2 −/− macrophages (1×10 6 ) were plated on 12 well plates. An artificial wound was introduced using a 200 ul pipette tip, and cells were imaged closing the wound every 10 minutes at four points per scratch for 24–48 hours using an Olympus-IX-70 inverted microscope with live cell imaging capability. The rate of change of the area of the scratch was measured over time using the NIH ImageJ program. The calculated area is in terms of pixels, and the rate of movement is calculated as pixels/sec and converted to micrometers/sec. Experiments were repeated two times with 6 wells per group, using cells from four different mice per group.
Figure Legend Snippet: Plxnb2 −/− cells demonstrate faster wound healing than wild type macrophages. A) F-actin distribution in sham and M-CSF treated macrophages. Wild type and Plxnb2 −/− macrophages were plated on glass coverslips, rested overnight, and fixed following sham (plain DMEM) treatment or treatment with 50 ng/ml M-CSF in DMEM for 2 or 10 minutes. Cells were imaged on an Olympus IX-70 (Center Valley, PA). Pictures are representatives of two different experiments with 10 pictures per treatment group, with cells from four mice per group. B) Migration of macrophages into extracellular matrix protein. Wild type and Plxnb2 −/− macrophages were seeded into the top chamber of a fibronectin coated transwell apparatus in plain DMEM. Cells were allowed to migrate towards plain DMEM, DMEM with 100 ng/ml M-CSF, and DMEM with 10% FBS for 20 hours and quantified by optical density. Graph is representative of two separate experiments performed with cells from four mice per group. C) Adhesion to extracellular matrix protein. Wild type and Plxnb2 −/− macrophages were plated on fibronectin (20 ug/ml) coated plates and allowed to adhere for 20 minutes. Plates were washed and adherent cells were quantified by ToxiLight Bioassay Kit (Lonza, Rockland, ME). Graph is representative of two separate experiments with cells from four mice per group. D) Wound healing assay. Wild type and Plxnb2 −/− macrophages (1×10 6 ) were plated on 12 well plates. An artificial wound was introduced using a 200 ul pipette tip, and cells were imaged closing the wound every 10 minutes at four points per scratch for 24–48 hours using an Olympus-IX-70 inverted microscope with live cell imaging capability. The rate of change of the area of the scratch was measured over time using the NIH ImageJ program. The calculated area is in terms of pixels, and the rate of movement is calculated as pixels/sec and converted to micrometers/sec. Experiments were repeated two times with 6 wells per group, using cells from four different mice per group.

Techniques Used: Mouse Assay, Migration, Wound Healing Assay, Transferring, Inverted Microscopy, Live Cell Imaging, Size-exclusion Chromatography

65) Product Images from "Transplantation of a bone marrow mesenchymal stem cell line increases neuronal progenitor cell migration in a cerebral ischemia animal model"

Article Title: Transplantation of a bone marrow mesenchymal stem cell line increases neuronal progenitor cell migration in a cerebral ischemia animal model

Journal: Scientific Reports

doi: 10.1038/s41598-018-33030-9

Effects of B10 cells on neuronal cell migration in culture. ( A ) To assess the effects of B10-secreted molecules on neuronal migration, B10 cells were cultured to confluency in complete medium. Then the medium was changed to 0.5% FBS containing DMEM, cultured for further 24 h and the culture supernatant was used for A1 migration assay. DMEM-only, DMEM containing 0.5% FBS (medium) or A1 culture supernatant was used as control. * p
Figure Legend Snippet: Effects of B10 cells on neuronal cell migration in culture. ( A ) To assess the effects of B10-secreted molecules on neuronal migration, B10 cells were cultured to confluency in complete medium. Then the medium was changed to 0.5% FBS containing DMEM, cultured for further 24 h and the culture supernatant was used for A1 migration assay. DMEM-only, DMEM containing 0.5% FBS (medium) or A1 culture supernatant was used as control. * p

Techniques Used: Migration, Cell Culture

66) Product Images from "Canine Platelet Lysate Is Inferior to Fetal Bovine Serum for the Isolation and Propagation of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells"

Article Title: Canine Platelet Lysate Is Inferior to Fetal Bovine Serum for the Isolation and Propagation of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0136621

MSC isolation and proliferation in PL vs. FBS culture. (A) Primary canine adipose tissue (AT)- and bone marrow (BM)-derived mesenchymal stromal cell (MSC) colonies isolated in 10% fetal bovine serum (FBS) or 10% platelet lysate (PL). Scale bar = 50 μm. (B) Proliferation assays (resazurin) of AT-MSC (n = 6) and (C) BM-MSC (n = 4) in FBS- or PL-enriched medium from 5% to 60%. Base medium (DMEM) was used as a negative control. (*P
Figure Legend Snippet: MSC isolation and proliferation in PL vs. FBS culture. (A) Primary canine adipose tissue (AT)- and bone marrow (BM)-derived mesenchymal stromal cell (MSC) colonies isolated in 10% fetal bovine serum (FBS) or 10% platelet lysate (PL). Scale bar = 50 μm. (B) Proliferation assays (resazurin) of AT-MSC (n = 6) and (C) BM-MSC (n = 4) in FBS- or PL-enriched medium from 5% to 60%. Base medium (DMEM) was used as a negative control. (*P

Techniques Used: Isolation, Derivative Assay, Negative Control

67) Product Images from "Activated ?-catenin Forces N2A Cell-derived Neurons Back to Tumor-like Neuroblasts and Positively Correlates with a Risk for Human Neuroblastoma"

Article Title: Activated ?-catenin Forces N2A Cell-derived Neurons Back to Tumor-like Neuroblasts and Positively Correlates with a Risk for Human Neuroblastoma

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.3520

BIO promotes N2A cell-derived neuron morphological changes. (A) N2A cells were induced to differentiate into neurons as previously described (b). Four days later, N2B27 medium was replaced by 10% FBS (c). The induced neurite outgrowth was shown by brightfield images. Sclar bar = 200μm. (B) After induction of N2A cells for 4 days, the cells were treated with 10% FBS (a) or 10% FBS+DKK1 (100ng/ml) (b). Sclar bar = 100μm. (C) After induction of N2A cells for 4 days, the cells were treated with DMSO (a), 2μM (b), 5μM (c) or 10μM (d) BIO for 12 hours, respectively. Sclar bar = 100μm
Figure Legend Snippet: BIO promotes N2A cell-derived neuron morphological changes. (A) N2A cells were induced to differentiate into neurons as previously described (b). Four days later, N2B27 medium was replaced by 10% FBS (c). The induced neurite outgrowth was shown by brightfield images. Sclar bar = 200μm. (B) After induction of N2A cells for 4 days, the cells were treated with 10% FBS (a) or 10% FBS+DKK1 (100ng/ml) (b). Sclar bar = 100μm. (C) After induction of N2A cells for 4 days, the cells were treated with DMSO (a), 2μM (b), 5μM (c) or 10μM (d) BIO for 12 hours, respectively. Sclar bar = 100μm

Techniques Used: Derivative Assay

68) Product Images from "Protein Corona in Response to Flow: Effect on Protein Concentration and Structure"

Article Title: Protein Corona in Response to Flow: Effect on Protein Concentration and Structure

Journal: Biophysical Journal

doi: 10.1016/j.bpj.2018.02.036

Flow cytometry was used to measure cellular binding of NPs incubated with 10% FBS under standard conditions and after 30 min of flow (8.5 cm/s). The NPs were added to the proteins after flow. ∗∗∗ p ≤ 0.001.
Figure Legend Snippet: Flow cytometry was used to measure cellular binding of NPs incubated with 10% FBS under standard conditions and after 30 min of flow (8.5 cm/s). The NPs were added to the proteins after flow. ∗∗∗ p ≤ 0.001.

Techniques Used: Flow Cytometry, Cytometry, Binding Assay, Incubation

69) Product Images from "Serum deprivation elevates the levels of microvesicles with different size distributions and selectively enriched proteins in human myeloma cells in vitro"

Article Title: Serum deprivation elevates the levels of microvesicles with different size distributions and selectively enriched proteins in human myeloma cells in vitro

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2013.166

SD treatment resulted in significantly decreased proliferation and G 0 /G 1 state arrest without increased apoptosis in RPMI 8226 cells. RPMI 8226 cells were cultured with 10% FBS and 1% FBS (SD) for 24 h, and proliferation and apoptosis were analyzed using MTT and flow cytometry, respectively. (A) For the growth assays, MTT was used and the results were indicated. (B) Cell cycle was determined by FACS analysis of DNA fragmentation of propidium iodide-stained nuclei. SD resulted in increased RPMI 8226 cells at the G 0 /G 1 phase (50.75%±1.13% vs 62.23%±1.26%, P
Figure Legend Snippet: SD treatment resulted in significantly decreased proliferation and G 0 /G 1 state arrest without increased apoptosis in RPMI 8226 cells. RPMI 8226 cells were cultured with 10% FBS and 1% FBS (SD) for 24 h, and proliferation and apoptosis were analyzed using MTT and flow cytometry, respectively. (A) For the growth assays, MTT was used and the results were indicated. (B) Cell cycle was determined by FACS analysis of DNA fragmentation of propidium iodide-stained nuclei. SD resulted in increased RPMI 8226 cells at the G 0 /G 1 phase (50.75%±1.13% vs 62.23%±1.26%, P

Techniques Used: Cell Culture, MTT Assay, Flow Cytometry, Cytometry, FACS, Staining

70) Product Images from "Surface ?-Enolase Promotes Extracellular Matrix Degradation and Tumor Metastasis and Represents a New Therapeutic Target"

Article Title: Surface ?-Enolase Promotes Extracellular Matrix Degradation and Tumor Metastasis and Represents a New Therapeutic Target

Journal: PLoS ONE

doi: 10.1371/journal.pone.0069354

Ab against ENO1 suppressed cell invasion. PE089 (A) or LLC/luc (B) cells were suspended in serum-free medium containing PBS, an ENO1-specific Ab (2 µg/ml), or an isotype-control Ab (2 µg/ml), and then added into transwell chambers consisting of matrigel-coated polycarbonate membranes. The transwell chambers were placed into wells containing medium with 5% FBS as a chemoattractant. After incubation for 24 h, the invading cells on the opposite side of the chambers were determined. (C) The growth of LLC/luc cells in a culture containing PBS, an isotype-control Ab (2 µg/ml), or an Ab against ENO1 (2 µg/ml) was determined by a MTT assay. * p
Figure Legend Snippet: Ab against ENO1 suppressed cell invasion. PE089 (A) or LLC/luc (B) cells were suspended in serum-free medium containing PBS, an ENO1-specific Ab (2 µg/ml), or an isotype-control Ab (2 µg/ml), and then added into transwell chambers consisting of matrigel-coated polycarbonate membranes. The transwell chambers were placed into wells containing medium with 5% FBS as a chemoattractant. After incubation for 24 h, the invading cells on the opposite side of the chambers were determined. (C) The growth of LLC/luc cells in a culture containing PBS, an isotype-control Ab (2 µg/ml), or an Ab against ENO1 (2 µg/ml) was determined by a MTT assay. * p

Techniques Used: Incubation, MTT Assay

Down-regulation of ENO1 by shRNA reduced collagen degradation and cell invasion. (A) LLC/luc cells were transfected with control (VC) and mENO1-specific (C1 and C4) shRNA plasmids, and the expression of ENO1 was determined by Western blotting analysis. Upper panel: whole cell lysate; middle panel: cytosolic fraction; lower panel: membrane fraction. Expression of actin, FAK, and α-catenin was used as loading controls. (B) Surface expression of mENO1 in various shRNA-transfected LLC/luc cells was further determined by FACS analysis with isotype-control Ab (dark lines) and Ab against mENO1 (red lines). (C) LLC/luc cells transfected with control and mENO1-specific shRNA plasmids were added into transwell chambers consisting of matrigel-coated polycarbonate membranes. The transwell chambers were placed into wells containing medium with 5% FBS as a chemoattractant. After incubation for 24 h, the invading cells on the opposite side of the chambers were determined. (D) The culture supernatant of LLC/luc cells transfected with control and mENO1-specific shRNA plasmids were collected at 30-min intervals and mixed with soluble DQ-collagen for detection of the fluorescence generated after the degradation of DQ-collagen. (E) The growth curve of LLC/luc cells transfected with control and mENO1-specific shRNA plasmids was determined by a MTT assay. ** p
Figure Legend Snippet: Down-regulation of ENO1 by shRNA reduced collagen degradation and cell invasion. (A) LLC/luc cells were transfected with control (VC) and mENO1-specific (C1 and C4) shRNA plasmids, and the expression of ENO1 was determined by Western blotting analysis. Upper panel: whole cell lysate; middle panel: cytosolic fraction; lower panel: membrane fraction. Expression of actin, FAK, and α-catenin was used as loading controls. (B) Surface expression of mENO1 in various shRNA-transfected LLC/luc cells was further determined by FACS analysis with isotype-control Ab (dark lines) and Ab against mENO1 (red lines). (C) LLC/luc cells transfected with control and mENO1-specific shRNA plasmids were added into transwell chambers consisting of matrigel-coated polycarbonate membranes. The transwell chambers were placed into wells containing medium with 5% FBS as a chemoattractant. After incubation for 24 h, the invading cells on the opposite side of the chambers were determined. (D) The culture supernatant of LLC/luc cells transfected with control and mENO1-specific shRNA plasmids were collected at 30-min intervals and mixed with soluble DQ-collagen for detection of the fluorescence generated after the degradation of DQ-collagen. (E) The growth curve of LLC/luc cells transfected with control and mENO1-specific shRNA plasmids was determined by a MTT assay. ** p

Techniques Used: shRNA, Transfection, Expressing, Western Blot, FACS, Incubation, Fluorescence, Generated, MTT Assay

ENO1 accumulated at the pericellular proteolytic site and Ab against ENO1 suppressed invasion and lung metastasis of human A549 lung cancer cells. (A) A549/luc cells were embedded in matrigel containing fluorescence DQ-collagen to allow the growth of cells in a 3D fashion, and then further stained with a PE-labeled Ab against ENO1 to detect the presence of ENO1. The cleaved product of DQ-collagen (green fluorescence) and the presence of ENO1 (red fluorescence) was observed under a confocal microscope. Cells were counterstained with DAPI. (B) In matrigel invasion assay, A549/luc cells in the upper chamber were suspended in serum free medium containing ENO1-specific IgY (ENO1 IgY) or control IgY (10 µg/ml) and medium containing 5% FBS was used as a chemoattractant in the bottom chamber. The number of invaded cells was determined after 24 h. (C) NOD-SCID mice (n = 5 in both experiments 1 and 2) were i.v. injected with A549/luc cells and adoptively transferred with control IgY or ENO1 IgY. Lung metastasis of A549/luc cells was determined by the presence of luminescence through the IVIS System. Results obtained at day 30 of experiment 1 were shown. (D) The percentage of mice without lung metastasis after adoptive transfer of control IgY or ENO1 IgY was determined in two independent experiments (experiments 1 and 2). The arrows indicated the period of Ab injection. * p
Figure Legend Snippet: ENO1 accumulated at the pericellular proteolytic site and Ab against ENO1 suppressed invasion and lung metastasis of human A549 lung cancer cells. (A) A549/luc cells were embedded in matrigel containing fluorescence DQ-collagen to allow the growth of cells in a 3D fashion, and then further stained with a PE-labeled Ab against ENO1 to detect the presence of ENO1. The cleaved product of DQ-collagen (green fluorescence) and the presence of ENO1 (red fluorescence) was observed under a confocal microscope. Cells were counterstained with DAPI. (B) In matrigel invasion assay, A549/luc cells in the upper chamber were suspended in serum free medium containing ENO1-specific IgY (ENO1 IgY) or control IgY (10 µg/ml) and medium containing 5% FBS was used as a chemoattractant in the bottom chamber. The number of invaded cells was determined after 24 h. (C) NOD-SCID mice (n = 5 in both experiments 1 and 2) were i.v. injected with A549/luc cells and adoptively transferred with control IgY or ENO1 IgY. Lung metastasis of A549/luc cells was determined by the presence of luminescence through the IVIS System. Results obtained at day 30 of experiment 1 were shown. (D) The percentage of mice without lung metastasis after adoptive transfer of control IgY or ENO1 IgY was determined in two independent experiments (experiments 1 and 2). The arrows indicated the period of Ab injection. * p

Techniques Used: Fluorescence, Staining, Labeling, Microscopy, Invasion Assay, Mouse Assay, Injection, Adoptive Transfer Assay

71) Product Images from "High Content Analysis Provides Mechanistic Insights on the Pathways of Toxicity Induced by Amine-Modified Polystyrene Nanoparticles"

Article Title: High Content Analysis Provides Mechanistic Insights on the Pathways of Toxicity Induced by Amine-Modified Polystyrene Nanoparticles

Journal: PLoS ONE

doi: 10.1371/journal.pone.0108025

Primary human astrocytes respond to PS-NH 2 and PS-COOH comparably to 1321N1 astrocytoma cells. 1321N1 and NHA cells were exposed to vehicle (ctrl) or increasing concentrations of PS-COOH or PS-NH 2 NPs for 24 hours and analysed using HCA. Comparison of results obtained between the two cell lines in DMEM supplemented with 10% FBS (A) or with 3% FBS (B). In the graphs 1321n1 cells are represented by the continuous line (▪) while HNA cells are represented by the dashed line (□) C. EC50/IC50 measurements for both cell lines exposed to NPs in presence of 10% or 3% FBS. NHA primary astrocytes showed slightly higher sensitivity than 1321N1 astrocytoma cells, with EC50/IC50 values in the same order of magnitude. Data are shown as average +/− SD of 30 acquired images from a representative experiment. Curve fitting and EC50/IC50 wer e obtained as described in the Methods. Fluo-4 and Lysotracker green dose response plots were fitted with a gaussian curve for visual purposes only.”
Figure Legend Snippet: Primary human astrocytes respond to PS-NH 2 and PS-COOH comparably to 1321N1 astrocytoma cells. 1321N1 and NHA cells were exposed to vehicle (ctrl) or increasing concentrations of PS-COOH or PS-NH 2 NPs for 24 hours and analysed using HCA. Comparison of results obtained between the two cell lines in DMEM supplemented with 10% FBS (A) or with 3% FBS (B). In the graphs 1321n1 cells are represented by the continuous line (▪) while HNA cells are represented by the dashed line (□) C. EC50/IC50 measurements for both cell lines exposed to NPs in presence of 10% or 3% FBS. NHA primary astrocytes showed slightly higher sensitivity than 1321N1 astrocytoma cells, with EC50/IC50 values in the same order of magnitude. Data are shown as average +/− SD of 30 acquired images from a representative experiment. Curve fitting and EC50/IC50 wer e obtained as described in the Methods. Fluo-4 and Lysotracker green dose response plots were fitted with a gaussian curve for visual purposes only.”

Techniques Used: High Content Screening

72) Product Images from "Pentabromopseudilin: a myosin V inhibitor suppresses TGF- β activity by recruiting the type II TGF- β receptor to lysosomal degradation"

Article Title: Pentabromopseudilin: a myosin V inhibitor suppresses TGF- β activity by recruiting the type II TGF- β receptor to lysosomal degradation

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

doi: 10.1080/14756366.2018.1465416

PBrP treatment resulted in the internalisation and rapid degradation of T β RII. Serum-starved Mv1Lu (A) and A549 (B) cells were treated with increasing concentrations of PBrP (0.01–1 μ M) for 6 h and then stimulated with 20 or 100 pM of TGF- β for 30 min. Cells were analysed through Western blotting for T β RII protein levels. Similarly, Mv1Lu (C), A549 (D), HepG2 (E) and Clone 9 (F) cells growing in a low-serum medium were treated with 0.2 or 0.5 μ M of PBrP for 0, 1, 2, 4 and 6 h followed by 50 or 100 pM of TGF- β stimulation for 30 min. Cell lysates were analysed for T β RII, T β RI or β -actin protein expression through Western blotting. The show results are those of the same samples from Figure 2 (A to G) and share the same β -actin for load control. Panels (A to D) show the representative images and illustrate quantitative analyses of ECL (mean ± SD) from at least three independent experiments (% of untreated control); * p < .05, ** p < .01. Mv1Lu cells transiently expressing T β RII-flag were treated with 0.01–0.2 μ M of PBrP for 6 h (G) or 0.2 μ M of PBrP for 0, 1, 2, 4 and 6 h (H). Subsequently, the cells lysates were subjected to Western blot analysis using an antibody against the flag tag. The mock showed no signal, which suggested that the flag signal from the T β RII-flag was not an artefact. (I) PBrP reduces T β RII protein stability. Mv1Lu cells were treated with 0.5 μ M of PBrP or a relevant amount of DMSO for the indicated time in the presence or absence of CHX. The amounts of T β RII protein from the cell lysates were analysed through Western blotting using anti-T β RII and anti- β -actin (as a loading control) antibodies. The percentage of the control indicates the T β RII amount at each time point relative to the control. (J) PBrP reduced T β R-II abundance at the cell surface, as assessed by affinity purification of cell surface proteins using NeutrAvidin-conjugated beads and Western blot. Cells expressing T β R-II-flag were pretreated with 0.2 μ M PBrP for 0, 1, 2, 3 and 4 h in DMEM containing 0.2% of FBS at 37 °C followed by cell surface biotinylation. NeutrAvidin-precipitated proteins and total lysates were subjected to immunoblotting to detect T β R-II-flag. Changes of T β R-II abundance were represented as % of control (mean ± SD) from three independent experiments; * p < .05, ** p < .01. (K) HEK293 cells co-expressing T β RII-flag and Rab5-RFP were treated with 0.2 μ M of PBrP for 1 h, followed by fixation and permeabilisation. T β RII-flag localisation was visualised by immunofluorescence staining using the anti-flag antibody and Alexa Fluor 488-conjugated secondary antibodies. Bar: 10 μ m.
Figure Legend Snippet: PBrP treatment resulted in the internalisation and rapid degradation of T β RII. Serum-starved Mv1Lu (A) and A549 (B) cells were treated with increasing concentrations of PBrP (0.01–1 μ M) for 6 h and then stimulated with 20 or 100 pM of TGF- β for 30 min. Cells were analysed through Western blotting for T β RII protein levels. Similarly, Mv1Lu (C), A549 (D), HepG2 (E) and Clone 9 (F) cells growing in a low-serum medium were treated with 0.2 or 0.5 μ M of PBrP for 0, 1, 2, 4 and 6 h followed by 50 or 100 pM of TGF- β stimulation for 30 min. Cell lysates were analysed for T β RII, T β RI or β -actin protein expression through Western blotting. The show results are those of the same samples from Figure 2 (A to G) and share the same β -actin for load control. Panels (A to D) show the representative images and illustrate quantitative analyses of ECL (mean ± SD) from at least three independent experiments (% of untreated control); * p < .05, ** p < .01. Mv1Lu cells transiently expressing T β RII-flag were treated with 0.01–0.2 μ M of PBrP for 6 h (G) or 0.2 μ M of PBrP for 0, 1, 2, 4 and 6 h (H). Subsequently, the cells lysates were subjected to Western blot analysis using an antibody against the flag tag. The mock showed no signal, which suggested that the flag signal from the T β RII-flag was not an artefact. (I) PBrP reduces T β RII protein stability. Mv1Lu cells were treated with 0.5 μ M of PBrP or a relevant amount of DMSO for the indicated time in the presence or absence of CHX. The amounts of T β RII protein from the cell lysates were analysed through Western blotting using anti-T β RII and anti- β -actin (as a loading control) antibodies. The percentage of the control indicates the T β RII amount at each time point relative to the control. (J) PBrP reduced T β R-II abundance at the cell surface, as assessed by affinity purification of cell surface proteins using NeutrAvidin-conjugated beads and Western blot. Cells expressing T β R-II-flag were pretreated with 0.2 μ M PBrP for 0, 1, 2, 3 and 4 h in DMEM containing 0.2% of FBS at 37 °C followed by cell surface biotinylation. NeutrAvidin-precipitated proteins and total lysates were subjected to immunoblotting to detect T β R-II-flag. Changes of T β R-II abundance were represented as % of control (mean ± SD) from three independent experiments; * p < .05, ** p < .01. (K) HEK293 cells co-expressing T β RII-flag and Rab5-RFP were treated with 0.2 μ M of PBrP for 1 h, followed by fixation and permeabilisation. T β RII-flag localisation was visualised by immunofluorescence staining using the anti-flag antibody and Alexa Fluor 488-conjugated secondary antibodies. Bar: 10 μ m.

Techniques Used: Western Blot, Expressing, FLAG-tag, Affinity Purification, Immunofluorescence, Staining

PBrP delays TGF- β -induced cell migration. Confluent monolayers of A549 cells in a 24-well cluster tissue culture plate were scratched and incubated at 37 °C for 20 h in DMEM containing 0.2% of FBS (Control; top). Cell motility was measured using a fully automatic microscope at 10× phase objective. Cell migration was observed by performing time-lapse microscopy and images of all four experimental conditions were captured simultaneously every 20 min. The red lines indicate the starting point of cell migration. Representative micrographs from three experiments are shown in panel (A). Right graphs illustrate of quantitative analyses of cell covering area (mean ± SD) from three independent experiments are shown (B) (% of TGF- β treatment alone); ** p
Figure Legend Snippet: PBrP delays TGF- β -induced cell migration. Confluent monolayers of A549 cells in a 24-well cluster tissue culture plate were scratched and incubated at 37 °C for 20 h in DMEM containing 0.2% of FBS (Control; top). Cell motility was measured using a fully automatic microscope at 10× phase objective. Cell migration was observed by performing time-lapse microscopy and images of all four experimental conditions were captured simultaneously every 20 min. The red lines indicate the starting point of cell migration. Representative micrographs from three experiments are shown in panel (A). Right graphs illustrate of quantitative analyses of cell covering area (mean ± SD) from three independent experiments are shown (B) (% of TGF- β treatment alone); ** p

Techniques Used: Migration, Incubation, Microscopy, Time-lapse Microscopy

PBrP blocks TGF- β -induced transcriptional responses and EMT protein production. (A) Inhibition of PAI-1 gene promoter-luciferase (PAI-1-Luc) activity in MLECs through a dose response of PBrP and the T β RI kinase inhibitor SB-431542 (SB) in response to TGF- β . (B to E) Control A549 cells (top) or cells pretreated with 0.5 μ M of PBrP for 6 h followed by 42 h of TGF- β stimulation were fixed and permeabilised. (B) Cells were incubated with TRITC-phalloidin (red) and DAPI (blue) to visualise the actin cytoskeleton and the nuclei, respectively. To visualize ZO-1 (C), fibronectin (D) and N-cadherin (E), cells pretreated with 0.5 μ M of PBrP for 6 h followed by 42 h of TGF- β stimulation were stained with specific antibodies and Alexa Fluor 488-conjugated secondary antibodies. Representative micrographs from three experiments are shown. Bar: 20 μ m. For Western blot analysis, A549 (F) and HepG2 (G) cells were treated with increasing doses of PBrP in DMEM containing 0.1% of FBS for 6 h and continually stimulated with or without 100 pM of TGF- β for 48 h. (H) A549 cells were pretreated with 0.5 μ M of PBrP for 6 h and continually stimulated with 0, 10 or 100 pM of TGF- β for 42 h. Cell lysates were then analysed through Western blotting with desired antibodies as indicated. The representative images (F to H) and right graphs illustrate of quantitative analyses of ECL (mean ± SD) from three independent experiments are shown (% of TGF- β treatment alone); * p < .05, ** p < .01.
Figure Legend Snippet: PBrP blocks TGF- β -induced transcriptional responses and EMT protein production. (A) Inhibition of PAI-1 gene promoter-luciferase (PAI-1-Luc) activity in MLECs through a dose response of PBrP and the T β RI kinase inhibitor SB-431542 (SB) in response to TGF- β . (B to E) Control A549 cells (top) or cells pretreated with 0.5 μ M of PBrP for 6 h followed by 42 h of TGF- β stimulation were fixed and permeabilised. (B) Cells were incubated with TRITC-phalloidin (red) and DAPI (blue) to visualise the actin cytoskeleton and the nuclei, respectively. To visualize ZO-1 (C), fibronectin (D) and N-cadherin (E), cells pretreated with 0.5 μ M of PBrP for 6 h followed by 42 h of TGF- β stimulation were stained with specific antibodies and Alexa Fluor 488-conjugated secondary antibodies. Representative micrographs from three experiments are shown. Bar: 20 μ m. For Western blot analysis, A549 (F) and HepG2 (G) cells were treated with increasing doses of PBrP in DMEM containing 0.1% of FBS for 6 h and continually stimulated with or without 100 pM of TGF- β for 48 h. (H) A549 cells were pretreated with 0.5 μ M of PBrP for 6 h and continually stimulated with 0, 10 or 100 pM of TGF- β for 42 h. Cell lysates were then analysed through Western blotting with desired antibodies as indicated. The representative images (F to H) and right graphs illustrate of quantitative analyses of ECL (mean ± SD) from three independent experiments are shown (% of TGF- β treatment alone); * p < .05, ** p < .01.

Techniques Used: Inhibition, Luciferase, Activity Assay, Incubation, Staining, Western Blot

PBrP induces T β RII turnover through the late endosome–lysosome pathway and is impaired by lysosome inhibitors. Mv1Lu cells grown in 0.2% of FBS-containing DMEM were incubated with PBrP (0.5 μ M) with chloroquine (100 μ M) or NH 4 Cl (20 mM) (A) for 1, 3 and 6 h or MG132 (20 μ M) and carfilzomib (0.5 μ M) (B). Subsequently, the cell lysates were subjected to SDS-PAGE, and T β RII expression was analysed through Western blotting and quantified through densitometry. (C) T β RII was enriched in the Rab11-positive endosomal compartment in PBrP-treated cells. Mv1Lu cells were treated with 0.5 μ M of PBrP for 1 h in low-serum DMEM. Endogenous T β RII and Rab11 were visualised through immunofluorescence staining using Alexa Fluor 488- and 594-conjugated secondary antibodies, respectively. (D) T β RII was internalised and directed into lysosomes in PBrP-treated cells. HEK293 cells transiently co-expressing T β RII-flag and Lamp-1-GFP were treated with 0.5 μ M of PBrP for 2.5 h in low-serum DMEM. After fixation and permeabilisation, T β RII-flag was visualised through immunofluorescence staining using anti-flag antibodies and an Alexa Fluor 594-conjugated secondary antibody. PBrP reduced membrane-associated T β RII-flag (red) and increased co-localisation with the lysosome marker Lamp-1 (green). Bar: 10 μ m.
Figure Legend Snippet: PBrP induces T β RII turnover through the late endosome–lysosome pathway and is impaired by lysosome inhibitors. Mv1Lu cells grown in 0.2% of FBS-containing DMEM were incubated with PBrP (0.5 μ M) with chloroquine (100 μ M) or NH 4 Cl (20 mM) (A) for 1, 3 and 6 h or MG132 (20 μ M) and carfilzomib (0.5 μ M) (B). Subsequently, the cell lysates were subjected to SDS-PAGE, and T β RII expression was analysed through Western blotting and quantified through densitometry. (C) T β RII was enriched in the Rab11-positive endosomal compartment in PBrP-treated cells. Mv1Lu cells were treated with 0.5 μ M of PBrP for 1 h in low-serum DMEM. Endogenous T β RII and Rab11 were visualised through immunofluorescence staining using Alexa Fluor 488- and 594-conjugated secondary antibodies, respectively. (D) T β RII was internalised and directed into lysosomes in PBrP-treated cells. HEK293 cells transiently co-expressing T β RII-flag and Lamp-1-GFP were treated with 0.5 μ M of PBrP for 2.5 h in low-serum DMEM. After fixation and permeabilisation, T β RII-flag was visualised through immunofluorescence staining using anti-flag antibodies and an Alexa Fluor 594-conjugated secondary antibody. PBrP reduced membrane-associated T β RII-flag (red) and increased co-localisation with the lysosome marker Lamp-1 (green). Bar: 10 μ m.

Techniques Used: Incubation, SDS Page, Expressing, Western Blot, Immunofluorescence, Staining, Marker

73) Product Images from "Low-affinity Nerve Growth Factor Receptor (CD271) Heterogeneous Expression in Adult and Fetal Mesenchymal Stromal Cells"

Article Title: Low-affinity Nerve Growth Factor Receptor (CD271) Heterogeneous Expression in Adult and Fetal Mesenchymal Stromal Cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-27587-8

Adult MSC harbor a CD271 + subpopulation. ( a ) Representative dot plots showing CD271 expression in additional MSC populations isolated by an independent laboratory. ( b ) Representative histograms showing NG2 expression in additional MSC populations isolated by an independent laboratory. ( c ) Validation of CD271 expression in n = 8 samples for each MSC source (adult MSC, n = 16; fetal MSC, n = 32). ( d ) Percentage of colony forming unit-fibroblasts (CFU-Fs) in sorted CD271 + (adult CD271 + ) and CD271 − (adult CD271 − ) adult MSC. ( e ) Representative cultures of sorted CD271 + (adult CD271 + ) and CD271 − (adult CD271 − ) adult MSC, which received osteogenic stimuli. ( f ) CD271 expression in adult and fetal MSC under different culture conditions. ( g ) CD271 expression in adult and fetal MSC at early and late passages. Statistical analysis was by unpaired two-sided Mann-Whitney-Wilcoxon test for c ( P = 0.0001) and g (Adult P1 vs P5, P = 0.0286; fetal P1 vs P5, P = 0.5143), by unpaired one-sided Mann-Whitney-Wilcoxon test for d ( P = 0.0429), by two-way ANOVA (α = 0.05) followed by Tukey’s multiple comparisons test for f (FBS vs PL, P
Figure Legend Snippet: Adult MSC harbor a CD271 + subpopulation. ( a ) Representative dot plots showing CD271 expression in additional MSC populations isolated by an independent laboratory. ( b ) Representative histograms showing NG2 expression in additional MSC populations isolated by an independent laboratory. ( c ) Validation of CD271 expression in n = 8 samples for each MSC source (adult MSC, n = 16; fetal MSC, n = 32). ( d ) Percentage of colony forming unit-fibroblasts (CFU-Fs) in sorted CD271 + (adult CD271 + ) and CD271 − (adult CD271 − ) adult MSC. ( e ) Representative cultures of sorted CD271 + (adult CD271 + ) and CD271 − (adult CD271 − ) adult MSC, which received osteogenic stimuli. ( f ) CD271 expression in adult and fetal MSC under different culture conditions. ( g ) CD271 expression in adult and fetal MSC at early and late passages. Statistical analysis was by unpaired two-sided Mann-Whitney-Wilcoxon test for c ( P = 0.0001) and g (Adult P1 vs P5, P = 0.0286; fetal P1 vs P5, P = 0.5143), by unpaired one-sided Mann-Whitney-Wilcoxon test for d ( P = 0.0429), by two-way ANOVA (α = 0.05) followed by Tukey’s multiple comparisons test for f (FBS vs PL, P

Techniques Used: Expressing, Isolation, MANN-WHITNEY

74) Product Images from "Nanoparticle engineered TRAIL-overexpressing adipose-derived stem cells target and eradicate glioblastoma via intracranial delivery"

Article Title: Nanoparticle engineered TRAIL-overexpressing adipose-derived stem cells target and eradicate glioblastoma via intracranial delivery

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

doi: 10.1073/pnas.1615396113

Optimization of the formulation of PBAE-pDNA nanoparticles. ( A ) Agarose gel electrophoresis retardation of plasmid EGFP by various weight ratios. When the weight ratio of polymer and pEGFP was > 10:1, the gene was completely condensed in polymer nanocomplexes. ( B and C ) Characterization of PBAE/pEGFP nanoparticles (NPs/pEGFP) with different formulations. ( B ) Dynamic light scattering indicated that the size of nanoparticles significantly decreased as the polymer concentration increased in deionizing water (DI-H 2 O) or full supplemented DMEM containing 10% (vol/vol) FBS. ( C ) Nanoparticles suspended in deionizing water had a positive charge, whereas the surface charge of nanoparticles was slightly negatively charged when suspended in cell culture medium.
Figure Legend Snippet: Optimization of the formulation of PBAE-pDNA nanoparticles. ( A ) Agarose gel electrophoresis retardation of plasmid EGFP by various weight ratios. When the weight ratio of polymer and pEGFP was > 10:1, the gene was completely condensed in polymer nanocomplexes. ( B and C ) Characterization of PBAE/pEGFP nanoparticles (NPs/pEGFP) with different formulations. ( B ) Dynamic light scattering indicated that the size of nanoparticles significantly decreased as the polymer concentration increased in deionizing water (DI-H 2 O) or full supplemented DMEM containing 10% (vol/vol) FBS. ( C ) Nanoparticles suspended in deionizing water had a positive charge, whereas the surface charge of nanoparticles was slightly negatively charged when suspended in cell culture medium.

Techniques Used: Agarose Gel Electrophoresis, Plasmid Preparation, Concentration Assay, Cell Culture

75) Product Images from "Alpl prevents bone ageing sensitivity by specifically regulating senescence and differentiation in mesenchymal stem cells"

Article Title: Alpl prevents bone ageing sensitivity by specifically regulating senescence and differentiation in mesenchymal stem cells

Journal: Bone Research

doi: 10.1038/s41413-018-0029-4

Alpl deficiency induces an elevation in extracellular ATP, which is internalized by MSCs and causes their dysfunction. a Extracellular ATP concentrations in Alpl +/+ and Alpl +/- MSC medium were assayed by a regular ATP concentration assay. b Apoptosis of Alpl +/+ and Alpl +/- MSCs was analyzed by flow cytometry. c , d Extracellular ATP concentrations were assayed 1 h after FBS deprivation and H 2 O 2 induction (50 mmol⋅L –1 ). e Extracellular ATP concentrations were assayed 48 h after transduction with different lentiviral vectors. f Expression levels of CD73 and CD39 in Alpl +/+ and Alpl +/- MSCs were analyzed by western blotting. g ATP concentrations were assayed after 2 min of treatment with 0, 1, 2, 5, 10 or 20 U⋅mL –1 of TNSALP and 2 U⋅mL –1 of ATP-apyrase in the presence of 20 nmol⋅L –1 ATP in double-distilled water (dd H 2 O, pH 7.5). h ATP concentrations were assayed at 0 min, 2 min, 10 min and 30 min after treatment with 10 U⋅mL –1 of TNSALP and 2 U⋅mL –1 of ATP-apyrase in the presence of 20 nmol⋅L –1 ATP in dd H 2 O. i , j Alpl +/+ and Alpl +/- MSCs were treated with 10 μmol⋅L –1 ATP in the presence or absence of 50 μmol⋅L –1 ethyl isopropyl amiloride (EIPA), 30 μmol⋅L –1 pyridoxal phosphate-6-azo (PPADS) or 100 μmol⋅L –1 suramin (Sur), and the intracellular ATP concentrations were assayed 1 h after treatment. k Intracellular radioactivity was examined after a 1-h treatment with ATP-γ- 32 P in the different lentivirus transduction groups. l Intracellular radioactivity was examined after a 1-h treatment with ATP-γ- 32 P in different lentivirus transduction groups treated with 100 μmol⋅L –1 suramin and 50 μmol⋅L –1 EIPA. m, n Alpl +/+ MSCs were treated with 10 μmol⋅L –1 ATP, and the ageing-specific genes were analyzed after 48 h. Expression levels of Runx2, OCN and PPARγ were examined by western blotting on day 7 after induction. n = 6 per group. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. * P
Figure Legend Snippet: Alpl deficiency induces an elevation in extracellular ATP, which is internalized by MSCs and causes their dysfunction. a Extracellular ATP concentrations in Alpl +/+ and Alpl +/- MSC medium were assayed by a regular ATP concentration assay. b Apoptosis of Alpl +/+ and Alpl +/- MSCs was analyzed by flow cytometry. c , d Extracellular ATP concentrations were assayed 1 h after FBS deprivation and H 2 O 2 induction (50 mmol⋅L –1 ). e Extracellular ATP concentrations were assayed 48 h after transduction with different lentiviral vectors. f Expression levels of CD73 and CD39 in Alpl +/+ and Alpl +/- MSCs were analyzed by western blotting. g ATP concentrations were assayed after 2 min of treatment with 0, 1, 2, 5, 10 or 20 U⋅mL –1 of TNSALP and 2 U⋅mL –1 of ATP-apyrase in the presence of 20 nmol⋅L –1 ATP in double-distilled water (dd H 2 O, pH 7.5). h ATP concentrations were assayed at 0 min, 2 min, 10 min and 30 min after treatment with 10 U⋅mL –1 of TNSALP and 2 U⋅mL –1 of ATP-apyrase in the presence of 20 nmol⋅L –1 ATP in dd H 2 O. i , j Alpl +/+ and Alpl +/- MSCs were treated with 10 μmol⋅L –1 ATP in the presence or absence of 50 μmol⋅L –1 ethyl isopropyl amiloride (EIPA), 30 μmol⋅L –1 pyridoxal phosphate-6-azo (PPADS) or 100 μmol⋅L –1 suramin (Sur), and the intracellular ATP concentrations were assayed 1 h after treatment. k Intracellular radioactivity was examined after a 1-h treatment with ATP-γ- 32 P in the different lentivirus transduction groups. l Intracellular radioactivity was examined after a 1-h treatment with ATP-γ- 32 P in different lentivirus transduction groups treated with 100 μmol⋅L –1 suramin and 50 μmol⋅L –1 EIPA. m, n Alpl +/+ MSCs were treated with 10 μmol⋅L –1 ATP, and the ageing-specific genes were analyzed after 48 h. Expression levels of Runx2, OCN and PPARγ were examined by western blotting on day 7 after induction. n = 6 per group. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. * P

Techniques Used: Concentration Assay, Flow Cytometry, Cytometry, Transduction, Expressing, Western Blot, Radioactivity

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MTT Assay:

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Article Snippet: Materials and Methods Chemicals The Dulbecco's Modified Eagle Medium (DMEM) as cell culture medium, Fetal Bovine Serum (FBS), penicillin and streptomycin were purchased from Gibco BRL (Life Technologies, Paisley, Scotland). .. The 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Roche Diagnostics GmbH (Germany).

Transfection:

Article Title: Trastuzumab-induced recruitment of Csk-homologous kinase (CHK) to ErbB2 receptor is associated with ErbB2-Y1248 phosphorylation and ErbB2 degradation to mediate cell growth inhibition
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In Vitro:

Article Title: Cat and Dog Primordial Follicles Enclosed in Ovarian Cortex Sustain Viability after In vitro Culture on Agarose Gel in a Protein-Free Medium
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Sulforhodamine B Assay:

Article Title: Antiproliferation potential of withaferin A on human osteosarcoma cells via the inhibition of G2/M checkpoint proteins
Article Snippet: WA, bovine serum albumin (BSA), ribonuclease A (RNase A), propidium iodide (PI), sulforhodamine B (SRB) and a rabbit polyclonal anti-β-actin antibody (1:1,000; cat. no. A2668) were purchased from Sigma-Aldrich (St. Louis, MO, USA). .. Fetal bovine serum (FBS), RPMI 1640 medium, trypsin-EDTA and antibiotic-antimycotic were purchased from Gibco Life Technologies (Grand Island, NY, USA).

Mutagenesis:

Article Title: IscR Is a Global Regulator Essential for Pathogenesis of Vibrio vulnificus and Induced by Host Cells
Article Snippet: The INT-407 cells were grown in minimum essential medium containing 1% (vol/vol) fetal bovine serum (MEMF) (Gibco-BRL, Gaithersburg, MD) in 96-well culture dishes (Nunc) as described previously ( ). .. Mortalities of mice infected with the wild-type and iscR mutant strains were compared as described elsewhere ( ).

Isolation:

Article Title: The Differential Proliferative Ability of Satellite Cells in Lantang and Landrace Pigs
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Cell Culture:

Article Title: The Differential Proliferative Ability of Satellite Cells in Lantang and Landrace Pigs
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Article Title: Trastuzumab-induced recruitment of Csk-homologous kinase (CHK) to ErbB2 receptor is associated with ErbB2-Y1248 phosphorylation and ErbB2 degradation to mediate cell growth inhibition
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Article Title: Cat and Dog Primordial Follicles Enclosed in Ovarian Cortex Sustain Viability after In vitro Culture on Agarose Gel in a Protein-Free Medium
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Purification:

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Enzyme-linked Immunosorbent Assay:

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Article Snippet: Materials Ham's F-12 and RPMI 1640 medium was purchased from Bio Whittaker Europe (Verviers, Belgium); 24-well culture plates from Costar (Cultek SL, Madrid, Spain); desloratadine and mometasone furoate from Schering Plough (New Jersey, USA); penicillin-streptomycin, fetal bovine serum (FBS) from Invitrogen Corporation (Paisley, Scotland, UK); and amphotericin B from Squibb (Esplugues de Llobregat, Catalonia, Spain). .. Hydrocortisone, N-formyl-methionyl-leucyl-phenylalanine, human transferrin, bovine insulin, 3,3',5-triiodo-l-tyrosine sodium salt, protease type XIV, light mineral oil, glutamine, trypan blue and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich Co. (Madrid, Spain); endothelial cell growth supplement and epidermal growth factor were supplied by Collaborative Research Inc. (Bedfort, MA, USA); cytokine ELISA kits from Amersham Biosciences Europe (Cerdanyola, Spain) and Diaclone (Stamford, CT, USA); and rat tail collagen type I from Upstate (Lake Placid, NY, USA).

Microarray:

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Incubation:

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Article Title: Cat and Dog Primordial Follicles Enclosed in Ovarian Cortex Sustain Viability after In vitro Culture on Agarose Gel in a Protein-Free Medium
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Activity Assay:

Article Title: IscR Is a Global Regulator Essential for Pathogenesis of Vibrio vulnificus and Induced by Host Cells
Article Snippet: Cytotoxicity was evaluated by measuring cytoplasmic lactate dehydrogenase (LDH) activity that is released from the INT-407 cells by damage of plasma membranes ( ). .. The INT-407 cells were grown in minimum essential medium containing 1% (vol/vol) fetal bovine serum (MEMF) (Gibco-BRL, Gaithersburg, MD) in 96-well culture dishes (Nunc) as described previously ( ).

Infection:

Article Title: IscR Is a Global Regulator Essential for Pathogenesis of Vibrio vulnificus and Induced by Host Cells
Article Snippet: The INT-407 cells were grown in minimum essential medium containing 1% (vol/vol) fetal bovine serum (MEMF) (Gibco-BRL, Gaithersburg, MD) in 96-well culture dishes (Nunc) as described previously ( ). .. Each well with 2 × 104 INT-407 cells was infected with the V. vulnificus strains at a multiplicity of infection (MOI) of 10 for various incubation times.

Expressing:

Article Title: Trastuzumab-induced recruitment of Csk-homologous kinase (CHK) to ErbB2 receptor is associated with ErbB2-Y1248 phosphorylation and ErbB2 degradation to mediate cell growth inhibition
Article Snippet: SKBR3 and BT474 cells were grown in DMEM (Lonza) containing 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic (Invitrogen). .. Transient expression of CHK in cells was done by Nucleofector technology (Lonza).

Modification:

Article Title: Establishment of Human Papillomavirus Infection Requires Cell Cycle Progression
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Article Title: Peripheral Nerve Regeneration by Secretomes of Stem Cells from Human Exfoliated Deciduous Teeth
Article Snippet: .. Single-cell suspensions were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Rockville, MD) supplemented with 10% fetal bovine serum (FBS) and antibiotic–antimycotic solution (100 units/mL penicillin G, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B; Gibco) and incubated at 37°C in an atmosphere of 5% CO2 /95% air. ..

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Mouse Assay:

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Article Snippet: The INT-407 cells were grown in minimum essential medium containing 1% (vol/vol) fetal bovine serum (MEMF) (Gibco-BRL, Gaithersburg, MD) in 96-well culture dishes (Nunc) as described previously ( ). .. Mortalities of mice infected with the wild-type and iscR mutant strains were compared as described elsewhere ( ).

Lactate Dehydrogenase Assay:

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    Thermo Fisher dialyzed fbs
    Phosphorylation of Girdin is required for its interaction with 4F2hc. (A) Amino acid sequences of Girdins from different species. Conserved serines at positions 233 (red) and 237 (green) that conform to the MAPK substrate motif are highlighted. (B) Endogenous interaction between MAPK1/2 and Girdin, as detected by co-IP in 293FT cells. (C) In vitro kinase assay showing that MAPK phosphorylates recombinant Girdin NT but not its mutants. (D) Lysates from 293FT cells transfected with the indicated plasmids were separated on Phos-tag gel. Arrowheads indicate multiple bands that may represent phosphorylated Girdin NT. (E) Lysates from 293FT cells stimulated with <t>FBS</t> in the presence or absence of U0126 were subjected to IP to enrich for endogenous Girdin, followed by the enrichment of phosphopeptides using a Titansphere Phos-TiO kit. The phosphopeptides were analyzed by mass spectrometry. The mass spectrum of a tryptic fragment at m/z 843.3431 (mass error, 0.46 ppm) that matched to the peptide 219-DGLHFLPHASSSAQpSPCGpSPGMK-241 containing phosphorylated S233 and S237 is shown. This phosphopeptide was detected with a high peptide confidence (false discovery rate of less than 1%) in cells stimulated with FBS (Mascot score, 28; expectation value, 0.431) but not in cells pretreated with U0126. (F) 293FT cells were transfected with the indicated plasmids, followed by co-IP, showing that mutation of MAPK phosphorylation sites in Girdin disrupts 4F2hc/Girdin interaction. (G) 293FT cells starved for serum were pretreated with DMSO or U0126 for 30 min and stimulated with <t>DMEM</t> containing 10% FBS for 30 min, followed by co-IP (left panel). 293FT cells were transfected with indicated plasmids, followed by co-IP to test endogenous 4F2hc/Girdin interaction (right panel). co-IP, co-immunoprecipitation; CT, carboxyl terminal; DMEM, Dulbecco’s Modified Eagle Medium; DMSO, dimethyl sulphoxide; FBS, fetal bovine serum; Flag-NT, Flag-tagged Girdin NT domain; GFP, green fluorescent protein; Girdin, girders of actin filaments; GST, glutathione S-transferase; IgG, immunoglobulin G; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKK CA, constitutively active mutant of MAPKK; Mr , molecular marker; NT, amino terminal; Rf , relative mobility; S233, serine at position 233; S237, serine at position 237; WT, wild-type; 4F2hc, 4F2 heavy chain.
    Dialyzed Fbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 305 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Thermo Fisher bcat gfp fb
    β-catenin interacts with YAP in human stromal fibroblasts. a – c Coimmunoprecipitation analysis. Total <t>GFP/Fb</t> and <t>bcat-GFP/FB</t> protein lysates were subjected to immunoprecipitation (IP) using the indicated antibodies: anti-β-catenin antibody and mouse IgG. a Immuno-complexes were analyzed by Western blotting with an anti-YAP antibody. b The middle panel shows the presence of YAP protein in cell lysates. c . Actin expression in the column wash through from IP. d – l PLA was performed to confirm the YAP and β-catenin interaction. GFP/Fb and bcat-GFP/Fb were seeded in chamber slides for PLA. A clear YAP-β-catenin interaction can be seen in GFP/Fb e but not bcat-GFP/Fb h . f Nuclear YAP-β-catenin interaction can be seen in the nuclei of GFP/Fb. N-cadherin was used as a positive control d , g , and keratin 14 was used as a negative control i . No signal could be detected in the β-catenin antibody-only treatment j , YAP antibody only treatment k and mouse IgG + rabbit IgG treatment l . m – v Wnt signaling regulates YAP nuclear translocation. GFP/Fb and bcat-GFP/Fb were seeded in chamber slides and treated with DMEM with 0.5% FBS m , n , SK-MEL-24 CM o , p , 100 μM ICG-00 q , r , 100 μM isoquercitrin s , t and 100 ng/ml WNT3A u , v . Yellow arrows indicate the cells expressing nuclear YAP. The pictures shown are representative of three independent experiments. w The mean fluorescence intensities of cytoplasmic YAP in individual GFP/Fb and bcat-GFP/Fb when cultured in DMEM with 0.5% FBS and SK-MEL-24 CM were determined using ImageJ and were normalized according to the intensity of cytoplasmic YAP in GFP/Fb cultured in DMEM with 0.5% FBS. A minimum of thirty cells in each group/culture condition were randomly selected from three independent experiments and were quantified. x Percentages of GFP/Fb and bcat-GFP/Fb expressing nuclear YAP were calculated by counting and dividing the number of nuclear YAP-positive cells by the total cell number under the following conditions: including DMEM with 0.5% FBS, SK-MEL-24 CM, 100 μM ICG-00, 100 μM isoquercitrin and 100 ng/ml WNT3A. At least fifty cells in each group/culture condition were randomly selected from three independent experiments and were counted.
    Bcat Gfp Fb, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher mv free fbs
    Induction of migration and tube formation of HUVECs by CdM from adipose-derived stem cells (ASCs). ASCs were incubated in endothelial basal medium/1% fetal bovine serum <t>(FBS)</t> for 2 days in the absence (A, B) or presence (C, D) of <t>GW4869,</t> an MV-formation inhibitor. CdM was collected and used to treat HUVECs. Cell migration (A, C) and tube formation (B, D) assays for treated HUVECs were performed. The endothelial basal medium/1% FBS without cells was incubated in parallel and was used as a control. The CdM with removal of MVs via ultracentrifugation was used as CdM-MV free. Representative images of cell migration and tube formation are displayed. Scale bar = 200 µm. ( A–D): n = 4. ∗, p
    Mv Free Fbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher arginine free dialysed fbs
    Neuroblastoma proliferation is dependent on arginine metabolism A) shRNA knock-out of ARG2 in SKNMC (high baseline ARG2 expression) decreases cell proliferation. Fold change in cell number after 72h compared to baseline. Experiment performed in duplicate. Corresponding Western blots for ARG2 in wild-type and knock-down cell lines shown below, with actin as a loading control B) Proliferation of tumour cell lines is inhibited by CAT1 inhibition with L-NAME, measured by 3 H-thymidine incorporation after 72 hours C) Cell lines were cultured with <t>RPMI+10%FBS</t> (R10%) or arginine-free RPMI+10%FBS (R10%-arginine). Metabolic activity was measured by MTT after 72h. n=7 replicates D) Sorted GD2+ neuroblastoma cells from patients were treated with BCT-100 (600ng/mL). Analysis of cell death was performed by transmission electron microscopy (Representative micrographs of 2 out of 6 patients shown). Upper panel show untreated cells. Lower panels show post treatment with 600ng/mL BCT-100. Features consistent with organelle enlargement, cell membrane permeablisation, and cellular fragmentation with 600ng/mL BCT-100. Experiments performed on 3 separate occasions E) Sorted GD2+ cells from TH-MYCN murine neuroblastomas were cultured with BCT-100 (600ng/mL) for 72 hours. The percentage of viable cells relative to untreated controls was determined by flow cytometry, using propidium iodide staining. BCT-100 leads to a decrease in murine neuroblastoma cell viability ex vivo F) Plasma from control (saline) and BCT-100 treated TH-MYCN mice was collected at the start (PRE), 16 days after (MID), and at tumour end-point (END). The concentration of arginine was determined by ELISA. BCT-100 maintains a significant reduction in the plasma arginine concentration in vivo . n=6 G) TH-MYCN mice were treated with BCT-100 (60mg/kg) twice weekly intraperitoneally ( ip ) from the time of weaning at 3 weeks of age before overt tumour formations (Prophylaxis). Kaplan-Meier curves show that the development of tumours is significantly delayed, and that survival is increased in BCT-100 treated mice H) TH-MYCN mice were treated with BCT-100 (60mg/kg) twice weekly ip once 5 mm tumours were palpable (Treatment). Kaplan-Meier curves show a significant prolongation of survival in BCT-100 treated mice.
    Arginine Free Dialysed Fbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Phosphorylation of Girdin is required for its interaction with 4F2hc. (A) Amino acid sequences of Girdins from different species. Conserved serines at positions 233 (red) and 237 (green) that conform to the MAPK substrate motif are highlighted. (B) Endogenous interaction between MAPK1/2 and Girdin, as detected by co-IP in 293FT cells. (C) In vitro kinase assay showing that MAPK phosphorylates recombinant Girdin NT but not its mutants. (D) Lysates from 293FT cells transfected with the indicated plasmids were separated on Phos-tag gel. Arrowheads indicate multiple bands that may represent phosphorylated Girdin NT. (E) Lysates from 293FT cells stimulated with FBS in the presence or absence of U0126 were subjected to IP to enrich for endogenous Girdin, followed by the enrichment of phosphopeptides using a Titansphere Phos-TiO kit. The phosphopeptides were analyzed by mass spectrometry. The mass spectrum of a tryptic fragment at m/z 843.3431 (mass error, 0.46 ppm) that matched to the peptide 219-DGLHFLPHASSSAQpSPCGpSPGMK-241 containing phosphorylated S233 and S237 is shown. This phosphopeptide was detected with a high peptide confidence (false discovery rate of less than 1%) in cells stimulated with FBS (Mascot score, 28; expectation value, 0.431) but not in cells pretreated with U0126. (F) 293FT cells were transfected with the indicated plasmids, followed by co-IP, showing that mutation of MAPK phosphorylation sites in Girdin disrupts 4F2hc/Girdin interaction. (G) 293FT cells starved for serum were pretreated with DMSO or U0126 for 30 min and stimulated with DMEM containing 10% FBS for 30 min, followed by co-IP (left panel). 293FT cells were transfected with indicated plasmids, followed by co-IP to test endogenous 4F2hc/Girdin interaction (right panel). co-IP, co-immunoprecipitation; CT, carboxyl terminal; DMEM, Dulbecco’s Modified Eagle Medium; DMSO, dimethyl sulphoxide; FBS, fetal bovine serum; Flag-NT, Flag-tagged Girdin NT domain; GFP, green fluorescent protein; Girdin, girders of actin filaments; GST, glutathione S-transferase; IgG, immunoglobulin G; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKK CA, constitutively active mutant of MAPKK; Mr , molecular marker; NT, amino terminal; Rf , relative mobility; S233, serine at position 233; S237, serine at position 237; WT, wild-type; 4F2hc, 4F2 heavy chain.

    Journal: PLoS Biology

    Article Title: Negative regulation of amino acid signaling by MAPK-regulated 4F2hc/Girdin complex

    doi: 10.1371/journal.pbio.2005090

    Figure Lengend Snippet: Phosphorylation of Girdin is required for its interaction with 4F2hc. (A) Amino acid sequences of Girdins from different species. Conserved serines at positions 233 (red) and 237 (green) that conform to the MAPK substrate motif are highlighted. (B) Endogenous interaction between MAPK1/2 and Girdin, as detected by co-IP in 293FT cells. (C) In vitro kinase assay showing that MAPK phosphorylates recombinant Girdin NT but not its mutants. (D) Lysates from 293FT cells transfected with the indicated plasmids were separated on Phos-tag gel. Arrowheads indicate multiple bands that may represent phosphorylated Girdin NT. (E) Lysates from 293FT cells stimulated with FBS in the presence or absence of U0126 were subjected to IP to enrich for endogenous Girdin, followed by the enrichment of phosphopeptides using a Titansphere Phos-TiO kit. The phosphopeptides were analyzed by mass spectrometry. The mass spectrum of a tryptic fragment at m/z 843.3431 (mass error, 0.46 ppm) that matched to the peptide 219-DGLHFLPHASSSAQpSPCGpSPGMK-241 containing phosphorylated S233 and S237 is shown. This phosphopeptide was detected with a high peptide confidence (false discovery rate of less than 1%) in cells stimulated with FBS (Mascot score, 28; expectation value, 0.431) but not in cells pretreated with U0126. (F) 293FT cells were transfected with the indicated plasmids, followed by co-IP, showing that mutation of MAPK phosphorylation sites in Girdin disrupts 4F2hc/Girdin interaction. (G) 293FT cells starved for serum were pretreated with DMSO or U0126 for 30 min and stimulated with DMEM containing 10% FBS for 30 min, followed by co-IP (left panel). 293FT cells were transfected with indicated plasmids, followed by co-IP to test endogenous 4F2hc/Girdin interaction (right panel). co-IP, co-immunoprecipitation; CT, carboxyl terminal; DMEM, Dulbecco’s Modified Eagle Medium; DMSO, dimethyl sulphoxide; FBS, fetal bovine serum; Flag-NT, Flag-tagged Girdin NT domain; GFP, green fluorescent protein; Girdin, girders of actin filaments; GST, glutathione S-transferase; IgG, immunoglobulin G; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKK CA, constitutively active mutant of MAPKK; Mr , molecular marker; NT, amino terminal; Rf , relative mobility; S233, serine at position 233; S237, serine at position 237; WT, wild-type; 4F2hc, 4F2 heavy chain.

    Article Snippet: Amino acid–free DMEM (Cell Science and Technology Institute, Inc., Sendai, Japan) supplemented with 10% dialyzed FBS (Thermo Fisher Scientific) was used to starve cells. siRNAs or plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

    Techniques: Co-Immunoprecipitation Assay, In Vitro, Kinase Assay, Recombinant, Transfection, Mass Spectrometry, Mutagenesis, Immunoprecipitation, Modification, Marker

    β-catenin interacts with YAP in human stromal fibroblasts. a – c Coimmunoprecipitation analysis. Total GFP/Fb and bcat-GFP/FB protein lysates were subjected to immunoprecipitation (IP) using the indicated antibodies: anti-β-catenin antibody and mouse IgG. a Immuno-complexes were analyzed by Western blotting with an anti-YAP antibody. b The middle panel shows the presence of YAP protein in cell lysates. c . Actin expression in the column wash through from IP. d – l PLA was performed to confirm the YAP and β-catenin interaction. GFP/Fb and bcat-GFP/Fb were seeded in chamber slides for PLA. A clear YAP-β-catenin interaction can be seen in GFP/Fb e but not bcat-GFP/Fb h . f Nuclear YAP-β-catenin interaction can be seen in the nuclei of GFP/Fb. N-cadherin was used as a positive control d , g , and keratin 14 was used as a negative control i . No signal could be detected in the β-catenin antibody-only treatment j , YAP antibody only treatment k and mouse IgG + rabbit IgG treatment l . m – v Wnt signaling regulates YAP nuclear translocation. GFP/Fb and bcat-GFP/Fb were seeded in chamber slides and treated with DMEM with 0.5% FBS m , n , SK-MEL-24 CM o , p , 100 μM ICG-00 q , r , 100 μM isoquercitrin s , t and 100 ng/ml WNT3A u , v . Yellow arrows indicate the cells expressing nuclear YAP. The pictures shown are representative of three independent experiments. w The mean fluorescence intensities of cytoplasmic YAP in individual GFP/Fb and bcat-GFP/Fb when cultured in DMEM with 0.5% FBS and SK-MEL-24 CM were determined using ImageJ and were normalized according to the intensity of cytoplasmic YAP in GFP/Fb cultured in DMEM with 0.5% FBS. A minimum of thirty cells in each group/culture condition were randomly selected from three independent experiments and were quantified. x Percentages of GFP/Fb and bcat-GFP/Fb expressing nuclear YAP were calculated by counting and dividing the number of nuclear YAP-positive cells by the total cell number under the following conditions: including DMEM with 0.5% FBS, SK-MEL-24 CM, 100 μM ICG-00, 100 μM isoquercitrin and 100 ng/ml WNT3A. At least fifty cells in each group/culture condition were randomly selected from three independent experiments and were counted.

    Journal: Signal Transduction and Targeted Therapy

    Article Title: The β-catenin/YAP signaling axis is a key regulator of melanoma-associated fibroblasts

    doi: 10.1038/s41392-019-0100-7

    Figure Lengend Snippet: β-catenin interacts with YAP in human stromal fibroblasts. a – c Coimmunoprecipitation analysis. Total GFP/Fb and bcat-GFP/FB protein lysates were subjected to immunoprecipitation (IP) using the indicated antibodies: anti-β-catenin antibody and mouse IgG. a Immuno-complexes were analyzed by Western blotting with an anti-YAP antibody. b The middle panel shows the presence of YAP protein in cell lysates. c . Actin expression in the column wash through from IP. d – l PLA was performed to confirm the YAP and β-catenin interaction. GFP/Fb and bcat-GFP/Fb were seeded in chamber slides for PLA. A clear YAP-β-catenin interaction can be seen in GFP/Fb e but not bcat-GFP/Fb h . f Nuclear YAP-β-catenin interaction can be seen in the nuclei of GFP/Fb. N-cadherin was used as a positive control d , g , and keratin 14 was used as a negative control i . No signal could be detected in the β-catenin antibody-only treatment j , YAP antibody only treatment k and mouse IgG + rabbit IgG treatment l . m – v Wnt signaling regulates YAP nuclear translocation. GFP/Fb and bcat-GFP/Fb were seeded in chamber slides and treated with DMEM with 0.5% FBS m , n , SK-MEL-24 CM o , p , 100 μM ICG-00 q , r , 100 μM isoquercitrin s , t and 100 ng/ml WNT3A u , v . Yellow arrows indicate the cells expressing nuclear YAP. The pictures shown are representative of three independent experiments. w The mean fluorescence intensities of cytoplasmic YAP in individual GFP/Fb and bcat-GFP/Fb when cultured in DMEM with 0.5% FBS and SK-MEL-24 CM were determined using ImageJ and were normalized according to the intensity of cytoplasmic YAP in GFP/Fb cultured in DMEM with 0.5% FBS. A minimum of thirty cells in each group/culture condition were randomly selected from three independent experiments and were quantified. x Percentages of GFP/Fb and bcat-GFP/Fb expressing nuclear YAP were calculated by counting and dividing the number of nuclear YAP-positive cells by the total cell number under the following conditions: including DMEM with 0.5% FBS, SK-MEL-24 CM, 100 μM ICG-00, 100 μM isoquercitrin and 100 ng/ml WNT3A. At least fifty cells in each group/culture condition were randomly selected from three independent experiments and were counted.

    Article Snippet: Confocal reflection microscopy GFP/Fb and bcat-GFP/Fb were harvested after induction with doxycycline for 48 h. Three mg/ml type I collagen (Thermo Fisher Scientific, Rochester, NY) was diluted in DMEM (with L-glutamine, w/o HEPES, Thermo Fisher Scientific, MA) and supplemented with 10% FBS to a final collagen gel concentration of one mg/ml, and then the cells were suspended at a final density of 1 × 105 cells/ml.

    Techniques: Immunoprecipitation, Western Blot, Expressing, Proximity Ligation Assay, Positive Control, Negative Control, Translocation Assay, Fluorescence, Cell Culture

    β-catenin is essential for the functional properties of stromal fibroblasts. a GFP/Fb and bcat-GFP/Fb were induced by addition of 500 ng/ml doxycycline to the culture medium for 72 h. Left: GFP expression in GFP/Fb and bcat-GFP/Fb; right: representative images of β-catenin immunofluorescence staining of GFP/Fb and bcat-GFP/Fb. Scale bar: 50 μm. b The viability of GFP/Fb and bcat-GFP/Fb was compared using the MTT assay. The cells were collected at 0, 24, 48, and 72 h. The data are representative of three independent experiments. c Expression of F-actin, paxillin, vimentin and fibronectin (red) in GFP/Fb and bcat-GFP/Fb was determined by immunostaining after a three-day doxycycline induction with DAPI counterstaining (blue). Scale bar: 50 μm. The mean fluorescence intensities of each immunostained protein in individual GFP/Fb and bcat-GFP/Fb cells were determined using ImageJ. d Expression of F-actin, paxillin, vimentin and fibronectin was measured in total protein extracted from 1 × 10 5 GFP/Fb and bcat-GFP/Fb cells by Western blotting. GAPDH was used as an internal control. e The intensities of the bands of the indicated proteins expressed in GFP/Fb and bcat-GFP/Fb were normalized to the GAPDH signal. The data are representative of three independent experiments. f – g α-SMA immunofluorescence staining of GFP/Fb, GFP/Fb + SK-MEL-24 coculture, bcat-GFP/Fb, and bcat-GFP/Fb + SK-MEL-24 coculture with DAPI nuclear costaining. GFP/Fb and bcat-GFP/Fb were tagged with GFP and are denoted with yellow arrows in coculture images. Scale bar: 50 μm. h Fluorescence intensities of α-SMA expression were determined and compared between four groups from three individual experiments. i–j FSP-1 immunofluorescence staining of GFP/Fb, GFP/Fb + SK-MEL-24 coculture, bcat-GFP/Fb, and bcat-GFP/Fb + SK-MEL-24 coculture with DAPI nuclear costaining. k Fluorescence intensities of FSP-1 expression were determined and compared between four groups from three individual experiments. l – o F-actin immunofluorescent staining of GFP/Fb and bcat-GFP/Fb cultured in DMEM with 0.5% FBS and SK-MEL-24 CM with DAPI nuclear costaining. Scale bar: 50 μm. q – t Paxillin immunofluorescent staining of GFP/Fb and bcat-GFP/Fb cultured in DMEM with 0.5% FBS and SK-MEL-24 CM with DAPI nuclear costaining. Scale bar: 50 μm. p Fluorescence intensities of F-actin expression and u . paxillin were determined and compared between four groups from three individual experiments. v Expression of F-actin and paxillin was measured in total protein extracts from 1 × 10 5 GFP/Fb and bcat-GFP/Fb cells cultured in DMEM with 0.5% FBS and SK-MEL-24 CM by Western blotting. GAPDH was used as an internal control. Statistical analysis of the intensities of the bands of indicated proteins expressed in GFP/Fb and bcat-GFP/Fb cultured in DMEM with 0.5% FBS and SK-MEL-24 CM were from three independent experiments and were normalized to the GAPDH band. w Representative images of the migration of GFP/Fb and bcat-GFP/Fb cultured in DMEM with 0.5% FBS at 0 and 30 h. Red lines indicate the starting edge of fibroblasts after the scratch. Yellow lines indicate the final distance that GFP/Fb and bcat-GFP/Fb migrated. x Representative images are shown of the migration of GFP/Fb and bcat-GFP/Fb cultured in SK-MEL-24 CM with 0.5% FBS at 0 and 30 h. Red lines indicate the starting points of fibroblasts after scratch. Yellow lines indicate the final distance that GFP/Fb and bcat-GFP/Fb migrated. The images shown are representative of at least three independently repeated experiments. y Statistical comparison was performed on the percentages of closed scratch areas between GFP/Fb and bcat-GFP/Fb under the same culture conditions. z Statistical comparison was performed on the percentages of closed scratch areas by GFP/Fb and bcat-GFP/Fb under different culture conditions.

    Journal: Signal Transduction and Targeted Therapy

    Article Title: The β-catenin/YAP signaling axis is a key regulator of melanoma-associated fibroblasts

    doi: 10.1038/s41392-019-0100-7

    Figure Lengend Snippet: β-catenin is essential for the functional properties of stromal fibroblasts. a GFP/Fb and bcat-GFP/Fb were induced by addition of 500 ng/ml doxycycline to the culture medium for 72 h. Left: GFP expression in GFP/Fb and bcat-GFP/Fb; right: representative images of β-catenin immunofluorescence staining of GFP/Fb and bcat-GFP/Fb. Scale bar: 50 μm. b The viability of GFP/Fb and bcat-GFP/Fb was compared using the MTT assay. The cells were collected at 0, 24, 48, and 72 h. The data are representative of three independent experiments. c Expression of F-actin, paxillin, vimentin and fibronectin (red) in GFP/Fb and bcat-GFP/Fb was determined by immunostaining after a three-day doxycycline induction with DAPI counterstaining (blue). Scale bar: 50 μm. The mean fluorescence intensities of each immunostained protein in individual GFP/Fb and bcat-GFP/Fb cells were determined using ImageJ. d Expression of F-actin, paxillin, vimentin and fibronectin was measured in total protein extracted from 1 × 10 5 GFP/Fb and bcat-GFP/Fb cells by Western blotting. GAPDH was used as an internal control. e The intensities of the bands of the indicated proteins expressed in GFP/Fb and bcat-GFP/Fb were normalized to the GAPDH signal. The data are representative of three independent experiments. f – g α-SMA immunofluorescence staining of GFP/Fb, GFP/Fb + SK-MEL-24 coculture, bcat-GFP/Fb, and bcat-GFP/Fb + SK-MEL-24 coculture with DAPI nuclear costaining. GFP/Fb and bcat-GFP/Fb were tagged with GFP and are denoted with yellow arrows in coculture images. Scale bar: 50 μm. h Fluorescence intensities of α-SMA expression were determined and compared between four groups from three individual experiments. i–j FSP-1 immunofluorescence staining of GFP/Fb, GFP/Fb + SK-MEL-24 coculture, bcat-GFP/Fb, and bcat-GFP/Fb + SK-MEL-24 coculture with DAPI nuclear costaining. k Fluorescence intensities of FSP-1 expression were determined and compared between four groups from three individual experiments. l – o F-actin immunofluorescent staining of GFP/Fb and bcat-GFP/Fb cultured in DMEM with 0.5% FBS and SK-MEL-24 CM with DAPI nuclear costaining. Scale bar: 50 μm. q – t Paxillin immunofluorescent staining of GFP/Fb and bcat-GFP/Fb cultured in DMEM with 0.5% FBS and SK-MEL-24 CM with DAPI nuclear costaining. Scale bar: 50 μm. p Fluorescence intensities of F-actin expression and u . paxillin were determined and compared between four groups from three individual experiments. v Expression of F-actin and paxillin was measured in total protein extracts from 1 × 10 5 GFP/Fb and bcat-GFP/Fb cells cultured in DMEM with 0.5% FBS and SK-MEL-24 CM by Western blotting. GAPDH was used as an internal control. Statistical analysis of the intensities of the bands of indicated proteins expressed in GFP/Fb and bcat-GFP/Fb cultured in DMEM with 0.5% FBS and SK-MEL-24 CM were from three independent experiments and were normalized to the GAPDH band. w Representative images of the migration of GFP/Fb and bcat-GFP/Fb cultured in DMEM with 0.5% FBS at 0 and 30 h. Red lines indicate the starting edge of fibroblasts after the scratch. Yellow lines indicate the final distance that GFP/Fb and bcat-GFP/Fb migrated. x Representative images are shown of the migration of GFP/Fb and bcat-GFP/Fb cultured in SK-MEL-24 CM with 0.5% FBS at 0 and 30 h. Red lines indicate the starting points of fibroblasts after scratch. Yellow lines indicate the final distance that GFP/Fb and bcat-GFP/Fb migrated. The images shown are representative of at least three independently repeated experiments. y Statistical comparison was performed on the percentages of closed scratch areas between GFP/Fb and bcat-GFP/Fb under the same culture conditions. z Statistical comparison was performed on the percentages of closed scratch areas by GFP/Fb and bcat-GFP/Fb under different culture conditions.

    Article Snippet: Confocal reflection microscopy GFP/Fb and bcat-GFP/Fb were harvested after induction with doxycycline for 48 h. Three mg/ml type I collagen (Thermo Fisher Scientific, Rochester, NY) was diluted in DMEM (with L-glutamine, w/o HEPES, Thermo Fisher Scientific, MA) and supplemented with 10% FBS to a final collagen gel concentration of one mg/ml, and then the cells were suspended at a final density of 1 × 105 cells/ml.

    Techniques: Functional Assay, Expressing, Immunofluorescence, Staining, MTT Assay, Immunostaining, Fluorescence, Western Blot, Cell Culture, Migration

    β-catenin is required by stromal fibroblasts to remodel the ECM. a–d Representative images of collagen gel contraction assays with GFP/Fb in either DMEM + 0.5% FBS a or SK-MEL-24 CM + 0.5% FBS d and with bcat-GFP/Fb in either DMEM + 0.5% FBS b or SK-MEL-24 CM + 0.5% FBS e after 48 h. The gel in each well is circled by a red line. The green double arrow line indicates the diameter of the contracted gel. c Statistical quantification is shown for the relative percentages of gel contraction by GFP/Fb and bcat-GFP/Fb cultured in DMEM + 0.5% FBS. f Statistical quantification is shown for the percentages of gel contraction by GFP/Fb and bcat-GFP/Fb cultured in SK-MEL-24 CM + 0.5% FBS. g Viability of GFP/Fb and bcat-GFP/Fb cultured in DMEM with 0.5% FBS after 72 h of culture was compared by the MTT assay. h – j CRM images of 1 mg/ml collagen gel alone h , gel with GFP/Fb i and gel with bcat-GFP/Fb j were acquired using a Zeiss LSM 510 Two-Photon microscope at 40X magnification three days after seeding. k – m CRM images were analyzed by ImageJ with the BoneJ plugin to compare fiber thickness k , connectivity l and spacing m among gels without cells, with GPF/Fb, and with bcat-GFP/Fb. n Illustration of the measurement of collagen gel stiffness using an atomic force microscope is shown. o Collagen gel stiffness was compared among collagen gel, collagen gel embedded with GFP/Fb and collagen gel embedded with bcat-GFP/Fb using AFM.

    Journal: Signal Transduction and Targeted Therapy

    Article Title: The β-catenin/YAP signaling axis is a key regulator of melanoma-associated fibroblasts

    doi: 10.1038/s41392-019-0100-7

    Figure Lengend Snippet: β-catenin is required by stromal fibroblasts to remodel the ECM. a–d Representative images of collagen gel contraction assays with GFP/Fb in either DMEM + 0.5% FBS a or SK-MEL-24 CM + 0.5% FBS d and with bcat-GFP/Fb in either DMEM + 0.5% FBS b or SK-MEL-24 CM + 0.5% FBS e after 48 h. The gel in each well is circled by a red line. The green double arrow line indicates the diameter of the contracted gel. c Statistical quantification is shown for the relative percentages of gel contraction by GFP/Fb and bcat-GFP/Fb cultured in DMEM + 0.5% FBS. f Statistical quantification is shown for the percentages of gel contraction by GFP/Fb and bcat-GFP/Fb cultured in SK-MEL-24 CM + 0.5% FBS. g Viability of GFP/Fb and bcat-GFP/Fb cultured in DMEM with 0.5% FBS after 72 h of culture was compared by the MTT assay. h – j CRM images of 1 mg/ml collagen gel alone h , gel with GFP/Fb i and gel with bcat-GFP/Fb j were acquired using a Zeiss LSM 510 Two-Photon microscope at 40X magnification three days after seeding. k – m CRM images were analyzed by ImageJ with the BoneJ plugin to compare fiber thickness k , connectivity l and spacing m among gels without cells, with GPF/Fb, and with bcat-GFP/Fb. n Illustration of the measurement of collagen gel stiffness using an atomic force microscope is shown. o Collagen gel stiffness was compared among collagen gel, collagen gel embedded with GFP/Fb and collagen gel embedded with bcat-GFP/Fb using AFM.

    Article Snippet: Confocal reflection microscopy GFP/Fb and bcat-GFP/Fb were harvested after induction with doxycycline for 48 h. Three mg/ml type I collagen (Thermo Fisher Scientific, Rochester, NY) was diluted in DMEM (with L-glutamine, w/o HEPES, Thermo Fisher Scientific, MA) and supplemented with 10% FBS to a final collagen gel concentration of one mg/ml, and then the cells were suspended at a final density of 1 × 105 cells/ml.

    Techniques: Cell Culture, MTT Assay, Microscopy

    The nuclear translocation of YAP is inhibited in melanoma-associated fibroblasts after β-catenin ablation. The expression of nuclear YAP (red) in fibroblasts (Fbs) in human melanoma tissue (indicated by white arrows) was visualized by coimmunostaining with a fibroblast-specific anti-TE7 antibody (green). a – c Surrounding Fbs. d – f Infiltrated Fbs. g – i Human skin Fbs. Images shown are typical of ten human melanoma samples for each type. j Comparison of the percentages of stromal fibroblasts expressing nuclear YAP among the following: fibroblasts surrounding melanoma, infiltrated fibroblasts in melanoma and stromal fibroblasts in normal human skin. The percentage of stromal fibroblasts expressing nuclear YAP was calculated by dividing the number of nuclear YAP-positive and TE7-positive cells by the total number of TE7-positive cell number in each group. Statistical analysis of differences was carried out by Student’s t -tests using GraphPad Prism. k Stromal fibroblasts in Braf V600E ; Pten lox5 -Fb tumors express cytoplasmic and nuclear YAP. Yellow arrows point to fibroblasts expressing nuclear YAP. l – m Enlarged images are shown of fibroblasts circled in k with single α-SMA staining l and double YAP/α-SMA staining m . n In Braf V600E ; Pten lox5 -bcat/Fb tumors, not only is the number of activated fibroblasts reduced but also the nuclear translocation of YAP was inhibited in fibroblasts. o – p Enlarged images are shown of fibroblasts circled in D with single α-SMA staining o and double YAP/α-SMA staining p The data are representative of three independent experiments. Scale bar: 100 μm. q The percentage of α-SMA-positive cells expressing nuclear YAP in Braf V600E ; Pten lox5 -Fb tumors and Braf V600E ; Pten lox5 -bcat/Fb tumors is shown. Five different pairs of Braf V600E , Pten lox5 -Fb tumors and Braf V600E , and Pten lox5 -bcat/Fb tumors were stained and counted. r – y YAP immunofluorescence staining of GFP/Fb, GFP/Fb + SK-MEL-24 coculture, bcat-GFP/Fb, and bcat-GFP/Fb + SK-MEL-24 coculture with DAPI nuclear costaining (blue). GFP/Fb and bcat-GFP/Fb were tagged with GFP and are denoted with yellow arrows in coculture pictures. Scale bar: 50 μm.

    Journal: Signal Transduction and Targeted Therapy

    Article Title: The β-catenin/YAP signaling axis is a key regulator of melanoma-associated fibroblasts

    doi: 10.1038/s41392-019-0100-7

    Figure Lengend Snippet: The nuclear translocation of YAP is inhibited in melanoma-associated fibroblasts after β-catenin ablation. The expression of nuclear YAP (red) in fibroblasts (Fbs) in human melanoma tissue (indicated by white arrows) was visualized by coimmunostaining with a fibroblast-specific anti-TE7 antibody (green). a – c Surrounding Fbs. d – f Infiltrated Fbs. g – i Human skin Fbs. Images shown are typical of ten human melanoma samples for each type. j Comparison of the percentages of stromal fibroblasts expressing nuclear YAP among the following: fibroblasts surrounding melanoma, infiltrated fibroblasts in melanoma and stromal fibroblasts in normal human skin. The percentage of stromal fibroblasts expressing nuclear YAP was calculated by dividing the number of nuclear YAP-positive and TE7-positive cells by the total number of TE7-positive cell number in each group. Statistical analysis of differences was carried out by Student’s t -tests using GraphPad Prism. k Stromal fibroblasts in Braf V600E ; Pten lox5 -Fb tumors express cytoplasmic and nuclear YAP. Yellow arrows point to fibroblasts expressing nuclear YAP. l – m Enlarged images are shown of fibroblasts circled in k with single α-SMA staining l and double YAP/α-SMA staining m . n In Braf V600E ; Pten lox5 -bcat/Fb tumors, not only is the number of activated fibroblasts reduced but also the nuclear translocation of YAP was inhibited in fibroblasts. o – p Enlarged images are shown of fibroblasts circled in D with single α-SMA staining o and double YAP/α-SMA staining p The data are representative of three independent experiments. Scale bar: 100 μm. q The percentage of α-SMA-positive cells expressing nuclear YAP in Braf V600E ; Pten lox5 -Fb tumors and Braf V600E ; Pten lox5 -bcat/Fb tumors is shown. Five different pairs of Braf V600E , Pten lox5 -Fb tumors and Braf V600E , and Pten lox5 -bcat/Fb tumors were stained and counted. r – y YAP immunofluorescence staining of GFP/Fb, GFP/Fb + SK-MEL-24 coculture, bcat-GFP/Fb, and bcat-GFP/Fb + SK-MEL-24 coculture with DAPI nuclear costaining (blue). GFP/Fb and bcat-GFP/Fb were tagged with GFP and are denoted with yellow arrows in coculture pictures. Scale bar: 50 μm.

    Article Snippet: Confocal reflection microscopy GFP/Fb and bcat-GFP/Fb were harvested after induction with doxycycline for 48 h. Three mg/ml type I collagen (Thermo Fisher Scientific, Rochester, NY) was diluted in DMEM (with L-glutamine, w/o HEPES, Thermo Fisher Scientific, MA) and supplemented with 10% FBS to a final collagen gel concentration of one mg/ml, and then the cells were suspended at a final density of 1 × 105 cells/ml.

    Techniques: Translocation Assay, Expressing, Staining, Immunofluorescence

    Stromal fibroblasts require β-catenin to support melanoma cell growth. SK-MEL-24; GFP/Fb spheroid and SK-MEL-24; bcat-GFP/Fb spheroid cultures were grown for 96 h. Representative bright-field images of SK-MEL-24; GFP/Fb spheroid a – d and SK-MEL-24; bcat-GFP/Fb spheroid cultures e – h were taken using a Carl Zeiss Axiovert 100 TV inverted microscope at ×5 magnification at 24, 48, 72, and 96 h. i Ki67 immunofluorescence staining (red) of SK-MEL-24; GFP/Fb and SK-MEL-24; bcat-GFP/Fb spheroids embedded in paraffin. The nuclei were stained with DAPI (blue). Scale bar: 200 μm. j Quantification of Ki67-positive cells per square millimeter of SK-MEL-24; GFP/Fb spheroid sections and SK-MEL-24; bcat-GFP/Fb spheroid sections. A minimum of ten randomly selected fields was counted for each spheroid type. k Flow cytometry was used to analyze apoptosis and death of SK-MEL-24 melanoma cells in both SK-MEL-24; GFP/Fb spheroids and SK-MEL-24; bcat-GFP/Fb spheroids using annexin V and PI staining. l Percentages of SK-MEL-24 cell subpopulations based on PI and annexin V staining in SK-MEL-24; GFP/Fb spheroids and SK-MEL-24; bcat-GFP/Fb spheroids are shown. m – p α-SMA m–n and FSP-1 o – p immunostaining was performed on 5-μm-thick paraffin sections of SK-MEL-24; GFP/Fb and SK-MEL-24; bcat-GFP/Fb spheroids. Pictures were taken using a Nikon microscope at ×20 magnification. Scale bar: 200 μm. q Collagen content in SK-MEL-24; GFP/Fb spheroids and SK-MEL-24; bcat-GFP/Fb spheroids was visualized by picrosirius red staining. Pictures were taken using an inverted light microscope at ×20 magnification. r Quantification of collagen content was performed by collagen extraction and colorimetric measurement. Collagen content was normalized to total protein content for each sample. s – v Fibronectin s – t and vimentin u – v immunostaining was performed on 5-μm-thick paraffin sections of SK-MEL-24; GFP/Fb and SK-MEL-24; bcat-GFP/Fb spheroids. Pictures were taken using a Nikon microscope at ×20 magnification. Scale bar: 200 μm. w – z Photographs were taken at ×40 magnification. Scale bar: 100 μm.

    Journal: Signal Transduction and Targeted Therapy

    Article Title: The β-catenin/YAP signaling axis is a key regulator of melanoma-associated fibroblasts

    doi: 10.1038/s41392-019-0100-7

    Figure Lengend Snippet: Stromal fibroblasts require β-catenin to support melanoma cell growth. SK-MEL-24; GFP/Fb spheroid and SK-MEL-24; bcat-GFP/Fb spheroid cultures were grown for 96 h. Representative bright-field images of SK-MEL-24; GFP/Fb spheroid a – d and SK-MEL-24; bcat-GFP/Fb spheroid cultures e – h were taken using a Carl Zeiss Axiovert 100 TV inverted microscope at ×5 magnification at 24, 48, 72, and 96 h. i Ki67 immunofluorescence staining (red) of SK-MEL-24; GFP/Fb and SK-MEL-24; bcat-GFP/Fb spheroids embedded in paraffin. The nuclei were stained with DAPI (blue). Scale bar: 200 μm. j Quantification of Ki67-positive cells per square millimeter of SK-MEL-24; GFP/Fb spheroid sections and SK-MEL-24; bcat-GFP/Fb spheroid sections. A minimum of ten randomly selected fields was counted for each spheroid type. k Flow cytometry was used to analyze apoptosis and death of SK-MEL-24 melanoma cells in both SK-MEL-24; GFP/Fb spheroids and SK-MEL-24; bcat-GFP/Fb spheroids using annexin V and PI staining. l Percentages of SK-MEL-24 cell subpopulations based on PI and annexin V staining in SK-MEL-24; GFP/Fb spheroids and SK-MEL-24; bcat-GFP/Fb spheroids are shown. m – p α-SMA m–n and FSP-1 o – p immunostaining was performed on 5-μm-thick paraffin sections of SK-MEL-24; GFP/Fb and SK-MEL-24; bcat-GFP/Fb spheroids. Pictures were taken using a Nikon microscope at ×20 magnification. Scale bar: 200 μm. q Collagen content in SK-MEL-24; GFP/Fb spheroids and SK-MEL-24; bcat-GFP/Fb spheroids was visualized by picrosirius red staining. Pictures were taken using an inverted light microscope at ×20 magnification. r Quantification of collagen content was performed by collagen extraction and colorimetric measurement. Collagen content was normalized to total protein content for each sample. s – v Fibronectin s – t and vimentin u – v immunostaining was performed on 5-μm-thick paraffin sections of SK-MEL-24; GFP/Fb and SK-MEL-24; bcat-GFP/Fb spheroids. Pictures were taken using a Nikon microscope at ×20 magnification. Scale bar: 200 μm. w – z Photographs were taken at ×40 magnification. Scale bar: 100 μm.

    Article Snippet: Confocal reflection microscopy GFP/Fb and bcat-GFP/Fb were harvested after induction with doxycycline for 48 h. Three mg/ml type I collagen (Thermo Fisher Scientific, Rochester, NY) was diluted in DMEM (with L-glutamine, w/o HEPES, Thermo Fisher Scientific, MA) and supplemented with 10% FBS to a final collagen gel concentration of one mg/ml, and then the cells were suspended at a final density of 1 × 105 cells/ml.

    Techniques: Inverted Microscopy, Immunofluorescence, Staining, Flow Cytometry, Cytometry, Immunostaining, Microscopy, Light Microscopy

    Induction of migration and tube formation of HUVECs by CdM from adipose-derived stem cells (ASCs). ASCs were incubated in endothelial basal medium/1% fetal bovine serum (FBS) for 2 days in the absence (A, B) or presence (C, D) of GW4869, an MV-formation inhibitor. CdM was collected and used to treat HUVECs. Cell migration (A, C) and tube formation (B, D) assays for treated HUVECs were performed. The endothelial basal medium/1% FBS without cells was incubated in parallel and was used as a control. The CdM with removal of MVs via ultracentrifugation was used as CdM-MV free. Representative images of cell migration and tube formation are displayed. Scale bar = 200 µm. ( A–D): n = 4. ∗, p

    Journal: Stem Cells Translational Medicine

    Article Title: Adipose-Derived Stem Cells Induce Angiogenesis via Microvesicle Transport of miRNA-31

    doi: 10.5966/sctm.2015-0177

    Figure Lengend Snippet: Induction of migration and tube formation of HUVECs by CdM from adipose-derived stem cells (ASCs). ASCs were incubated in endothelial basal medium/1% fetal bovine serum (FBS) for 2 days in the absence (A, B) or presence (C, D) of GW4869, an MV-formation inhibitor. CdM was collected and used to treat HUVECs. Cell migration (A, C) and tube formation (B, D) assays for treated HUVECs were performed. The endothelial basal medium/1% FBS without cells was incubated in parallel and was used as a control. The CdM with removal of MVs via ultracentrifugation was used as CdM-MV free. Representative images of cell migration and tube formation are displayed. Scale bar = 200 µm. ( A–D): n = 4. ∗, p

    Article Snippet: Cells were then washed with phosphate-buffered saline (PBS) and incubated in fresh endothelial basal medium/1% MV-free FBS with or without GW4869 (Thermo Fisher Scientific), an inhibitor of MV formation [ ], for an additional 2 days.

    Techniques: Migration, Derivative Assay, Incubation

    Induction of migration and tube formation of HUVECs by MVs from adipose-derived stem cells (ASCs). (A): Conditioned medium was subjected to standard serial centrifugation for MV isolation. The isolated pellet was examined with scanning electron microscopy (scale bar = 1 µm). (B): ASCs were maintained in growth medium or preconditioned with endothelial differentiation medium for 4 days. After washing, the ASCs were then incubated for 2 days in endothelial basal medium/1% FBS with or without the presence of GW4869. Western blot analysis of the MVs was performed with Alix, an MV marker. Each lane represented an MV lysate from 3 × 10 6 cells. (C): The protein content of MVs and MV-P from ASCs were determined. (D–F): HUVECs were treated with 30 µg/ml (protein concentration) MV or MV-P. HUVECs in fresh endothelial basal medium/1% FBS was used as a control. Cell migration (D) , tube formation (E) , and proliferation (F) assays of the HUVECs were performed as described in Material and Methods. (C–F): n = 4. ## , p

    Journal: Stem Cells Translational Medicine

    Article Title: Adipose-Derived Stem Cells Induce Angiogenesis via Microvesicle Transport of miRNA-31

    doi: 10.5966/sctm.2015-0177

    Figure Lengend Snippet: Induction of migration and tube formation of HUVECs by MVs from adipose-derived stem cells (ASCs). (A): Conditioned medium was subjected to standard serial centrifugation for MV isolation. The isolated pellet was examined with scanning electron microscopy (scale bar = 1 µm). (B): ASCs were maintained in growth medium or preconditioned with endothelial differentiation medium for 4 days. After washing, the ASCs were then incubated for 2 days in endothelial basal medium/1% FBS with or without the presence of GW4869. Western blot analysis of the MVs was performed with Alix, an MV marker. Each lane represented an MV lysate from 3 × 10 6 cells. (C): The protein content of MVs and MV-P from ASCs were determined. (D–F): HUVECs were treated with 30 µg/ml (protein concentration) MV or MV-P. HUVECs in fresh endothelial basal medium/1% FBS was used as a control. Cell migration (D) , tube formation (E) , and proliferation (F) assays of the HUVECs were performed as described in Material and Methods. (C–F): n = 4. ## , p

    Article Snippet: Cells were then washed with phosphate-buffered saline (PBS) and incubated in fresh endothelial basal medium/1% MV-free FBS with or without GW4869 (Thermo Fisher Scientific), an inhibitor of MV formation [ ], for an additional 2 days.

    Techniques: Migration, Derivative Assay, Centrifugation, Isolation, Electron Microscopy, Incubation, Western Blot, Marker, Protein Concentration

    Neuroblastoma proliferation is dependent on arginine metabolism A) shRNA knock-out of ARG2 in SKNMC (high baseline ARG2 expression) decreases cell proliferation. Fold change in cell number after 72h compared to baseline. Experiment performed in duplicate. Corresponding Western blots for ARG2 in wild-type and knock-down cell lines shown below, with actin as a loading control B) Proliferation of tumour cell lines is inhibited by CAT1 inhibition with L-NAME, measured by 3 H-thymidine incorporation after 72 hours C) Cell lines were cultured with RPMI+10%FBS (R10%) or arginine-free RPMI+10%FBS (R10%-arginine). Metabolic activity was measured by MTT after 72h. n=7 replicates D) Sorted GD2+ neuroblastoma cells from patients were treated with BCT-100 (600ng/mL). Analysis of cell death was performed by transmission electron microscopy (Representative micrographs of 2 out of 6 patients shown). Upper panel show untreated cells. Lower panels show post treatment with 600ng/mL BCT-100. Features consistent with organelle enlargement, cell membrane permeablisation, and cellular fragmentation with 600ng/mL BCT-100. Experiments performed on 3 separate occasions E) Sorted GD2+ cells from TH-MYCN murine neuroblastomas were cultured with BCT-100 (600ng/mL) for 72 hours. The percentage of viable cells relative to untreated controls was determined by flow cytometry, using propidium iodide staining. BCT-100 leads to a decrease in murine neuroblastoma cell viability ex vivo F) Plasma from control (saline) and BCT-100 treated TH-MYCN mice was collected at the start (PRE), 16 days after (MID), and at tumour end-point (END). The concentration of arginine was determined by ELISA. BCT-100 maintains a significant reduction in the plasma arginine concentration in vivo . n=6 G) TH-MYCN mice were treated with BCT-100 (60mg/kg) twice weekly intraperitoneally ( ip ) from the time of weaning at 3 weeks of age before overt tumour formations (Prophylaxis). Kaplan-Meier curves show that the development of tumours is significantly delayed, and that survival is increased in BCT-100 treated mice H) TH-MYCN mice were treated with BCT-100 (60mg/kg) twice weekly ip once 5 mm tumours were palpable (Treatment). Kaplan-Meier curves show a significant prolongation of survival in BCT-100 treated mice.

    Journal: Cancer research

    Article Title: Macrophage-derived IL-1β and TNF-α regulate arginine metabolism in neuroblastoma

    doi: 10.1158/0008-5472.CAN-18-2139

    Figure Lengend Snippet: Neuroblastoma proliferation is dependent on arginine metabolism A) shRNA knock-out of ARG2 in SKNMC (high baseline ARG2 expression) decreases cell proliferation. Fold change in cell number after 72h compared to baseline. Experiment performed in duplicate. Corresponding Western blots for ARG2 in wild-type and knock-down cell lines shown below, with actin as a loading control B) Proliferation of tumour cell lines is inhibited by CAT1 inhibition with L-NAME, measured by 3 H-thymidine incorporation after 72 hours C) Cell lines were cultured with RPMI+10%FBS (R10%) or arginine-free RPMI+10%FBS (R10%-arginine). Metabolic activity was measured by MTT after 72h. n=7 replicates D) Sorted GD2+ neuroblastoma cells from patients were treated with BCT-100 (600ng/mL). Analysis of cell death was performed by transmission electron microscopy (Representative micrographs of 2 out of 6 patients shown). Upper panel show untreated cells. Lower panels show post treatment with 600ng/mL BCT-100. Features consistent with organelle enlargement, cell membrane permeablisation, and cellular fragmentation with 600ng/mL BCT-100. Experiments performed on 3 separate occasions E) Sorted GD2+ cells from TH-MYCN murine neuroblastomas were cultured with BCT-100 (600ng/mL) for 72 hours. The percentage of viable cells relative to untreated controls was determined by flow cytometry, using propidium iodide staining. BCT-100 leads to a decrease in murine neuroblastoma cell viability ex vivo F) Plasma from control (saline) and BCT-100 treated TH-MYCN mice was collected at the start (PRE), 16 days after (MID), and at tumour end-point (END). The concentration of arginine was determined by ELISA. BCT-100 maintains a significant reduction in the plasma arginine concentration in vivo . n=6 G) TH-MYCN mice were treated with BCT-100 (60mg/kg) twice weekly intraperitoneally ( ip ) from the time of weaning at 3 weeks of age before overt tumour formations (Prophylaxis). Kaplan-Meier curves show that the development of tumours is significantly delayed, and that survival is increased in BCT-100 treated mice H) TH-MYCN mice were treated with BCT-100 (60mg/kg) twice weekly ip once 5 mm tumours were palpable (Treatment). Kaplan-Meier curves show a significant prolongation of survival in BCT-100 treated mice.

    Article Snippet: The effects of arginine deprivation were tested on cells cultured in arginine-free RPMI 1640 for SILAC (ThermoFisher Scientific) supplemented with 10% v/v arginine-free dialysed FBS (ThermoFisher Scientific).

    Techniques: shRNA, Knock-Out, Expressing, Western Blot, Inhibition, Cell Culture, Activity Assay, MTT Assay, Transmission Assay, Electron Microscopy, Flow Cytometry, Cytometry, Staining, Ex Vivo, Mouse Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, In Vivo