syringic aldehyde  (Millipore)


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    Structured Review

    Millipore syringic aldehyde
    HPLC chromatograms of (A) the authentic compounds, (B) the IRG active fraction F 7 and (C) the IRG active fraction F 8 . Peaks: 1, (+)-catechin; 2, caffeic acid; 3, ferulic acid; 4, p -coumaric acid; 5, <t>syringic</t> aldehyde; 6, myricetin; 7, propyl gallate; 8, quercetin; 9, kaempferol. Detection was performed at 280 nm.
    Syringic Aldehyde, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 8183 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/syringic aldehyde/product/Millipore
    Average 90 stars, based on 8183 article reviews
    Price from $9.99 to $1999.99
    syringic aldehyde - by Bioz Stars, 2020-08
    90/100 stars

    Images

    1) Product Images from "Antioxidant, anti-inflammatory and anti-septic potential of phenolic acids and flavonoid fractions isolated from Lolium multiflorum"

    Article Title: Antioxidant, anti-inflammatory and anti-septic potential of phenolic acids and flavonoid fractions isolated from Lolium multiflorum

    Journal: Pharmaceutical Biology

    doi: 10.1080/13880209.2016.1266673

    HPLC chromatograms of (A) the authentic compounds, (B) the IRG active fraction F 7 and (C) the IRG active fraction F 8 . Peaks: 1, (+)-catechin; 2, caffeic acid; 3, ferulic acid; 4, p -coumaric acid; 5, syringic aldehyde; 6, myricetin; 7, propyl gallate; 8, quercetin; 9, kaempferol. Detection was performed at 280 nm.
    Figure Legend Snippet: HPLC chromatograms of (A) the authentic compounds, (B) the IRG active fraction F 7 and (C) the IRG active fraction F 8 . Peaks: 1, (+)-catechin; 2, caffeic acid; 3, ferulic acid; 4, p -coumaric acid; 5, syringic aldehyde; 6, myricetin; 7, propyl gallate; 8, quercetin; 9, kaempferol. Detection was performed at 280 nm.

    Techniques Used: High Performance Liquid Chromatography

    2) Product Images from "Staufen1 links RNA stress granules and autophagy in a model of neurodegeneration"

    Article Title: Staufen1 links RNA stress granules and autophagy in a model of neurodegeneration

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06041-3

    Staufen1 protein but not mRNA steady-state levels are increased in neurodegenerative disease cells and tissues. Western blot analysis of SCA2- FBs ( a ) and LBCs ( b ) show increased STAU1 levels compared with normal controls. DDX6 levels are unchanged. HD and SCA3 patient (polyQ expanded) FBs were used as additional controls. Four normal and five SCA2 FBs, and two normal and three SCA2 LBCs were used. c , d Western blot analyses of ATXN2 Q127 ( c ) and BAC-Q72 ( d ) mouse cerebellar extracts (24 weeks of age) showing increased Stau1 levels compared with wild-type or BAC-Q22 controls ( n = 2–3 animals per group). e Western blot of FB extracts from an ALS patient with the TDP-43 G298S mutation show increased STAU1 levels. β-Actin was used as loading control and representative blots of three independent experiments are shown. f – h STAU1 RNA levels are unaltered in SCA2 and ALS cells and SCA2 mice. qRT-PCR analyses of STAU1 mRNA in SCA2 FBs and ALS FB with TDP-43 G298S mutation ( f ) or SCA2 LBCs ( g ). h qRT-PCR analyses of cerebellar RNAs from ATXN2 Q127 and BAC-Q72 mice compared to wild-type littermates (24 weeks of age; n = animals per group). Gene expression levels were normalized to Actb
    Figure Legend Snippet: Staufen1 protein but not mRNA steady-state levels are increased in neurodegenerative disease cells and tissues. Western blot analysis of SCA2- FBs ( a ) and LBCs ( b ) show increased STAU1 levels compared with normal controls. DDX6 levels are unchanged. HD and SCA3 patient (polyQ expanded) FBs were used as additional controls. Four normal and five SCA2 FBs, and two normal and three SCA2 LBCs were used. c , d Western blot analyses of ATXN2 Q127 ( c ) and BAC-Q72 ( d ) mouse cerebellar extracts (24 weeks of age) showing increased Stau1 levels compared with wild-type or BAC-Q22 controls ( n = 2–3 animals per group). e Western blot of FB extracts from an ALS patient with the TDP-43 G298S mutation show increased STAU1 levels. β-Actin was used as loading control and representative blots of three independent experiments are shown. f – h STAU1 RNA levels are unaltered in SCA2 and ALS cells and SCA2 mice. qRT-PCR analyses of STAU1 mRNA in SCA2 FBs and ALS FB with TDP-43 G298S mutation ( f ) or SCA2 LBCs ( g ). h qRT-PCR analyses of cerebellar RNAs from ATXN2 Q127 and BAC-Q72 mice compared to wild-type littermates (24 weeks of age; n = animals per group). Gene expression levels were normalized to Actb

    Techniques Used: Western Blot, BAC Assay, Mutagenesis, Mouse Assay, Quantitative RT-PCR, Expressing

    3) Product Images from "Fibronectin rescues estrogen receptor α from lysosomal degradation in breast cancer cells"

    Article Title: Fibronectin rescues estrogen receptor α from lysosomal degradation in breast cancer cells

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201703037

    Endosomes containing ERα are present in normal and tumor human breast tissues. (a) Top: Confocal images of a normal human breast tissue (reduction mammoplasty; sample N211) stained for ERα (HC-20 clone), β1-integrin, and DAPI. In the inset, arrows indicate the presence of ERα + endosomes. Similar results were obtained in the four different specimens analyzed. Bottom: Confocal images of a human breast tumor (Luminal A subtype adenocarcinoma; sample T171) stained for ERα (HC-20 clone), β1-integrin, and DAPI. Yellow arrows indicate ERα + endosomes. In the inset, arrows indicate the presence of ERα + endosomes. Similar results were obtained in the three different specimens analyzed. (b) Magnification from the inset shown in panel a (top). Diameters of ER + vesicles are shown on the right. (c) Table showing mean and SD of Pearson’s correlation index calculated for the overall colocalization between ERα and β1-integrin. Differences between groups were analyzed by two-tailed Student’s t test (per replicate: n normal = six fields; n tumor = seven fields). (d) OncoPrint from http://www.cbioportal.org ( Cerami et al., 2012 ; Gao et al., 2013 ) showing the alterations found in ERα ( ESR1 ) and β1-integrin ( ITGB1 ) genes in different patients obtained from the search in four different datasets: British Columbia, Nature 2014 ( Eirew et al., 2015 ); TCGA, Nature 2012 ( Cancer Genome Atlas Network, 2012 ); TCGA, Cell 2015 ( Ciriello et al., 2015 ); and Nature 2012 and Nature Communications 2016 ( Pereira et al., 2016 ). (e) Kaplan–Meier plot of the overall survival of patients with alterations in ESR1 or ITGB1 genes using thelargest and newest dataset available in http://www.cbioportal.org (Breast Cancer-METABRIC; Cerami et al., 2012 ; Gao et al., 2013 ). Significance level after the log-rank test is shown in the plot. *, P
    Figure Legend Snippet: Endosomes containing ERα are present in normal and tumor human breast tissues. (a) Top: Confocal images of a normal human breast tissue (reduction mammoplasty; sample N211) stained for ERα (HC-20 clone), β1-integrin, and DAPI. In the inset, arrows indicate the presence of ERα + endosomes. Similar results were obtained in the four different specimens analyzed. Bottom: Confocal images of a human breast tumor (Luminal A subtype adenocarcinoma; sample T171) stained for ERα (HC-20 clone), β1-integrin, and DAPI. Yellow arrows indicate ERα + endosomes. In the inset, arrows indicate the presence of ERα + endosomes. Similar results were obtained in the three different specimens analyzed. (b) Magnification from the inset shown in panel a (top). Diameters of ER + vesicles are shown on the right. (c) Table showing mean and SD of Pearson’s correlation index calculated for the overall colocalization between ERα and β1-integrin. Differences between groups were analyzed by two-tailed Student’s t test (per replicate: n normal = six fields; n tumor = seven fields). (d) OncoPrint from http://www.cbioportal.org ( Cerami et al., 2012 ; Gao et al., 2013 ) showing the alterations found in ERα ( ESR1 ) and β1-integrin ( ITGB1 ) genes in different patients obtained from the search in four different datasets: British Columbia, Nature 2014 ( Eirew et al., 2015 ); TCGA, Nature 2012 ( Cancer Genome Atlas Network, 2012 ); TCGA, Cell 2015 ( Ciriello et al., 2015 ); and Nature 2012 and Nature Communications 2016 ( Pereira et al., 2016 ). (e) Kaplan–Meier plot of the overall survival of patients with alterations in ESR1 or ITGB1 genes using thelargest and newest dataset available in http://www.cbioportal.org (Breast Cancer-METABRIC; Cerami et al., 2012 ; Gao et al., 2013 ). Significance level after the log-rank test is shown in the plot. *, P

    Techniques Used: Staining, Two Tailed Test

    Model for endocytic transport of ERα and β1-integrin regulated by FN in breast cancer cells. Estrogens induce rapid endocytosis of membrane ERα–β1-integrin complexes, generating EEA1 + vesicles. In the absence of FN, vesicles containing β1-integrin and ERα could either fuse to the nuclear membrane where ERα exerts its action or follow the lysosomal pathway, where ERα colocalizes with Rab7. After 60 min, ERα and β1-integrin are degraded in lysosomes and the signal ends. In the presence of FN, ERα and β1-integrin are localized in Rab11 + vesicles, suggesting that they might be recycled and therefore avoid the lysosomal pathway. ERα and β1-integrin levels are maintained over time and the cycle continues, keeping ERα transcriptionally active.
    Figure Legend Snippet: Model for endocytic transport of ERα and β1-integrin regulated by FN in breast cancer cells. Estrogens induce rapid endocytosis of membrane ERα–β1-integrin complexes, generating EEA1 + vesicles. In the absence of FN, vesicles containing β1-integrin and ERα could either fuse to the nuclear membrane where ERα exerts its action or follow the lysosomal pathway, where ERα colocalizes with Rab7. After 60 min, ERα and β1-integrin are degraded in lysosomes and the signal ends. In the presence of FN, ERα and β1-integrin are localized in Rab11 + vesicles, suggesting that they might be recycled and therefore avoid the lysosomal pathway. ERα and β1-integrin levels are maintained over time and the cycle continues, keeping ERα transcriptionally active.

    Techniques Used:

    ERα is degraded in lysosomes and rescued by FN. (a) Top: Western blot of T47D cells seeded on BSA or FN pretreated with BZ 8nM or its vehicle (saline) for 4 h and treated as indicated. Bottom: Densitometry. For each subcellular fraction, the mean ERα/β-actin density ratio is shown normalized to the mean control group. (b) Top: Western blot of a subcellular fractionation of T47D cells pretreated for 90 min with 25 nM BAF or its vehicle (DMSO) and then treated for 60 min with 10 −8 M E 2 or its vehicle (ethanol). Blotting antibodies are shown on the left. Bottom: Densitometry. For each experimental condition, the ERα/β-actin density ratio is shown, normalized to the mean control group. Each symbol represents a different experiment. Differences between groups were analyzed by one-tailed paired Student’s t test ( n = 3 replicates). (c) Confocal images of T47D cells expressing GFP-Rab7 seeded on BSA treated for 60 min with vehicle or E 2 , and stained for ERα. In the inset, arrows indicate points of colocalization. (d) Quantification of c. For each experimental condition, Pearson’s correlation index and Manders’ coefficients (M1 and M2) were calculated within the areas of colocalization using Fiji. Data are represented as mean ± SD. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: Pearson’s: n vehicle = 11 fields, n E2 = 12 fields; Manders’: n vehicle = 8 fields, n E2 = 9 fields). (e) Confocal images of T47D cells expressing GFP-Rab7 seeded on FN treated for 60 min with vehicle or E 2 , and stained for ERα. (f) Quantification of e. For each experimental condition, Pearson’s correlation index and Manders’ coefficients (M1 and M2) were calculated within the areas of colocalization using Fiji. Data are represented as mean ± SD. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: n vehicle = 9 fields, n E2 = 7 fields). Treatments: ethanol (vehicle) or 10 −8 M E 2 , 8 nM BZ. *, P
    Figure Legend Snippet: ERα is degraded in lysosomes and rescued by FN. (a) Top: Western blot of T47D cells seeded on BSA or FN pretreated with BZ 8nM or its vehicle (saline) for 4 h and treated as indicated. Bottom: Densitometry. For each subcellular fraction, the mean ERα/β-actin density ratio is shown normalized to the mean control group. (b) Top: Western blot of a subcellular fractionation of T47D cells pretreated for 90 min with 25 nM BAF or its vehicle (DMSO) and then treated for 60 min with 10 −8 M E 2 or its vehicle (ethanol). Blotting antibodies are shown on the left. Bottom: Densitometry. For each experimental condition, the ERα/β-actin density ratio is shown, normalized to the mean control group. Each symbol represents a different experiment. Differences between groups were analyzed by one-tailed paired Student’s t test ( n = 3 replicates). (c) Confocal images of T47D cells expressing GFP-Rab7 seeded on BSA treated for 60 min with vehicle or E 2 , and stained for ERα. In the inset, arrows indicate points of colocalization. (d) Quantification of c. For each experimental condition, Pearson’s correlation index and Manders’ coefficients (M1 and M2) were calculated within the areas of colocalization using Fiji. Data are represented as mean ± SD. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: Pearson’s: n vehicle = 11 fields, n E2 = 12 fields; Manders’: n vehicle = 8 fields, n E2 = 9 fields). (e) Confocal images of T47D cells expressing GFP-Rab7 seeded on FN treated for 60 min with vehicle or E 2 , and stained for ERα. (f) Quantification of e. For each experimental condition, Pearson’s correlation index and Manders’ coefficients (M1 and M2) were calculated within the areas of colocalization using Fiji. Data are represented as mean ± SD. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: n vehicle = 9 fields, n E2 = 7 fields). Treatments: ethanol (vehicle) or 10 −8 M E 2 , 8 nM BZ. *, P

    Techniques Used: Western Blot, Fractionation, One-tailed Test, Expressing, Staining

    Effect of E 2 treatment on the conditional distribution of ERα versus β1-integrin. (a) Images from STORM of filopodia of MCF7 cells treated as indicated for 15 min and stained for ERα or β1-integrin. Insets in the top left corners show the same images taken with widefield microscopy. Inside the zoomed areas, arrows show regions of superposition of the two markers (yellow pixels). Blue squares outline representative areas of 500 × 500 pixels used for subsequent analyses. In the treated cell, arrow inside the blue square shows a region of dense clustering between ERα and β1-integrin. (b) Tables showing the IF calculated as described previously ( Bermudez-Hernandez et al., 2017 ) for 10 representative frames of filopodia of MCF7 cells under control (top) or treated (bottom) conditions. R-G, red–green correlation; G-R, green–red correlation. Red, β1-integrin; green, ERα. (c) IF calculated for one treated cell (frame 7) and for two sub-ROIs of this frame, showing how this index changes between areas of different β1-integrin/ERα densities. Bars 2 µm. (d) Histogram for normalized frequencies of MD between β1-integrin and ERα in filopodia of MCF7 cells among all the analyzed frames for each condition. For each domain detected, centroids were identified, and MDs were calculated from each β1-integrin to its nearest ERα domain throughout each 500 × 500–pixel frame. Frequencies were normalized to the highest value. The graph shows a slight shift toward smaller MDs for treated cells. (e) Mean density covariance between ERα and β1-integrin domains. Each frame was divided into square ROI of different sizes (window lengths ranging from 130 nm [10 pixels] to 2,000 nm [150 pixels] in side length). For each ROI size, the densities of ERα and β1-integrin were obtained, and the correlation coefficient (C) between these densities was calculated for all datasets. The mean of C among all the control (black full circles) or treated (pink empty squares) cells was plotted as a function of the window side length. Light-blue crosses show the z score (defined by the difference between the mean of the control group (for each window) and the mean of the treated group, and further divided by the square root of the sum of the SD of each group normalized by n ). Thus, because the z score expresses, in units of SD, the distance between the two distributions, one may safely conclude that here there is no significant difference in density covariance between control and treated cells. (f) Two examples of a Voronoi partition for control (left) or treated (right) cells using the centroids of the ERα domains to compute the transformation. Colors indicated in the color bar on the right represent the size of each Voronoi shell (in square nanometers). Black small dots indicate the location of the centroids for β1-integrin domains. Empty big circles indicate the centroids of ERα domains; red circles denote those ERα that have β1-integrins closer than 160 nm, and blue circles indicate those ERα that have β1-integrins further than 160 nm away on the mean. The examples in these panels reveal a clear difference in ERα–β1-integrin bunching between control and treated conditions. (g) Histogram of frequencies for the ADs from each ERα centroid to the β1-integrins inside its Voronoi shell among all the analyzed data for each condition (note the semilog axis for presentation purposes). The graph shows a shift toward smaller distances for treated cells. Green arrows indicate as an example a region of the plot where the difference between treated and control fields is almost double. The inset shows the histogram for frequencies of the areas of the Voronoi regions among all the analyzed fields for each condition, demonstrating that treated cells present also relatively smaller Voronoi shells. (h) Graph of the bunching index for each cell, which is the ratio between the number of shells (normalized) that contains mean ERα–β1-integrin distances smaller than a threshold value of 160 nm. We named it bunching index as it quantifies the proportion of ERα–β1-integrin complexes among all the domains localized. Control image 1 and E 2 -treated image 4 are the ones represented in f. Inset shows the z score, which was calculated as the difference between each bunching index for the treated cell and the mean of the bunching indexes for the control group divided by the SD of the control group. Z score results demonstrate for six cells a significant difference (abs[z score] > 1) in the bunching index between control and treated cells. In all plots, control cells are represented with black full circles and treated cells with pink empty squares. Treatments: ethanol (vehicle) or 10 −8 M E 2 .
    Figure Legend Snippet: Effect of E 2 treatment on the conditional distribution of ERα versus β1-integrin. (a) Images from STORM of filopodia of MCF7 cells treated as indicated for 15 min and stained for ERα or β1-integrin. Insets in the top left corners show the same images taken with widefield microscopy. Inside the zoomed areas, arrows show regions of superposition of the two markers (yellow pixels). Blue squares outline representative areas of 500 × 500 pixels used for subsequent analyses. In the treated cell, arrow inside the blue square shows a region of dense clustering between ERα and β1-integrin. (b) Tables showing the IF calculated as described previously ( Bermudez-Hernandez et al., 2017 ) for 10 representative frames of filopodia of MCF7 cells under control (top) or treated (bottom) conditions. R-G, red–green correlation; G-R, green–red correlation. Red, β1-integrin; green, ERα. (c) IF calculated for one treated cell (frame 7) and for two sub-ROIs of this frame, showing how this index changes between areas of different β1-integrin/ERα densities. Bars 2 µm. (d) Histogram for normalized frequencies of MD between β1-integrin and ERα in filopodia of MCF7 cells among all the analyzed frames for each condition. For each domain detected, centroids were identified, and MDs were calculated from each β1-integrin to its nearest ERα domain throughout each 500 × 500–pixel frame. Frequencies were normalized to the highest value. The graph shows a slight shift toward smaller MDs for treated cells. (e) Mean density covariance between ERα and β1-integrin domains. Each frame was divided into square ROI of different sizes (window lengths ranging from 130 nm [10 pixels] to 2,000 nm [150 pixels] in side length). For each ROI size, the densities of ERα and β1-integrin were obtained, and the correlation coefficient (C) between these densities was calculated for all datasets. The mean of C among all the control (black full circles) or treated (pink empty squares) cells was plotted as a function of the window side length. Light-blue crosses show the z score (defined by the difference between the mean of the control group (for each window) and the mean of the treated group, and further divided by the square root of the sum of the SD of each group normalized by n ). Thus, because the z score expresses, in units of SD, the distance between the two distributions, one may safely conclude that here there is no significant difference in density covariance between control and treated cells. (f) Two examples of a Voronoi partition for control (left) or treated (right) cells using the centroids of the ERα domains to compute the transformation. Colors indicated in the color bar on the right represent the size of each Voronoi shell (in square nanometers). Black small dots indicate the location of the centroids for β1-integrin domains. Empty big circles indicate the centroids of ERα domains; red circles denote those ERα that have β1-integrins closer than 160 nm, and blue circles indicate those ERα that have β1-integrins further than 160 nm away on the mean. The examples in these panels reveal a clear difference in ERα–β1-integrin bunching between control and treated conditions. (g) Histogram of frequencies for the ADs from each ERα centroid to the β1-integrins inside its Voronoi shell among all the analyzed data for each condition (note the semilog axis for presentation purposes). The graph shows a shift toward smaller distances for treated cells. Green arrows indicate as an example a region of the plot where the difference between treated and control fields is almost double. The inset shows the histogram for frequencies of the areas of the Voronoi regions among all the analyzed fields for each condition, demonstrating that treated cells present also relatively smaller Voronoi shells. (h) Graph of the bunching index for each cell, which is the ratio between the number of shells (normalized) that contains mean ERα–β1-integrin distances smaller than a threshold value of 160 nm. We named it bunching index as it quantifies the proportion of ERα–β1-integrin complexes among all the domains localized. Control image 1 and E 2 -treated image 4 are the ones represented in f. Inset shows the z score, which was calculated as the difference between each bunching index for the treated cell and the mean of the bunching indexes for the control group divided by the SD of the control group. Z score results demonstrate for six cells a significant difference (abs[z score] > 1) in the bunching index between control and treated cells. In all plots, control cells are represented with black full circles and treated cells with pink empty squares. Treatments: ethanol (vehicle) or 10 −8 M E 2 .

    Techniques Used: Staining, Microscopy, Transformation Assay

    ERα + is localized in Rab11 + vesicles in the presence of FN. (a) Images of confocal microscopy of MCF7 cells seeded on BSA or FN treated for 15 min as indicated and stained for ERα. Panels show the cytoplasmic/plasma membrane (basal) plane (z2). Nuclear/cytoplasmic (apical) plane (z1) is shown in Fig. S3 b. White arrows indicate ERα + vesicles determined as punctae of 10–15 pixels in diameter (∼200 nm). (b) Quantification of a. Apical (nuclear/cytoplasmic) versus basal (cytoplasmic/plasma membrane) distribution of ERα + vesicles. Structures of 10–15 pixels in diameter were quantified using Fiji. Mean number of endosomes in each fraction and for each condition is shown. (c) Heatmaps of T47D cells seeded on BSA or FN treated for 60 min as indicated and stained for ERα. Cells are outlined in white. Dashed line outlines the nucleus. Intensity bars are shown on the right (red, maximum pixel intensity; blue, minimum pixel intensity). Original images are shown in the insets. (d) Images of confocal microscopy of MCF7 cells seeded on BSA or FN treated for 60 min with E 2 and stained for ERα and Rab11. Arrows indicate areas of colocalization within filopodia. Pearson’s colocalization maps are shown in the insets. (e) Quantification of d. For each experimental condition, Pearson’s correlation index was calculated within filopodia protrusions using Fiji. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: n BSA = 16 fields, n FN = 15 fields). (f) Quantification of d. For each experimental condition, overall Rab11 intensity was calculated using Fiji. Data are represented as mean ± SD. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: n BSA = 5 fields, n FN = 4 fields). (g) Images of confocal microscopy of MCF7 cells seeded on BSA or FN treated for 15 min with E 2 in the presence of dextran-CF543 and stained for Rab11. Arrows indicate areas of colocalization between the two fluorophores. Higher magnification is shown in the inset. ***, P
    Figure Legend Snippet: ERα + is localized in Rab11 + vesicles in the presence of FN. (a) Images of confocal microscopy of MCF7 cells seeded on BSA or FN treated for 15 min as indicated and stained for ERα. Panels show the cytoplasmic/plasma membrane (basal) plane (z2). Nuclear/cytoplasmic (apical) plane (z1) is shown in Fig. S3 b. White arrows indicate ERα + vesicles determined as punctae of 10–15 pixels in diameter (∼200 nm). (b) Quantification of a. Apical (nuclear/cytoplasmic) versus basal (cytoplasmic/plasma membrane) distribution of ERα + vesicles. Structures of 10–15 pixels in diameter were quantified using Fiji. Mean number of endosomes in each fraction and for each condition is shown. (c) Heatmaps of T47D cells seeded on BSA or FN treated for 60 min as indicated and stained for ERα. Cells are outlined in white. Dashed line outlines the nucleus. Intensity bars are shown on the right (red, maximum pixel intensity; blue, minimum pixel intensity). Original images are shown in the insets. (d) Images of confocal microscopy of MCF7 cells seeded on BSA or FN treated for 60 min with E 2 and stained for ERα and Rab11. Arrows indicate areas of colocalization within filopodia. Pearson’s colocalization maps are shown in the insets. (e) Quantification of d. For each experimental condition, Pearson’s correlation index was calculated within filopodia protrusions using Fiji. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: n BSA = 16 fields, n FN = 15 fields). (f) Quantification of d. For each experimental condition, overall Rab11 intensity was calculated using Fiji. Data are represented as mean ± SD. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: n BSA = 5 fields, n FN = 4 fields). (g) Images of confocal microscopy of MCF7 cells seeded on BSA or FN treated for 15 min with E 2 in the presence of dextran-CF543 and stained for Rab11. Arrows indicate areas of colocalization between the two fluorophores. Higher magnification is shown in the inset. ***, P

    Techniques Used: Confocal Microscopy, Staining, One-tailed Test

    E 2 stimulates endocytosis of vesicles containing ERα. (a) Confocal images of MCF7 cells seeded on BSA (left) or FN (right) treated for 15 min as indicated and stained for ERα. Arrows indicate ERα + endosomes. (b) Quantification of a. For each experimental condition, structures of ∼200-nm diameter (10–15 pixels) were quantified using Fiji. Shown is the number of ERα + puncta per cell. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: BSA: n vehicle = 81 cells, n E2 = 87 cells; FN: n vehicle = 50 cells, n E2 = 64 cells). (c) Confocal images of MCF7 cells seeded on BSA (left) or FN (right) treated for 15 min as indicated and stained for EEA1. Cells are delineated in white. Arrows indicate early endosomes. (d) Quantification of c. For each experimental condition, shown is EEA1 intensity (mean gray value) per cell calculated using Fiji relative to the highest intensity recorded. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: BSA: n EtOH = 68 cells; n E2 = 39 cells; FN: n EtOH = 94 cells; n E2 = 108 cells). (e) Confocal images of MCF7 cells seeded on FN treated for 15 min as indicated and stained for EEA1. Merges between differential interference contrast (DIC) microscopy and the green channel are shown. Arrows indicate early endosomes present either in the nuclear membrane or inside the nucleus. (f) Quantification of e. For each experimental condition, structures of 10–15 pixels in diameter were quantified using Fiji. Shown is the number of nuclear early endosomes per cell. It was calculated as the total number of EEA1 + vesicles in the nuclear membrane or inside the nucleus, per cell. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: n EtOH = 59 cells; n E2 = 54 cells). (g) Top: Outline of the protocol followed and Western blot of a subcellular fractionation of MCF7 cells treated as indicated. Blotting antibodies are shown on the left. Bottom: Densitometry. For each subcellular fraction, shown is the ERα/β-actin density ratio normalized to the mean control group. Each symbol represents a different experiment. Differences between groups were analyzed by one-tailed paired Student’s t test ( n = 3 replicates). (h) Top: Western blot of a subcellular fractionation of MCF7 cells treated for 15 min as indicated. Blotting antibodies are shown on the left. Bottom: Densitometry. For each subcellular fraction, shown is the ERα/β-actin density ratio normalized to the control group. Differences between groups were analyzed by one-tailed paired Student’s t test ( n = 3 replicates). (i) Luciferase assay in MCF7 cells transiently transfected with pTK-ERE-Luc and pTK-Renilla and treated for 14 h as indicated. Differences between groups were analyzed by two-way ANOVA followed by Bonferroni contrasts adjusted for multiple comparisons ( n = 3 replicates). Data are represented as mean ± SD. *, P
    Figure Legend Snippet: E 2 stimulates endocytosis of vesicles containing ERα. (a) Confocal images of MCF7 cells seeded on BSA (left) or FN (right) treated for 15 min as indicated and stained for ERα. Arrows indicate ERα + endosomes. (b) Quantification of a. For each experimental condition, structures of ∼200-nm diameter (10–15 pixels) were quantified using Fiji. Shown is the number of ERα + puncta per cell. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: BSA: n vehicle = 81 cells, n E2 = 87 cells; FN: n vehicle = 50 cells, n E2 = 64 cells). (c) Confocal images of MCF7 cells seeded on BSA (left) or FN (right) treated for 15 min as indicated and stained for EEA1. Cells are delineated in white. Arrows indicate early endosomes. (d) Quantification of c. For each experimental condition, shown is EEA1 intensity (mean gray value) per cell calculated using Fiji relative to the highest intensity recorded. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: BSA: n EtOH = 68 cells; n E2 = 39 cells; FN: n EtOH = 94 cells; n E2 = 108 cells). (e) Confocal images of MCF7 cells seeded on FN treated for 15 min as indicated and stained for EEA1. Merges between differential interference contrast (DIC) microscopy and the green channel are shown. Arrows indicate early endosomes present either in the nuclear membrane or inside the nucleus. (f) Quantification of e. For each experimental condition, structures of 10–15 pixels in diameter were quantified using Fiji. Shown is the number of nuclear early endosomes per cell. It was calculated as the total number of EEA1 + vesicles in the nuclear membrane or inside the nucleus, per cell. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: n EtOH = 59 cells; n E2 = 54 cells). (g) Top: Outline of the protocol followed and Western blot of a subcellular fractionation of MCF7 cells treated as indicated. Blotting antibodies are shown on the left. Bottom: Densitometry. For each subcellular fraction, shown is the ERα/β-actin density ratio normalized to the mean control group. Each symbol represents a different experiment. Differences between groups were analyzed by one-tailed paired Student’s t test ( n = 3 replicates). (h) Top: Western blot of a subcellular fractionation of MCF7 cells treated for 15 min as indicated. Blotting antibodies are shown on the left. Bottom: Densitometry. For each subcellular fraction, shown is the ERα/β-actin density ratio normalized to the control group. Differences between groups were analyzed by one-tailed paired Student’s t test ( n = 3 replicates). (i) Luciferase assay in MCF7 cells transiently transfected with pTK-ERE-Luc and pTK-Renilla and treated for 14 h as indicated. Differences between groups were analyzed by two-way ANOVA followed by Bonferroni contrasts adjusted for multiple comparisons ( n = 3 replicates). Data are represented as mean ± SD. *, P

    Techniques Used: Staining, One-tailed Test, Microscopy, Western Blot, Fractionation, Luciferase, Transfection

    ERα is endocytosed through a caveolin 1–dependent pathway. (a) Top: Confocal images of MCF7 cells treated for 15 min as indicated and stained for caveolin 1. Bottom: Merge between caveolin 1 signal and differential interference contrast (DIC) images. Arrows indicate internal or peripheral localization of caveolin 1. (b) Top: Confocal images of MCF7 cells, treated for 15 min as indicated, and stained for clathrin. Bottom: Merge between caveolin 1 signal and DIC images. Arrows indicate internal or peripheral localization of clathrin. (c) Confocal images of MCF7 cells treated for 15 min as indicated and stained for caveolin 1 or EEA1. Arrows indicate regions of colocalization between the two markers. (d) Western blots of MCF7 cells transfected with siRNAs against caveolin 1, clathrin HC, or scrambled for 48 h. Blotting antibodies are shown on the right. Fold change relative to scrambled siRNA is shown on the bottom. (e) Luciferase assay in MCF7 cells transiently transfected with pTK-ERE-Luc and pTK-Renilla and the respective siRNAs and treated for 14 h as indicated. Differences between groups were analyzed by two-way ANOVA followed by Bonferroni contrasts adjusted for multiple comparisons ( n = 3 replicates). Data are represented as mean ± SD. **, P
    Figure Legend Snippet: ERα is endocytosed through a caveolin 1–dependent pathway. (a) Top: Confocal images of MCF7 cells treated for 15 min as indicated and stained for caveolin 1. Bottom: Merge between caveolin 1 signal and differential interference contrast (DIC) images. Arrows indicate internal or peripheral localization of caveolin 1. (b) Top: Confocal images of MCF7 cells, treated for 15 min as indicated, and stained for clathrin. Bottom: Merge between caveolin 1 signal and DIC images. Arrows indicate internal or peripheral localization of clathrin. (c) Confocal images of MCF7 cells treated for 15 min as indicated and stained for caveolin 1 or EEA1. Arrows indicate regions of colocalization between the two markers. (d) Western blots of MCF7 cells transfected with siRNAs against caveolin 1, clathrin HC, or scrambled for 48 h. Blotting antibodies are shown on the right. Fold change relative to scrambled siRNA is shown on the bottom. (e) Luciferase assay in MCF7 cells transiently transfected with pTK-ERE-Luc and pTK-Renilla and the respective siRNAs and treated for 14 h as indicated. Differences between groups were analyzed by two-way ANOVA followed by Bonferroni contrasts adjusted for multiple comparisons ( n = 3 replicates). Data are represented as mean ± SD. **, P

    Techniques Used: Staining, Western Blot, Transfection, Luciferase

    FN stimulates ERα’s transcriptional activity. (a) Luciferase assay in MCF7 cells transiently transfected with pTK-ERE-Luc and pTK-Renilla, seeded on BSA or FN and treated for 14 h as indicated. Data are represented as mean ± SD. Differences between groups were analyzed by two-way ANOVA followed by Bonferroni contrasts adjusted for multiple comparisons ( n = 3 replicates). (b and c) Top: Western blot of a subcellular fractionation of MCF7 cells seeded on BSA and treated for 15 min (b) or 60 min (c) as indicated. Blotting antibodies are shown on the left. Bottom: densitometry. For each subcellular fraction, shown is the ERα/β-actin density ratio normalized to the mean control group. Each symbol represents a different experiment. Differences between groups were analyzed by a one-tailed paired Student’s t test ( n = 3 replicates). (d and e) Top: Western blot of a subcellular fractionation of MCF7 cells seeded on FN and treated for 15 (d) or 60 min (e) as indicated. Blotting antibodies are shown on the left. Bottom: Densitometry. For each subcellular fraction, shown is the ERα/β-actin density ratio normalized to the mean control group. Each symbol represents a different experiment. (f) Western blot of the cytosolic + membrane fraction of MCF7 cells seeded on BSA or FN and treated with E 2 for the indicated times. Below the blots, the ERα/β-actin density ratio is shown, normalized to the control group. (g) Western blot of the nuclear fraction of MCF7 cells seeded on BSA or FN and treated with E 2 for the indicated times. Below the blots, the ERα/PCNA density ratio is shown, normalized to the control group. Differences between groups were analyzed by one-tailed paired Student’s t test ( n = 3 replicates). *, P
    Figure Legend Snippet: FN stimulates ERα’s transcriptional activity. (a) Luciferase assay in MCF7 cells transiently transfected with pTK-ERE-Luc and pTK-Renilla, seeded on BSA or FN and treated for 14 h as indicated. Data are represented as mean ± SD. Differences between groups were analyzed by two-way ANOVA followed by Bonferroni contrasts adjusted for multiple comparisons ( n = 3 replicates). (b and c) Top: Western blot of a subcellular fractionation of MCF7 cells seeded on BSA and treated for 15 min (b) or 60 min (c) as indicated. Blotting antibodies are shown on the left. Bottom: densitometry. For each subcellular fraction, shown is the ERα/β-actin density ratio normalized to the mean control group. Each symbol represents a different experiment. Differences between groups were analyzed by a one-tailed paired Student’s t test ( n = 3 replicates). (d and e) Top: Western blot of a subcellular fractionation of MCF7 cells seeded on FN and treated for 15 (d) or 60 min (e) as indicated. Blotting antibodies are shown on the left. Bottom: Densitometry. For each subcellular fraction, shown is the ERα/β-actin density ratio normalized to the mean control group. Each symbol represents a different experiment. (f) Western blot of the cytosolic + membrane fraction of MCF7 cells seeded on BSA or FN and treated with E 2 for the indicated times. Below the blots, the ERα/β-actin density ratio is shown, normalized to the control group. (g) Western blot of the nuclear fraction of MCF7 cells seeded on BSA or FN and treated with E 2 for the indicated times. Below the blots, the ERα/PCNA density ratio is shown, normalized to the control group. Differences between groups were analyzed by one-tailed paired Student’s t test ( n = 3 replicates). *, P

    Techniques Used: Activity Assay, Luciferase, Transfection, Western Blot, Fractionation, One-tailed Test

    ERα is spatially associated with β1-integrin and they are endocytosed together. (a) Widefield (top) and TIRFM (bottom) images of a coimmunofluorescence in MCF7 cells, using antibodies against β1-integrin (live-stained) and ERα. In the inset, white arrowheads indicate points of colocalization. Pearson’s correlation maps corresponding with the white box shown on the right. White arrowheads indicate points of positive Pearson’s correlation. (b) Quantification of a. Top left: Polar transformation of TIRFM images was performed using Fiji to align areas of the cell periphery where colocalization is found. For each experimental condition, Pearson’s correlation index and Manders’ coefficients (M1 and M2) were calculated within the areas of colocalization (ROI) and compared with random areas without colocalization (Null), using Fiji. For Pearson’s correlation, datasets are plotted and mean ± SD are shown on the graph. For Manders’ coefficients, the table shows mean and SD for each dataset. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: Pearson’s: n null = 9 fields, n ROI = 9 fields; Manders’: n null = 14 fields, n ROI = 9 fields). (c) Western blot of a coimmunoprecipitation in MCF7 cells, using antibodies against β1-integrin or ERα. Blotting antibodies are shown on the right. Input, whole lysate. IP, immunoprecipitated fraction. (d) ClustalW alignment of the eight β-integrins present in humans. The sequence of SRC1 is shown on top. NR-box motif is indicated in red. On the sequences of β1-integrin and β3-integrin, underlined in black is the region corresponding with their transmembrane domain, and in green is their cytoplasmic domain. The topology was predicted using the algorithm TMpred from the website ExPASy and the algorithm from the website TOPCONS. (e) Cartoon showing β1-integrin structure and putative interaction site with ERα. Black box indicates the localization of NR-box motif (LXXLL) within β1-integrin transmembrane domain. Red dot shows ERα palmitoylation site, and the arrow indicates where its helix 12 would be localized within the AF-2 domain. (f and g) Top: Western blot of total lysates of MCF7 cells, seeded on BSA (f) or FN (g) and treated for 60 min as indicated. Blotting antibodies are shown on the left. Bottom: Densitometry. For each experimental condition, shown is the β1-integrin/β-actin density ratio normalized to the mean control group. Each symbol represents a different experiment. Differences between groups were analyzed by one-tailed paired Student’s t test ( n = 3 replicates). (h) Confocal images of MCF7 cells treated for 15 min as indicated and stained for β1-integrin (live stained) and ERα. Arrows indicate points of colocalization. Corresponding Pearson’s correlation maps are shown on the right, respectively. White arrows indicate points of positive Pearson’s correlation. (i) Quantification of h. For each experimental condition, Pearson’s correlation index and Manders’ coefficients (M1 and M2) were calculated within the areas of colocalization (ROI) and compared with random areas without colocalization (Null) using Fiji. For Pearson’s correlation, datasets are plotted and mean ± SD are shown on the graph. For Manders’ coefficients, the table shows mean and SD for each dataset. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: Pearson’s: n null = 14 fields, n ROI = 15 fields; Manders’: n null = 10 fields, n ROI = 11 fields). (j) Confocal images of MCF7 cells seeded on BSA or FN treated with E 2 for 15 min and stained for β1-integrin and Rab11. Full images are shown in the insets. (k) Quantification of j. For each experimental condition, Pearson’s correlation index was calculated within the areas of colocalization using Fiji. Data are represented as mean ± SD. Differences between groups were analyzed by a one-tailed Student’s t test (per replicate: n BSA = 4 fields, n FN = 4 fields). *, P
    Figure Legend Snippet: ERα is spatially associated with β1-integrin and they are endocytosed together. (a) Widefield (top) and TIRFM (bottom) images of a coimmunofluorescence in MCF7 cells, using antibodies against β1-integrin (live-stained) and ERα. In the inset, white arrowheads indicate points of colocalization. Pearson’s correlation maps corresponding with the white box shown on the right. White arrowheads indicate points of positive Pearson’s correlation. (b) Quantification of a. Top left: Polar transformation of TIRFM images was performed using Fiji to align areas of the cell periphery where colocalization is found. For each experimental condition, Pearson’s correlation index and Manders’ coefficients (M1 and M2) were calculated within the areas of colocalization (ROI) and compared with random areas without colocalization (Null), using Fiji. For Pearson’s correlation, datasets are plotted and mean ± SD are shown on the graph. For Manders’ coefficients, the table shows mean and SD for each dataset. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: Pearson’s: n null = 9 fields, n ROI = 9 fields; Manders’: n null = 14 fields, n ROI = 9 fields). (c) Western blot of a coimmunoprecipitation in MCF7 cells, using antibodies against β1-integrin or ERα. Blotting antibodies are shown on the right. Input, whole lysate. IP, immunoprecipitated fraction. (d) ClustalW alignment of the eight β-integrins present in humans. The sequence of SRC1 is shown on top. NR-box motif is indicated in red. On the sequences of β1-integrin and β3-integrin, underlined in black is the region corresponding with their transmembrane domain, and in green is their cytoplasmic domain. The topology was predicted using the algorithm TMpred from the website ExPASy and the algorithm from the website TOPCONS. (e) Cartoon showing β1-integrin structure and putative interaction site with ERα. Black box indicates the localization of NR-box motif (LXXLL) within β1-integrin transmembrane domain. Red dot shows ERα palmitoylation site, and the arrow indicates where its helix 12 would be localized within the AF-2 domain. (f and g) Top: Western blot of total lysates of MCF7 cells, seeded on BSA (f) or FN (g) and treated for 60 min as indicated. Blotting antibodies are shown on the left. Bottom: Densitometry. For each experimental condition, shown is the β1-integrin/β-actin density ratio normalized to the mean control group. Each symbol represents a different experiment. Differences between groups were analyzed by one-tailed paired Student’s t test ( n = 3 replicates). (h) Confocal images of MCF7 cells treated for 15 min as indicated and stained for β1-integrin (live stained) and ERα. Arrows indicate points of colocalization. Corresponding Pearson’s correlation maps are shown on the right, respectively. White arrows indicate points of positive Pearson’s correlation. (i) Quantification of h. For each experimental condition, Pearson’s correlation index and Manders’ coefficients (M1 and M2) were calculated within the areas of colocalization (ROI) and compared with random areas without colocalization (Null) using Fiji. For Pearson’s correlation, datasets are plotted and mean ± SD are shown on the graph. For Manders’ coefficients, the table shows mean and SD for each dataset. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: Pearson’s: n null = 14 fields, n ROI = 15 fields; Manders’: n null = 10 fields, n ROI = 11 fields). (j) Confocal images of MCF7 cells seeded on BSA or FN treated with E 2 for 15 min and stained for β1-integrin and Rab11. Full images are shown in the insets. (k) Quantification of j. For each experimental condition, Pearson’s correlation index was calculated within the areas of colocalization using Fiji. Data are represented as mean ± SD. Differences between groups were analyzed by a one-tailed Student’s t test (per replicate: n BSA = 4 fields, n FN = 4 fields). *, P

    Techniques Used: Staining, Transformation Assay, One-tailed Test, Western Blot, Immunoprecipitation, Sequencing

    4) Product Images from "Reduced synaptic function of Kainate receptors in the insular cortex of Fmr1 Knock-out mice"

    Article Title: Reduced synaptic function of Kainate receptors in the insular cortex of Fmr1 Knock-out mice

    Journal: Molecular Brain

    doi: 10.1186/s13041-018-0396-1

    The expression of KARs in the cultured cortical neurons from Fmr1 WT and Fmr1 KO mice. a , b , The abundance of GluK1 or GluK2/3 in the homogenate of the cultured cortical neurons from Fmr1 WT mice and Fmr1 KO mice showed no change. c , d , Surface expression levels of GluK2/3 in insular cortex neurons obtained from Fmr1 WT and Fmr1 KO mice were detected by western blot analysis. The surface expression levels of GluK2/3 were not altered between Fmr1 WT and Fmr1 KO mice (n = 3 independent experiments). Actin was used as negative control for surface biotinylation
    Figure Legend Snippet: The expression of KARs in the cultured cortical neurons from Fmr1 WT and Fmr1 KO mice. a , b , The abundance of GluK1 or GluK2/3 in the homogenate of the cultured cortical neurons from Fmr1 WT mice and Fmr1 KO mice showed no change. c , d , Surface expression levels of GluK2/3 in insular cortex neurons obtained from Fmr1 WT and Fmr1 KO mice were detected by western blot analysis. The surface expression levels of GluK2/3 were not altered between Fmr1 WT and Fmr1 KO mice (n = 3 independent experiments). Actin was used as negative control for surface biotinylation

    Techniques Used: Expressing, Cell Culture, Mouse Assay, Western Blot, Negative Control

    Distribution of KARs in the insular cortex from Fmr1 WT mice. Presynaptic marker synaptophysin, postsynaptic marker PSD95, NMDAR subunits GluN2B and GluN2A, KAR subunits GluK1 and GluK2/3 or synaptic protein Rab5 were analyzed by Western blot in the homogenates (H1, 10 μg), postnuclear supernatant (S1, 5 μg), nuclei and large debris pellet (P1, 10 μg), cytosomes (S2, 5 μg), crude synaptosomal membrane (P2, 5 μg), non-PSD (5 μg) or PSD (10 μg) fractions of the insular cortex in Fmr1 WT mice. This experiment was repeated three times
    Figure Legend Snippet: Distribution of KARs in the insular cortex from Fmr1 WT mice. Presynaptic marker synaptophysin, postsynaptic marker PSD95, NMDAR subunits GluN2B and GluN2A, KAR subunits GluK1 and GluK2/3 or synaptic protein Rab5 were analyzed by Western blot in the homogenates (H1, 10 μg), postnuclear supernatant (S1, 5 μg), nuclei and large debris pellet (P1, 10 μg), cytosomes (S2, 5 μg), crude synaptosomal membrane (P2, 5 μg), non-PSD (5 μg) or PSD (10 μg) fractions of the insular cortex in Fmr1 WT mice. This experiment was repeated three times

    Techniques Used: Mouse Assay, Marker, Western Blot

    The abundance of KARs in the synaptosome is decreased in Fmr1 KO mice. a , b , Total expression levels of GluK1 and GluK2/3 in the homogenates fraction of the insular cortex conducted from Fmr1 WT and Fmr1 KO mice were detected by Western blot. The expression levels of GluK1 and GluK2/3 in the homogenates were not altered between Fmr1 WT and Fmr1 KO mice ( n = 3 mice for each group). c , d , Expression levels of GluK1 and GluK2/3 in the synaptosome of the insular cortex obtained from Fmr1 WT and Fmr1 KO mice were detected by western blot anaylsis. The expression levels of GluK1 and GluK2/3 in the homogenates was significantly reduced in Fmr1 KO mice compare to Fmr1 WT mice ( n = 5 mice for each group). * P
    Figure Legend Snippet: The abundance of KARs in the synaptosome is decreased in Fmr1 KO mice. a , b , Total expression levels of GluK1 and GluK2/3 in the homogenates fraction of the insular cortex conducted from Fmr1 WT and Fmr1 KO mice were detected by Western blot. The expression levels of GluK1 and GluK2/3 in the homogenates were not altered between Fmr1 WT and Fmr1 KO mice ( n = 3 mice for each group). c , d , Expression levels of GluK1 and GluK2/3 in the synaptosome of the insular cortex obtained from Fmr1 WT and Fmr1 KO mice were detected by western blot anaylsis. The expression levels of GluK1 and GluK2/3 in the homogenates was significantly reduced in Fmr1 KO mice compare to Fmr1 WT mice ( n = 5 mice for each group). * P

    Techniques Used: Mouse Assay, Expressing, Western Blot

    5) Product Images from "The effects and mechanism of peiminine-induced apoptosis in human hepatocellular carcinoma HepG2 cells"

    Article Title: The effects and mechanism of peiminine-induced apoptosis in human hepatocellular carcinoma HepG2 cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201864

    Expression of peiminine-induced proteins related to proliferation in HCC cells. (A) Expressions of p53, Bcl-2, Bax, PARP, and cleaved PARP in HepG2 cells treated with peiminine were determined by Western blot analysis. (B) Expressions of procaspase-3, -8, and -9, caspase-3, -8, and -9 in HepG2 cells treated with peiminine were determined by Western blot analysis. Data are presented as the mean±SD from three independent experiments.* p
    Figure Legend Snippet: Expression of peiminine-induced proteins related to proliferation in HCC cells. (A) Expressions of p53, Bcl-2, Bax, PARP, and cleaved PARP in HepG2 cells treated with peiminine were determined by Western blot analysis. (B) Expressions of procaspase-3, -8, and -9, caspase-3, -8, and -9 in HepG2 cells treated with peiminine were determined by Western blot analysis. Data are presented as the mean±SD from three independent experiments.* p

    Techniques Used: Expressing, Western Blot

    6) Product Images from "Transferrin is responsible for mediating the effects of iron ions on the regulation of anterior pharynx-defective-1α/β and Presenilin 1 expression via PGE2 and PGD2 at the early stage of Alzheimer’s Disease"

    Article Title: Transferrin is responsible for mediating the effects of iron ions on the regulation of anterior pharynx-defective-1α/β and Presenilin 1 expression via PGE2 and PGD2 at the early stage of Alzheimer’s Disease

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101615

    Tf-TfR mediated the effects of Fe on the stimulation of the expression and metabolic activity of mPGES-1 and L-PGDS, which result in the synthesis of APH-1α/1β and PS1 in neurons. n2a cells were treated with Fe (10 μM) in the absence or presence of transfection with Tf or TfR siRNA. The mRNA and protein levels of mPGES-1, L-PGDS, APH-1α/1β and PS1 were determined by qRT-PCR and western blots, respectively. GAPDH and β-actin served as internal controls. The data represent the means±S.E. of all the experiments. ** p
    Figure Legend Snippet: Tf-TfR mediated the effects of Fe on the stimulation of the expression and metabolic activity of mPGES-1 and L-PGDS, which result in the synthesis of APH-1α/1β and PS1 in neurons. n2a cells were treated with Fe (10 μM) in the absence or presence of transfection with Tf or TfR siRNA. The mRNA and protein levels of mPGES-1, L-PGDS, APH-1α/1β and PS1 were determined by qRT-PCR and western blots, respectively. GAPDH and β-actin served as internal controls. The data represent the means±S.E. of all the experiments. ** p

    Techniques Used: Expressing, Activity Assay, Transfection, Quantitative RT-PCR, Western Blot

    PGE 2 and PGD 2 antagonistically regulated the expression of APH-1α/1β and PS1 in n2a cells. ( A, C ) n2a cells were treated with Fe (10 μM) in the absence or presence of siRNA-targeted mPGES-1 or L-PGDS cDNA. ( B, D ) n2a cells were treated with PGE 2 (10 μM) or PGD 2 (1 μM) for 48 h. APH-1α/1β and PS1 were determined by qRT-PCR and western blots, respectively. GAPDH and β-actin served as internal controls. * p
    Figure Legend Snippet: PGE 2 and PGD 2 antagonistically regulated the expression of APH-1α/1β and PS1 in n2a cells. ( A, C ) n2a cells were treated with Fe (10 μM) in the absence or presence of siRNA-targeted mPGES-1 or L-PGDS cDNA. ( B, D ) n2a cells were treated with PGE 2 (10 μM) or PGD 2 (1 μM) for 48 h. APH-1α/1β and PS1 were determined by qRT-PCR and western blots, respectively. GAPDH and β-actin served as internal controls. * p

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

    7) Product Images from "Autophosphorylation-dependent degradation of Pak1, triggered by the Rho-family GTPase, Chp"

    Article Title: Autophosphorylation-dependent degradation of Pak1, triggered by the Rho-family GTPase, Chp

    Journal: The Biochemical Journal

    doi: 10.1042/BJ20061696

    Pak1 protein level is reduced in Chp-transfected cells ( A ) Cells were transfected with Chp//H-2K k or empty H-2K k vector, and H-2K k -expressing cells were purified using magnetic beads. The following day, cell lysates were subjected to Western blotting with anti-Pak1, anti-Pak2, anti-α-tubulin or anti-ERK1/2, as indicated. Cells were Jurkat tTA (left- and right-hand panels) or HEK-293 tTA (middle panels). ( B ) HEK-293T cells were transfected with a pcDNA-based expression plasmid encoding activated Chp or control vector, and lysates were probed with anti-Pak1 antibody (upper panel) and anti-α-tubulin (lower panel). ( C ) HEK-293T (left-hand panel) or Jurkat TAg (right-hand panel) were co-transfected with HA–Pak1 together with activated Chp, dominant-negative Chp or vector control, and total cell lysates were probed with anti-HA antibody (upper panel) and anti-α-tubulin antibody (lower panel). ( D ) The amount of Pak1 protein found in the lysates of transfected Jurkat TAg cells was quantified by densitometry (arbitrary units). For each transfection, Pak1 was normalized to the amount present in vector-transfected cells in the same experiment, to yield the fold change in Pak1 expression. Results are means±S.E.M. for three independent experiments. ( E ) HEK-293T cells were co-transfected with HA–Pak1 together with Myc-tagged activated Chp or vector control. Total cell lysates were prepared using Jurkat lysis buffer (lanes 1 and 2), RIPA buffer (lanes 3 and 4), WCE buffer (lanes 5 and 6) or sample buffer (lanes 7 and 8), and probed with anti-HA (top panel) and anti-α-tubulin (middle panel), followed by anti-Myc (bottom panel). The positions of the molecular-mass markers and the non-specific bands (N.S.) are indicated (values are in kDa). Chp Ac., activated Chp; Chp Dn., dominant-negative Chp.
    Figure Legend Snippet: Pak1 protein level is reduced in Chp-transfected cells ( A ) Cells were transfected with Chp//H-2K k or empty H-2K k vector, and H-2K k -expressing cells were purified using magnetic beads. The following day, cell lysates were subjected to Western blotting with anti-Pak1, anti-Pak2, anti-α-tubulin or anti-ERK1/2, as indicated. Cells were Jurkat tTA (left- and right-hand panels) or HEK-293 tTA (middle panels). ( B ) HEK-293T cells were transfected with a pcDNA-based expression plasmid encoding activated Chp or control vector, and lysates were probed with anti-Pak1 antibody (upper panel) and anti-α-tubulin (lower panel). ( C ) HEK-293T (left-hand panel) or Jurkat TAg (right-hand panel) were co-transfected with HA–Pak1 together with activated Chp, dominant-negative Chp or vector control, and total cell lysates were probed with anti-HA antibody (upper panel) and anti-α-tubulin antibody (lower panel). ( D ) The amount of Pak1 protein found in the lysates of transfected Jurkat TAg cells was quantified by densitometry (arbitrary units). For each transfection, Pak1 was normalized to the amount present in vector-transfected cells in the same experiment, to yield the fold change in Pak1 expression. Results are means±S.E.M. for three independent experiments. ( E ) HEK-293T cells were co-transfected with HA–Pak1 together with Myc-tagged activated Chp or vector control. Total cell lysates were prepared using Jurkat lysis buffer (lanes 1 and 2), RIPA buffer (lanes 3 and 4), WCE buffer (lanes 5 and 6) or sample buffer (lanes 7 and 8), and probed with anti-HA (top panel) and anti-α-tubulin (middle panel), followed by anti-Myc (bottom panel). The positions of the molecular-mass markers and the non-specific bands (N.S.) are indicated (values are in kDa). Chp Ac., activated Chp; Chp Dn., dominant-negative Chp.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Purification, Magnetic Beads, Western Blot, Dominant Negative Mutation, Lysis

    Overexpression of Chp inhibits Jurkat T-cell chemotaxis through restrictive pores ( A ]. The migration of EGFP-expressing cells was normalized to the migration of non-expressing cells, from the same transfection and migrating in the same well, and then multiplied by 100 to yield relative migration. Results are the means±S.E.M. for three independent experiments. ( B ) Migration of Jurkat tTA cells overexpressing wild-type Chp was examined as in ( A ), but using transwell insets with the indicated pore size (3, 5 and 8 μm).
    Figure Legend Snippet: Overexpression of Chp inhibits Jurkat T-cell chemotaxis through restrictive pores ( A ]. The migration of EGFP-expressing cells was normalized to the migration of non-expressing cells, from the same transfection and migrating in the same well, and then multiplied by 100 to yield relative migration. Results are the means±S.E.M. for three independent experiments. ( B ) Migration of Jurkat tTA cells overexpressing wild-type Chp was examined as in ( A ), but using transwell insets with the indicated pore size (3, 5 and 8 μm).

    Techniques Used: Over Expression, Chemotaxis Assay, Migration, Expressing, Transfection

    8) Product Images from "Interactions between the APP C-terminal domain and G-proteins mediate calcium dysregulation and Aβ toxicity in Alzheimer disease"

    Article Title: Interactions between the APP C-terminal domain and G-proteins mediate calcium dysregulation and Aβ toxicity in Alzheimer disease

    Journal: The FEBS journal

    doi: 10.1111/j.1742-4658.2009.06997.x

    The APP:Go Interaction and G-protein activation is Modulated During AD Progression (A) Brain samples were homogenized and immunoprecipitated with the APP C-terminal specific CT15 antibody, run on a SDS-PAGE gel and Go protein binding was assessed by immunoblot with a monoclonal Go antibody. Signal intensity of the 40kDa band (corresponding to the interaction between APP and Go) was analyzed for each sample at each Braak stage. A decrease in the signal intensity of the 40kDa band was observed with increasing disease severity (mean ± SEM). * indicates a significant difference between expression levels at Braak stage 0 and Braak stage V–VI (p
    Figure Legend Snippet: The APP:Go Interaction and G-protein activation is Modulated During AD Progression (A) Brain samples were homogenized and immunoprecipitated with the APP C-terminal specific CT15 antibody, run on a SDS-PAGE gel and Go protein binding was assessed by immunoblot with a monoclonal Go antibody. Signal intensity of the 40kDa band (corresponding to the interaction between APP and Go) was analyzed for each sample at each Braak stage. A decrease in the signal intensity of the 40kDa band was observed with increasing disease severity (mean ± SEM). * indicates a significant difference between expression levels at Braak stage 0 and Braak stage V–VI (p

    Techniques Used: Activation Assay, Immunoprecipitation, SDS Page, Protein Binding, Expressing

    9) Product Images from "The 37 kDa/67 kDa laminin receptor is required for PrPSc propagation in scrapie-infected neuronal cells"

    Article Title: The 37 kDa/67 kDa laminin receptor is required for PrPSc propagation in scrapie-infected neuronal cells

    Journal: EMBO Reports

    doi: 10.1038/sj.embor.embor768

    Abolition of PrP Sc propagation using laminin receptor precursor (LRP) antisense RNA. ( A ) Analysis by PCR with reverse transcription of total RNA extracts from transfected ScMNB cells. Oligodesoxythymidine-primed complementary DNA was amplified by PCR using specific primers for the pCI–neo plasmid. This gave a 322-bp cDNA fragment for the pCI–neo transfected cells and a 1,115-bp cDNA fragment for the pCI–neo–asLRP transfected cells. ( B ) A ribonuclease protection assay was carried out on total RNA from cells transfected with either pCI–neo or pCI–neo–asLRP; the RNA was then separated using a 5% acrylamide/urea gel. 5 μg or 10 μg of total RNA was used, and in both cases the level of LRP messenger RNA was reduced by 80–85% in cells transfected with pCI–neo–asLRP (quantified by posphorimaging). ( C ) Western blot analysis of cell lysates from pCI–neo- and pCI–neo–asLRP-transfected ScMNB cells assayed 48 hours after transfection. LRP was detected using the polyclonal anti-LRP/LR antibody, W3. β-actin was detected using an anti-β-actin antibody as loading control. ( D ) ScMNB cells were transfected with pCI–neo and pCI–neo–asLRP. The PrP Sc content of ScMNB cells was analysed 72 h after transfection. The monoclonal anti-PrP antibody SAF70 was used for PrP Sc detection and the SAF32 antibody was used for detection of PrP C .
    Figure Legend Snippet: Abolition of PrP Sc propagation using laminin receptor precursor (LRP) antisense RNA. ( A ) Analysis by PCR with reverse transcription of total RNA extracts from transfected ScMNB cells. Oligodesoxythymidine-primed complementary DNA was amplified by PCR using specific primers for the pCI–neo plasmid. This gave a 322-bp cDNA fragment for the pCI–neo transfected cells and a 1,115-bp cDNA fragment for the pCI–neo–asLRP transfected cells. ( B ) A ribonuclease protection assay was carried out on total RNA from cells transfected with either pCI–neo or pCI–neo–asLRP; the RNA was then separated using a 5% acrylamide/urea gel. 5 μg or 10 μg of total RNA was used, and in both cases the level of LRP messenger RNA was reduced by 80–85% in cells transfected with pCI–neo–asLRP (quantified by posphorimaging). ( C ) Western blot analysis of cell lysates from pCI–neo- and pCI–neo–asLRP-transfected ScMNB cells assayed 48 hours after transfection. LRP was detected using the polyclonal anti-LRP/LR antibody, W3. β-actin was detected using an anti-β-actin antibody as loading control. ( D ) ScMNB cells were transfected with pCI–neo and pCI–neo–asLRP. The PrP Sc content of ScMNB cells was analysed 72 h after transfection. The monoclonal anti-PrP antibody SAF70 was used for PrP Sc detection and the SAF32 antibody was used for detection of PrP C .

    Techniques Used: Polymerase Chain Reaction, Transfection, Amplification, Plasmid Preparation, Western Blot

    10) Product Images from "Platelet-derived SDF1 primes pulmonary capillary vascular niche to drive lung alveolar regeneration"

    Article Title: Platelet-derived SDF1 primes pulmonary capillary vascular niche to drive lung alveolar regeneration

    Journal: Nature cell biology

    doi: 10.1038/ncb3096

    Regulation of SDF1 signaling in PCEC niche by VEGFR2/FGFR1 pathway a) Strategy to test the activation of VEGFR2, FGFR1 and expression of SDF1 receptor CXCR4 in PCECs of indicated mouse groups. b-c ) Activation of VEGFR2 and FGFR1 in PCECs of Thpo −/− mice after PNX. Phosphorylation of VEGFR2 (p-VEGFR2) was tested by Western blot. To test FGFR1 activation, phosphorylation of downstream effector FRS2 (p-FRS2) was similarly assessed. Protein levels of VEGFR2 and FRS2 and β-actin were also measured as control. n = 4 mice in all tested groups; P = 0.0172 (VEGFR2 activation) and 0.0153 (FGFR1 activation) between WT and Thpo −/− mice. d-e ) Expression of CXCR4 protein in mice that are deficient of Vegfr2 and Fgfr1 in ECs ( Vegfr2 iΔEC/iΔEC Fgfr1 iΔEC/+) . Vegfr2 iΔEC/iΔEC Fgfr1 iΔEC/+ . n = 4 animals in both groups, and P = 0.0005 between two genotypes. f-g ) Rescue effect of VEGF 164 and SDF1 in pneumonectomized Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC and Vegfr2 iΔEC/iΔEC Fgfr1 iΔEC/+ mice, respectively. Restoration of gas exchange function (f) and PCEC Akt activation (g) were tested. P = 0.0319 between Vegfr2 iΔEC/iΔEC Fgfr1 iΔEC/+ mice injected with SDF1 and vehicle, and P = 0.28 between Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC mice injected with VEGF 164 and vehicle; n = 4 mice per group.. h-i ) PCEC Akt activation in indicated mice was determined. n = 6 mice in all tested groups. In the presented immunoblot image, each lane indicates individual mouse sample. Error bar stands for s.e.m., and line represents mean for panels c, d, g, i. Statistical difference between groups was determined by unpaired two tail t-test. j ) Regulation of SDF1 signaling in PCEC niche by VEGFR2/FGFR1 pathway after PNX. Activation of VEGFR2 and FGFR1 is mediated by both platelet-dependent and independent mechanisms. Activation of VEGFR2/FGFR1 synergistically acts with SDF1 receptors (CXCR4/CXCR7) on PCECs to trigger vascular niche-mediated neo-alveologenesis. k ) Recruitment of activated platelets via mobilizing SDF1 primes PCEC niche and drives lung alveolar regeneration/regrowth. Upon PNX, activated platelets supply SDF1 to activate CXCR4 and CXCR7 on PCECs. Platelet-mediated CXCR4/7-Akt activation deploys MMP14 in PCEC niche and causes release of alveologenic ligand HB-EGF, eliciting propagation of AEC2s and driving neo-alveologenesis.
    Figure Legend Snippet: Regulation of SDF1 signaling in PCEC niche by VEGFR2/FGFR1 pathway a) Strategy to test the activation of VEGFR2, FGFR1 and expression of SDF1 receptor CXCR4 in PCECs of indicated mouse groups. b-c ) Activation of VEGFR2 and FGFR1 in PCECs of Thpo −/− mice after PNX. Phosphorylation of VEGFR2 (p-VEGFR2) was tested by Western blot. To test FGFR1 activation, phosphorylation of downstream effector FRS2 (p-FRS2) was similarly assessed. Protein levels of VEGFR2 and FRS2 and β-actin were also measured as control. n = 4 mice in all tested groups; P = 0.0172 (VEGFR2 activation) and 0.0153 (FGFR1 activation) between WT and Thpo −/− mice. d-e ) Expression of CXCR4 protein in mice that are deficient of Vegfr2 and Fgfr1 in ECs ( Vegfr2 iΔEC/iΔEC Fgfr1 iΔEC/+) . Vegfr2 iΔEC/iΔEC Fgfr1 iΔEC/+ . n = 4 animals in both groups, and P = 0.0005 between two genotypes. f-g ) Rescue effect of VEGF 164 and SDF1 in pneumonectomized Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC and Vegfr2 iΔEC/iΔEC Fgfr1 iΔEC/+ mice, respectively. Restoration of gas exchange function (f) and PCEC Akt activation (g) were tested. P = 0.0319 between Vegfr2 iΔEC/iΔEC Fgfr1 iΔEC/+ mice injected with SDF1 and vehicle, and P = 0.28 between Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC mice injected with VEGF 164 and vehicle; n = 4 mice per group.. h-i ) PCEC Akt activation in indicated mice was determined. n = 6 mice in all tested groups. In the presented immunoblot image, each lane indicates individual mouse sample. Error bar stands for s.e.m., and line represents mean for panels c, d, g, i. Statistical difference between groups was determined by unpaired two tail t-test. j ) Regulation of SDF1 signaling in PCEC niche by VEGFR2/FGFR1 pathway after PNX. Activation of VEGFR2 and FGFR1 is mediated by both platelet-dependent and independent mechanisms. Activation of VEGFR2/FGFR1 synergistically acts with SDF1 receptors (CXCR4/CXCR7) on PCECs to trigger vascular niche-mediated neo-alveologenesis. k ) Recruitment of activated platelets via mobilizing SDF1 primes PCEC niche and drives lung alveolar regeneration/regrowth. Upon PNX, activated platelets supply SDF1 to activate CXCR4 and CXCR7 on PCECs. Platelet-mediated CXCR4/7-Akt activation deploys MMP14 in PCEC niche and causes release of alveologenic ligand HB-EGF, eliciting propagation of AEC2s and driving neo-alveologenesis.

    Techniques Used: Activation Assay, Expressing, Mouse Assay, Western Blot, Injection

    Platelets activate CXCR4 and CXCR7 in PCEC niche to drive alveolar regeneration a ) Endothelial cell (EC)-specific inducible deletion of Cxcr4 and Cxcr7 in adult mice. VE-cadherin-Cre ERT2 /Cdh5-PAC-Cre ERT2 mice were crossed with floxed Cxcr4 and Cxcr7 mice to induce EC-specific deletion of Cxcr4 ( Cxcr4 iΔEC/iΔEC ) and both Cxcr4 and Cxcr7 ( Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC ) in adult mice. Cxcr4 iΔEC/+ mice was used as control. b-e ) Proliferation rate of AEC2s and PCECs in control, Cxcr4 iΔEC/iΔEC , and Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC mice after PNX. n = 4 mice in both control and Cxcr4 iΔEC/iΔEC group, and n = 3 mice in Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC group; Scale bar = 50 μm. In panel (c), P = 0.0066 (control versus Cxcr4 iΔEC/iΔEC ) and 0.0015 (control versus Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC ). In panel (d), P = 0.0013 (control versus Cxcr4 iΔEC/iΔEC ) and 0.00055 (control versus Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC ). f-g) Functional alveolar regrowth in indicated mouse groups following PNX. In panels (f) (g), n = 3 mice in both control and Cxcr4 iΔEC/iΔEC group; n = 4 mice in Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC group. P = 0.00069 (f) and 0.014 (g) between control and Cxcr4 iΔEC/iΔEC mice. P = 0.00026 (f) and 0.0017 (g) between control and Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC group. h ) PCEC MMP14 protein level in control, Cxcr4 iΔEC/iΔEC , and Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC mice_was tested by flow cytometry. i-k ) Weight (i), volume (j) and PCEC Akt activation (k) in right lungs after PNX. Western blot was used to test p-Akt level in PCECs. n = 5, 4, 4, 5, 4, 4 mice (i) and = 5, 4, 4, 3, 5, 4 animals (j) in control Cxcr4 iΔEC/+ , Cxcr4 iΔEC/iΔEC , Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC groups that underwent sham and PNX, respectively. l-n ) BALF level of HB-EGF in pneumonectomized Cxcr4 iΔEC/iΔEC and Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC mice after Infusion of Sdf1 +/+ platelet. Influence of platelets on alveolar regeneration of indicated mouse groups was performed as described in (l), HB-EGF in BALF was tested by Western blot (m) and quantified (n). In (n), n = 5 mice in control Cxcr4 iΔEC/+ group, n= 4 mice in other shown groups. In the shown immunoblot image, each lane indicates sample from individual mouse. Error bar denotes s.e.m., and line defines mean for panels c, d, f, g, i, j, n. Statistical difference between groups was analyzed by unpaired two tail t-test.
    Figure Legend Snippet: Platelets activate CXCR4 and CXCR7 in PCEC niche to drive alveolar regeneration a ) Endothelial cell (EC)-specific inducible deletion of Cxcr4 and Cxcr7 in adult mice. VE-cadherin-Cre ERT2 /Cdh5-PAC-Cre ERT2 mice were crossed with floxed Cxcr4 and Cxcr7 mice to induce EC-specific deletion of Cxcr4 ( Cxcr4 iΔEC/iΔEC ) and both Cxcr4 and Cxcr7 ( Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC ) in adult mice. Cxcr4 iΔEC/+ mice was used as control. b-e ) Proliferation rate of AEC2s and PCECs in control, Cxcr4 iΔEC/iΔEC , and Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC mice after PNX. n = 4 mice in both control and Cxcr4 iΔEC/iΔEC group, and n = 3 mice in Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC group; Scale bar = 50 μm. In panel (c), P = 0.0066 (control versus Cxcr4 iΔEC/iΔEC ) and 0.0015 (control versus Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC ). In panel (d), P = 0.0013 (control versus Cxcr4 iΔEC/iΔEC ) and 0.00055 (control versus Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC ). f-g) Functional alveolar regrowth in indicated mouse groups following PNX. In panels (f) (g), n = 3 mice in both control and Cxcr4 iΔEC/iΔEC group; n = 4 mice in Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC group. P = 0.00069 (f) and 0.014 (g) between control and Cxcr4 iΔEC/iΔEC mice. P = 0.00026 (f) and 0.0017 (g) between control and Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC group. h ) PCEC MMP14 protein level in control, Cxcr4 iΔEC/iΔEC , and Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC mice_was tested by flow cytometry. i-k ) Weight (i), volume (j) and PCEC Akt activation (k) in right lungs after PNX. Western blot was used to test p-Akt level in PCECs. n = 5, 4, 4, 5, 4, 4 mice (i) and = 5, 4, 4, 3, 5, 4 animals (j) in control Cxcr4 iΔEC/+ , Cxcr4 iΔEC/iΔEC , Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC groups that underwent sham and PNX, respectively. l-n ) BALF level of HB-EGF in pneumonectomized Cxcr4 iΔEC/iΔEC and Cxcr4 iΔEC/iΔEC Cxcr7 iΔEC/iΔEC mice after Infusion of Sdf1 +/+ platelet. Influence of platelets on alveolar regeneration of indicated mouse groups was performed as described in (l), HB-EGF in BALF was tested by Western blot (m) and quantified (n). In (n), n = 5 mice in control Cxcr4 iΔEC/+ group, n= 4 mice in other shown groups. In the shown immunoblot image, each lane indicates sample from individual mouse. Error bar denotes s.e.m., and line defines mean for panels c, d, f, g, i, j, n. Statistical difference between groups was analyzed by unpaired two tail t-test.

    Techniques Used: Mouse Assay, Functional Assay, Flow Cytometry, Cytometry, Activation Assay, Western Blot

    Platelets recruited by PNX derive SDF1 to elicit alveolar re-growth/regeneration a-c ) Platelet (PLT)-specific deletion of Sdf1 ( Sdf1 ΔPLT/ΔPLT ) in mice. Mouse line with platelet/platelet progenitor-specific Platelet factor 4 promoter-driven Cre ( PF4-Cre ) was crossed with Sdf1 loxP/loxP mice to delete Sdf1 specifically in platelets (a). Sdf1 ΔPLT/+ mice harboring haplodeficiency of Sdf1 in platelets served as control. SDF1 protein in platelets was analyzed by Western blot to determine Sdf1 deletion efficiency (b-c). SDF1 protein was decreased by 95% in Sdf1 ΔPLT/ΔPLT platelets, compared to wild type control (n = 6 mice per group). d-e ) Right lung weight (d) and volume (e) in Sdf1 ΔPLT/ΔPLT and control mice after PNX. (d): n = 4 and 3 mice in control and Sdf1 ΔPLT/ΔPLT mice (sham), n = 5 mice in both control and Sdf1 Δ/Δ mice (PNX). (e): n = 5 and 4 mice in control and Sdf1 ΔPLT/ΔPLT mouse groups (sham), n = 4 and 5 mice in pneumonectomized control and Sdf1 Δ/Δ mice groups. Scale bar = 50 μm. f-i ) Proliferation of AEC2s and PCECs in Sdf1 ΔPLT/ΔPLT and control mice after PNX. Expansion of AEC2s and PCECs at day 7 after PNX was determined by immunostaining (f, g) and flow cytometry (h, i), respectively; n = 4 mice per group in (f) and (i); P = 0.001 (f) and 0.0011 (i) between two genotypes. Scale bar = 50 μm. j ) Restoration of AEC1s in Sdf1 ΔPLT/ΔPLT and control mice after PNX. Scale bar= 50 μm. k ) After PNX, activated platelets adhere to PCECs and supply SDF1 to drive regenerative alveolarization. Error bar denotes s.e.m., and line describes mean for panels c, d, e, i. Unpaired two-tail t-test was used to determine the difference between individual groups.
    Figure Legend Snippet: Platelets recruited by PNX derive SDF1 to elicit alveolar re-growth/regeneration a-c ) Platelet (PLT)-specific deletion of Sdf1 ( Sdf1 ΔPLT/ΔPLT ) in mice. Mouse line with platelet/platelet progenitor-specific Platelet factor 4 promoter-driven Cre ( PF4-Cre ) was crossed with Sdf1 loxP/loxP mice to delete Sdf1 specifically in platelets (a). Sdf1 ΔPLT/+ mice harboring haplodeficiency of Sdf1 in platelets served as control. SDF1 protein in platelets was analyzed by Western blot to determine Sdf1 deletion efficiency (b-c). SDF1 protein was decreased by 95% in Sdf1 ΔPLT/ΔPLT platelets, compared to wild type control (n = 6 mice per group). d-e ) Right lung weight (d) and volume (e) in Sdf1 ΔPLT/ΔPLT and control mice after PNX. (d): n = 4 and 3 mice in control and Sdf1 ΔPLT/ΔPLT mice (sham), n = 5 mice in both control and Sdf1 Δ/Δ mice (PNX). (e): n = 5 and 4 mice in control and Sdf1 ΔPLT/ΔPLT mouse groups (sham), n = 4 and 5 mice in pneumonectomized control and Sdf1 Δ/Δ mice groups. Scale bar = 50 μm. f-i ) Proliferation of AEC2s and PCECs in Sdf1 ΔPLT/ΔPLT and control mice after PNX. Expansion of AEC2s and PCECs at day 7 after PNX was determined by immunostaining (f, g) and flow cytometry (h, i), respectively; n = 4 mice per group in (f) and (i); P = 0.001 (f) and 0.0011 (i) between two genotypes. Scale bar = 50 μm. j ) Restoration of AEC1s in Sdf1 ΔPLT/ΔPLT and control mice after PNX. Scale bar= 50 μm. k ) After PNX, activated platelets adhere to PCECs and supply SDF1 to drive regenerative alveolarization. Error bar denotes s.e.m., and line describes mean for panels c, d, e, i. Unpaired two-tail t-test was used to determine the difference between individual groups.

    Techniques Used: Mouse Assay, Western Blot, Immunostaining, Flow Cytometry, Cytometry

    Intravascular transfusion of Sdf1 +/+ but not Sdf1 -deficient ( Sdf1 −/− ) platelets promotes alveolar regeneration in Thpo −/− mice a-c ) Platelets accumulate in the lungs of pneumonectomized Thpo −/− mice after intravascular infusion. Platelets were isolated from β-actin promoter driven- tdTomato ( ACTB-tdTomato ) mice and infused into Thpo −/− mice (a). Accumulation of tdTomato + CD41 + platelets in the lungs of Thpo −/− mice was determined by flow cytometry and immunostaining; Scale bar = 50 μm. d-g) Strategy to examine the influence of platelet-derived SDF1 on lung alveolar regeneration. Sdf1 +/+ and Sdf1 −/− platelets were isolated from wild type and Sdf1 Δ/Δ mice and infused into pneumonectomized Thpo −/− mice, respectively. Recovery of right lung volume (e), mass (f) and respiratory function (g) were determined in recipient mice. In panels (e) and (f), n =5 mice in all groups; P = 0.011 (e) and 0.026 (f) between mice transplanted with Sdf1 +/+ and Sdf1 −/− platelets. In panel (g), n = 6 mice ( Sdf1 +/+ ) and 4 mice ( Sdf1 −/− ) . P = 0.0013 (left) and 0.0042 (right) between two groups. h-k ) Expansion of AEC2s (h, j) and PCECs (i, k) in Thpo −/− mice after infusion of Sdf1 +/+ and Sdf1 −/− platelets. Representative immunostaining image of two transplanted groups and flow cytometry graph are shown; n = 5 mixw in all groups; P = 0.00011 (j) and 0.00081 (k) between two platelet types. Scale bar = 50 μm. l ) AEC1s in Thpo −/− mice following PNX and Sdf1 +/+ and Sdf1 −/− platelet transplantation. Scale bar = 50 μm. m-o ) Alveolar architecture of Thpo −/− mice receiving platelet infusion after PNX. Alveolar morphology was assessed by H E staining (m), and alveolar mean linear intercept (n) and number (o) were compared; n = 5 mice in both groups; P = 0.027 (n) and 0.0015 (o) between mice transplanted with Sdf1 +/+ and Sdf1 −/− platelets. Scale bar = 50 um. Error bar defines s.e.m., and line represents mean for panels e, f, g, j, k, n, o. Statistical difference between groups was assessed by unpaired two tail t-test.
    Figure Legend Snippet: Intravascular transfusion of Sdf1 +/+ but not Sdf1 -deficient ( Sdf1 −/− ) platelets promotes alveolar regeneration in Thpo −/− mice a-c ) Platelets accumulate in the lungs of pneumonectomized Thpo −/− mice after intravascular infusion. Platelets were isolated from β-actin promoter driven- tdTomato ( ACTB-tdTomato ) mice and infused into Thpo −/− mice (a). Accumulation of tdTomato + CD41 + platelets in the lungs of Thpo −/− mice was determined by flow cytometry and immunostaining; Scale bar = 50 μm. d-g) Strategy to examine the influence of platelet-derived SDF1 on lung alveolar regeneration. Sdf1 +/+ and Sdf1 −/− platelets were isolated from wild type and Sdf1 Δ/Δ mice and infused into pneumonectomized Thpo −/− mice, respectively. Recovery of right lung volume (e), mass (f) and respiratory function (g) were determined in recipient mice. In panels (e) and (f), n =5 mice in all groups; P = 0.011 (e) and 0.026 (f) between mice transplanted with Sdf1 +/+ and Sdf1 −/− platelets. In panel (g), n = 6 mice ( Sdf1 +/+ ) and 4 mice ( Sdf1 −/− ) . P = 0.0013 (left) and 0.0042 (right) between two groups. h-k ) Expansion of AEC2s (h, j) and PCECs (i, k) in Thpo −/− mice after infusion of Sdf1 +/+ and Sdf1 −/− platelets. Representative immunostaining image of two transplanted groups and flow cytometry graph are shown; n = 5 mixw in all groups; P = 0.00011 (j) and 0.00081 (k) between two platelet types. Scale bar = 50 μm. l ) AEC1s in Thpo −/− mice following PNX and Sdf1 +/+ and Sdf1 −/− platelet transplantation. Scale bar = 50 μm. m-o ) Alveolar architecture of Thpo −/− mice receiving platelet infusion after PNX. Alveolar morphology was assessed by H E staining (m), and alveolar mean linear intercept (n) and number (o) were compared; n = 5 mice in both groups; P = 0.027 (n) and 0.0015 (o) between mice transplanted with Sdf1 +/+ and Sdf1 −/− platelets. Scale bar = 50 um. Error bar defines s.e.m., and line represents mean for panels e, f, g, j, k, n, o. Statistical difference between groups was assessed by unpaired two tail t-test.

    Techniques Used: Mouse Assay, Isolation, Flow Cytometry, Cytometry, Immunostaining, Derivative Assay, Transplantation Assay, Staining

    Expression of MMP14 in PCEC niche is essential for evoking functional recovery of AECs after PNX a ) Endothelial cell (EC)-specific inducible deletion of Mmp14 in adult mice. Mice carrying EC-specific promoter VE-cadherin-driven tamoxifen-responsive Cre ( Cdh5-PAC-Cre ERT2 ) were bred with mice harboring LoxP site-flanked Mmp14 to generate mice with EC-specific deletion of Mmp14 ( Mmp14 iΔEC/iΔEC ). Mmp14 iΔEC/+ mice harboring EC-specific Mmp14 haplodeficiency were used as control. b, c ) Lung mass (b) and volume (c) in Mmp14 iΔEC/iΔEC and control mice after sham and PNX. n =3 mice in all groups. d-f ) Proliferation of AEC2s (d) and PCECs (f) in Mmp14 iΔEC/iΔEC and control mice. (e): n = 3 animals in all groups; P = 0.0029 (AEC2 proliferation, top panel) and 0.88 (PCEC proliferation, bottom panel) between Mmp14 iΔEC/iΔEC and control group; Scale bar = 50 μm. g-j ) Effect of platelet infusion on the alveolarization in Mmp14 iΔEC/iΔEC mice. Platelets were infused into pneumonectomized Mmp14 iΔEC/iΔEC and compared with control mice (g). Recovery of pulmonary respiratory function (h, i) and AECs (j) was determined. Platelet infusion failed to rescue the defective alveolar regeneration in Mmp14 iΔEC/iΔEC mice. n = 3 mice per group in (h) and (i); P = 0.003 (h), = 0.011 (i) between control and Mmp14 iΔEC/iΔEC mice, Scale bar = 50 μm. k-o) Influence of recombinant epidermal growth factor (EGF) on Thpo −/− mice after PNX. EGF was injected pneumonectomized Thpo −/− mice (k). Proliferation of AEC2s (l) and PCECs (m), recovery of AEC1s (n), and blood oxygenation (o) were assessed. (o) n = 4 mice in both groups; P = 0.006 between two EGF and vehicle injected groups; Scale bar = 50 μm. Error bar defines s.e.m., and line depicts mean for panels b, c, e, h, i, o. Unpaired two tail t-test was used to determine statistical difference between groups. p ) Activated platelets via depositing SDF1 activate MMP14 pathway in PCEC, functionalizing a hemo-vascular niche promoting lung neo-alveologenesis. Upon PNX, activated platelets supply SDF1 to deploy MMP14 in PCEC niche and alveologenic ligand HB-EGF, eliciting propagation of AEC2s and driving alveologenesis.
    Figure Legend Snippet: Expression of MMP14 in PCEC niche is essential for evoking functional recovery of AECs after PNX a ) Endothelial cell (EC)-specific inducible deletion of Mmp14 in adult mice. Mice carrying EC-specific promoter VE-cadherin-driven tamoxifen-responsive Cre ( Cdh5-PAC-Cre ERT2 ) were bred with mice harboring LoxP site-flanked Mmp14 to generate mice with EC-specific deletion of Mmp14 ( Mmp14 iΔEC/iΔEC ). Mmp14 iΔEC/+ mice harboring EC-specific Mmp14 haplodeficiency were used as control. b, c ) Lung mass (b) and volume (c) in Mmp14 iΔEC/iΔEC and control mice after sham and PNX. n =3 mice in all groups. d-f ) Proliferation of AEC2s (d) and PCECs (f) in Mmp14 iΔEC/iΔEC and control mice. (e): n = 3 animals in all groups; P = 0.0029 (AEC2 proliferation, top panel) and 0.88 (PCEC proliferation, bottom panel) between Mmp14 iΔEC/iΔEC and control group; Scale bar = 50 μm. g-j ) Effect of platelet infusion on the alveolarization in Mmp14 iΔEC/iΔEC mice. Platelets were infused into pneumonectomized Mmp14 iΔEC/iΔEC and compared with control mice (g). Recovery of pulmonary respiratory function (h, i) and AECs (j) was determined. Platelet infusion failed to rescue the defective alveolar regeneration in Mmp14 iΔEC/iΔEC mice. n = 3 mice per group in (h) and (i); P = 0.003 (h), = 0.011 (i) between control and Mmp14 iΔEC/iΔEC mice, Scale bar = 50 μm. k-o) Influence of recombinant epidermal growth factor (EGF) on Thpo −/− mice after PNX. EGF was injected pneumonectomized Thpo −/− mice (k). Proliferation of AEC2s (l) and PCECs (m), recovery of AEC1s (n), and blood oxygenation (o) were assessed. (o) n = 4 mice in both groups; P = 0.006 between two EGF and vehicle injected groups; Scale bar = 50 μm. Error bar defines s.e.m., and line depicts mean for panels b, c, e, h, i, o. Unpaired two tail t-test was used to determine statistical difference between groups. p ) Activated platelets via depositing SDF1 activate MMP14 pathway in PCEC, functionalizing a hemo-vascular niche promoting lung neo-alveologenesis. Upon PNX, activated platelets supply SDF1 to deploy MMP14 in PCEC niche and alveologenic ligand HB-EGF, eliciting propagation of AEC2s and driving alveologenesis.

    Techniques Used: Expressing, Functional Assay, Mouse Assay, Recombinant, Injection

    11) Product Images from "Differential effects of catecholamines on vascular rings from ductus venosus and intrahepatic veins of fetal sheep"

    Article Title: Differential effects of catecholamines on vascular rings from ductus venosus and intrahepatic veins of fetal sheep

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2002.034470

    Expression of β-adrenergic receptors in an intrahepatic artery and negative control of immunostaining of the DV with antibody against α-adrenergic receptors A , layers of muscle cells are shown in the media of a hepatic artery. The stratum stains positively with an antibody against β-adrenergic receptors (×82). B , negative control in the DV for immunostaining with antibody against α-adrenergic receptors. The negative controls do not reveal any staining (×221).
    Figure Legend Snippet: Expression of β-adrenergic receptors in an intrahepatic artery and negative control of immunostaining of the DV with antibody against α-adrenergic receptors A , layers of muscle cells are shown in the media of a hepatic artery. The stratum stains positively with an antibody against β-adrenergic receptors (×82). B , negative control in the DV for immunostaining with antibody against α-adrenergic receptors. The negative controls do not reveal any staining (×221).

    Techniques Used: Expressing, Negative Control, Immunostaining, Staining

    12) Product Images from "Hypoxia and defective apoptosis drive genomic instability and tumorigenesis"

    Article Title: Hypoxia and defective apoptosis drive genomic instability and tumorigenesis

    Journal: Genes & Development

    doi: 10.1101/gad.1204904

    Formation of tumor giant cells in tumors defective for apoptosis. ( A ) Hematoxylin and eosin-stained sections reveal numerous tumor giant cells in tumors from transformed BMK cells expressing BCL-2 or E1B 19K. Typical sections of carcinomas as described in the text are shown at a magnification of 200× and highlight areas enriched for tumor giant cells in tumors from animals injected with transformed BMK cells expressing BCL-2 or E1B 19K, with insets of grossly polyploid cells in mitosis (at 1000× magnification). Several tumor giant cells in each image are indicated by white arrows. Note the absence of tumor giant cells in tumors formed by the W2.3.1–5 cells. A typical section of a tumor area enriched for tumor giant cells was also immunostained for adenovirus E1A to demonstrate that the tumor giant cells are derived from the input-transformed BMK cells. Note the numerous tumor giant cells that stain brown in the E1A immunohistochemistry, including several examples indicated by arrows. A tumor section (20 μm) enriched for tumor giant cells (arrows) was stained with YOYO-1 to reveal DNA content as described in the text. This image is shown at 630×. ( B ) Aberrant metaphases and polyploid cells accumulate during tumor progression. Tumor sections were developed by immunohistochemistry using antibodies specific for phospho-histone H3 and are shown at 200×. Black arrows indicate aberrant polyploid mitotic arrays, and mitotic arrays presented at 600× in the insets are boxed. Top panels represent images of sections from mature tumors. Phosphohistone H3 immunohistochemistry of sections of transformed BMK cell masses excised from mice on days 2 and 9 after injection are shown in the bottom two rows (200×). Insets in the phospho-histone H3 panels were photographed at 600×, and areas present in the insets are boxed. Grossly aberrant mitotic arrays stained for phospho-histone H3 that are evident in the W2.Bcl2–3 and D3.zeo-2, but not W2.3.1–5, cells on day 9 are indicated in the insets by white arrows. Necrotic centers are indicated (N).
    Figure Legend Snippet: Formation of tumor giant cells in tumors defective for apoptosis. ( A ) Hematoxylin and eosin-stained sections reveal numerous tumor giant cells in tumors from transformed BMK cells expressing BCL-2 or E1B 19K. Typical sections of carcinomas as described in the text are shown at a magnification of 200× and highlight areas enriched for tumor giant cells in tumors from animals injected with transformed BMK cells expressing BCL-2 or E1B 19K, with insets of grossly polyploid cells in mitosis (at 1000× magnification). Several tumor giant cells in each image are indicated by white arrows. Note the absence of tumor giant cells in tumors formed by the W2.3.1–5 cells. A typical section of a tumor area enriched for tumor giant cells was also immunostained for adenovirus E1A to demonstrate that the tumor giant cells are derived from the input-transformed BMK cells. Note the numerous tumor giant cells that stain brown in the E1A immunohistochemistry, including several examples indicated by arrows. A tumor section (20 μm) enriched for tumor giant cells (arrows) was stained with YOYO-1 to reveal DNA content as described in the text. This image is shown at 630×. ( B ) Aberrant metaphases and polyploid cells accumulate during tumor progression. Tumor sections were developed by immunohistochemistry using antibodies specific for phospho-histone H3 and are shown at 200×. Black arrows indicate aberrant polyploid mitotic arrays, and mitotic arrays presented at 600× in the insets are boxed. Top panels represent images of sections from mature tumors. Phosphohistone H3 immunohistochemistry of sections of transformed BMK cell masses excised from mice on days 2 and 9 after injection are shown in the bottom two rows (200×). Insets in the phospho-histone H3 panels were photographed at 600×, and areas present in the insets are boxed. Grossly aberrant mitotic arrays stained for phospho-histone H3 that are evident in the W2.Bcl2–3 and D3.zeo-2, but not W2.3.1–5, cells on day 9 are indicated in the insets by white arrows. Necrotic centers are indicated (N).

    Techniques Used: Staining, Transformation Assay, Expressing, Injection, Derivative Assay, Immunohistochemistry, Mouse Assay

    Antiapoptotic BCL-2 family proteins block apoptosis and promote tumor formation. ( A ) Generation of stable cell lines. Cell extracts made from stable BMK cells that express both BAX and BAK (W2), or that are deficient for BAX and BAK (D3), were subjected to Western blotting with antibodies specific for BCL-2 ( left top panel) or E1B 19K ( right top panel). Note the similar expression levels of each exogenous protein in three independent clones (depicted numerically) and undetectable levels of each exogenous protein in the vector-only control cell lines (W2.3.1–2,5,6 or D3.zeo-1,2,3). Blots were then reprobed with an antibody to actin to verify nearly equivalent levels of protein in all lanes, shown below the BCL-2 and E1B 19K panels. ( B ) BCL-2 and E1B 19K block apoptosis in response to staurosporine. Stable BMK cell lines expressing BCL-2, E1B 19K, and controls were treated with media alone (open bars) or media containing 0.4 μM staurosporine (filled bars) for 24 h, and the viable cell number was determined by trypan blue exclusion. Results are presented as the percent of viable cells in each condition, which in each case represents the average of three independent plates. ( C ) BCL-2 and E1B 19K antagonize BAX and BAK to promote tumor formation. Three independent stable BMK cell lines (circles, squares, and diamonds) expressing BCL-2 (green symbols), E1B 19K (blue symbols), or controls (red symbols) were injected subcutaneously into nude mice, and tumor formation was monitored over time. Each point represents the average tumor volume for five injected animals. W2 cells, which express both BAX and BAK, are shown in the left panel. D3 cells, which are deficient for both BAX and BAK, are shown in the right panel. Note that BCL-2 or E1B 19K expression caused a profound acceleration of tumor formation in the W2 cells, whereas the kinetics of tumor formation in the D3 cells, which are deficient for both BAX and BAK, were unchanged by BCL-2 or E1B 19K expression.
    Figure Legend Snippet: Antiapoptotic BCL-2 family proteins block apoptosis and promote tumor formation. ( A ) Generation of stable cell lines. Cell extracts made from stable BMK cells that express both BAX and BAK (W2), or that are deficient for BAX and BAK (D3), were subjected to Western blotting with antibodies specific for BCL-2 ( left top panel) or E1B 19K ( right top panel). Note the similar expression levels of each exogenous protein in three independent clones (depicted numerically) and undetectable levels of each exogenous protein in the vector-only control cell lines (W2.3.1–2,5,6 or D3.zeo-1,2,3). Blots were then reprobed with an antibody to actin to verify nearly equivalent levels of protein in all lanes, shown below the BCL-2 and E1B 19K panels. ( B ) BCL-2 and E1B 19K block apoptosis in response to staurosporine. Stable BMK cell lines expressing BCL-2, E1B 19K, and controls were treated with media alone (open bars) or media containing 0.4 μM staurosporine (filled bars) for 24 h, and the viable cell number was determined by trypan blue exclusion. Results are presented as the percent of viable cells in each condition, which in each case represents the average of three independent plates. ( C ) BCL-2 and E1B 19K antagonize BAX and BAK to promote tumor formation. Three independent stable BMK cell lines (circles, squares, and diamonds) expressing BCL-2 (green symbols), E1B 19K (blue symbols), or controls (red symbols) were injected subcutaneously into nude mice, and tumor formation was monitored over time. Each point represents the average tumor volume for five injected animals. W2 cells, which express both BAX and BAK, are shown in the left panel. D3 cells, which are deficient for both BAX and BAK, are shown in the right panel. Note that BCL-2 or E1B 19K expression caused a profound acceleration of tumor formation in the W2 cells, whereas the kinetics of tumor formation in the D3 cells, which are deficient for both BAX and BAK, were unchanged by BCL-2 or E1B 19K expression.

    Techniques Used: Blocking Assay, Stable Transfection, Western Blot, Expressing, Clone Assay, Plasmid Preparation, Injection, Mouse Assay

    13) Product Images from "Proteomic and functional evidence for a P2X7 receptor signalling complex"

    Article Title: Proteomic and functional evidence for a P2X7 receptor signalling complex

    Journal: The EMBO Journal

    doi: 10.1093/emboj/20.22.6347

    Fig. 2. Tyrosine phosphorylation of P2X 7 receptor. ( A ) Membrane extracts from untransfected (lane 1, negative control) or P2X 7 receptor-expressing HEK293 cells were immunoprecipitated with ecto-P2X 7 Ab, and phosphotyrosine incorporation into the P2X 7 protein was detected by probing with anti-phosphotyrosine Ab PY20. The blot was then stripped and re-probed with C-terminal anti-P2X 7 Ab to confirm that the phosphotyrosine bands (lanes 3, 4 and 6) were the P2X 7 receptor. No tyrosine phosphorylation was detected in control P2X 7 -expressing cells (lanes 2 and 5) but after 10 min incubation with the tyrosine phosphatase inhibitor bpV (100 µM) phosphotyrosine was clearly detected (lanes 3 and 6). The level was much reduced if the P2X 7 receptor was activated for 10 min with BzATP (100 µM) prior to incubation with bpV (lane 4). ( B ) Similar experiments using phosphatase inhibitors mpV (100 µM, lanes 5 and 6) or 3,4-dephostatin (100 µM, lanes 10 and 11) also show tyrosine phosphorylation of P2X 7 subunit in the presence of the phosphatase inhibitors (lanes 5 and 10), which is reduced when BzATP is added prior to application of phosphatase inhibitor (lane 11), but not when BzATP is added after the phosphatase inhibitor (lanes 3 and 6). ( C ) Tyrosine phosphorylation occurs on P2X 7 receptor but not P2X 2 receptor. Similar experiment to others performed on HEK cells transiently transfected with P2X 7 -EE (lanes 1 and 2) or P2X 2 -EE (lanes 3 and 4) receptors. Immunoprecipitation was with anti-EE Ab in the absence or presence of bpV as indicated. Anti-PY20 blotting detected phosphotyrosine-P2X 7 after bpV treatment (b, lane 2) but no tyrosine phosphorylation of P2X 2 receptor (b, lanes 3 and 4). Stripping and re-probing with C-terminal anti-P2X 7 (a, lanes 1 and 2) or anti-P2X 2 (a, lanes 3 and 4) confirms tyrosine phosphorylation of P2X 7 but not P2X 2 receptor even though there is greater expression of P2X 2 protein.
    Figure Legend Snippet: Fig. 2. Tyrosine phosphorylation of P2X 7 receptor. ( A ) Membrane extracts from untransfected (lane 1, negative control) or P2X 7 receptor-expressing HEK293 cells were immunoprecipitated with ecto-P2X 7 Ab, and phosphotyrosine incorporation into the P2X 7 protein was detected by probing with anti-phosphotyrosine Ab PY20. The blot was then stripped and re-probed with C-terminal anti-P2X 7 Ab to confirm that the phosphotyrosine bands (lanes 3, 4 and 6) were the P2X 7 receptor. No tyrosine phosphorylation was detected in control P2X 7 -expressing cells (lanes 2 and 5) but after 10 min incubation with the tyrosine phosphatase inhibitor bpV (100 µM) phosphotyrosine was clearly detected (lanes 3 and 6). The level was much reduced if the P2X 7 receptor was activated for 10 min with BzATP (100 µM) prior to incubation with bpV (lane 4). ( B ) Similar experiments using phosphatase inhibitors mpV (100 µM, lanes 5 and 6) or 3,4-dephostatin (100 µM, lanes 10 and 11) also show tyrosine phosphorylation of P2X 7 subunit in the presence of the phosphatase inhibitors (lanes 5 and 10), which is reduced when BzATP is added prior to application of phosphatase inhibitor (lane 11), but not when BzATP is added after the phosphatase inhibitor (lanes 3 and 6). ( C ) Tyrosine phosphorylation occurs on P2X 7 receptor but not P2X 2 receptor. Similar experiment to others performed on HEK cells transiently transfected with P2X 7 -EE (lanes 1 and 2) or P2X 2 -EE (lanes 3 and 4) receptors. Immunoprecipitation was with anti-EE Ab in the absence or presence of bpV as indicated. Anti-PY20 blotting detected phosphotyrosine-P2X 7 after bpV treatment (b, lane 2) but no tyrosine phosphorylation of P2X 2 receptor (b, lanes 3 and 4). Stripping and re-probing with C-terminal anti-P2X 7 (a, lanes 1 and 2) or anti-P2X 2 (a, lanes 3 and 4) confirms tyrosine phosphorylation of P2X 7 but not P2X 2 receptor even though there is greater expression of P2X 2 protein.

    Techniques Used: Negative Control, Expressing, Immunoprecipitation, Incubation, Transfection, Stripping Membranes

    14) Product Images from "A Novel Bivalent Vaccine Based on a PB2-Knockout Influenza Virus Protects Mice from Pandemic H1N1 and Highly Pathogenic H5N1 Virus Challenges"

    Article Title: A Novel Bivalent Vaccine Based on a PB2-Knockout Influenza Virus Protects Mice from Pandemic H1N1 and Highly Pathogenic H5N1 Virus Challenges

    Journal: Journal of Virology

    doi: 10.1128/JVI.00076-13

    Characterization of PR8/PB2-Ca04HA and PR8/PB2-VN1203HA virus. (A) Schematic diagrams of wild-type PB2, PB2(120)Ca04HA(336), and PB2(120)VN1203HA(336) vRNAs. PB2(120)Ca04HA(336) and PB2(120)VN1203HA(336) vRNAs possess the 3′ noncoding region, 120 nucleotides (nt) of the coding sequence of PB2 vRNA, the Ca04HA or the VN1203HA gene, and 336 nt of the 3′ and 5′ noncoding regions of PB2 vRNA. The noncoding region and coding regions of PB2 vRNA are represented by shaded and filled bars, respectively. (B) Growth kinetics of PR8/PB2–Ca04HA or PR8/PB2-VN1203HA virus. AX4 and AX4/PB2 cells were infected with PR8, PR8/PB2–Ca04HA, or PR8/PB2–VN1203HA virus at an MOI of 0.001. Supernatant were collected at 12 h, 24 h, 36 h, 48 h, and 72 h postinfection for virus titration by plaque assays in AX4/PB2 cells. (C and D) HA expression in AX4/PB2 cells infected with PR8/PB2-Ca04HA or PR8/PB2-VN1203HA virus. (C) AX4 and AX4/PB2 cells were mock infected or infected with PR8, PR8/PB2-Ca04HA (left panels), or PR8/PB2-VN1203HA (right panels) virus at an MOI of 5. At 24 h after infection, the cells were collected and Ca04HA (left), VN1203 HA (right), M1, and beta-actin were detected by Western blotting with specific antibodies. (D) AX4 and AX4/PB2 cells were mock infected or infected with PR8, PR8/PB2-Ca04HA (left panels), or PR8/PB2-VN1203HA (right panels) virus. At 48 h after infection, the cells were fixed and stained with anti-influenza virus polyclonal (R309), -H1N1pdm09 HA monoclonal (9C4C11), and -H5 HA monoclonal (9B2) antibodies.
    Figure Legend Snippet: Characterization of PR8/PB2-Ca04HA and PR8/PB2-VN1203HA virus. (A) Schematic diagrams of wild-type PB2, PB2(120)Ca04HA(336), and PB2(120)VN1203HA(336) vRNAs. PB2(120)Ca04HA(336) and PB2(120)VN1203HA(336) vRNAs possess the 3′ noncoding region, 120 nucleotides (nt) of the coding sequence of PB2 vRNA, the Ca04HA or the VN1203HA gene, and 336 nt of the 3′ and 5′ noncoding regions of PB2 vRNA. The noncoding region and coding regions of PB2 vRNA are represented by shaded and filled bars, respectively. (B) Growth kinetics of PR8/PB2–Ca04HA or PR8/PB2-VN1203HA virus. AX4 and AX4/PB2 cells were infected with PR8, PR8/PB2–Ca04HA, or PR8/PB2–VN1203HA virus at an MOI of 0.001. Supernatant were collected at 12 h, 24 h, 36 h, 48 h, and 72 h postinfection for virus titration by plaque assays in AX4/PB2 cells. (C and D) HA expression in AX4/PB2 cells infected with PR8/PB2-Ca04HA or PR8/PB2-VN1203HA virus. (C) AX4 and AX4/PB2 cells were mock infected or infected with PR8, PR8/PB2-Ca04HA (left panels), or PR8/PB2-VN1203HA (right panels) virus at an MOI of 5. At 24 h after infection, the cells were collected and Ca04HA (left), VN1203 HA (right), M1, and beta-actin were detected by Western blotting with specific antibodies. (D) AX4 and AX4/PB2 cells were mock infected or infected with PR8, PR8/PB2-Ca04HA (left panels), or PR8/PB2-VN1203HA (right panels) virus. At 48 h after infection, the cells were fixed and stained with anti-influenza virus polyclonal (R309), -H1N1pdm09 HA monoclonal (9C4C11), and -H5 HA monoclonal (9B2) antibodies.

    Techniques Used: Sequencing, Infection, Titration, Expressing, Western Blot, Staining

    15) Product Images from "Trans-arachidonic acids generated during nitrative stress induce a thrombospondin-1-dependent microvascular degeneration"

    Article Title: Trans-arachidonic acids generated during nitrative stress induce a thrombospondin-1-dependent microvascular degeneration

    Journal:

    doi: 10.1038/nm1336

    TAA-induced upregulation of TSP-1 in vivo and importance in hyperoxia-induced retinal microvascular degeneration. ( a ) Representative western blots of TSP-1 and VEGFR2 expression in rat pup retinas injected with TAAs (left). TSP-1 was localized in microvessels
    Figure Legend Snippet: TAA-induced upregulation of TSP-1 in vivo and importance in hyperoxia-induced retinal microvascular degeneration. ( a ) Representative western blots of TSP-1 and VEGFR2 expression in rat pup retinas injected with TAAs (left). TSP-1 was localized in microvessels

    Techniques Used: In Vivo, Western Blot, Expressing, Injection

    TAAs induce endothelial cell death by an ERK 1/2-dependent upregulation of TSP-1. ( a ) Representative western blot of TSP-1 expression in endothelial cells exposed to each TAA for 18 h (left), and of TSP-1 and VEGFR2 immunoreactivity in response to 14
    Figure Legend Snippet: TAAs induce endothelial cell death by an ERK 1/2-dependent upregulation of TSP-1. ( a ) Representative western blot of TSP-1 expression in endothelial cells exposed to each TAA for 18 h (left), and of TSP-1 and VEGFR2 immunoreactivity in response to 14

    Techniques Used: Western Blot, Expressing

    TAAs induce a TSP-1–dependent microvascular degeneration and inhibit of angiogenesis in tissue explants. ( a ) Representative retinal explants from newborn pigs treated with 14 E -AA in the presence or absence of TSP-1– (TSP-1 Ab ) and CD36-specific
    Figure Legend Snippet: TAAs induce a TSP-1–dependent microvascular degeneration and inhibit of angiogenesis in tissue explants. ( a ) Representative retinal explants from newborn pigs treated with 14 E -AA in the presence or absence of TSP-1– (TSP-1 Ab ) and CD36-specific

    Techniques Used:

    16) Product Images from "Glycerol-3-Phosphate Acyltransferase-2 Is Expressed in Spermatic Germ Cells and Incorporates Arachidonic Acid into Triacylglycerols"

    Article Title: Glycerol-3-Phosphate Acyltransferase-2 Is Expressed in Spermatic Germ Cells and Incorporates Arachidonic Acid into Triacylglycerols

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042986

    GPAT2 overexpression increased TAG storage in CHO-K1 cells. CHO-K1 cells were transiently transfected with pcDNA3.1 empty vector (control), pcDNA3.1-GPAT1 (GPAT1) or pcDNA3.1-GPAT2 (GPAT2) constructs tagged with a FLAG epitope (Lanes 1–3 and 4–6 correspond to two different transient transfections). The expression of GPAT1 and GPAT2 was confirmed by western blot. Total particulate protein (50 µg) from GPAT1, GPAT2 and control cells was probed with anti-FLAG (A) and anti-GPAT2 (B) antibodies. The molecular mass of the expressed protein was 90 kDa (GPAT1) and 80 kDa (GPAT2). The membranes were probed with anti-β-actin antibody as a loading control. C) Lipid droplets were visualized in control, GPAT1, and GPAT2-overexpressing CHO-K1 cells by Oil-Red O staining. D) The average size of cellular lipid droplets and the average number of lipid droplets in each cell were quantified by Image Pro plus v5.1 software. Data represent mean ± SD of three independent experiments. (* p
    Figure Legend Snippet: GPAT2 overexpression increased TAG storage in CHO-K1 cells. CHO-K1 cells were transiently transfected with pcDNA3.1 empty vector (control), pcDNA3.1-GPAT1 (GPAT1) or pcDNA3.1-GPAT2 (GPAT2) constructs tagged with a FLAG epitope (Lanes 1–3 and 4–6 correspond to two different transient transfections). The expression of GPAT1 and GPAT2 was confirmed by western blot. Total particulate protein (50 µg) from GPAT1, GPAT2 and control cells was probed with anti-FLAG (A) and anti-GPAT2 (B) antibodies. The molecular mass of the expressed protein was 90 kDa (GPAT1) and 80 kDa (GPAT2). The membranes were probed with anti-β-actin antibody as a loading control. C) Lipid droplets were visualized in control, GPAT1, and GPAT2-overexpressing CHO-K1 cells by Oil-Red O staining. D) The average size of cellular lipid droplets and the average number of lipid droplets in each cell were quantified by Image Pro plus v5.1 software. Data represent mean ± SD of three independent experiments. (* p

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Construct, FLAG-tag, Expressing, Western Blot, Staining, Software

    17) Product Images from "Beneficial Effect of Shikonin on Experimental Colitis Induced by Dextran Sulfate Sodium in Balb/C Mice"

    Article Title: Beneficial Effect of Shikonin on Experimental Colitis Induced by Dextran Sulfate Sodium in Balb/C Mice

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2012/271606

    Effects of shikonin on cyclooxygenase-2 expression and on nuclear translocation of p65 and phosphorylated signal transducer and activator of transcription 3. The left panels show an example of western blot following probing with the corresponding antibody. The histograms at the right represent the data derived from the western blots following densitometry analysis. Levels were normalized against  β -actin or PARP-1 antibody. ** P
    Figure Legend Snippet: Effects of shikonin on cyclooxygenase-2 expression and on nuclear translocation of p65 and phosphorylated signal transducer and activator of transcription 3. The left panels show an example of western blot following probing with the corresponding antibody. The histograms at the right represent the data derived from the western blots following densitometry analysis. Levels were normalized against β -actin or PARP-1 antibody. ** P

    Techniques Used: Expressing, Translocation Assay, Western Blot, Derivative Assay

    Expression of proinflammatory genes in peritoneal macrophages isolated from Balb/C mice. Proinflammatory gene expression levels were measured using RT-PCR, as described in  Section 2 . Representative photographs from ten independent experiments with each gene are shown.  β -Actin served as the internal control. The expression seen in the lipopolysaccharide-stimulated group (control) was standardized as 100% expression. * P
    Figure Legend Snippet: Expression of proinflammatory genes in peritoneal macrophages isolated from Balb/C mice. Proinflammatory gene expression levels were measured using RT-PCR, as described in Section 2 . Representative photographs from ten independent experiments with each gene are shown. β -Actin served as the internal control. The expression seen in the lipopolysaccharide-stimulated group (control) was standardized as 100% expression. * P

    Techniques Used: Expressing, Isolation, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

    18) Product Images from "Plasma membrane calcium ATPase regulates bone mass by fine-tuning osteoclast differentiation and survival"

    Article Title: Plasma membrane calcium ATPase regulates bone mass by fine-tuning osteoclast differentiation and survival

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201204067

    PMCA reduced the RANKL-dependent Ca 2+ oscillations and subsequent NFATc1 nuclear localization in preosteoclasts. (A–C) BMMs on glass coverslips were transfected with scrambled control siRNA or PMCA siRNA and further incubated for 2 d with 30 ng/ml M-CSF and 100 ng/ml RANKL. (A) Cells were loaded with Fura-2/AM for the recording of Ca 2+ oscillations (presented as a ratio of maximal fluorescence) in individual preosteoclast (BMMs treated with RANKL for 2 d). Each colored line represents Ca 2+ oscillations in a single cell. 10 µM ionomycin was added at the end of the experiment (arrow) to determine the maximal Ca 2+ -induced fluorescence. (B and C) Cells were stained with NFATc1 antibody (FITC labeled) and lamin B antibody (Cy3 labeled) to detect the nuclear localization of NFATc1 (arrows). Treatment of 1 µM cyclosporineA (CyA) for 3 h induced exclusive translocation of NFATc1 to the cytosol (arrowheads). Bars, 50 µm. (D) NFATc1 localization was examined biochemically by subjecting nuclear fractions from the cells treated as in B to Western blotting. (E and F) PMCA1 (pcDNA-rPMCA1) or PMCA4 (pMX-PMCA4) were overexpressed in BMMs. (E) Ca 2+ oscillations were monitored in BMMs after RANKL treatment for 2 d (top). TRAP staining was performed after culturing the cells for 4 d (bottom). Bars, 200 µm. (F) PMCA1 or PMCA4 expression levels were examined by Western blotting after culturing the cells for 2 d. (G) The number of TRAP-positive multinucleated cells was counted from E. Data are means ± SD, representative of more than three experiments performed in triplicate (**, P
    Figure Legend Snippet: PMCA reduced the RANKL-dependent Ca 2+ oscillations and subsequent NFATc1 nuclear localization in preosteoclasts. (A–C) BMMs on glass coverslips were transfected with scrambled control siRNA or PMCA siRNA and further incubated for 2 d with 30 ng/ml M-CSF and 100 ng/ml RANKL. (A) Cells were loaded with Fura-2/AM for the recording of Ca 2+ oscillations (presented as a ratio of maximal fluorescence) in individual preosteoclast (BMMs treated with RANKL for 2 d). Each colored line represents Ca 2+ oscillations in a single cell. 10 µM ionomycin was added at the end of the experiment (arrow) to determine the maximal Ca 2+ -induced fluorescence. (B and C) Cells were stained with NFATc1 antibody (FITC labeled) and lamin B antibody (Cy3 labeled) to detect the nuclear localization of NFATc1 (arrows). Treatment of 1 µM cyclosporineA (CyA) for 3 h induced exclusive translocation of NFATc1 to the cytosol (arrowheads). Bars, 50 µm. (D) NFATc1 localization was examined biochemically by subjecting nuclear fractions from the cells treated as in B to Western blotting. (E and F) PMCA1 (pcDNA-rPMCA1) or PMCA4 (pMX-PMCA4) were overexpressed in BMMs. (E) Ca 2+ oscillations were monitored in BMMs after RANKL treatment for 2 d (top). TRAP staining was performed after culturing the cells for 4 d (bottom). Bars, 200 µm. (F) PMCA1 or PMCA4 expression levels were examined by Western blotting after culturing the cells for 2 d. (G) The number of TRAP-positive multinucleated cells was counted from E. Data are means ± SD, representative of more than three experiments performed in triplicate (**, P

    Techniques Used: Transfection, Incubation, Fluorescence, Staining, Labeling, Translocation Assay, Western Blot, Expressing

    19) Product Images from "Lewis y antigen promotes the proliferation of ovarian carcinoma-derived RMG-I cells through the PI3K/Akt signaling pathway"

    Article Title: Lewis y antigen promotes the proliferation of ovarian carcinoma-derived RMG-I cells through the PI3K/Akt signaling pathway

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/1756-9966-28-154

    Effects of α-L-fucosidase on the proliferation of the cells before and after the transfection . (A) The cell growth curves of each group before and after the process by α-L-fucosidase (B) The colony formation rates of each group before and after the process by α-L-fucosidase. * p
    Figure Legend Snippet: Effects of α-L-fucosidase on the proliferation of the cells before and after the transfection . (A) The cell growth curves of each group before and after the process by α-L-fucosidase (B) The colony formation rates of each group before and after the process by α-L-fucosidase. * p

    Techniques Used: Transfection

    20) Product Images from "Protective Roles of Gadd45 and MDM2 in Blueberry Anthocyanins Mediated DNA Repair of Fragmented and Non-Fragmented DNA Damage in UV-Irradiated HepG2 Cells"

    Article Title: Protective Roles of Gadd45 and MDM2 in Blueberry Anthocyanins Mediated DNA Repair of Fragmented and Non-Fragmented DNA Damage in UV-Irradiated HepG2 Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms141121447

    Western blot detection of Gadd45, MDM2, p53 and p21 protein expression in UV-irradiated and BA pre-treated HepG2 cells. ( a ) Western blotting with HepG2 cells that were treated with or without BA, irradiated or non irradiated with a UV dose of 30 mJ/cm 2 and harvested at the indicated times following treatment; ( b ) The relative expression of Gadd45 value after normalizing to β-actin; ( c ) The relative expression of MDM2 value after normalizing to β-actin; ( d ) The relative expression of p21 value after normalizing to β-actin; ( e ) The relative expression of p53 value after normalizing to β-actin. * p
    Figure Legend Snippet: Western blot detection of Gadd45, MDM2, p53 and p21 protein expression in UV-irradiated and BA pre-treated HepG2 cells. ( a ) Western blotting with HepG2 cells that were treated with or without BA, irradiated or non irradiated with a UV dose of 30 mJ/cm 2 and harvested at the indicated times following treatment; ( b ) The relative expression of Gadd45 value after normalizing to β-actin; ( c ) The relative expression of MDM2 value after normalizing to β-actin; ( d ) The relative expression of p21 value after normalizing to β-actin; ( e ) The relative expression of p53 value after normalizing to β-actin. * p

    Techniques Used: Western Blot, Expressing, Irradiation

    Gene expression of Gadd45 and MDM2 by RT-PCR in UV-irradiated HepG2 cells that were pre-treated for 12 h with BA. ( a ) Lane M, marker; lane 1 control (no UV radiation); lane 2, UV radiation; lane 3, UV+ 25 μg/mL; lane 4, UV+ 50 μg/mL; lane 5, UV+ 75 μg/mL; ( b ) The relative expression of Gadd45 and MDM2 value after normalizing to β-actin. * p
    Figure Legend Snippet: Gene expression of Gadd45 and MDM2 by RT-PCR in UV-irradiated HepG2 cells that were pre-treated for 12 h with BA. ( a ) Lane M, marker; lane 1 control (no UV radiation); lane 2, UV radiation; lane 3, UV+ 25 μg/mL; lane 4, UV+ 50 μg/mL; lane 5, UV+ 75 μg/mL; ( b ) The relative expression of Gadd45 and MDM2 value after normalizing to β-actin. * p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Irradiation, Marker

    21) Product Images from "MSH3 Polymorphisms and Protein Levels Affect CAG Repeat Instability in Huntington's Disease Mice"

    Article Title: MSH3 Polymorphisms and Protein Levels Affect CAG Repeat Instability in Huntington's Disease Mice

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003280

    Western blot analysis of MMR and DHFR protein levels. MMR expression in liver and striatum from 4 and 16 week-old mouse. Actin was used as a loading control. MSH2: 104 kD, MSH6: 160 kD, MSH3: 127 kD (Ab = 2F11) and actin: 42 kD. DHFR expression in cortex from 4 and 16 week-old mice DHFR: 21 kD. A) Simultaneous Western blot using MSH2-, MSH3-, MSH6- and actin-specific antibodies in liver. For antibody dilutions see Materials and methods . B) Western blot using only anti-MSH3 (Ab = 2F11) and actin antibodies in liver and striatum. C) Western blot analysis of DHFR in cortex from 4 and 16 week-old mice.
    Figure Legend Snippet: Western blot analysis of MMR and DHFR protein levels. MMR expression in liver and striatum from 4 and 16 week-old mouse. Actin was used as a loading control. MSH2: 104 kD, MSH6: 160 kD, MSH3: 127 kD (Ab = 2F11) and actin: 42 kD. DHFR expression in cortex from 4 and 16 week-old mice DHFR: 21 kD. A) Simultaneous Western blot using MSH2-, MSH3-, MSH6- and actin-specific antibodies in liver. For antibody dilutions see Materials and methods . B) Western blot using only anti-MSH3 (Ab = 2F11) and actin antibodies in liver and striatum. C) Western blot analysis of DHFR in cortex from 4 and 16 week-old mice.

    Techniques Used: Western Blot, Expressing, Mouse Assay

    22) Product Images from "Antagonism of Bradykinin B2 Receptor Prevents Inflammatory Responses in Human Endothelial Cells by Quenching the NF-kB Pathway Activation"

    Article Title: Antagonism of Bradykinin B2 Receptor Prevents Inflammatory Responses in Human Endothelial Cells by Quenching the NF-kB Pathway Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0084358

    BK stimulates translocation/phosphorylation of NF-κB in circulating proangiogenic cells. (A) p65 (NF-κB) phosphorylation following exposure to BK (1 µM, 15 min) in presence/absence of fasitibant (0.1 µM). Gel are representative of three experiments. The ratio between p-p65 over p65 is reported. *p
    Figure Legend Snippet: BK stimulates translocation/phosphorylation of NF-κB in circulating proangiogenic cells. (A) p65 (NF-κB) phosphorylation following exposure to BK (1 µM, 15 min) in presence/absence of fasitibant (0.1 µM). Gel are representative of three experiments. The ratio between p-p65 over p65 is reported. *p

    Techniques Used: Translocation Assay

    23) Product Images from "Netrin-1 protects hypoxia-induced mitochondrial apoptosis through HSP27 expression via DCC- and integrin α6β4-dependent Akt, GSK-3β, and HSF-1 in mesenchymal stem cells"

    Article Title: Netrin-1 protects hypoxia-induced mitochondrial apoptosis through HSP27 expression via DCC- and integrin α6β4-dependent Akt, GSK-3β, and HSF-1 in mesenchymal stem cells

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.94

    Hypothetical model of Ntn-1-related anti-apoptotic effect. Ntn-1 increased APPL-1/Akt2 complex formation and Akt phosphorylation via lipid raft-independent DCC and -dependent IN α 6 β 4. This signal increased GSK-3 β phosphorylation and HSF-1 expression, and subsequently increased HSP27 expression. Finally, Ntn-1-induced HSP27 inhibited Bax expression and cyt c release thereby contributing to the protecting of hypoxia-induced UCB-MSC apoptosis
    Figure Legend Snippet: Hypothetical model of Ntn-1-related anti-apoptotic effect. Ntn-1 increased APPL-1/Akt2 complex formation and Akt phosphorylation via lipid raft-independent DCC and -dependent IN α 6 β 4. This signal increased GSK-3 β phosphorylation and HSF-1 expression, and subsequently increased HSP27 expression. Finally, Ntn-1-induced HSP27 inhibited Bax expression and cyt c release thereby contributing to the protecting of hypoxia-induced UCB-MSC apoptosis

    Techniques Used: Droplet Countercurrent Chromatography, Expressing

    Involvement of DCC/caspase-3, APPL-1/Akt2 complexes and Akt phosphorylation. ( a and b ) Cells were pretreated with Ntn-1 (10 ng/ml) or DCC function-blocking antibody (2.5 μ l/ml) for 30 min prior to 72 h incubation in hypoxic condition. Cell lysates were analyzed by western blotting with antibodies that recognize caspase-3 or APPL-1. Immunoprecipitation of anti-DCC was analyzed by western blotting with antibodies that recognize caspase-3 or APPL-1. ( c ) Cells were pretreated with Ntn-1 (10 ng/ml) for 30 min prior to 72 h incubation in hypoxic condition. Cell lysates were analyzed by western blotting with antibody that recognize Akt2. Immunoprecipitation of anti-APPL-1 was analyzed by western blotting with antibody that recognize Akt2. ( d and e ) Cells were pretreated with DCC function-blocking antibody (2.5 μ l/ml), or combination of INs α 6 and β 4 (2.5 μ l/ml) for 30 min prior to a 30-min Ntn-1 (10 ng/ml) treatment. And then, the cell incubated prior to 72 h in hypoxic condition. Total protein was extracted and blotted with phospho-Akt thr308 , phospho-Akt ser473 , or Akt antibody. ( a – e ) Each of the examples is representative of four independent experiments. The right or lower part ( a – e ) depicting the bars denotes the mean±S.E. of four independent experiments for each condition determined from densitometry relative to β -actin or total Akt. * P
    Figure Legend Snippet: Involvement of DCC/caspase-3, APPL-1/Akt2 complexes and Akt phosphorylation. ( a and b ) Cells were pretreated with Ntn-1 (10 ng/ml) or DCC function-blocking antibody (2.5 μ l/ml) for 30 min prior to 72 h incubation in hypoxic condition. Cell lysates were analyzed by western blotting with antibodies that recognize caspase-3 or APPL-1. Immunoprecipitation of anti-DCC was analyzed by western blotting with antibodies that recognize caspase-3 or APPL-1. ( c ) Cells were pretreated with Ntn-1 (10 ng/ml) for 30 min prior to 72 h incubation in hypoxic condition. Cell lysates were analyzed by western blotting with antibody that recognize Akt2. Immunoprecipitation of anti-APPL-1 was analyzed by western blotting with antibody that recognize Akt2. ( d and e ) Cells were pretreated with DCC function-blocking antibody (2.5 μ l/ml), or combination of INs α 6 and β 4 (2.5 μ l/ml) for 30 min prior to a 30-min Ntn-1 (10 ng/ml) treatment. And then, the cell incubated prior to 72 h in hypoxic condition. Total protein was extracted and blotted with phospho-Akt thr308 , phospho-Akt ser473 , or Akt antibody. ( a – e ) Each of the examples is representative of four independent experiments. The right or lower part ( a – e ) depicting the bars denotes the mean±S.E. of four independent experiments for each condition determined from densitometry relative to β -actin or total Akt. * P

    Techniques Used: Droplet Countercurrent Chromatography, Blocking Assay, Incubation, Western Blot, Immunoprecipitation

    Involvement of DCC, IN α 6 β 4, Akt, GSK-3 β , HSF-1, HSP27, and HSP70 on Ntn-1-induced protection of apoptosis in hypoxic condition. Cells were pretreated with DCC- and IN α 6 β 4 function-blocking antibodies, combination of DCC and IN α 6 β 4 function-blocking antibodies, Akt inhibitor, HSF-1 -, HSP27 -, HSP70 -specific siRNA, and non-targeting control siRNA for 30 min or 24 h prior to hypoxia with Ntn-1 exposure for 72 h. ( a ) Cells were analyzed for their viability by MTT assay. The values are reported as a mean±S.E. of three independent experiments with triplicate dishes. * P
    Figure Legend Snippet: Involvement of DCC, IN α 6 β 4, Akt, GSK-3 β , HSF-1, HSP27, and HSP70 on Ntn-1-induced protection of apoptosis in hypoxic condition. Cells were pretreated with DCC- and IN α 6 β 4 function-blocking antibodies, combination of DCC and IN α 6 β 4 function-blocking antibodies, Akt inhibitor, HSF-1 -, HSP27 -, HSP70 -specific siRNA, and non-targeting control siRNA for 30 min or 24 h prior to hypoxia with Ntn-1 exposure for 72 h. ( a ) Cells were analyzed for their viability by MTT assay. The values are reported as a mean±S.E. of three independent experiments with triplicate dishes. * P

    Techniques Used: Droplet Countercurrent Chromatography, Blocking Assay, MTT Assay

    24) Product Images from "Cinnamic acid induces apoptotic cell death and cytoskeleton disruption in human melanoma cells"

    Article Title: Cinnamic acid induces apoptotic cell death and cytoskeleton disruption in human melanoma cells

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/1756-9966-32-31

    Cytoskeleton organization in NGM cells treated with 3.2 mM cinnamic acid. The cells were treated with the drug for 48 hours. F-actin (green) was stained with phalloidin FITC-conjugated. Microtubules (blue) were labeled with anti-α and β tubulin and secondary antibody CY-5-conjugated. DNA was counterstained with propidium iodide (red). The images were obtained by Laser Scanning Confocal Microscopy. Note that there are cells with normal cytoskeletal organization (left column) and cells with drastic morphological changes (intermediate and right columns).
    Figure Legend Snippet: Cytoskeleton organization in NGM cells treated with 3.2 mM cinnamic acid. The cells were treated with the drug for 48 hours. F-actin (green) was stained with phalloidin FITC-conjugated. Microtubules (blue) were labeled with anti-α and β tubulin and secondary antibody CY-5-conjugated. DNA was counterstained with propidium iodide (red). The images were obtained by Laser Scanning Confocal Microscopy. Note that there are cells with normal cytoskeletal organization (left column) and cells with drastic morphological changes (intermediate and right columns).

    Techniques Used: Staining, Labeling, Confocal Microscopy

    M30 and tubulin labeling in HT-144 cells. HT-144 cells were treated with 0.4 or 3.2 mM cinnamic acid for 24 or 48 hours. Fragmented cytokeratin 18 (green) were labeled with M30 antibody FITC and microtubules (blue) were labeled with anti-α and β tubulin and secondary antibody TRITC-conjugated. A,B) cells with intact microtubules and M30(+); C) cells with microtubule disruption and M30(+); D) cells with microtubule disruption and M30(–). Arrows = M30 staining. The results demonstrate that cell death and microtubule disorganization are independent events in our system. The images were obtained by Laser Scanning Confocal Microscopy.
    Figure Legend Snippet: M30 and tubulin labeling in HT-144 cells. HT-144 cells were treated with 0.4 or 3.2 mM cinnamic acid for 24 or 48 hours. Fragmented cytokeratin 18 (green) were labeled with M30 antibody FITC and microtubules (blue) were labeled with anti-α and β tubulin and secondary antibody TRITC-conjugated. A,B) cells with intact microtubules and M30(+); C) cells with microtubule disruption and M30(+); D) cells with microtubule disruption and M30(–). Arrows = M30 staining. The results demonstrate that cell death and microtubule disorganization are independent events in our system. The images were obtained by Laser Scanning Confocal Microscopy.

    Techniques Used: Labeling, Staining, Confocal Microscopy

    Cytoskeleton organization in NGM control cells. F-actin (green) was stained with phalloidin FITC-conjugated. Microtubules (blue) were labeled with anti-α and β tubulin and secondary antibody CY-5-conjugated. DNA was counterstained with propidium iodide (red). Note the stress fiber formation (actin filaments). The cells showed a microtubule network that was very finely departed from the centrosome region near the nucleus. We can also observe a mitotic cell (right column). The images were obtained by Laser Scanning Confocal Microscopy.
    Figure Legend Snippet: Cytoskeleton organization in NGM control cells. F-actin (green) was stained with phalloidin FITC-conjugated. Microtubules (blue) were labeled with anti-α and β tubulin and secondary antibody CY-5-conjugated. DNA was counterstained with propidium iodide (red). Note the stress fiber formation (actin filaments). The cells showed a microtubule network that was very finely departed from the centrosome region near the nucleus. We can also observe a mitotic cell (right column). The images were obtained by Laser Scanning Confocal Microscopy.

    Techniques Used: Staining, Labeling, Confocal Microscopy

    25) Product Images from "Cyclin E1 is a common target of BMI1 and MYCN and a prognostic marker for neuroblastoma progression"

    Article Title: Cyclin E1 is a common target of BMI1 and MYCN and a prognostic marker for neuroblastoma progression

    Journal: Oncogene

    doi: 10.1038/onc.2011.536

    BMI1 suppresses cell death by stabilizing cyclin E1. a , Immunoblot analysis of cyclins and CDKs in pooled BMI1-sensitive clones following BMI1 knockdown. α-tubulin levels are shown as loading control. b , Immunoblot analysis of cyclin E1 levels in BE(2)-C cells either uninfected (parental) or infected with retroviruses expressing shRNA sequences against GFP (GFPsh) or different regions of CCNE1 (CCNE1sh-80 and -81). Cyclin E1 levels were quantified against α-tubulin and are presented as the fraction of the cyclin E1 level in parental cells. c , Crystal violet staining of BE(2)-C cells expressing either GFPsh or CCNE1sh-80. d , Immunoblot analysis of BMI1 and cyclin E1 levels in pooled BMI1-sensitive clones infected with retroviruses expressing either GFP or Myc-cyclin E1 and cultured in the presence or absence of Doxy for 3 days. α-tubulin levels are shown as loading control. e , Phase contrast imaging of pooled BMI1-sensitive clones expressing either GFP or Myc-cyclin E1 and cultured in the presence or absence of Doxy for 6 days. Scale bars, 100 μm. f , qRT-PCR analysis of CCND1 and CCNE1 mRNA levels in pooled BMI1-sensitive cells cultured in the presence or absence of Doxy for 3 days (error bars, s.d., n=3). g , Quantification of cyclin E1 half-life in pooled BMI1-sensitive clones cultured in the presence or absence of Doxy for 3 days. Samples were collected at various time points following addition of cycloheximide (CHX) for immunoblot analysis. Cyclin E1 levels were quantified against α-tubulin and are presented as the fraction of the initial levels at time zero (error bars, s.d., n=4). h , In vivo ubiquitination assay of pooled BMI1-sensitive cells cultured in the presence or absence of Doxy for 3 days and co-transfected with Flag-ubiquitin and Myc-cyclin E1 expression plasmids. Polyubiquitinated cyclin E1 was detected by immunoprecipitation of Myccyclin E1, followed by immunoblotting for Flag-ubiquitin.
    Figure Legend Snippet: BMI1 suppresses cell death by stabilizing cyclin E1. a , Immunoblot analysis of cyclins and CDKs in pooled BMI1-sensitive clones following BMI1 knockdown. α-tubulin levels are shown as loading control. b , Immunoblot analysis of cyclin E1 levels in BE(2)-C cells either uninfected (parental) or infected with retroviruses expressing shRNA sequences against GFP (GFPsh) or different regions of CCNE1 (CCNE1sh-80 and -81). Cyclin E1 levels were quantified against α-tubulin and are presented as the fraction of the cyclin E1 level in parental cells. c , Crystal violet staining of BE(2)-C cells expressing either GFPsh or CCNE1sh-80. d , Immunoblot analysis of BMI1 and cyclin E1 levels in pooled BMI1-sensitive clones infected with retroviruses expressing either GFP or Myc-cyclin E1 and cultured in the presence or absence of Doxy for 3 days. α-tubulin levels are shown as loading control. e , Phase contrast imaging of pooled BMI1-sensitive clones expressing either GFP or Myc-cyclin E1 and cultured in the presence or absence of Doxy for 6 days. Scale bars, 100 μm. f , qRT-PCR analysis of CCND1 and CCNE1 mRNA levels in pooled BMI1-sensitive cells cultured in the presence or absence of Doxy for 3 days (error bars, s.d., n=3). g , Quantification of cyclin E1 half-life in pooled BMI1-sensitive clones cultured in the presence or absence of Doxy for 3 days. Samples were collected at various time points following addition of cycloheximide (CHX) for immunoblot analysis. Cyclin E1 levels were quantified against α-tubulin and are presented as the fraction of the initial levels at time zero (error bars, s.d., n=4). h , In vivo ubiquitination assay of pooled BMI1-sensitive cells cultured in the presence or absence of Doxy for 3 days and co-transfected with Flag-ubiquitin and Myc-cyclin E1 expression plasmids. Polyubiquitinated cyclin E1 was detected by immunoprecipitation of Myccyclin E1, followed by immunoblotting for Flag-ubiquitin.

    Techniques Used: Clone Assay, Infection, Expressing, shRNA, Staining, Cell Culture, Imaging, Quantitative RT-PCR, In Vivo, Ubiquitin Assay, Transfection, Immunoprecipitation

    26) Product Images from "N-acetylcysteine potentiates doxorubicin-induced ATM and p53 activation in ovarian cancer cells"

    Article Title: N-acetylcysteine potentiates doxorubicin-induced ATM and p53 activation in ovarian cancer cells

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2012.1680

    Effect of NAC on doxorubicin-induced activation of ATM, p53 and H2AX in cultured ovarian cancer cells. CaOV3 cells were cultured in 8-well chamber slides, pretreated with NAC, PDTC, or Wortmannin, for 2 h and then treated with doxorubicin. Cells were fixed 2 h post-doxorubicin treatment. Antibodies were used to detect (A) phospho-ATM, (B) phospho-p53, (C) acetylated p53 and (D) phospho-H2AX. Confocal microscopy was performed.
    Figure Legend Snippet: Effect of NAC on doxorubicin-induced activation of ATM, p53 and H2AX in cultured ovarian cancer cells. CaOV3 cells were cultured in 8-well chamber slides, pretreated with NAC, PDTC, or Wortmannin, for 2 h and then treated with doxorubicin. Cells were fixed 2 h post-doxorubicin treatment. Antibodies were used to detect (A) phospho-ATM, (B) phospho-p53, (C) acetylated p53 and (D) phospho-H2AX. Confocal microscopy was performed.

    Techniques Used: Activation Assay, Cell Culture, Confocal Microscopy

    Effect of doxorubicin on cell viability in cultured ovarian cancer cells. CaOV3 cells were cultured in 96-well plates and treated with various concentrations of doxorubicin. (A) Cells were observed 24 h after treatment under an inverted microscope and (B) cell viability was measured by MTT assay. Cells were treated with doxorubicin at the concentration of 10 μ M and (C) observed at different time points and (D) cell viability was assayed by MTT.
    Figure Legend Snippet: Effect of doxorubicin on cell viability in cultured ovarian cancer cells. CaOV3 cells were cultured in 96-well plates and treated with various concentrations of doxorubicin. (A) Cells were observed 24 h after treatment under an inverted microscope and (B) cell viability was measured by MTT assay. Cells were treated with doxorubicin at the concentration of 10 μ M and (C) observed at different time points and (D) cell viability was assayed by MTT.

    Techniques Used: Cell Culture, Inverted Microscopy, MTT Assay, Concentration Assay

    Effect of doxorubicin on cytoskeletal proteins in cultured ovarian cancer cells. CaOV3 cells were cultured in 8-well chamber slides, treated with 10 μ M o f doxurubicin and fixed at different time points. Tubulin, vimentin and actin filaments were stained with antibodies and visualized under a confocal microscope.
    Figure Legend Snippet: Effect of doxorubicin on cytoskeletal proteins in cultured ovarian cancer cells. CaOV3 cells were cultured in 8-well chamber slides, treated with 10 μ M o f doxurubicin and fixed at different time points. Tubulin, vimentin and actin filaments were stained with antibodies and visualized under a confocal microscope.

    Techniques Used: Cell Culture, Staining, Microscopy

    Effect of doxorubicin on ovarian cancer cell migration. CaOV3 cells were cultured and treated with or without doxorubicin or EGF and cell migration was monitored 24 h after treatment by phagokinetic mobility track assay.
    Figure Legend Snippet: Effect of doxorubicin on ovarian cancer cell migration. CaOV3 cells were cultured and treated with or without doxorubicin or EGF and cell migration was monitored 24 h after treatment by phagokinetic mobility track assay.

    Techniques Used: Migration, Cell Culture

    27) Product Images from "Altered Lipid Homeostasis in Sertoli Cells Stressed by Mild Hyperthermia"

    Article Title: Altered Lipid Homeostasis in Sertoli Cells Stressed by Mild Hyperthermia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0091127

    Effects of hyperthermia on specific functional and structural SC proteins. The depicted proteins were obtained from cells cultured for 5 days at 37°C and from cells cultured similarly but exposed once a day for 15 min to 43°C. A) Intensities of the WT1 and transferrin bands, as normalized with respect to the intensity of β-actin. B) β-actin and α-tubulin levels, as normalized with respect to total cell protein. C) Effects of the repeated brief exposures to 43°C, on α-tubulin microtubules and f -actin networks, as observed by confocal microscopy. The areas indicated by the white rectangles are shown enlarged on the right panels.
    Figure Legend Snippet: Effects of hyperthermia on specific functional and structural SC proteins. The depicted proteins were obtained from cells cultured for 5 days at 37°C and from cells cultured similarly but exposed once a day for 15 min to 43°C. A) Intensities of the WT1 and transferrin bands, as normalized with respect to the intensity of β-actin. B) β-actin and α-tubulin levels, as normalized with respect to total cell protein. C) Effects of the repeated brief exposures to 43°C, on α-tubulin microtubules and f -actin networks, as observed by confocal microscopy. The areas indicated by the white rectangles are shown enlarged on the right panels.

    Techniques Used: Functional Assay, Cell Culture, Confocal Microscopy

    28) Product Images from "The Role of STAT1 for Crosstalk between Fibroblasts and Colon Cancer Cells"

    Article Title: The Role of STAT1 for Crosstalk between Fibroblasts and Colon Cancer Cells

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2014.00088

    Fibroblasts induce STAT1 signaling in tumor cells . (A) HCT116 cells transfected with GAS-LUC were treated as indicated, (B) HCT116 and Hke-3 cells were transfected with GAS-LUC and were co-cultured with 18Co cells, fibroblasts isolated from a normal donor (HIF ND), a patient with ulcerative colitis (HIF UC) or Crohn’s disease (CD). (C) HCT116 and Hke-3 cells were co-cultured with 18Co, HIF, or CD myofibroblasts and the levels of pSTAT1 and IRF1 were determined by immunoblotting. * p =
    Figure Legend Snippet: Fibroblasts induce STAT1 signaling in tumor cells . (A) HCT116 cells transfected with GAS-LUC were treated as indicated, (B) HCT116 and Hke-3 cells were transfected with GAS-LUC and were co-cultured with 18Co cells, fibroblasts isolated from a normal donor (HIF ND), a patient with ulcerative colitis (HIF UC) or Crohn’s disease (CD). (C) HCT116 and Hke-3 cells were co-cultured with 18Co, HIF, or CD myofibroblasts and the levels of pSTAT1 and IRF1 were determined by immunoblotting. * p =

    Techniques Used: Transfection, Cell Culture, Isolation

    29) Product Images from "Stra13 regulates satellite cell activation by antagonizing Notch signaling"

    Article Title: Stra13 regulates satellite cell activation by antagonizing Notch signaling

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200609007

    Stra13 inhibits Notch signaling. (A) 100 ng of a CBF-1 reporter construct was transfected in 10T1/2 cells along with 10 ng N1IC in the absence or presence of increasing amounts of Stra13 (10, 25, 50, and 100 ng). (B) 100 ng Hes-1 promoter was transfected with 10 ng N1IC in the absence or presence of 100 ng Stra13. Reporter assays were repeated at least three times, each with duplicates. Data are means ± SEM (error bars). (C) 10T1/2 cells were transfected with vector (pCS2), Stra13, N1IC, or both N1IC and Stra13. The expression of Stra13, N1IC, and Hes1 was detected by Western blotting (left). Hey1 mRNA level was analyzed by quantitative PCR (right). (D) Control C2C12 cells or stable cell lines expressing Notch (C2C12-N1IC) were stained for MHC 4 d after culturing in differentiation medium. C2C12-N1IC cells were blocked in myogenic differentiation. C2C12-N1IC cells were transduced with an empty retroviral vector (Babe) or one expressing Stra13 and stained for MHC after 4 d in differentiation medium (left). The expression of Notch and Stra13 was detected in N1IC-vector and N1IC-Stra13 cell lines by Western blot analysis (right).
    Figure Legend Snippet: Stra13 inhibits Notch signaling. (A) 100 ng of a CBF-1 reporter construct was transfected in 10T1/2 cells along with 10 ng N1IC in the absence or presence of increasing amounts of Stra13 (10, 25, 50, and 100 ng). (B) 100 ng Hes-1 promoter was transfected with 10 ng N1IC in the absence or presence of 100 ng Stra13. Reporter assays were repeated at least three times, each with duplicates. Data are means ± SEM (error bars). (C) 10T1/2 cells were transfected with vector (pCS2), Stra13, N1IC, or both N1IC and Stra13. The expression of Stra13, N1IC, and Hes1 was detected by Western blotting (left). Hey1 mRNA level was analyzed by quantitative PCR (right). (D) Control C2C12 cells or stable cell lines expressing Notch (C2C12-N1IC) were stained for MHC 4 d after culturing in differentiation medium. C2C12-N1IC cells were blocked in myogenic differentiation. C2C12-N1IC cells were transduced with an empty retroviral vector (Babe) or one expressing Stra13 and stained for MHC after 4 d in differentiation medium (left). The expression of Notch and Stra13 was detected in N1IC-vector and N1IC-Stra13 cell lines by Western blot analysis (right).

    Techniques Used: Construct, Transfection, Plasmid Preparation, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Stable Transfection, Staining, Transduction

    Reexpression of Stra13 in Stra13 −/− myoblasts rescues the proliferation and differentiation defect. Stra13 −/− myoblasts were transduced with retrovirus expressing Stra13 (pBabe-Stra13) or with vector alone (pBabe). After selection, infected cells were either pulsed with BrdU and analyzed for cell proliferation (A) or changed to differentiation medium for 4 d and analyzed for myotube formation by MHC staining (B). The fusion index was calculated as described in Materials and methods. Data are means ± SEM (error bars). * , P
    Figure Legend Snippet: Reexpression of Stra13 in Stra13 −/− myoblasts rescues the proliferation and differentiation defect. Stra13 −/− myoblasts were transduced with retrovirus expressing Stra13 (pBabe-Stra13) or with vector alone (pBabe). After selection, infected cells were either pulsed with BrdU and analyzed for cell proliferation (A) or changed to differentiation medium for 4 d and analyzed for myotube formation by MHC staining (B). The fusion index was calculated as described in Materials and methods. Data are means ± SEM (error bars). * , P

    Techniques Used: Transduction, Expressing, Plasmid Preparation, Selection, Infection, Staining

    30) Product Images from "L-Ascorbate Attenuates the Endotoxin-Induced Production of Inflammatory Mediators by Inhibiting MAPK Activation and NF-?B Translocation in Cortical Neurons/Glia Cocultures"

    Article Title: L-Ascorbate Attenuates the Endotoxin-Induced Production of Inflammatory Mediators by Inhibiting MAPK Activation and NF-?B Translocation in Cortical Neurons/Glia Cocultures

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0097276

    Effects of MAPK inhibitors on the levels of LPS-induced nitrite accumulation and iNOS expression. Cocultured cells were treated for 24 µM of SB203580 (a p38 MAPK inhibitor) or PD98059 (an ERK MAPK inhibitor), which were simultaneously with LPS (100 ng/ml). SB203580 (A) and PD98059 (B) significantly suppressed the LPS-induced accumulation of nitrite and expression of iNOS (C). The data are represented as mean ± SEM based on 3–5 independent experiments. * p
    Figure Legend Snippet: Effects of MAPK inhibitors on the levels of LPS-induced nitrite accumulation and iNOS expression. Cocultured cells were treated for 24 µM of SB203580 (a p38 MAPK inhibitor) or PD98059 (an ERK MAPK inhibitor), which were simultaneously with LPS (100 ng/ml). SB203580 (A) and PD98059 (B) significantly suppressed the LPS-induced accumulation of nitrite and expression of iNOS (C). The data are represented as mean ± SEM based on 3–5 independent experiments. * p

    Techniques Used: Expressing

    MAPK Inhibitors and Vit. C inhibited the LPS-induced phosphorylation of p38 and ERK. Cells were treated for 180(control), LPS (100 ng/ml) alone, or LPS combined with 20 µM of the p38 inhibitor SB203580 or ERK inhibitor PD98059, or 10 mM of Vit. C, and then harvested to conduct western blot analyses of p-p38 (A) and p-ERK (p-p42/44) (B). The data are represented as the mean ± SEM based on 3–4 independent experiments. ** p
    Figure Legend Snippet: MAPK Inhibitors and Vit. C inhibited the LPS-induced phosphorylation of p38 and ERK. Cells were treated for 180(control), LPS (100 ng/ml) alone, or LPS combined with 20 µM of the p38 inhibitor SB203580 or ERK inhibitor PD98059, or 10 mM of Vit. C, and then harvested to conduct western blot analyses of p-p38 (A) and p-ERK (p-p42/44) (B). The data are represented as the mean ± SEM based on 3–4 independent experiments. ** p

    Techniques Used: Western Blot

    Time course of the LPS-stimulated phosphorylation of p38 (p-p38) and ERK (p-ERK). Cocultured cells were treated with LPS (100 ng/ml) for the indicated times (30, 60, 120, 180, and 300 min). The cells were harvested to analyze the levels of p-p38 (A) and p-ERK (p-p42/44) (B) protein expression by using western blotting. LPS stimulated the phosphorylation of both p38 and ERK, but the phosphorylation of p38 appeared at 60-min post LPS, whereas that of ERK appeared substantially later at 180 min. The data are represented as mean ± SEM based on 3–4 independent experiments. * p
    Figure Legend Snippet: Time course of the LPS-stimulated phosphorylation of p38 (p-p38) and ERK (p-ERK). Cocultured cells were treated with LPS (100 ng/ml) for the indicated times (30, 60, 120, 180, and 300 min). The cells were harvested to analyze the levels of p-p38 (A) and p-ERK (p-p42/44) (B) protein expression by using western blotting. LPS stimulated the phosphorylation of both p38 and ERK, but the phosphorylation of p38 appeared at 60-min post LPS, whereas that of ERK appeared substantially later at 180 min. The data are represented as mean ± SEM based on 3–4 independent experiments. * p

    Techniques Used: Expressing, Western Blot

    Effects of MAPK inhibitors on LPS-induced IL-6 and MIP-2 production. Cocultured cells were treated with 20 µM of the p38 inhibitor SB203580 or the ERK inhibitor PD98059, which were simultaneously added with LPS (100 ng/ml) for 24 h. The levels of IL-6 (A) and MIP-2 (B) in the medium were measured using ELISA kits. Both inhibitors significantly suppressed both the LPS stimulated release of IL-6 and MIP-2. The data are represented as mean ± SEM based on 4–6 independent experiments. * p
    Figure Legend Snippet: Effects of MAPK inhibitors on LPS-induced IL-6 and MIP-2 production. Cocultured cells were treated with 20 µM of the p38 inhibitor SB203580 or the ERK inhibitor PD98059, which were simultaneously added with LPS (100 ng/ml) for 24 h. The levels of IL-6 (A) and MIP-2 (B) in the medium were measured using ELISA kits. Both inhibitors significantly suppressed both the LPS stimulated release of IL-6 and MIP-2. The data are represented as mean ± SEM based on 4–6 independent experiments. * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    31) Product Images from "Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis"

    Article Title: Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200606016

    TLR8 stimulation in neurons does not activate the canonical TLR–NF-κB signaling pathway, but rather down-regulates IκBα and IRAK4. (A) ELISA assay for NF-κB (p65) transactivation using nuclear extracts from cortical neurons stimulated with 100 μM R-848, 500 μM loxoribine, 5 μg/ml LPS, or 10 ng/ml TNFα for the indicated times. LPS and TNFα serve as negative and positive controls, respectively. (B) Western blotting of the hallmarks of the conventional TLR-signaling pathway with lysates from neurons and Raw264.7 macrophages treated with 100 μM R-848 for the indicated times. (C) Quantification of changes in IκBα levels in R-848–stimulated neurons by band densitometry. A representative blot is shown in B. (D) Western blotting of IRAK4 in neurons stimulated with 100 μM R-848 for the indicated times. Note that TLR8 levels remain unchanged. (E) Quantification of changes in IRAK4 levels by band densitometry. A representative blot is shown in D. Data in C and E, expressed as percentage normalized to controls (100%), are the mean ± the SEM for pooled Western-blots from three independent cultures. Statistical analysis was done by t test. *, P
    Figure Legend Snippet: TLR8 stimulation in neurons does not activate the canonical TLR–NF-κB signaling pathway, but rather down-regulates IκBα and IRAK4. (A) ELISA assay for NF-κB (p65) transactivation using nuclear extracts from cortical neurons stimulated with 100 μM R-848, 500 μM loxoribine, 5 μg/ml LPS, or 10 ng/ml TNFα for the indicated times. LPS and TNFα serve as negative and positive controls, respectively. (B) Western blotting of the hallmarks of the conventional TLR-signaling pathway with lysates from neurons and Raw264.7 macrophages treated with 100 μM R-848 for the indicated times. (C) Quantification of changes in IκBα levels in R-848–stimulated neurons by band densitometry. A representative blot is shown in B. (D) Western blotting of IRAK4 in neurons stimulated with 100 μM R-848 for the indicated times. Note that TLR8 levels remain unchanged. (E) Quantification of changes in IRAK4 levels by band densitometry. A representative blot is shown in D. Data in C and E, expressed as percentage normalized to controls (100%), are the mean ± the SEM for pooled Western-blots from three independent cultures. Statistical analysis was done by t test. *, P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Western Blot

    32) Product Images from "BCA2/Rabring7 Targets HIV-1 Gag for Lysosomal Degradation in a Tetherin-Independent Manner"

    Article Title: BCA2/Rabring7 Targets HIV-1 Gag for Lysosomal Degradation in a Tetherin-Independent Manner

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004151

    The E3 ligase activity of BCA2 is required for antiviral activity. (A) Schematic representation of BCA2 with the N-terminal BCA2 Zinc-finger domain (BZF), the AKT-phosphorylation sites, and the C-terminal RING-finger domain. Key residues are indicated. (B) Virus release assays for HIV-1 NL4-3, SIV mac 239 and Mo-MLV were performed from cells expressing wild-type HA-BCA2 or the indicated BCA2 mutants, and expressed as the percentage of maximal release, as previously described. The whole cell lysates (WCL) and the virions produced were also analyzed by western blot to assess HA-BCA2, Gag, Nef, CA and β-actin expression. Red asterisk indicates MW of 37 KDa. (C) The tetherin-independent and tetherin-dependent antiviral activity of BCA2 was examined in a virus release assay from parental 293T and 293T cells stably expressing tetherin. Results were also corroborated by western blot of the whole cell lysates (WCL) and pelleted virions. Error bars represent standard deviation of independent experiments.
    Figure Legend Snippet: The E3 ligase activity of BCA2 is required for antiviral activity. (A) Schematic representation of BCA2 with the N-terminal BCA2 Zinc-finger domain (BZF), the AKT-phosphorylation sites, and the C-terminal RING-finger domain. Key residues are indicated. (B) Virus release assays for HIV-1 NL4-3, SIV mac 239 and Mo-MLV were performed from cells expressing wild-type HA-BCA2 or the indicated BCA2 mutants, and expressed as the percentage of maximal release, as previously described. The whole cell lysates (WCL) and the virions produced were also analyzed by western blot to assess HA-BCA2, Gag, Nef, CA and β-actin expression. Red asterisk indicates MW of 37 KDa. (C) The tetherin-independent and tetherin-dependent antiviral activity of BCA2 was examined in a virus release assay from parental 293T and 293T cells stably expressing tetherin. Results were also corroborated by western blot of the whole cell lysates (WCL) and pelleted virions. Error bars represent standard deviation of independent experiments.

    Techniques Used: Activity Assay, Expressing, Produced, Western Blot, Release Assay, Stable Transfection, Standard Deviation

    The N-terminus of BCA2 interacts with the Matrix region of Gag. (A) To investigate if retroviral Gag proteins interact with BCA2, 293T cells were co-transfected with HIV-1, SIV and Mo-MLV Gag constructs and either empty vector or a vector encoding HA-BCA2. Cell lysates were immunoprecipitated with a CA-specific antibody and membranes were probed with an anti-HA antibody. Results obtained from these assays were corroborated independently twice. (B) Schematic representation of the Gag and HA-BCA2 deleted constructs used. Similar to panel A, HIV (C) and SIV (D) Gag deleted mutants were tested for an interaction with HA-BCA2 by co-immunoprecipitation. Likewise, HA-BCA2 deleted mutants were tested for an interaction with HIV (E) and SIV (F) Gag proteins by co-immunoprecipitation. In this case, lysates were immunoprecipitated with an anti-HA antibody and western blot membranes were probed with a GFP-specific antibody. Whole cell lysates (WCL) were set aside to check the input levels of these proteins and β-actin. IP: immunoprecipitation. V: empty vector.
    Figure Legend Snippet: The N-terminus of BCA2 interacts with the Matrix region of Gag. (A) To investigate if retroviral Gag proteins interact with BCA2, 293T cells were co-transfected with HIV-1, SIV and Mo-MLV Gag constructs and either empty vector or a vector encoding HA-BCA2. Cell lysates were immunoprecipitated with a CA-specific antibody and membranes were probed with an anti-HA antibody. Results obtained from these assays were corroborated independently twice. (B) Schematic representation of the Gag and HA-BCA2 deleted constructs used. Similar to panel A, HIV (C) and SIV (D) Gag deleted mutants were tested for an interaction with HA-BCA2 by co-immunoprecipitation. Likewise, HA-BCA2 deleted mutants were tested for an interaction with HIV (E) and SIV (F) Gag proteins by co-immunoprecipitation. In this case, lysates were immunoprecipitated with an anti-HA antibody and western blot membranes were probed with a GFP-specific antibody. Whole cell lysates (WCL) were set aside to check the input levels of these proteins and β-actin. IP: immunoprecipitation. V: empty vector.

    Techniques Used: Transfection, Construct, Plasmid Preparation, Immunoprecipitation, Western Blot

    The targeted depletion of endogenous BCA2 results in increased virus release and replication. Endogenous BCA2 was depleted by shRNA from 293T cells (A) and HOS cells (B) by transient transfection. Cells were subsequently transfected with HIV-1 NL4-3 or SIV mac 239 proviral DNA, and virus release was measured 48 hours later. Depletion of endogenous BCA2 was also achieved in CD4 + T cells by transduction. Next, virus replication was assessed for HIV-1 in Jurkat cells (C) and for SIV in 221 T cells (D) by HIV-1 p24 and SIV p27 antigen-capture ELISA, respectively, at selected time points. The depletion of endogenous BCA2 was confirmed by western blot for each of these cell lines, by comparing the levels of endogenous BCA2 in the whole cell lysate (WCL) to those of β-actin. Error bars represent standard deviation of independent experiments.
    Figure Legend Snippet: The targeted depletion of endogenous BCA2 results in increased virus release and replication. Endogenous BCA2 was depleted by shRNA from 293T cells (A) and HOS cells (B) by transient transfection. Cells were subsequently transfected with HIV-1 NL4-3 or SIV mac 239 proviral DNA, and virus release was measured 48 hours later. Depletion of endogenous BCA2 was also achieved in CD4 + T cells by transduction. Next, virus replication was assessed for HIV-1 in Jurkat cells (C) and for SIV in 221 T cells (D) by HIV-1 p24 and SIV p27 antigen-capture ELISA, respectively, at selected time points. The depletion of endogenous BCA2 was confirmed by western blot for each of these cell lines, by comparing the levels of endogenous BCA2 in the whole cell lysate (WCL) to those of β-actin. Error bars represent standard deviation of independent experiments.

    Techniques Used: shRNA, Transfection, Transduction, Enzyme-linked Immunosorbent Assay, Western Blot, Standard Deviation

    33) Product Images from "Stable silencing of SNAP-25 in PC12 cells by RNA interference"

    Article Title: Stable silencing of SNAP-25 in PC12 cells by RNA interference

    Journal: BMC Neuroscience

    doi: 10.1186/1471-2202-7-9

    Specific silencing of SNAP-25 by RNA interference . A) Immunoblots were done to assess the levels of SNAP-25, SNAP-23, synaptotagmin I, syntaxin 1A, tyrosine hydroxylase and β-actin in wild type PC12 cells, SNAP-25 knockdown cells and in control transfected cells (PC12 cells stably transfected with pG418-shRNA lacking an shRNA insert). Equal amounts of protein were loaded per lane. B) The SNAP-25 phenotype is maintained for at least 10 weeks in culture in both the parent PC12 cell line and the SNAP-25 knockdown cell line. C) Human and zebrafish SNAP-25 mRNAs are resistant to RNA interference. The specificity of the SNAP-25 shRNA was demonstrated by transiently transfecting SNAP-25 knockdown cells with plasmids designed to express SNAP-25 cDNA of rat, human, or zebrafish origin. The 19 nucleotide region of rat SNAP-25 mRNA which is targeted by the SNAP-25 shRNA differs from human SNAP-25 RNA at only 2 positions and from zebrafish SNAP-25 mRNA in 4 positions. The immunoblot shows that expression of rat SNAP-25 was silenced in the SNAP-25 knockdown cells, but human and zebrafish SNAP-25 were expressed. All three SNAP-25 cDNAs appeared to be expressed in the control transfected cells in that there was more SNAP-25 in the control transfected cells than in untransfected PC12 cells. The immunoblot was stripped and reprobed for β-actin to demonstrate approximately equal amounts of protein in each sample. D) SNAP-25 mRNA is reduced in SNAP-25 knockdown cells. RT-PCR was carried out with RNA isolated from wild type PC12 cells, SNAP-25 knockdown cells and in control transfected cells. SNAP-25 mRNA was easily detected in wild type and control transfected PC12 cells, but no SNAP-25 mRNA was detected in the SNAP-25 knockdown cells in this PCR experiments. However, if more of the reverse-transcription product was used for the PCR or if more cycles were done in the PCR reaction, some SNAP-25 mRNA was detectable in the SNAP-25 knockdown cells.
    Figure Legend Snippet: Specific silencing of SNAP-25 by RNA interference . A) Immunoblots were done to assess the levels of SNAP-25, SNAP-23, synaptotagmin I, syntaxin 1A, tyrosine hydroxylase and β-actin in wild type PC12 cells, SNAP-25 knockdown cells and in control transfected cells (PC12 cells stably transfected with pG418-shRNA lacking an shRNA insert). Equal amounts of protein were loaded per lane. B) The SNAP-25 phenotype is maintained for at least 10 weeks in culture in both the parent PC12 cell line and the SNAP-25 knockdown cell line. C) Human and zebrafish SNAP-25 mRNAs are resistant to RNA interference. The specificity of the SNAP-25 shRNA was demonstrated by transiently transfecting SNAP-25 knockdown cells with plasmids designed to express SNAP-25 cDNA of rat, human, or zebrafish origin. The 19 nucleotide region of rat SNAP-25 mRNA which is targeted by the SNAP-25 shRNA differs from human SNAP-25 RNA at only 2 positions and from zebrafish SNAP-25 mRNA in 4 positions. The immunoblot shows that expression of rat SNAP-25 was silenced in the SNAP-25 knockdown cells, but human and zebrafish SNAP-25 were expressed. All three SNAP-25 cDNAs appeared to be expressed in the control transfected cells in that there was more SNAP-25 in the control transfected cells than in untransfected PC12 cells. The immunoblot was stripped and reprobed for β-actin to demonstrate approximately equal amounts of protein in each sample. D) SNAP-25 mRNA is reduced in SNAP-25 knockdown cells. RT-PCR was carried out with RNA isolated from wild type PC12 cells, SNAP-25 knockdown cells and in control transfected cells. SNAP-25 mRNA was easily detected in wild type and control transfected PC12 cells, but no SNAP-25 mRNA was detected in the SNAP-25 knockdown cells in this PCR experiments. However, if more of the reverse-transcription product was used for the PCR or if more cycles were done in the PCR reaction, some SNAP-25 mRNA was detectable in the SNAP-25 knockdown cells.

    Techniques Used: Western Blot, Transfection, Stable Transfection, shRNA, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction

    34) Product Images from "Cdc20 Is Critical for Meiosis I and Fertility of Female Mice"

    Article Title: Cdc20 Is Critical for Meiosis I and Fertility of Female Mice

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1001147

    Generation of mice with graded reduction in Cdc20 dosage. (A) Schematic representation of the primary Cdc20 gene targeting strategy. Part of the Cdc20 locus (+), the targeting vector, the hypomorphic allele ( Cdc20 H ), EcoR1 restriction sites and the Southern probe are indicated. (B) Schematic representation of the Cdc20 − allele was from gene trap mouse embryonic stem (ES) cell clone XE368. (C) Southern-blot analysis of mice with indicated Cdc20 genotypes. (D) PCR-based genotype analysis of Cdc20 mutant mice. Positions of PCR primers (a–e) are indicated in (A,B). (E–G) Western blot analysis of whole ovary (E), testis (F), spleen and bone marrow (G) extracts of the indicated genotypes for Cdc20. Actin and tubulin served as loading controls. Cdc20 protein signals were quantified using ImageJ software and normalized to background and either actin or tubulin. For details see materials and methods .
    Figure Legend Snippet: Generation of mice with graded reduction in Cdc20 dosage. (A) Schematic representation of the primary Cdc20 gene targeting strategy. Part of the Cdc20 locus (+), the targeting vector, the hypomorphic allele ( Cdc20 H ), EcoR1 restriction sites and the Southern probe are indicated. (B) Schematic representation of the Cdc20 − allele was from gene trap mouse embryonic stem (ES) cell clone XE368. (C) Southern-blot analysis of mice with indicated Cdc20 genotypes. (D) PCR-based genotype analysis of Cdc20 mutant mice. Positions of PCR primers (a–e) are indicated in (A,B). (E–G) Western blot analysis of whole ovary (E), testis (F), spleen and bone marrow (G) extracts of the indicated genotypes for Cdc20. Actin and tubulin served as loading controls. Cdc20 protein signals were quantified using ImageJ software and normalized to background and either actin or tubulin. For details see materials and methods .

    Techniques Used: Mouse Assay, Plasmid Preparation, Southern Blot, Polymerase Chain Reaction, Mutagenesis, Western Blot, Software

    35) Product Images from "Nuclear expression of Survivin in paediatric ependymomas and choroid plexus tumours correlates with morphologic tumour grade"

    Article Title: Nuclear expression of Survivin in paediatric ependymomas and choroid plexus tumours correlates with morphologic tumour grade

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6601334

    Survivin and  β -actin protein expression in normal human ependyma and cortex.
    Figure Legend Snippet: Survivin and β -actin protein expression in normal human ependyma and cortex.

    Techniques Used: Expressing

    36) Product Images from "Head-to-Tail Intramolecular Interaction of Herpes Simplex Virus Type 1 Regulatory Protein ICP27 Is Important for Its Interaction with Cellular mRNA Export Receptor TAP/NXF1"

    Article Title: Head-to-Tail Intramolecular Interaction of Herpes Simplex Virus Type 1 Regulatory Protein ICP27 Is Important for Its Interaction with Cellular mRNA Export Receptor TAP/NXF1

    Journal: mBio

    doi: 10.1128/mBio.00268-10

    Interaction between CFP-ICP27 and YFP-TAP/NXF1 was not observed by FRET after acceptor photobleaching. (A) RSF were transfected with CFP-ICP27-YFP and were infected with 27-LacZ for 6 h. Merged images depicting the colocalization of YFP-ICP27 and CFP-ICP27 are shown, with the regions of interest for donor and acceptor images before and after bleaching circled. Quantification of FRET for the circled regions is displayed graphically as fluorescence intensity over time. (B) RSF were transfected with CFP-ICP27 and YFP-ICP27 and were then infected with 27-LacZ for 6 h. Merged images depicting the colocalization of CFP-ICP27 and YFP-TAP/NXF1 are shown, with the regions of interest for bleaching circled. Quantification of FRET for the circled regions is displayed graphically as fluorescence intensity over time, as described in Materials and Methods. FRET analysis was performed by using an LSM confocal microscope at a magnification of ×63.
    Figure Legend Snippet: Interaction between CFP-ICP27 and YFP-TAP/NXF1 was not observed by FRET after acceptor photobleaching. (A) RSF were transfected with CFP-ICP27-YFP and were infected with 27-LacZ for 6 h. Merged images depicting the colocalization of YFP-ICP27 and CFP-ICP27 are shown, with the regions of interest for donor and acceptor images before and after bleaching circled. Quantification of FRET for the circled regions is displayed graphically as fluorescence intensity over time. (B) RSF were transfected with CFP-ICP27 and YFP-ICP27 and were then infected with 27-LacZ for 6 h. Merged images depicting the colocalization of CFP-ICP27 and YFP-TAP/NXF1 are shown, with the regions of interest for bleaching circled. Quantification of FRET for the circled regions is displayed graphically as fluorescence intensity over time, as described in Materials and Methods. FRET analysis was performed by using an LSM confocal microscope at a magnification of ×63.

    Techniques Used: Transfection, Infection, Fluorescence, Microscopy

    CFP–ICP27-C483,488S is confined to the nucleus during infection. (A) Vero cells were infected with HSV-1 KOS, 27-GFP, vICP27-C–CFP, and vN-CFP–ICP27-C483,488S at an MOI of 1. Experiments were performed in triplicate, and virus was harvested at 0, 4, 8, 16, and 24 h after infection. Plaque assays were performed in duplicate on Vero cells. (B) RSF were infected with vICP27-C–CFP or vN-CFP–ICP27-C483,488S at an MOI of 10 for 4 and 10 h after infection. CFP fluorescence was viewed with a Zeiss LSM 510 Meta confocal microscope at a magnification of ×63.
    Figure Legend Snippet: CFP–ICP27-C483,488S is confined to the nucleus during infection. (A) Vero cells were infected with HSV-1 KOS, 27-GFP, vICP27-C–CFP, and vN-CFP–ICP27-C483,488S at an MOI of 1. Experiments were performed in triplicate, and virus was harvested at 0, 4, 8, 16, and 24 h after infection. Plaque assays were performed in duplicate on Vero cells. (B) RSF were infected with vICP27-C–CFP or vN-CFP–ICP27-C483,488S at an MOI of 10 for 4 and 10 h after infection. CFP fluorescence was viewed with a Zeiss LSM 510 Meta confocal microscope at a magnification of ×63.

    Techniques Used: Infection, Fluorescence, Microscopy

    Hsc70 interacts directly with ICP27. RSF were cotransfected with N-Venus–Hsc70 and ICP27–C-Venus or with N-Venus–Hsc70 and ICP27-C483,488S–C-Venus as depicted and were subsequently infected with 27-LacZ for 6 h (A) or 8 h (B). Venus fluorescence was viewed with a Zeiss LSM 510 Meta confocal microscope at a magnification of ×20.
    Figure Legend Snippet: Hsc70 interacts directly with ICP27. RSF were cotransfected with N-Venus–Hsc70 and ICP27–C-Venus or with N-Venus–Hsc70 and ICP27-C483,488S–C-Venus as depicted and were subsequently infected with 27-LacZ for 6 h (A) or 8 h (B). Venus fluorescence was viewed with a Zeiss LSM 510 Meta confocal microscope at a magnification of ×20.

    Techniques Used: Infection, Fluorescence, Microscopy

    ICP27 interacts directly with TAP/NXF1. (A) RSF were cotransfected with GFP-TAP/NXF1 DNA and with ICP27 constructs carrying wild-type ICP27 or ICP27-C483,488S and were subsequently infected with 27-LacZ for 8 h. Immunofluorescent staining was performed with anti-ICP27 antibody, and GFP fluorescence was viewed directly. (B to D) Cells were transfected with NC-Venus-ICP27 (B) or were cotransfected with N-Venus–TAP/NXF1 and ICP27–C-Venus (C) or with N-Venus–TAP/NXF1 and ICP27-C483,488S–C-Venus (D). Twenty-four hours later, cells were infected with 27-LacZ for 8 h. Cells were viewed directly for Venus fluorescence using a Zeiss LSM 510 Meta confocal microscope at a magnification of ×20.
    Figure Legend Snippet: ICP27 interacts directly with TAP/NXF1. (A) RSF were cotransfected with GFP-TAP/NXF1 DNA and with ICP27 constructs carrying wild-type ICP27 or ICP27-C483,488S and were subsequently infected with 27-LacZ for 8 h. Immunofluorescent staining was performed with anti-ICP27 antibody, and GFP fluorescence was viewed directly. (B to D) Cells were transfected with NC-Venus-ICP27 (B) or were cotransfected with N-Venus–TAP/NXF1 and ICP27–C-Venus (C) or with N-Venus–TAP/NXF1 and ICP27-C483,488S–C-Venus (D). Twenty-four hours later, cells were infected with 27-LacZ for 8 h. Cells were viewed directly for Venus fluorescence using a Zeiss LSM 510 Meta confocal microscope at a magnification of ×20.

    Techniques Used: Construct, Infection, Staining, Fluorescence, Transfection, Microscopy

    BiFC-FRET after photobleaching demonstrates the interaction of TAP/NXF1 with ICP27 upon head-to-tail association. (A) Schematic representation of BiFC for NC-Venus-ICP27. (B) Model showing FRET between CFP-TAP/NXF1 and NC-Venus-ICP27. (C) RSF were transfected with CFP-TAP/NXF1 and were subsequently infected with vNC-Venus-ICP27 at an MOI of 10. Merged images depicting the colocalization of ICP27 with TAP/NXF1 are shown, with the regions of interest circled before and after bleaching of the acceptor. Quantification of FRET for the circled regions is displayed graphically as fluorescence intensity over time. FRET analysis was performed as described in the legend to Fig. 6 . (D) RSF were transfected with CFP-TAP/NXF1 and were subsequently infected with vNC-Venus-ICP27 for 6 h. Venus and CFP fluorescence were viewed directly using an LSM confocal microscope at a magnification of ×63.
    Figure Legend Snippet: BiFC-FRET after photobleaching demonstrates the interaction of TAP/NXF1 with ICP27 upon head-to-tail association. (A) Schematic representation of BiFC for NC-Venus-ICP27. (B) Model showing FRET between CFP-TAP/NXF1 and NC-Venus-ICP27. (C) RSF were transfected with CFP-TAP/NXF1 and were subsequently infected with vNC-Venus-ICP27 at an MOI of 10. Merged images depicting the colocalization of ICP27 with TAP/NXF1 are shown, with the regions of interest circled before and after bleaching of the acceptor. Quantification of FRET for the circled regions is displayed graphically as fluorescence intensity over time. FRET analysis was performed as described in the legend to Fig. 6 . (D) RSF were transfected with CFP-TAP/NXF1 and were subsequently infected with vNC-Venus-ICP27 for 6 h. Venus and CFP fluorescence were viewed directly using an LSM confocal microscope at a magnification of ×63.

    Techniques Used: Bimolecular Fluorescence Complementation Assay, Transfection, Infection, Fluorescence, Microscopy

    Analysis of percent FRET over time. RSF were transfected with the constructs indicated and were subsequently infected with 27-LacZ or with vNC-Venus-ICP27, as indicated. FRET after acceptor photobleaching was performed by photobleaching the acceptor protein Venus or YFP at a specific location within the cell using the 514-nm laser line at 100% transmission. The change in donor fluorescence was quantified by comparing prebleach and postbleach levels of CFP fluorescence from the images. The FRET efficiency (EF) obtained from at least 30 different bleached regions of interest was calculated as described in Materials and Methods. FRET efficiency is shown as percent FRET over time. Experiments were performed in triplicate. Error bars are shown.
    Figure Legend Snippet: Analysis of percent FRET over time. RSF were transfected with the constructs indicated and were subsequently infected with 27-LacZ or with vNC-Venus-ICP27, as indicated. FRET after acceptor photobleaching was performed by photobleaching the acceptor protein Venus or YFP at a specific location within the cell using the 514-nm laser line at 100% transmission. The change in donor fluorescence was quantified by comparing prebleach and postbleach levels of CFP fluorescence from the images. The FRET efficiency (EF) obtained from at least 30 different bleached regions of interest was calculated as described in Materials and Methods. FRET efficiency is shown as percent FRET over time. Experiments were performed in triplicate. Error bars are shown.

    Techniques Used: Transfection, Construct, Infection, Transmission Assay, Fluorescence

    Mutation of the ICP27 zinc finger affects the functional interaction of ICP27 and Hsc70. (A) RSF were either mock infected or infected with HSV-1 KOS or 27-LacZ as indicated for 6 h. Cells were fixed and stained with anti-Hsc70 antibody as described ( 5 ). (B, C) RSF were cotransfected with N-Venus–Hsc70 and ICP27–C-Venus (B) or with ICP27-C483,488S–C-Venus (C) as indicated and infected with 27-LacZ. At 8 h after infection, Venus fluorescence was viewed using a Zeiss LSM 510 Meta confocal microscope at a magnification of ×63. White arrows point to Hsc70 nuclear foci.
    Figure Legend Snippet: Mutation of the ICP27 zinc finger affects the functional interaction of ICP27 and Hsc70. (A) RSF were either mock infected or infected with HSV-1 KOS or 27-LacZ as indicated for 6 h. Cells were fixed and stained with anti-Hsc70 antibody as described ( 5 ). (B, C) RSF were cotransfected with N-Venus–Hsc70 and ICP27–C-Venus (B) or with ICP27-C483,488S–C-Venus (C) as indicated and infected with 27-LacZ. At 8 h after infection, Venus fluorescence was viewed using a Zeiss LSM 510 Meta confocal microscope at a magnification of ×63. White arrows point to Hsc70 nuclear foci.

    Techniques Used: Mutagenesis, Functional Assay, Infection, Staining, Fluorescence, Microscopy

    Western blot analysis of TAP/NXF1-Venus and ICP27-Venus fusion proteins. (A) RSF were transfected with plasmid DNA encoding the proteins indicated and infected with 27-LacZ to induce expression of ICP27 constructs. Cells were harvested 8 h after infection, and proteins were fractionated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose. Membranes were probed with antibodies against GFP, ICP27, and β-actin as described previously ( 27 ). Asterisks mark the protein bands. (B) RSF were transfected with NC-Venus-ICP27 or cotransfected with ICP27–C-Venus and N-Venus–TAP/NXF1 and later infected with 27-LacZ for 6 and 8 h, as indicated. Venus fluorescence was viewed directly using a Zeiss LSM 510 Meta confocal microscope at a magnification of ×63. White arrows point to Venus cytoplasmic fluorescence.
    Figure Legend Snippet: Western blot analysis of TAP/NXF1-Venus and ICP27-Venus fusion proteins. (A) RSF were transfected with plasmid DNA encoding the proteins indicated and infected with 27-LacZ to induce expression of ICP27 constructs. Cells were harvested 8 h after infection, and proteins were fractionated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose. Membranes were probed with antibodies against GFP, ICP27, and β-actin as described previously ( 27 ). Asterisks mark the protein bands. (B) RSF were transfected with NC-Venus-ICP27 or cotransfected with ICP27–C-Venus and N-Venus–TAP/NXF1 and later infected with 27-LacZ for 6 and 8 h, as indicated. Venus fluorescence was viewed directly using a Zeiss LSM 510 Meta confocal microscope at a magnification of ×63. White arrows point to Venus cytoplasmic fluorescence.

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Infection, Expressing, Construct, Fluorescence, Microscopy

    37) Product Images from "Rac1 activation inhibits E-cadherin-mediated adherens junctions via binding to IQGAP1 in pancreatic carcinoma cells"

    Article Title: Rac1 activation inhibits E-cadherin-mediated adherens junctions via binding to IQGAP1 in pancreatic carcinoma cells

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/1478-811X-7-23

    Interaction of IQGAP1 with Rac1 or β-catenin. (A) IQGAP1 was precipitated from lysates of EGFP-, EGFP-Rac1(V12)- and EGFP-Rac1(N17)-expressing PANC-1 cells and coprecipitated β-catenin was determined by Western blotting (left panel) . In parallel, immunoprecipitates of EGFP/EGFP-Rac1 proteins were analysed for coprecipitated IQGAP1 (right panel). Staining of the precipitated proteins verified equal amounts of protein in each immunoprecipitation (lower panels) (B) Binding of IQGAP1 to β-catenin was confirmed by GST-pull down assays. GST-tagged β-catenin was incubated with 500 μg of lysate prepared from EGFP-, EGFP-Rac1(V12)- or EGFP-Rac1(N17)-expressing PANC-1 cells and GST-β-catenin complexes were precipitated by binding to glutathione sepharose. Precipitated IQGAP1 and GST-β-catenin was determined by Western blotting. Determination of IQGAP1 and EGFP proteins in 30 μg of the cell lysate served to demonstrate equal amounts of protein in each sample. One representative blot of three independent experiments is shown.
    Figure Legend Snippet: Interaction of IQGAP1 with Rac1 or β-catenin. (A) IQGAP1 was precipitated from lysates of EGFP-, EGFP-Rac1(V12)- and EGFP-Rac1(N17)-expressing PANC-1 cells and coprecipitated β-catenin was determined by Western blotting (left panel) . In parallel, immunoprecipitates of EGFP/EGFP-Rac1 proteins were analysed for coprecipitated IQGAP1 (right panel). Staining of the precipitated proteins verified equal amounts of protein in each immunoprecipitation (lower panels) (B) Binding of IQGAP1 to β-catenin was confirmed by GST-pull down assays. GST-tagged β-catenin was incubated with 500 μg of lysate prepared from EGFP-, EGFP-Rac1(V12)- or EGFP-Rac1(N17)-expressing PANC-1 cells and GST-β-catenin complexes were precipitated by binding to glutathione sepharose. Precipitated IQGAP1 and GST-β-catenin was determined by Western blotting. Determination of IQGAP1 and EGFP proteins in 30 μg of the cell lysate served to demonstrate equal amounts of protein in each sample. One representative blot of three independent experiments is shown.

    Techniques Used: Expressing, Western Blot, Staining, Immunoprecipitation, Binding Assay, Incubation

    EGFP-Rac1-induced differences in the amount of cell-cell adhesion protein concentration . (A) The amount of E-cadherin, α- and β-catenin in 30 μg of total cell lysate was analysed by Western blotting. Individual proteins were detected by using specific antibodies. Detection of β-actin served as control to document equal amounts of protein in each lane. (B+C) The amount of the E-cadherin/catenin complex was analysed by immunoprecipitation of β-catenin (B) or E-cadherin (C) from 300 μg of total cell lysate. Coprecipitated E-cadherin and α-catenin (B) or α-catenin and β-catenin (C) , respectively, were identified by Western blotting. (D) Reverse transcription-PCR analysis was performed using total RNA isolated from PANC-1 cells stably expressing EGFP, EGFP-Rac1(N17) or EGFP-Rac1(V12). E-cadherin mRNA was amplified using specific primers. Amplification of β-actin cDNA served as control to document equal amounts of mRNA. (E) Association of the E-cadherin/catenin complex with the actin cytoskeleton was analysed using Triton X-100-fractionated cell extracts. Amounts of E-cadherin and β-catenin were analysed by Western blotting using 10 μg of Triton X-100-soluble and Triton X-100-insoluble fraction. (F) To investigate the influence of endogenous active Rac1 on the E-cadherin/catenin complex, PANC-1 cells were treated with 50 μM of the Rac1-inhibitor NSC23766 or stimulated for 5 min with PDGF AB (10 ng/ml). Active Rac1-GTP was precipitated from 800 μg of cell lysates by affinity precipitation assays (upper panel). To control for equal loading, aliquots of the samples were analysed in parallel (lower panel). The amount of E-cadherin/catenin complexes was analysed by detection of E-cadherin, which was coprecipitated with β-catenin from 500 μg of total cell lysates. (G) To investigate E-cadherin degradation in Rac1(V12)-expressing PANC-1 cells, cells were incubated with MG132 (1 μM) or E64 (1.5 μM) to suppress proteolytic degradation of proteins. The amount of E-cadherin was estimated by Western blotting using 30 μg of total protein lysates. Determination of β-actin served to control for comparable loading. In each figure one representative blot/agarose gel of three to four independent experiments is shown.
    Figure Legend Snippet: EGFP-Rac1-induced differences in the amount of cell-cell adhesion protein concentration . (A) The amount of E-cadherin, α- and β-catenin in 30 μg of total cell lysate was analysed by Western blotting. Individual proteins were detected by using specific antibodies. Detection of β-actin served as control to document equal amounts of protein in each lane. (B+C) The amount of the E-cadherin/catenin complex was analysed by immunoprecipitation of β-catenin (B) or E-cadherin (C) from 300 μg of total cell lysate. Coprecipitated E-cadherin and α-catenin (B) or α-catenin and β-catenin (C) , respectively, were identified by Western blotting. (D) Reverse transcription-PCR analysis was performed using total RNA isolated from PANC-1 cells stably expressing EGFP, EGFP-Rac1(N17) or EGFP-Rac1(V12). E-cadherin mRNA was amplified using specific primers. Amplification of β-actin cDNA served as control to document equal amounts of mRNA. (E) Association of the E-cadherin/catenin complex with the actin cytoskeleton was analysed using Triton X-100-fractionated cell extracts. Amounts of E-cadherin and β-catenin were analysed by Western blotting using 10 μg of Triton X-100-soluble and Triton X-100-insoluble fraction. (F) To investigate the influence of endogenous active Rac1 on the E-cadherin/catenin complex, PANC-1 cells were treated with 50 μM of the Rac1-inhibitor NSC23766 or stimulated for 5 min with PDGF AB (10 ng/ml). Active Rac1-GTP was precipitated from 800 μg of cell lysates by affinity precipitation assays (upper panel). To control for equal loading, aliquots of the samples were analysed in parallel (lower panel). The amount of E-cadherin/catenin complexes was analysed by detection of E-cadherin, which was coprecipitated with β-catenin from 500 μg of total cell lysates. (G) To investigate E-cadherin degradation in Rac1(V12)-expressing PANC-1 cells, cells were incubated with MG132 (1 μM) or E64 (1.5 μM) to suppress proteolytic degradation of proteins. The amount of E-cadherin was estimated by Western blotting using 30 μg of total protein lysates. Determination of β-actin served to control for comparable loading. In each figure one representative blot/agarose gel of three to four independent experiments is shown.

    Techniques Used: Protein Concentration, Western Blot, Immunoprecipitation, Polymerase Chain Reaction, Isolation, Stable Transfection, Expressing, Amplification, Affinity Precipitation, Incubation, Agarose Gel Electrophoresis

    Immunofluorescence localisation of E-cadherin, β-catenin, IQGAP1 and filamentous actin (F-actin) . (A) PANC-1 cells stably expressing EGFP, EGFP-Rac1(V12) or EGFP-Rac1(N17) were incubated with specific antibodies against E-cadherin, β-catenin, IQGAP1 or CPTIC-conjugated phalloidin. The staining was examined by fluorescence microscopy (bar = 20 μm). (B) EGFP-Rac1(V12)-expressing PANC-1 cells were stained for IQGAP1 and F-actin with CPTIC-conjugated phalloidin. The merged image shows in yellow the colocalisation of IQGAP1 and filamentous actin (bar = 10 μm).
    Figure Legend Snippet: Immunofluorescence localisation of E-cadherin, β-catenin, IQGAP1 and filamentous actin (F-actin) . (A) PANC-1 cells stably expressing EGFP, EGFP-Rac1(V12) or EGFP-Rac1(N17) were incubated with specific antibodies against E-cadherin, β-catenin, IQGAP1 or CPTIC-conjugated phalloidin. The staining was examined by fluorescence microscopy (bar = 20 μm). (B) EGFP-Rac1(V12)-expressing PANC-1 cells were stained for IQGAP1 and F-actin with CPTIC-conjugated phalloidin. The merged image shows in yellow the colocalisation of IQGAP1 and filamentous actin (bar = 10 μm).

    Techniques Used: Immunofluorescence, Stable Transfection, Expressing, Incubation, Staining, Fluorescence, Microscopy

    Analyses of Rac1/IQGAP1/β-catenin complexes in EGFP-Rac1(V12)- or EGFP-Rac1(N17)-expressing PANC-1 cells . (A) Soluble (S100) and particulate (P100) fractions of EGFP-, EGFP-Rac1(V12)- and EGFP-Rac1(N17)-expressing PANC-1 cells were prepared from total cell lysates by centrifugation at 100000 × g. Subcellular localisation of Rac1, IQGAP1 and β-catenin was analysed in aliquots of 50 μg by Western blotting. To control for comparable loading β-actin was determined (lower panel). (B) Immunoprecipitation was performed using 300 μg of soluble (S100) or particulate, membrane-containing (P100) fractions of PANC-1 cells stably expressing EGFP, EGFP-Rac1(V12) or EGFP-Rac1(N17). After immunoprecipitation of IQGAP1 coprecipitated β-catenin and EGFP-Rac1 was determined by Western blotting. Detection of the immunoprecipitated IQGAP1 demonstrated equal amounts of protein in each precipitation. (C) Immunoprecipitation of β-catenin was performed in parallel experiments. Coprecipitated IQGAP1 and EGFP-Rac1 was determined by Western blotting and detection of the immunoprecipitated β-catenin verified equal amounts of protein. (D) The concentration of IQGAP1 in PANC-1 cells was reduced by transfection of two different siRNA oligonucleotides targeting IQGAP1. The amount of E-cadherin, β-catenin and IQGAP1 was demonstrated by Western blotting. Staining of β-actin served to demonstrate equal amounts of cell lysate. In each figure one representative blot out of three independent experiments is shown.
    Figure Legend Snippet: Analyses of Rac1/IQGAP1/β-catenin complexes in EGFP-Rac1(V12)- or EGFP-Rac1(N17)-expressing PANC-1 cells . (A) Soluble (S100) and particulate (P100) fractions of EGFP-, EGFP-Rac1(V12)- and EGFP-Rac1(N17)-expressing PANC-1 cells were prepared from total cell lysates by centrifugation at 100000 × g. Subcellular localisation of Rac1, IQGAP1 and β-catenin was analysed in aliquots of 50 μg by Western blotting. To control for comparable loading β-actin was determined (lower panel). (B) Immunoprecipitation was performed using 300 μg of soluble (S100) or particulate, membrane-containing (P100) fractions of PANC-1 cells stably expressing EGFP, EGFP-Rac1(V12) or EGFP-Rac1(N17). After immunoprecipitation of IQGAP1 coprecipitated β-catenin and EGFP-Rac1 was determined by Western blotting. Detection of the immunoprecipitated IQGAP1 demonstrated equal amounts of protein in each precipitation. (C) Immunoprecipitation of β-catenin was performed in parallel experiments. Coprecipitated IQGAP1 and EGFP-Rac1 was determined by Western blotting and detection of the immunoprecipitated β-catenin verified equal amounts of protein. (D) The concentration of IQGAP1 in PANC-1 cells was reduced by transfection of two different siRNA oligonucleotides targeting IQGAP1. The amount of E-cadherin, β-catenin and IQGAP1 was demonstrated by Western blotting. Staining of β-actin served to demonstrate equal amounts of cell lysate. In each figure one representative blot out of three independent experiments is shown.

    Techniques Used: Expressing, Centrifugation, Western Blot, Immunoprecipitation, Stable Transfection, Concentration Assay, Transfection, Staining

    38) Product Images from "FAK alters invadopodia and focal adhesion composition and dynamics to regulate breast cancer invasion"

    Article Title: FAK alters invadopodia and focal adhesion composition and dynamics to regulate breast cancer invasion

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200809110

    FAK differentially regulates tyrosine phosphorylation at focal adhesions and invadopodia. (A) MTLn3 (parental) cells or v-Src–transformed MTLn3 cells transiently transfected with control siRNA (Ctlsi) or FAK siRNA (FAKsi) and cotransfected with GFP-paxillin were cultured on fibronectin-gelatin coverslips and stained with anti-p34Arc (Arp2/3 subunit) antibody and antiphosphotyrosine antibody. Two sets of overlays are depicted to illustrate localization of GFP-paxillin or Arp2/3 (green) with phosphotyrosine (pY) staining (red). (B) MTLn3 cells transiently transfected with control siRNA or FAK siRNA and cotransfected with YFP-dSH2 were cultured on fibronectin-gelatin coverslips and stained with anti-p34Arc antibody (red) and antiphosphotyrosine antibody (blue). Regions outlined by boxes indicate images of invadopodia and focal adhesions. Arrows indicate representative invadopodia, and arrowheads indicate representative focal adhesions. Bars, 10 µm.
    Figure Legend Snippet: FAK differentially regulates tyrosine phosphorylation at focal adhesions and invadopodia. (A) MTLn3 (parental) cells or v-Src–transformed MTLn3 cells transiently transfected with control siRNA (Ctlsi) or FAK siRNA (FAKsi) and cotransfected with GFP-paxillin were cultured on fibronectin-gelatin coverslips and stained with anti-p34Arc (Arp2/3 subunit) antibody and antiphosphotyrosine antibody. Two sets of overlays are depicted to illustrate localization of GFP-paxillin or Arp2/3 (green) with phosphotyrosine (pY) staining (red). (B) MTLn3 cells transiently transfected with control siRNA or FAK siRNA and cotransfected with YFP-dSH2 were cultured on fibronectin-gelatin coverslips and stained with anti-p34Arc antibody (red) and antiphosphotyrosine antibody (blue). Regions outlined by boxes indicate images of invadopodia and focal adhesions. Arrows indicate representative invadopodia, and arrowheads indicate representative focal adhesions. Bars, 10 µm.

    Techniques Used: Transformation Assay, Transfection, Cell Culture, Staining

    39) Product Images from "Differential effects of 24-hydroxycholesterol and 27-hydroxycholesterol on ?-amyloid precursor protein levels and processing in human neuroblastoma SH-SY5Y cells"

    Article Title: Differential effects of 24-hydroxycholesterol and 27-hydroxycholesterol on ?-amyloid precursor protein levels and processing in human neuroblastoma SH-SY5Y cells

    Journal: Molecular Neurodegeneration

    doi: 10.1186/1750-1326-4-1

    24-OHC increases processing of APP via the non-amyloidogenic pathway . Western blot (a) and densitometric analyses (b) demonstrating increased levels of sAPPα in medium of 24-OHC-treated cells. Treatment with 27-OHC or a mixture of 24-OHC + 27-OHC did not influence sAPPα levels. Levels of Aβ40 were not affected by treatment with 24-OHC, 27-OHC, or a mixture of 24-OHC + 27-OHC compared to levels in control cells (c). *p
    Figure Legend Snippet: 24-OHC increases processing of APP via the non-amyloidogenic pathway . Western blot (a) and densitometric analyses (b) demonstrating increased levels of sAPPα in medium of 24-OHC-treated cells. Treatment with 27-OHC or a mixture of 24-OHC + 27-OHC did not influence sAPPα levels. Levels of Aβ40 were not affected by treatment with 24-OHC, 27-OHC, or a mixture of 24-OHC + 27-OHC compared to levels in control cells (c). *p

    Techniques Used: Western Blot

    40) Product Images from "Albumin fibrillization induces apoptosis via integrin/FAK/Akt pathway"

    Article Title: Albumin fibrillization induces apoptosis via integrin/FAK/Akt pathway

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-9-2

    Interaction between fibrillar BSA and integrin α5β1 . (A) T47D cell lines were pre-treated with or without 0.67 μM goat IgG or 0.67 μM goat anti-integrin α5β1 antibody for 30 min as indicated, then incubated with 2 μM F-BSA (BSA-S200) in serum-free medium for 8 h. Cell viability was determined by the MTT assay. Data are means ± S.D. (n = 3). (B) Integrin α5β1 protein was linked to protein A/G beads by use of anti-integrin α5β1 antibody, then incubated with F-BSA (BSA-S200) or G-BSA (BSA) overnight. The immunocomplexes were separated by SDS-PAGE and immunoblotted with anti-integrin α5 and anti-BSA antibodies.
    Figure Legend Snippet: Interaction between fibrillar BSA and integrin α5β1 . (A) T47D cell lines were pre-treated with or without 0.67 μM goat IgG or 0.67 μM goat anti-integrin α5β1 antibody for 30 min as indicated, then incubated with 2 μM F-BSA (BSA-S200) in serum-free medium for 8 h. Cell viability was determined by the MTT assay. Data are means ± S.D. (n = 3). (B) Integrin α5β1 protein was linked to protein A/G beads by use of anti-integrin α5β1 antibody, then incubated with F-BSA (BSA-S200) or G-BSA (BSA) overnight. The immunocomplexes were separated by SDS-PAGE and immunoblotted with anti-integrin α5 and anti-BSA antibodies.

    Techniques Used: Incubation, MTT Assay, SDS Page

    Fibrillar BSA induced cytotoxicity via the integrin/FAK/Akt pathway . (A) BHK-21 cells were treated with 3 μM F-BSA (BSA-S200) in serum-free medium for the indicated time, and cell lysates were analyzed by western blotting with anti-phospho-FAK(Tyr576/577), anti-phospho-FAK(Tyr397), and anti-phospho-Akt (p-Akt) antibodies. (B) BHK-21 cells were pre-treated for 30 min with or without 1 μM goat IgG or 1 μM goat anti-integrin α5β1 antibody as indicated, then treated with 3 μM F-BSA (BSA-S200) in serum-free medium for 15 min. Cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) and anti-phospho-GSK-3β (p-GSK-3β) antibodies. (C) BHK-21 cells were treated with increasing concentrations of G-BSA (BSA) in serum-free medium as indicated, and cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) antibody. (D) BHK-21 cells were treated with or without 1 μM anti-integrin α5β1 antibody in serum-free medium for 30 min, and cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) antibody.
    Figure Legend Snippet: Fibrillar BSA induced cytotoxicity via the integrin/FAK/Akt pathway . (A) BHK-21 cells were treated with 3 μM F-BSA (BSA-S200) in serum-free medium for the indicated time, and cell lysates were analyzed by western blotting with anti-phospho-FAK(Tyr576/577), anti-phospho-FAK(Tyr397), and anti-phospho-Akt (p-Akt) antibodies. (B) BHK-21 cells were pre-treated for 30 min with or without 1 μM goat IgG or 1 μM goat anti-integrin α5β1 antibody as indicated, then treated with 3 μM F-BSA (BSA-S200) in serum-free medium for 15 min. Cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) and anti-phospho-GSK-3β (p-GSK-3β) antibodies. (C) BHK-21 cells were treated with increasing concentrations of G-BSA (BSA) in serum-free medium as indicated, and cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) antibody. (D) BHK-21 cells were treated with or without 1 μM anti-integrin α5β1 antibody in serum-free medium for 30 min, and cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) antibody.

    Techniques Used: Western Blot

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    Western Blot:

    Article Title: Staufen1 links RNA stress granules and autophagy in a model of neurodegeneration
    Article Snippet: .. Antibodies The antibodies used for western blotting and their dilutions were as follows: mouse anti-Ataxin-2 antibody (Clone 22/Ataxin-2) [(1:4000), BD Biosciences, Cat# 611378], rabbit anti-Staufen antibody [(1:5000), Novus biologicals, NBP1-33202], DDX6 antibody [(1:5000), Novus biologicals, NB200-191], RGS8 antibody [(1:5000), Novus Biologicals, NBP2-20153], LC3B Antibody [(1:7000), Novus biologicals, NB100-2220], TDP-43 antibody [(1:7000), Proteintech, Cat# 10782-2-AP], monoclonal anti-FLAG M2 antibody [(1:10,000), Sigma-Aldrich, F3165], monoclonal Anti-Calbindin-D-28K antibody [(1:5000), Sigma-Aldrich, C9848], monoclonal anti-β-Actin−peroxidase antibody (clone AC-15) [(1:20,000), Sigma-Aldrich, A3854], PCP-2 antibody (F-3) [(1:3000), Santa Cruz, sc-137064], Homer-3 antibody (E-6) [(1:2000), Santa Cruz, sc-376155], Anti-PCP4 antibody [(1:5000), Abcam, ab197377], Anti-FAM107B antibody [(1:5000), Abcam, ab175148], rabbit anti-PABP antibody [(1:4000), Abcam, ab153930], p21 Waf1/Cip1 (12D1) rabbit mAb [(1:7000), Cell Signaling, Cat# 2947], SQSTM1/p62 antibody [(1:4000), Cell Signaling, Cat# 5114], Cyclin B1 (V152) mouse mAb [(1:5000), Cell Signaling, Cat# 4135], anti-Polyglutamine-Expansion diseases marker antibody, clone 5TF1-1C2 [(1:3000), EMD Millipore, MAB1574], rabbit anti-neomycin phosphotransferase II (NPTII) antibody [(1:5000), EMD Millipore, AC113], anti-Myc-HRP antibody [(1:5000), Invitrogen, P/N 46-0709], 6 × -His Tag Monoclonal Antibody (HIS.H8), HRP [(1:10,000), ThermoFisher Scientific, MA1-21315-HRP] and sheep-anti-Digoxigenin-POD, Fab fragments [(1:10,000), Roche Life Science, Cat# 11207733910]. .. The secondary antibodies were: Peroxidase-conjugated horse anti-mouse IgG (H + L) antibody [(1:5000), Vector laboratories, PI-2000] and Peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H + L) antibody [(1:5000), Jackson ImmunoResearch Laboratories, Cat# 111-035-144].

    Article Title: Fibronectin rescues estrogen receptor α from lysosomal degradation in breast cancer cells
    Article Snippet: .. Antibodies The following antibodies were used in this study and were purchased from Santa Cruz Biotechnology unless otherwise noted (including dilutions/amounts used for immunofluorescence, Western blot [WB], and immunoprecipitation [IP]): ERα (HC-20 rabbit; 1:100 immunofluorescence, 1:200 WB; 3 µg IP), ERα (F-10 mouse; 3 µg IP), β1-integrin (LM534 mouse; 1:100 immunofluorescence; EMD Millipore), β1-integrin (M-106 rabbit; 1:300 WB; 3 µg IP), E-cadherin (H-108 rabbit; 1:1,000 WB), β-actin (C4 mouse; 1:10,000 WB), Rab11 (H-87 rabbit; 1:200 WB), Rab7 (sc-376362 mouse; 1:100 immunofluorescence), and caveolin 1 (sc-53564 mouse; 1:600 immunofluorescence; 1:200 WB). .. LAMP-1 (ab25630 mouse; 1:20 immunofluorescence), clathrin (ab2731 mouse; 1:500 immunofluorescence), and Lamin B1 (ab133741 rabbit; 1:243 immunofluorescence) were purchased from Abcam; and clathrin-HC (clone 23 mouse; 610500; 1:1,000 WB) was purchased from BD.

    Marker:

    Article Title: Staufen1 links RNA stress granules and autophagy in a model of neurodegeneration
    Article Snippet: .. Antibodies The antibodies used for western blotting and their dilutions were as follows: mouse anti-Ataxin-2 antibody (Clone 22/Ataxin-2) [(1:4000), BD Biosciences, Cat# 611378], rabbit anti-Staufen antibody [(1:5000), Novus biologicals, NBP1-33202], DDX6 antibody [(1:5000), Novus biologicals, NB200-191], RGS8 antibody [(1:5000), Novus Biologicals, NBP2-20153], LC3B Antibody [(1:7000), Novus biologicals, NB100-2220], TDP-43 antibody [(1:7000), Proteintech, Cat# 10782-2-AP], monoclonal anti-FLAG M2 antibody [(1:10,000), Sigma-Aldrich, F3165], monoclonal Anti-Calbindin-D-28K antibody [(1:5000), Sigma-Aldrich, C9848], monoclonal anti-β-Actin−peroxidase antibody (clone AC-15) [(1:20,000), Sigma-Aldrich, A3854], PCP-2 antibody (F-3) [(1:3000), Santa Cruz, sc-137064], Homer-3 antibody (E-6) [(1:2000), Santa Cruz, sc-376155], Anti-PCP4 antibody [(1:5000), Abcam, ab197377], Anti-FAM107B antibody [(1:5000), Abcam, ab175148], rabbit anti-PABP antibody [(1:4000), Abcam, ab153930], p21 Waf1/Cip1 (12D1) rabbit mAb [(1:7000), Cell Signaling, Cat# 2947], SQSTM1/p62 antibody [(1:4000), Cell Signaling, Cat# 5114], Cyclin B1 (V152) mouse mAb [(1:5000), Cell Signaling, Cat# 4135], anti-Polyglutamine-Expansion diseases marker antibody, clone 5TF1-1C2 [(1:3000), EMD Millipore, MAB1574], rabbit anti-neomycin phosphotransferase II (NPTII) antibody [(1:5000), EMD Millipore, AC113], anti-Myc-HRP antibody [(1:5000), Invitrogen, P/N 46-0709], 6 × -His Tag Monoclonal Antibody (HIS.H8), HRP [(1:10,000), ThermoFisher Scientific, MA1-21315-HRP] and sheep-anti-Digoxigenin-POD, Fab fragments [(1:10,000), Roche Life Science, Cat# 11207733910]. .. The secondary antibodies were: Peroxidase-conjugated horse anti-mouse IgG (H + L) antibody [(1:5000), Vector laboratories, PI-2000] and Peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H + L) antibody [(1:5000), Jackson ImmunoResearch Laboratories, Cat# 111-035-144].

    Staining:

    Article Title: B cell depletion reduces T cell activation in pancreatic islets in a murine autoimmune diabetes model
    Article Snippet: .. After an overnight resting period, 3 h before antibody staining, phorbal 12-myristrate-13-acetate (PMA) (50 ng/ml), ionomycin (500 ng/ml) and monensin (3 μg/ml) (all from Sigma-Aldrich) were added to the cells. ..

    Immunofluorescence:

    Article Title: Fibronectin rescues estrogen receptor α from lysosomal degradation in breast cancer cells
    Article Snippet: .. Antibodies The following antibodies were used in this study and were purchased from Santa Cruz Biotechnology unless otherwise noted (including dilutions/amounts used for immunofluorescence, Western blot [WB], and immunoprecipitation [IP]): ERα (HC-20 rabbit; 1:100 immunofluorescence, 1:200 WB; 3 µg IP), ERα (F-10 mouse; 3 µg IP), β1-integrin (LM534 mouse; 1:100 immunofluorescence; EMD Millipore), β1-integrin (M-106 rabbit; 1:300 WB; 3 µg IP), E-cadherin (H-108 rabbit; 1:1,000 WB), β-actin (C4 mouse; 1:10,000 WB), Rab11 (H-87 rabbit; 1:200 WB), Rab7 (sc-376362 mouse; 1:100 immunofluorescence), and caveolin 1 (sc-53564 mouse; 1:600 immunofluorescence; 1:200 WB). .. LAMP-1 (ab25630 mouse; 1:20 immunofluorescence), clathrin (ab2731 mouse; 1:500 immunofluorescence), and Lamin B1 (ab133741 rabbit; 1:243 immunofluorescence) were purchased from Abcam; and clathrin-HC (clone 23 mouse; 610500; 1:1,000 WB) was purchased from BD.

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  • 86
    Millipore microglial sa β gal activity
    Microglia aged in culture display signs of senescence, including increased senescent-associated β-galactosidase <t>(SA-β-gal)</t> activity and microRNA (miR)-146a expression. Microglial cells were kept in culture for 2 and 16 days in vitro (DIV). Activity of SA-β-gal was determined using a commercial kit. (A) Representative images of 2 and 16 DIV microglia showing SA-β-gal staining. (B) SA-β-gal-positive cells were counted and results expressed in graph bars as mean ± SEM. (C) miR-146a expression was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. t -test, * p
    Microglial Sa β Gal Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/microglial sa β gal activity/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    microglial sa β gal activity - by Bioz Stars, 2020-08
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    99
    Millipore β actin
    Effects of repeated TM stimulation on fibroblasts’ morphology and GRP78/BiP expression. Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. After this repeated TM or DMSO stimulation, medium was changed to DMEM with 2% horse serum (Basal medium condition) and incubated for 12h to induce differentiation. Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. (a) Just after this repeated TM or DMSO stimulation, the cells were observed (upper panels) and stained by anti-Bip antibody (bottom panels). (b) The cells treated with TM (Rep-TM) or DMSO (Rep-DM/Rep-DMSO) cultured in the culture condition medium (C.C.) or in the Basal medium condition for differentiation (M.C.) were collected and lysed. Western blot analysis was performed using an anti-Bip or <t>anti-β-actin</t> primary antibody (upper panels). Quantitative data were obtained by densitometry of the bands. Data are expressed as the mean ± SEM for at least three independent experiments (shown as a ratio of the Rep-DM C.C.). The P value was compared with the control and calculated by Student's T test. (c) Left and middle panels show the cells treated with TM (Rep-TM) or DMSO (Rep-DMSO) cultured at Basal medium condition. Right panel shows the cells treated with TGF-β1 after the incubation at the basal medium condition.
    β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2095 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    β actin - by Bioz Stars, 2020-08
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    Image Search Results


    Microglia aged in culture display signs of senescence, including increased senescent-associated β-galactosidase (SA-β-gal) activity and microRNA (miR)-146a expression. Microglial cells were kept in culture for 2 and 16 days in vitro (DIV). Activity of SA-β-gal was determined using a commercial kit. (A) Representative images of 2 and 16 DIV microglia showing SA-β-gal staining. (B) SA-β-gal-positive cells were counted and results expressed in graph bars as mean ± SEM. (C) miR-146a expression was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. t -test, * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Microglia change from a reactive to an age-like phenotype with the time in culture

    doi: 10.3389/fncel.2014.00152

    Figure Lengend Snippet: Microglia aged in culture display signs of senescence, including increased senescent-associated β-galactosidase (SA-β-gal) activity and microRNA (miR)-146a expression. Microglial cells were kept in culture for 2 and 16 days in vitro (DIV). Activity of SA-β-gal was determined using a commercial kit. (A) Representative images of 2 and 16 DIV microglia showing SA-β-gal staining. (B) SA-β-gal-positive cells were counted and results expressed in graph bars as mean ± SEM. (C) miR-146a expression was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. t -test, * p

    Article Snippet: Microglial SA-β-gal activity was determined using the Cellular senescence assay kit (Millipore), according to the manufacturer instructions.

    Techniques: Activity Assay, Expressing, In Vitro, Staining, Real-time Polymerase Chain Reaction

    Effects of repeated TM stimulation on fibroblasts’ morphology and GRP78/BiP expression. Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. After this repeated TM or DMSO stimulation, medium was changed to DMEM with 2% horse serum (Basal medium condition) and incubated for 12h to induce differentiation. Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. (a) Just after this repeated TM or DMSO stimulation, the cells were observed (upper panels) and stained by anti-Bip antibody (bottom panels). (b) The cells treated with TM (Rep-TM) or DMSO (Rep-DM/Rep-DMSO) cultured in the culture condition medium (C.C.) or in the Basal medium condition for differentiation (M.C.) were collected and lysed. Western blot analysis was performed using an anti-Bip or anti-β-actin primary antibody (upper panels). Quantitative data were obtained by densitometry of the bands. Data are expressed as the mean ± SEM for at least three independent experiments (shown as a ratio of the Rep-DM C.C.). The P value was compared with the control and calculated by Student's T test. (c) Left and middle panels show the cells treated with TM (Rep-TM) or DMSO (Rep-DMSO) cultured at Basal medium condition. Right panel shows the cells treated with TGF-β1 after the incubation at the basal medium condition.

    Journal: PLoS ONE

    Article Title: Physiological ER Stress Mediates the Differentiation of Fibroblasts

    doi: 10.1371/journal.pone.0123578

    Figure Lengend Snippet: Effects of repeated TM stimulation on fibroblasts’ morphology and GRP78/BiP expression. Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. After this repeated TM or DMSO stimulation, medium was changed to DMEM with 2% horse serum (Basal medium condition) and incubated for 12h to induce differentiation. Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. (a) Just after this repeated TM or DMSO stimulation, the cells were observed (upper panels) and stained by anti-Bip antibody (bottom panels). (b) The cells treated with TM (Rep-TM) or DMSO (Rep-DM/Rep-DMSO) cultured in the culture condition medium (C.C.) or in the Basal medium condition for differentiation (M.C.) were collected and lysed. Western blot analysis was performed using an anti-Bip or anti-β-actin primary antibody (upper panels). Quantitative data were obtained by densitometry of the bands. Data are expressed as the mean ± SEM for at least three independent experiments (shown as a ratio of the Rep-DM C.C.). The P value was compared with the control and calculated by Student's T test. (c) Left and middle panels show the cells treated with TM (Rep-TM) or DMSO (Rep-DMSO) cultured at Basal medium condition. Right panel shows the cells treated with TGF-β1 after the incubation at the basal medium condition.

    Article Snippet: Equal amounts of protein were subjected to 5–20% gradient SDS-PAGE, e-PAGEL (ATTO CO., Tokyo, Japan) for GRP78/Bip or β-actin and transferred to PVDF membrane (Millipore, Bedford, MA).

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

    Anti-proliferative effects of Dp44mT and the effects of Dp44mT, DFO, and deferasirox on protein expressions of NDRG1, NDRG2, NDRG3, Maspin, and cyclin D1 in OECM-1 cells. ( A ) OECM-1 cells were treated with various concentrations of Dp44mt as indicated for 24 h and growth inhibitory effect was determined by the CyQUANT cell proliferation assay. The data shown in each bar chart represent the mean percentage ± SE of cells in each dose of the iron chelators treatment and are compared with the control solvent-treated group ( n = 8). The OECM-1 cells were treated with various concentrations of Dp44mt ( B ), DFO ( C ), and deferasirox ( D ) as indicated for 24 h, the expressions of NDRG1, NDRG2, NDRG3, Maspin, cyclin D1 proteins, and β-actin were determined by Western-blot assays. Data of quantitative analysis were expressed as the intensity of protein bands produced from the expressions of the target genes/β-actin (±SE; n = 3) relative to the control solvent-treated group, + p

    Journal: International Journal of Molecular Sciences

    Article Title: The Iron Chelator, Dp44mT, Effectively Inhibits Human Oral Squamous Cell Carcinoma Cell Growth in Vitro and in Vivo

    doi: 10.3390/ijms17091435

    Figure Lengend Snippet: Anti-proliferative effects of Dp44mT and the effects of Dp44mT, DFO, and deferasirox on protein expressions of NDRG1, NDRG2, NDRG3, Maspin, and cyclin D1 in OECM-1 cells. ( A ) OECM-1 cells were treated with various concentrations of Dp44mt as indicated for 24 h and growth inhibitory effect was determined by the CyQUANT cell proliferation assay. The data shown in each bar chart represent the mean percentage ± SE of cells in each dose of the iron chelators treatment and are compared with the control solvent-treated group ( n = 8). The OECM-1 cells were treated with various concentrations of Dp44mt ( B ), DFO ( C ), and deferasirox ( D ) as indicated for 24 h, the expressions of NDRG1, NDRG2, NDRG3, Maspin, cyclin D1 proteins, and β-actin were determined by Western-blot assays. Data of quantitative analysis were expressed as the intensity of protein bands produced from the expressions of the target genes/β-actin (±SE; n = 3) relative to the control solvent-treated group, + p

    Article Snippet: The β-actin (MAB1501; Millipore, Temecula, CA, USA) was used as the internal positive control.

    Techniques: CyQUANT Assay, Proliferation Assay, Western Blot, Produced

    Effects of Dp44mT, DFO, and deferasirox on protein expressions of NDRG1, NDRG2, NDRG3, Maspin, and cyclin D1 in SAS cells. The SAS cells were treated with various concentrations of Dp44mt ( A ), DFO ( B ), and deferasirox ( C ) as indicated for 24 h, the expressions of NDRG1, NDRG2, NDRG3, Maspin, cyclin D1 proteins, and β-actin were determined by Western-blot assays. Data of quantitative analysis were expressed as the intensity of protein bands produced from the expressions of the target genes/β-actin (±SE; n = 3) relative to the control solvent-treated group ( D – F ), + p

    Journal: International Journal of Molecular Sciences

    Article Title: The Iron Chelator, Dp44mT, Effectively Inhibits Human Oral Squamous Cell Carcinoma Cell Growth in Vitro and in Vivo

    doi: 10.3390/ijms17091435

    Figure Lengend Snippet: Effects of Dp44mT, DFO, and deferasirox on protein expressions of NDRG1, NDRG2, NDRG3, Maspin, and cyclin D1 in SAS cells. The SAS cells were treated with various concentrations of Dp44mt ( A ), DFO ( B ), and deferasirox ( C ) as indicated for 24 h, the expressions of NDRG1, NDRG2, NDRG3, Maspin, cyclin D1 proteins, and β-actin were determined by Western-blot assays. Data of quantitative analysis were expressed as the intensity of protein bands produced from the expressions of the target genes/β-actin (±SE; n = 3) relative to the control solvent-treated group ( D – F ), + p

    Article Snippet: The β-actin (MAB1501; Millipore, Temecula, CA, USA) was used as the internal positive control.

    Techniques: Western Blot, Produced

    The Bmi1 siRNA delivery efficiency by FA-siRNA-L and repression effects on Bmi1 expression in KB cells. For western blotting and qRT-PCR assay, cells cultured in DMEM medium were harvested and protein or mRNA was extracted for the determinations. For the uptake study, KB and LO2 cells grown in a monolayer were incubated with FA-siRNA-L, siRNA-L, and Chol-siRNA for 1 h at 37°C and were then used for the next assays. A. The Bmi1 protein expression in cancer or normal cell lines detected by western blot. B. QRT-PCR quantitative analysis of the expression of Bmi1 in cancer cells in mRNA level. C. Cellular uptake and intracellular distribution of Bmi1 siRNA in the KB cells. D. Western blotting of Bmi1 expression in the cells treated with Bmi1 siRNA complex or liposomes. LO2 cells were used as the normal cell control and β-actin was used as the loading control. E. Quantitative analysis of the western blotting bands. Data are expressed as mean ± SD of 3 independent samples. **: P

    Journal: Theranostics

    Article Title: Co-delivery of Doxorubicin and Bmi1 siRNA by Folate Receptor Targeted Liposomes Exhibits Enhanced Anti-Tumor Effects in vitro and in vivo

    doi: 10.7150/thno.9423

    Figure Lengend Snippet: The Bmi1 siRNA delivery efficiency by FA-siRNA-L and repression effects on Bmi1 expression in KB cells. For western blotting and qRT-PCR assay, cells cultured in DMEM medium were harvested and protein or mRNA was extracted for the determinations. For the uptake study, KB and LO2 cells grown in a monolayer were incubated with FA-siRNA-L, siRNA-L, and Chol-siRNA for 1 h at 37°C and were then used for the next assays. A. The Bmi1 protein expression in cancer or normal cell lines detected by western blot. B. QRT-PCR quantitative analysis of the expression of Bmi1 in cancer cells in mRNA level. C. Cellular uptake and intracellular distribution of Bmi1 siRNA in the KB cells. D. Western blotting of Bmi1 expression in the cells treated with Bmi1 siRNA complex or liposomes. LO2 cells were used as the normal cell control and β-actin was used as the loading control. E. Quantitative analysis of the western blotting bands. Data are expressed as mean ± SD of 3 independent samples. **: P

    Article Snippet: Membranes were blocked in 5% skim milk for 1 h and then incubated with monoclonal antibody against Bmi1 (1:1000, Millipore, USA) or β-actin (1:5000, Millipore, USA) overnight.

    Techniques: Expressing, Western Blot, Quantitative RT-PCR, Cell Culture, Incubation