Structured Review

Thermo Fisher hek 293
Compositional analysis of the mammalian GID complex. ( A ) AP-MS analysis of N-terminally HSS-tagged WDR26 in <t>HEK-293</t> cells from a doxycycline-inducible promoter using lentiviral transduction. HSS-WDR26 expression was induced for 24 hr and immunoprecipitated using HA-antibodies. The number of peptide spectral matches (PSM) of HSS-WDR26 and selected proteins in control - and WDR26 samples are listed in the table below. Note that in contrast to multiple hGID/CTLH subunits, the CRL4 core component Ddb1 is not enriched. ( B ) Immunoprecipitation of N-terminally HSS-tagged Ddb1 stably expressed in HeLa cells. The HA-peptide eluate (HA-IP) was probed for the presence of the canonical DCAF protein WDR23 and the putative CRL4 adapter WDR26. ( C ) SAINT analysis plot of previously annotated and novel Rmnd5a-HCIPs corresponding to Figure 1C . ( D ) Immunoprecipitation of a transiently expressed HSS-GID4 construct in HEK-293 cells was analyzed for the presence of the GID proteins RanBP9, WDR26 and MAEA. ( E ) Schematic illustration of the yeast GID E3 ligase (adapted from Menssen et al., 2012 ) and the potential architecture of the human GID complex (this study). ( F ) S. cerevisiae wild-type (WT), moh1∆ and gid4∆ deletion strains expressing Fbp1-TAP were grown o/n in YP + 3% EtOH before shifting into YP medium containing 2% glucose. Samples for immunoblot analysis were taken at the indicated time points and relative Fbp1-TAP levels were quantified using ImageJ.
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Images

1) Product Images from "The multi-subunit GID/CTLH E3 ubiquitin ligase promotes cell proliferation and targets the transcription factor Hbp1 for degradation"

Article Title: The multi-subunit GID/CTLH E3 ubiquitin ligase promotes cell proliferation and targets the transcription factor Hbp1 for degradation

Journal: eLife

doi: 10.7554/eLife.35528

Compositional analysis of the mammalian GID complex. ( A ) AP-MS analysis of N-terminally HSS-tagged WDR26 in HEK-293 cells from a doxycycline-inducible promoter using lentiviral transduction. HSS-WDR26 expression was induced for 24 hr and immunoprecipitated using HA-antibodies. The number of peptide spectral matches (PSM) of HSS-WDR26 and selected proteins in control - and WDR26 samples are listed in the table below. Note that in contrast to multiple hGID/CTLH subunits, the CRL4 core component Ddb1 is not enriched. ( B ) Immunoprecipitation of N-terminally HSS-tagged Ddb1 stably expressed in HeLa cells. The HA-peptide eluate (HA-IP) was probed for the presence of the canonical DCAF protein WDR23 and the putative CRL4 adapter WDR26. ( C ) SAINT analysis plot of previously annotated and novel Rmnd5a-HCIPs corresponding to Figure 1C . ( D ) Immunoprecipitation of a transiently expressed HSS-GID4 construct in HEK-293 cells was analyzed for the presence of the GID proteins RanBP9, WDR26 and MAEA. ( E ) Schematic illustration of the yeast GID E3 ligase (adapted from Menssen et al., 2012 ) and the potential architecture of the human GID complex (this study). ( F ) S. cerevisiae wild-type (WT), moh1∆ and gid4∆ deletion strains expressing Fbp1-TAP were grown o/n in YP + 3% EtOH before shifting into YP medium containing 2% glucose. Samples for immunoblot analysis were taken at the indicated time points and relative Fbp1-TAP levels were quantified using ImageJ.
Figure Legend Snippet: Compositional analysis of the mammalian GID complex. ( A ) AP-MS analysis of N-terminally HSS-tagged WDR26 in HEK-293 cells from a doxycycline-inducible promoter using lentiviral transduction. HSS-WDR26 expression was induced for 24 hr and immunoprecipitated using HA-antibodies. The number of peptide spectral matches (PSM) of HSS-WDR26 and selected proteins in control - and WDR26 samples are listed in the table below. Note that in contrast to multiple hGID/CTLH subunits, the CRL4 core component Ddb1 is not enriched. ( B ) Immunoprecipitation of N-terminally HSS-tagged Ddb1 stably expressed in HeLa cells. The HA-peptide eluate (HA-IP) was probed for the presence of the canonical DCAF protein WDR23 and the putative CRL4 adapter WDR26. ( C ) SAINT analysis plot of previously annotated and novel Rmnd5a-HCIPs corresponding to Figure 1C . ( D ) Immunoprecipitation of a transiently expressed HSS-GID4 construct in HEK-293 cells was analyzed for the presence of the GID proteins RanBP9, WDR26 and MAEA. ( E ) Schematic illustration of the yeast GID E3 ligase (adapted from Menssen et al., 2012 ) and the potential architecture of the human GID complex (this study). ( F ) S. cerevisiae wild-type (WT), moh1∆ and gid4∆ deletion strains expressing Fbp1-TAP were grown o/n in YP + 3% EtOH before shifting into YP medium containing 2% glucose. Samples for immunoblot analysis were taken at the indicated time points and relative Fbp1-TAP levels were quantified using ImageJ.

Techniques Used: Mass Spectrometry, Transduction, Expressing, Immunoprecipitation, Stable Transfection, Construct

GID activity is required for cell growth in RPE cells. ( A ) Gallery of HA-immunofluorescence images showing the respective subcellular localization of HSS-tagged GID subunits MAEA, Twa1 and WDR26, stably expressed from a doxycycline-inducible promoter. Co-staining of DNA with DAPI visualizes the nuclei. The proteins are predominantly nuclear with some diffuse staining in the cytoplasm. Scale bar, 5 μM. ( B ) Immunoprecipitation of HEK-293 control and MAEA-KO cells transiently expressing HSS-tagged Rmnd5a. The presence of associated proteins was examined by immunoblotting with the indicated antibodies. The Twa1 antibody also recognizes an unspecific band at approximately the same size in WCEs (asterisks). Note that the assembly of the GID complex requires the RING protein MAEA. ( C ) Cell extracts prepared from RPE control (ctrl), MAEA-KO (targeted by two distinct sets of gRNAs, left panel) or WDR26-KO cells (right panel) were analyzed 6 days after lentiviral treatment for the indicated cell cycle and growth pathway markers by immunoblotting with the indicated antibodies. Note that cells deficient for GID activity show reduced levels of phosphorylated (780 and 807/811) and total levels of the tumor suppressor protein Rb, Cyclin A and phosphorylated Histone H3 indicating that they stopped proliferating. The autoregulated novel GID subunit YPEL5 is stabilized in catalytically inactive MAEA-KO cells. ( D ) Depletion efficiency of individual GID subunits in mouse primary hepatocytes analyzed by qRT-PCR using gene-specific primers. Averaged data plotted from independent experiments ± SEM, n = 2. ( E ) Relative mRNA expression of FBP1 in murine primary hepatocytes in gluconeogenic and glycolytic conditions in control siRNA or GID siRNA samples analyzed by qRT-PCR. Graphs present the averaged data from two independent experiments performed in duplicates ± SEM, n = 2. ( F ) The ability of hepatic glucose production was measured in control- and GID siRNA-treated cells starved for 6 hr before processing. Data expressed as mean mg/dl of glucose per μg protein relative to control ± SD, n = 6. ( G ) Glucose levels in the medium of control and GID-depleted primary hepatocytes were determined 4 hr after they were shifted from gluconeogenesis to glycolysis. Data presented as mean fold change relative to control ± SD, n = 3.
Figure Legend Snippet: GID activity is required for cell growth in RPE cells. ( A ) Gallery of HA-immunofluorescence images showing the respective subcellular localization of HSS-tagged GID subunits MAEA, Twa1 and WDR26, stably expressed from a doxycycline-inducible promoter. Co-staining of DNA with DAPI visualizes the nuclei. The proteins are predominantly nuclear with some diffuse staining in the cytoplasm. Scale bar, 5 μM. ( B ) Immunoprecipitation of HEK-293 control and MAEA-KO cells transiently expressing HSS-tagged Rmnd5a. The presence of associated proteins was examined by immunoblotting with the indicated antibodies. The Twa1 antibody also recognizes an unspecific band at approximately the same size in WCEs (asterisks). Note that the assembly of the GID complex requires the RING protein MAEA. ( C ) Cell extracts prepared from RPE control (ctrl), MAEA-KO (targeted by two distinct sets of gRNAs, left panel) or WDR26-KO cells (right panel) were analyzed 6 days after lentiviral treatment for the indicated cell cycle and growth pathway markers by immunoblotting with the indicated antibodies. Note that cells deficient for GID activity show reduced levels of phosphorylated (780 and 807/811) and total levels of the tumor suppressor protein Rb, Cyclin A and phosphorylated Histone H3 indicating that they stopped proliferating. The autoregulated novel GID subunit YPEL5 is stabilized in catalytically inactive MAEA-KO cells. ( D ) Depletion efficiency of individual GID subunits in mouse primary hepatocytes analyzed by qRT-PCR using gene-specific primers. Averaged data plotted from independent experiments ± SEM, n = 2. ( E ) Relative mRNA expression of FBP1 in murine primary hepatocytes in gluconeogenic and glycolytic conditions in control siRNA or GID siRNA samples analyzed by qRT-PCR. Graphs present the averaged data from two independent experiments performed in duplicates ± SEM, n = 2. ( F ) The ability of hepatic glucose production was measured in control- and GID siRNA-treated cells starved for 6 hr before processing. Data expressed as mean mg/dl of glucose per μg protein relative to control ± SD, n = 6. ( G ) Glucose levels in the medium of control and GID-depleted primary hepatocytes were determined 4 hr after they were shifted from gluconeogenesis to glycolysis. Data presented as mean fold change relative to control ± SD, n = 3.

Techniques Used: Activity Assay, Immunofluorescence, Stable Transfection, Staining, Immunoprecipitation, Expressing, Quantitative RT-PCR

The transcription factor Hbp1 is a novel reciprocal GID-interacting protein. ( A ) The protein levels of the GID-interactor and mitochondrial protease HTRA2 was compared by immunoblotting of extracts prepared from HeLa Kyoto and HEK-293 cells deleted for WDR26 or MAEA using different gRNAs. ( B ) HA-immunoprecipitation of stably expressed HSS-Rmnd5a (upper panel) or HSS-Armc8 (lower panel) from HEK-293 cells. Where indicated (+), the cells were treated for 30 min with MG132 prior to cell lysis to prevent proteasomal degradation. Note that the mammalian GET complex protein BAG6 and the chaperone HSPA2 only weakly interact with the hGID complex, and the interaction of Rmnd5a with BAG6 is only marginally increased in the presence of MG132. ( C ) Graphical (left) and network (right) view of Hbp1-HCIPs mapped by AP-MS and subsequent SAINT analysis in HEK-293 cells (SAINT probability score ≥0.9, fold change over control (FC-B) ≥4, n = 2). Hbp1 shows two strong independent interaction modules comprising multiple subunits of the Sin3a co-repressor complex and the GID E3 ligase with all subunits present. Previously reported hits documented in the interaction database (iRefIndex) are displayed by red dots and novel hits are colored in blue.
Figure Legend Snippet: The transcription factor Hbp1 is a novel reciprocal GID-interacting protein. ( A ) The protein levels of the GID-interactor and mitochondrial protease HTRA2 was compared by immunoblotting of extracts prepared from HeLa Kyoto and HEK-293 cells deleted for WDR26 or MAEA using different gRNAs. ( B ) HA-immunoprecipitation of stably expressed HSS-Rmnd5a (upper panel) or HSS-Armc8 (lower panel) from HEK-293 cells. Where indicated (+), the cells were treated for 30 min with MG132 prior to cell lysis to prevent proteasomal degradation. Note that the mammalian GET complex protein BAG6 and the chaperone HSPA2 only weakly interact with the hGID complex, and the interaction of Rmnd5a with BAG6 is only marginally increased in the presence of MG132. ( C ) Graphical (left) and network (right) view of Hbp1-HCIPs mapped by AP-MS and subsequent SAINT analysis in HEK-293 cells (SAINT probability score ≥0.9, fold change over control (FC-B) ≥4, n = 2). Hbp1 shows two strong independent interaction modules comprising multiple subunits of the Sin3a co-repressor complex and the GID E3 ligase with all subunits present. Previously reported hits documented in the interaction database (iRefIndex) are displayed by red dots and novel hits are colored in blue.

Techniques Used: Immunoprecipitation, Stable Transfection, Lysis, Mass Spectrometry

2) Product Images from "Engulfment, persistence and fate of Bdellovibrio bacteriovorus predators inside human phagocytic cells informs their future therapeutic potential"

Article Title: Engulfment, persistence and fate of Bdellovibrio bacteriovorus predators inside human phagocytic cells informs their future therapeutic potential

Journal: Scientific Reports

doi: 10.1038/s41598-019-40223-3

Viability of U937 cells exposed to B. bacteriovorus . BbHD100CFP were exposed to U937 cells for 2 h at a MOE of 50:1 and the exposed U937 cells were stained with Calcein (live cells, green) and EthD-1 (dead cells, red) and imaged live at 4, 8 and 24 h. ( a ) Shown are representative images of live/dead stained control and BbHD100CFP containing U937 cells at 4, 8 and 24 h. The images were generated by merging snapshots of live and dead cells stained with Calcein AM (green) and EthD-1 (red) with/without BbHD100CFP, imaged as z-stacks, restored and 2D projected at maximum intensity (blue). Scale bar −10 µm. The images were acquired using 60x lens ( a.1 ) and 100x lens ( a.2 ). ( b ) Percentage of live and dead cells: the stained live and dead cells that were exposed to BbHD100CFP were counted and the percentages of each population of cells present were calculated. The percentage of live and dead cells counted at different time points in the control and BbHD100CFP exposed U937 cells were not significantly different (Live cells – P
Figure Legend Snippet: Viability of U937 cells exposed to B. bacteriovorus . BbHD100CFP were exposed to U937 cells for 2 h at a MOE of 50:1 and the exposed U937 cells were stained with Calcein (live cells, green) and EthD-1 (dead cells, red) and imaged live at 4, 8 and 24 h. ( a ) Shown are representative images of live/dead stained control and BbHD100CFP containing U937 cells at 4, 8 and 24 h. The images were generated by merging snapshots of live and dead cells stained with Calcein AM (green) and EthD-1 (red) with/without BbHD100CFP, imaged as z-stacks, restored and 2D projected at maximum intensity (blue). Scale bar −10 µm. The images were acquired using 60x lens ( a.1 ) and 100x lens ( a.2 ). ( b ) Percentage of live and dead cells: the stained live and dead cells that were exposed to BbHD100CFP were counted and the percentages of each population of cells present were calculated. The percentage of live and dead cells counted at different time points in the control and BbHD100CFP exposed U937 cells were not significantly different (Live cells – P

Techniques Used: Staining, Ethidium Homodimer Assay, Generated

Role of cytoskeleton in the uptake of B. bacteriovorus by U937 cells. ( a ) U937 cells pretreated with actin depolymerising agent, cytochalasin D (10 µM) for 1 h were exposed to BbHD100TFP (green) for 2 h at an MOE of 50 bacteria per cell in the presence of inhibitors or carrier (DMSO) and fixed. The actin filaments of fixed cells were stained with Rhodamine-phalloidin (false coloured in cyan), the nuclei were stained with SiR-DNA (red) and imaged. Shown are the maximum intensity 2D-projections (Stacks 1–20 used for both control and cytochalasin D treated cells) of the restored z-stack images. Scale bar −5 µm. Images are representative of two independent experiments. ( b ) U937 cells pretreated with microtubule inhibitor, Nocodazole (2.5 µM) for 1 h were exposed to BbHD100TFP (green) for 2 h at a MOE of 50 bacteria per cell in the presence of inhibitor or carrier (DMSO) and fixed. The microtubules of the fixed cells were stained with anti-tubulin primary antibody and Alexa 555 secondary antibody (false coloured in magenta), nuclei were stained with SiR-DNA (red) and imaged. Shown are the maximum intensity 2D-projections (Stacks 5–25 for both control and Nocodazole treated cells) of the restored images. Scale bar −5 µm. Images are representative of two independent experiments. ( c ) B. bacteriovorus detected inside U937 cells that were exposed to BbHD100TFP for 2 hours at a MOE of 50 bacteria per cell in the presence of cytoskeletal inhibitors or carrier, DMSO were counted from the restored z-stack images of fixed and immunostained cells. Data shown, as number of BbHD100TFP visualised per cell, are representative of one of the two independent experiments, each set up in duplicate and a minimum of 125 cells were analysed from each experiment. ****corresponds to P
Figure Legend Snippet: Role of cytoskeleton in the uptake of B. bacteriovorus by U937 cells. ( a ) U937 cells pretreated with actin depolymerising agent, cytochalasin D (10 µM) for 1 h were exposed to BbHD100TFP (green) for 2 h at an MOE of 50 bacteria per cell in the presence of inhibitors or carrier (DMSO) and fixed. The actin filaments of fixed cells were stained with Rhodamine-phalloidin (false coloured in cyan), the nuclei were stained with SiR-DNA (red) and imaged. Shown are the maximum intensity 2D-projections (Stacks 1–20 used for both control and cytochalasin D treated cells) of the restored z-stack images. Scale bar −5 µm. Images are representative of two independent experiments. ( b ) U937 cells pretreated with microtubule inhibitor, Nocodazole (2.5 µM) for 1 h were exposed to BbHD100TFP (green) for 2 h at a MOE of 50 bacteria per cell in the presence of inhibitor or carrier (DMSO) and fixed. The microtubules of the fixed cells were stained with anti-tubulin primary antibody and Alexa 555 secondary antibody (false coloured in magenta), nuclei were stained with SiR-DNA (red) and imaged. Shown are the maximum intensity 2D-projections (Stacks 5–25 for both control and Nocodazole treated cells) of the restored images. Scale bar −5 µm. Images are representative of two independent experiments. ( c ) B. bacteriovorus detected inside U937 cells that were exposed to BbHD100TFP for 2 hours at a MOE of 50 bacteria per cell in the presence of cytoskeletal inhibitors or carrier, DMSO were counted from the restored z-stack images of fixed and immunostained cells. Data shown, as number of BbHD100TFP visualised per cell, are representative of one of the two independent experiments, each set up in duplicate and a minimum of 125 cells were analysed from each experiment. ****corresponds to P

Techniques Used: Staining

Cytokine responses induced by B. bacteriovorus in comparison to pathogens S . Typhimurium LT2 and K. pneumoniae KPC. BbHD100, S . Typhimurium LT2 and K. pneumoniae KPC were exposed to U937 cells by synchronous spin-assisted uptake at MOEs of 50:1 and 10:1 and cell culture supernatants were collected at various time points as illustrated in Fig. S1 . The levels of cytokines (IL-1β ( a ), TNF-α ( b ), IL-6 ( c ), IL-10 ( d ) and IL-8 ( e )) present in the supernatants of the bacteria-exposed U937 cells, collected at 2, 4, 8, 24 and 48 h, were measured by ELISA set up in triplicates for each individual supernatant sample collected. For pathogens, the 2-hour time points in all panels represent cytokines produced during gentamycin treatment and the subsequent time points show cumulative cytokine production from 2 h onwards. The cytokine concentrations shown as pg/mL are representative of mean ± standard error of values from three (BbHD100 and K. pneumoniae KPC (n = 18)) or two ( S . Typhimurium LT2 (n = 12)) independent experiments, each set up with two technical replicates for U937-predatory/pathogenic bacteria exposures.
Figure Legend Snippet: Cytokine responses induced by B. bacteriovorus in comparison to pathogens S . Typhimurium LT2 and K. pneumoniae KPC. BbHD100, S . Typhimurium LT2 and K. pneumoniae KPC were exposed to U937 cells by synchronous spin-assisted uptake at MOEs of 50:1 and 10:1 and cell culture supernatants were collected at various time points as illustrated in Fig. S1 . The levels of cytokines (IL-1β ( a ), TNF-α ( b ), IL-6 ( c ), IL-10 ( d ) and IL-8 ( e )) present in the supernatants of the bacteria-exposed U937 cells, collected at 2, 4, 8, 24 and 48 h, were measured by ELISA set up in triplicates for each individual supernatant sample collected. For pathogens, the 2-hour time points in all panels represent cytokines produced during gentamycin treatment and the subsequent time points show cumulative cytokine production from 2 h onwards. The cytokine concentrations shown as pg/mL are representative of mean ± standard error of values from three (BbHD100 and K. pneumoniae KPC (n = 18)) or two ( S . Typhimurium LT2 (n = 12)) independent experiments, each set up with two technical replicates for U937-predatory/pathogenic bacteria exposures.

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Produced

Trafficking of B. bacteriovorus inside U937 cells. BbHD100TFP were exposed to U937 cells for 2 hours at a MOE of 50:1 and the acidic vacuoles of the cells were labelled with LysoTracker red DND-99 and imaged live at 4, 8 and 24 h. ( a ) Shown are representative images (single slices from restored z-stacks) of U937 cells (Nuclei stained with Vybrant DyeCycle Violet, blue) containing BbHD100TFP (green) with labelled acidic vacuoles (red) at 4, 8 and 24 h from one of the two independent experiments. The red and green arrows in the LysoTracker and BbHD100TFP channels point to specific acidic vacuoles and BbHD100TFP respectively. The yellow/white arrows in the merged images show colocalisation/no colocalisation respectively of predatory bacteria with the acidic vacuoles. Scale bar −5 µm. ( b ) Box plots with each point corresponding to the fraction of bacteria colocalising with the labelled acidic vacuoles in a cell at the time points, 4, 8 and 24 h. A minimum of 100 cells with predatory bacteria were analysed in each experiment at each time point and the data shown are the combined values from two independent experiments (n = 100 cells per experiment). ****corresponds to P
Figure Legend Snippet: Trafficking of B. bacteriovorus inside U937 cells. BbHD100TFP were exposed to U937 cells for 2 hours at a MOE of 50:1 and the acidic vacuoles of the cells were labelled with LysoTracker red DND-99 and imaged live at 4, 8 and 24 h. ( a ) Shown are representative images (single slices from restored z-stacks) of U937 cells (Nuclei stained with Vybrant DyeCycle Violet, blue) containing BbHD100TFP (green) with labelled acidic vacuoles (red) at 4, 8 and 24 h from one of the two independent experiments. The red and green arrows in the LysoTracker and BbHD100TFP channels point to specific acidic vacuoles and BbHD100TFP respectively. The yellow/white arrows in the merged images show colocalisation/no colocalisation respectively of predatory bacteria with the acidic vacuoles. Scale bar −5 µm. ( b ) Box plots with each point corresponding to the fraction of bacteria colocalising with the labelled acidic vacuoles in a cell at the time points, 4, 8 and 24 h. A minimum of 100 cells with predatory bacteria were analysed in each experiment at each time point and the data shown are the combined values from two independent experiments (n = 100 cells per experiment). ****corresponds to P

Techniques Used: Staining

Persistence and survival of B. bacteriovorus inside U937 cells. ( a ) BbHD100 were exposed to U937 cells for 2 h at MOEs of 50:1 and 10:1. The predatory bacteria recovered from the U937 cells were enumerated at 2, 4, 8, 24 and 48 h. Data shown, as PFU/mL, are representative of mean ± standard deviation of two independent experiments, each set up in duplicate for U937-HD100 exposures and included technical replicates for bacterial-plaque enumerations (n = 8). ***corresponds to P = 0.0002. ( b ) Representative images of U937 cells containing BbHD100CFP at 2 and 24 hours. BbHD100CFP were exposed to U937 cells for 2 hours at a MOE of 50:1 and the U937 cells with predatory bacteria were fixed at 2 and 24 h. The images shown constitute whole cells (phase), snapshots of nuclei (stained with SiR-DNA, red) and maximum intensity 2D-projections of restored z-stack images of BbHD100CFP (blue). Scale bar −10 µm. Images are representative of two independent experiments, each set up in duplicate. In analysing images, no BbHD100CFP were observed as attached to the outside of the cells after the 2 h uptake. ( c ) BbHD100CFP inside U937 cells were counted from the restored z-stack images of fixed U937 cells with BbHD100CFP at 2 and 24 h from MOE 50:1 exposures. Data shown, as number of BbHD100CFP visualised per cell, are representative of one of the two independent experiments, each set up in duplicate and a minimum of 150 cells were analysed at each time point from each experiment (n = 150 cells per experiment). **corresponds to P = 0.0060.
Figure Legend Snippet: Persistence and survival of B. bacteriovorus inside U937 cells. ( a ) BbHD100 were exposed to U937 cells for 2 h at MOEs of 50:1 and 10:1. The predatory bacteria recovered from the U937 cells were enumerated at 2, 4, 8, 24 and 48 h. Data shown, as PFU/mL, are representative of mean ± standard deviation of two independent experiments, each set up in duplicate for U937-HD100 exposures and included technical replicates for bacterial-plaque enumerations (n = 8). ***corresponds to P = 0.0002. ( b ) Representative images of U937 cells containing BbHD100CFP at 2 and 24 hours. BbHD100CFP were exposed to U937 cells for 2 hours at a MOE of 50:1 and the U937 cells with predatory bacteria were fixed at 2 and 24 h. The images shown constitute whole cells (phase), snapshots of nuclei (stained with SiR-DNA, red) and maximum intensity 2D-projections of restored z-stack images of BbHD100CFP (blue). Scale bar −10 µm. Images are representative of two independent experiments, each set up in duplicate. In analysing images, no BbHD100CFP were observed as attached to the outside of the cells after the 2 h uptake. ( c ) BbHD100CFP inside U937 cells were counted from the restored z-stack images of fixed U937 cells with BbHD100CFP at 2 and 24 h from MOE 50:1 exposures. Data shown, as number of BbHD100CFP visualised per cell, are representative of one of the two independent experiments, each set up in duplicate and a minimum of 150 cells were analysed at each time point from each experiment (n = 150 cells per experiment). **corresponds to P = 0.0060.

Techniques Used: Standard Deviation, Staining

Colocalisation of B. bacteriovorus with early and late phagosomal markers. BbHD100CFP were exposed to U937 cells for 15 min at a MOE of 200:1 in a smaller volume (200 µl) to promote rapid uptake of the predatory bacteria. The cells fixed at time points, 20 min, 40 min, 1 h and 2 h were immunostained for the early phagosomal marker EEA1 and late phagosomal markers Rab7 and LAMP1. Shown are the representative maximum intensity 2D-projections of restored z-stack images of U937 cells with BbHD100CFP (blue), immunostained for the phagosomal markers (false coloured in red), taken at time points 20 minutes (EEA1) and 1 h (Rab7 and LAMP1). The magnified images of the merged panel are representative regions showing the predatory bacteria colocalising with the phagosomal markers. A minimum of 100 cells with BbHD100CFP were analysed at each time point to study colocalisation of predators with phagosomal markers. Scale bar −5 µm.
Figure Legend Snippet: Colocalisation of B. bacteriovorus with early and late phagosomal markers. BbHD100CFP were exposed to U937 cells for 15 min at a MOE of 200:1 in a smaller volume (200 µl) to promote rapid uptake of the predatory bacteria. The cells fixed at time points, 20 min, 40 min, 1 h and 2 h were immunostained for the early phagosomal marker EEA1 and late phagosomal markers Rab7 and LAMP1. Shown are the representative maximum intensity 2D-projections of restored z-stack images of U937 cells with BbHD100CFP (blue), immunostained for the phagosomal markers (false coloured in red), taken at time points 20 minutes (EEA1) and 1 h (Rab7 and LAMP1). The magnified images of the merged panel are representative regions showing the predatory bacteria colocalising with the phagosomal markers. A minimum of 100 cells with BbHD100CFP were analysed at each time point to study colocalisation of predators with phagosomal markers. Scale bar −5 µm.

Techniques Used: Marker

3) Product Images from "Oral co-administration of a bacterial protease inhibitor in the vaccine formulation increases antigen delivery at the intestinal epithelial barrier"

Article Title: Oral co-administration of a bacterial protease inhibitor in the vaccine formulation increases antigen delivery at the intestinal epithelial barrier

Journal: Journal of Controlled Release

doi: 10.1016/j.jconrel.2018.11.025

U-Omp19 co-delivery induces antigen accumulation at enterocytes lysosomes. Confocal scanning microscopy analysis of Caco-2 cells treated with OVA–Alexa Fluor 647 (25 μg/ml) alone or plus U-Omp19 (25 or 50 μg/ml) and Leupeptin (10 μg/ml). After 3 h of incubation cells were fixed, permeabilized, and stained with mAb anti–human Lamp-2. Anti-mouse IgG coupled to Alexa Fluor 546 (red) was used as secondary Ab. Images are representative of most cells examined by confocal microscopy. The merge between Lamp-2/OVA is shown. Quantification of colocalization OVA/Lamp-2 (M1) and Lamp-2/OVA (M2) was analyzed by Manders overlap coefficient. Data are means of Manders coefficients ± SEM present in 4–5 images of each condition. Scale bars 20 μm. * p
Figure Legend Snippet: U-Omp19 co-delivery induces antigen accumulation at enterocytes lysosomes. Confocal scanning microscopy analysis of Caco-2 cells treated with OVA–Alexa Fluor 647 (25 μg/ml) alone or plus U-Omp19 (25 or 50 μg/ml) and Leupeptin (10 μg/ml). After 3 h of incubation cells were fixed, permeabilized, and stained with mAb anti–human Lamp-2. Anti-mouse IgG coupled to Alexa Fluor 546 (red) was used as secondary Ab. Images are representative of most cells examined by confocal microscopy. The merge between Lamp-2/OVA is shown. Quantification of colocalization OVA/Lamp-2 (M1) and Lamp-2/OVA (M2) was analyzed by Manders overlap coefficient. Data are means of Manders coefficients ± SEM present in 4–5 images of each condition. Scale bars 20 μm. * p

Techniques Used: Microscopy, Incubation, Staining, Confocal Microscopy

U-Omp19 inhibits cathepsin L activity in human intestinal epithelial cells. Caco-2 (A) and HT29 (B) cells were incubated during 1 h with different concentrations of U-Omp19 or Leupeptin. Then, a specific fluorescence quenched substrate for cathepsin L was added and the reaction was measured in a microplate fluorescence reader for 4 h. Data are shown as arbitrary fluorescence units or percentage of remaining cathepsin L activity over time. C. Confocal microscopy analysis of Caco-2 cells after incubation with the specific quenched substrate for cathepsin L (green) and Lysotracker (red) in presence of medium, U-Omp19 or Leupeptin. Images are representative of most cells examined by confocal microscopy. Colocalization was analyzed by Manders colocalization coefficient (M1 and M2). Data are means of Manders coefficient ± SEM. Scale bars 5 μm. * p
Figure Legend Snippet: U-Omp19 inhibits cathepsin L activity in human intestinal epithelial cells. Caco-2 (A) and HT29 (B) cells were incubated during 1 h with different concentrations of U-Omp19 or Leupeptin. Then, a specific fluorescence quenched substrate for cathepsin L was added and the reaction was measured in a microplate fluorescence reader for 4 h. Data are shown as arbitrary fluorescence units or percentage of remaining cathepsin L activity over time. C. Confocal microscopy analysis of Caco-2 cells after incubation with the specific quenched substrate for cathepsin L (green) and Lysotracker (red) in presence of medium, U-Omp19 or Leupeptin. Images are representative of most cells examined by confocal microscopy. Colocalization was analyzed by Manders colocalization coefficient (M1 and M2). Data are means of Manders coefficient ± SEM. Scale bars 5 μm. * p

Techniques Used: Activity Assay, Incubation, Fluorescence, Confocal Microscopy

U-Omp19 facilitates Ag transport through epithelial cell monolayers. Transwell plates cultured Caco-2 cell monolayers were incubated with OVA-Alexa Fluor 647 alone (0.5 mg/ml) or in presence of U-Omp19 or Leupeptin during 3 h. Then, fluorescence in cell lysates (A) and basolateral medium (B) were measured in a microplate fluorescence reader. Result were pooled from 3 different experiments and data are means of percentage of OVA ± SEM. * p
Figure Legend Snippet: U-Omp19 facilitates Ag transport through epithelial cell monolayers. Transwell plates cultured Caco-2 cell monolayers were incubated with OVA-Alexa Fluor 647 alone (0.5 mg/ml) or in presence of U-Omp19 or Leupeptin during 3 h. Then, fluorescence in cell lysates (A) and basolateral medium (B) were measured in a microplate fluorescence reader. Result were pooled from 3 different experiments and data are means of percentage of OVA ± SEM. * p

Techniques Used: Cell Culture, Incubation, Fluorescence

U-Omp19 increases CTB half-life and immunogenicity after oral co-delivery. A. Caco-2 epithelial cell line was incubated with CTB-Alexa Fluor 647 (10 μg/ml) alone or plus U-Omp19 (50 μg/ml) or Leupeptin (10 μg/ml) during 3 h and then MFI was determined by flow cytometry. Data are presented as histograms and bar graphs ± SEM. B. U-Omp19 co-delivery with CTB increases CTB accumulation inside the lysosome of intestinal epithelial cells. Confocal microscopy analysis of Caco-2 cells treated with CTB-Alexa Fluor 647 (10 μg/ml, green) alone or plus U-Omp19 (25 or 50 μg/ml) and Leupeptin (10 μg/ml). After 3 h of incubation cells were fixed, permeabilized, and stained with mAb anti–human Lamp-2. Anti-mouse IgG coupled to Alexa Fluor 546 (red) was used as secondary Ab. Images are representative of most cells examined by confocal microscopy. C. Quantification of colocalization was analyzed by Manders overlap coefficient. Data are means of Manders coefficients ± SEM present in 4–5 images of each condition. * p
Figure Legend Snippet: U-Omp19 increases CTB half-life and immunogenicity after oral co-delivery. A. Caco-2 epithelial cell line was incubated with CTB-Alexa Fluor 647 (10 μg/ml) alone or plus U-Omp19 (50 μg/ml) or Leupeptin (10 μg/ml) during 3 h and then MFI was determined by flow cytometry. Data are presented as histograms and bar graphs ± SEM. B. U-Omp19 co-delivery with CTB increases CTB accumulation inside the lysosome of intestinal epithelial cells. Confocal microscopy analysis of Caco-2 cells treated with CTB-Alexa Fluor 647 (10 μg/ml, green) alone or plus U-Omp19 (25 or 50 μg/ml) and Leupeptin (10 μg/ml). After 3 h of incubation cells were fixed, permeabilized, and stained with mAb anti–human Lamp-2. Anti-mouse IgG coupled to Alexa Fluor 546 (red) was used as secondary Ab. Images are representative of most cells examined by confocal microscopy. C. Quantification of colocalization was analyzed by Manders overlap coefficient. Data are means of Manders coefficients ± SEM present in 4–5 images of each condition. * p

Techniques Used: CtB Assay, Incubation, Flow Cytometry, Cytometry, Confocal Microscopy, Staining

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

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

Journal: Scientific Reports

doi: 10.1038/s41598-019-39650-z

Analysis of U87MG secretome from serum-containing (SCM) and serum-free media (SFM). ( a ) Schematic workflow for quantitative analysis of U87MG secretome between SCM and SFM. DDA: data-dependent acquisition. DDA-EL: DDA with exclusion list. ( b ) The number of identified proteins in the secretome. ( c ) The distribution of differentially secreted proteins between SFM and SCM. The H/M ratios are log2-transformed after normalization by the difference in growth rate. ( d , e ) Secretion pathways and subcellular localization of the differentially secreted proteins. Secretion pathways were analyzed by using SignalP, SecretomeP, and TMHMM (d) , and subcellular localization by using Cello v2.5 ( e ).
Figure Legend Snippet: Analysis of U87MG secretome from serum-containing (SCM) and serum-free media (SFM). ( a ) Schematic workflow for quantitative analysis of U87MG secretome between SCM and SFM. DDA: data-dependent acquisition. DDA-EL: DDA with exclusion list. ( b ) The number of identified proteins in the secretome. ( c ) The distribution of differentially secreted proteins between SFM and SCM. The H/M ratios are log2-transformed after normalization by the difference in growth rate. ( d , e ) Secretion pathways and subcellular localization of the differentially secreted proteins. Secretion pathways were analyzed by using SignalP, SecretomeP, and TMHMM (d) , and subcellular localization by using Cello v2.5 ( e ).

Techniques Used: Transformation Assay

5) Product Images from "Gelatin/Nanohyroxyapatite Cryogel Embedded Poly(lactic-co-glycolic Acid)/Nanohydroxyapatite Microsphere Hybrid Scaffolds for Simultaneous Bone Regeneration and Load-Bearing"

Article Title: Gelatin/Nanohyroxyapatite Cryogel Embedded Poly(lactic-co-glycolic Acid)/Nanohydroxyapatite Microsphere Hybrid Scaffolds for Simultaneous Bone Regeneration and Load-Bearing

Journal: Polymers

doi: 10.3390/polym10060620

The DNA content ( A ) and ALP activity ( B ) of rBMSCs cultured in cryogel and hybrid scaffolds. ( C ) Qualitative assessment of mineralization through SEM and quantitative estimation of mineral composition by EDS of the cryogel scaffold, the cryogel part in the hybrid scaffold and the microsphere cavity in the hybrid scaffold. The blue arrows indicate places where SEM/EDS samples were taken. The red arrows indicate mineral deposition from cell differentiation. * p
Figure Legend Snippet: The DNA content ( A ) and ALP activity ( B ) of rBMSCs cultured in cryogel and hybrid scaffolds. ( C ) Qualitative assessment of mineralization through SEM and quantitative estimation of mineral composition by EDS of the cryogel scaffold, the cryogel part in the hybrid scaffold and the microsphere cavity in the hybrid scaffold. The blue arrows indicate places where SEM/EDS samples were taken. The red arrows indicate mineral deposition from cell differentiation. * p

Techniques Used: ALP Assay, Activity Assay, Cell Culture, Cell Differentiation

6) Product Images from "Chromosomal instability and cytoskeletal defects in oral cancer cells"

Article Title: Chromosomal instability and cytoskeletal defects in oral cancer cells

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

doi:

Anaphase bridges containing centromeres and chromosome 11. Immunolabeling with Abs to tubulin (yellow), centromeres (red), and with DAPI (blue) and FISH with a chromosome 11 paint probe (green). ( B ) Arrows point to centromeres trapped in the forming midbody as these late telophase cells divide. ( D ) Arrow points to the trapped lagging chromosome excluded from the reforming nucleus of the cell on the right. ( E ) Some micronuclei are immunonegative for anti-centromere Abs. Arrow points to negative micronucleus, and arrowhead points to positive. Examples are from UPCI:SCC131.
Figure Legend Snippet: Anaphase bridges containing centromeres and chromosome 11. Immunolabeling with Abs to tubulin (yellow), centromeres (red), and with DAPI (blue) and FISH with a chromosome 11 paint probe (green). ( B ) Arrows point to centromeres trapped in the forming midbody as these late telophase cells divide. ( D ) Arrow points to the trapped lagging chromosome excluded from the reforming nucleus of the cell on the right. ( E ) Some micronuclei are immunonegative for anti-centromere Abs. Arrow points to negative micronucleus, and arrowhead points to positive. Examples are from UPCI:SCC131.

Techniques Used: Immunolabeling, Fluorescence In Situ Hybridization

7) Product Images from "Regulation of Cripto-1 Signaling and Biological Activity by Caveolin-1 in Mammary Epithelial Cells"

Article Title: Regulation of Cripto-1 Signaling and Biological Activity by Caveolin-1 in Mammary Epithelial Cells

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2008.070696

Stable Cav-1 expression in EpH4 mouse mammary epithelial cells. Cell lysates from EpH4 WT, EpH4 Cav-1, EpH4 Cr-1, and EpH4 Cr-1/Cav-1 cells were analyzed by Western blotting to confirm gene expression using anti-CR-1 mouse monoclonal antibody, anti-V5 mouse monoclonal antibody to detect Cav-1-V5/His fusion protein, or anti-β-actin monoclonal antibody to ensure equal loading of the protein samples. WB, Western blot.
Figure Legend Snippet: Stable Cav-1 expression in EpH4 mouse mammary epithelial cells. Cell lysates from EpH4 WT, EpH4 Cav-1, EpH4 Cr-1, and EpH4 Cr-1/Cav-1 cells were analyzed by Western blotting to confirm gene expression using anti-CR-1 mouse monoclonal antibody, anti-V5 mouse monoclonal antibody to detect Cav-1-V5/His fusion protein, or anti-β-actin monoclonal antibody to ensure equal loading of the protein samples. WB, Western blot.

Techniques Used: Expressing, Western Blot

Binding of Cripto-1 to Cav-1 in coimmunoprecipitation experiments in COS7 cells and in FVB/N and MCF-10A mammary epithelial cells. A: COS7 cells were transiently transfected with Cav-1-myc/His, CR-1 and c-src expression vectors either alone or in various combinations, and 48 hours after transfection the cells were lysed and proteins were immunoprecipitated with anti-myc monoclonal antibody agarose beads. The myc-tagged immunoprecipitated proteins were then analyzed by Western blot analysis using anti-CR-1 rabbit polyclonal antibody, anti-c-src mouse monoclonal antibody or anti-His mouse monoclonal antibody to detect Cav-1-myc/His fusion protein. B: COS7 cells were transiently transfected with Cav-1-myc/His, CR-1 and glypican-1-Fc expression vectors either alone or in various combinations and 48 hours after transfection the cells were lysed and protein G-agarose beads were used to immunoprecipitate glypican-1-Fc fusion protein. The bound proteins were analyzed by Western blotting with anti-CR-1 rabbit polyclonal antibody, anti-His mouse monoclonal antibody, or anti-human IgG horseradish peroxidase-linked whole antibody to detect glypican-1-Fc fusion protein. C and D: Western blot analysis for CR-1, Cav-1, c-src, and glypican-1 on cell lysates of COS7 cells transiently transfected with various expression vectors as described above. E: FVB/N primary mouse mammary epithelial cells and MCF-10A human mammary epithelial cells were lysed and immunoprecipitated with an anti-CR-1 rabbit polyclonal antibody. The immunoprecipitated proteins were then analyzed by Western blotting using an anti-Cav-1 rabbit polyclonal antibody and anti-CR-1 rabbit polyclonal antibody. IP, immunoprecipitation; WB, Western blot.
Figure Legend Snippet: Binding of Cripto-1 to Cav-1 in coimmunoprecipitation experiments in COS7 cells and in FVB/N and MCF-10A mammary epithelial cells. A: COS7 cells were transiently transfected with Cav-1-myc/His, CR-1 and c-src expression vectors either alone or in various combinations, and 48 hours after transfection the cells were lysed and proteins were immunoprecipitated with anti-myc monoclonal antibody agarose beads. The myc-tagged immunoprecipitated proteins were then analyzed by Western blot analysis using anti-CR-1 rabbit polyclonal antibody, anti-c-src mouse monoclonal antibody or anti-His mouse monoclonal antibody to detect Cav-1-myc/His fusion protein. B: COS7 cells were transiently transfected with Cav-1-myc/His, CR-1 and glypican-1-Fc expression vectors either alone or in various combinations and 48 hours after transfection the cells were lysed and protein G-agarose beads were used to immunoprecipitate glypican-1-Fc fusion protein. The bound proteins were analyzed by Western blotting with anti-CR-1 rabbit polyclonal antibody, anti-His mouse monoclonal antibody, or anti-human IgG horseradish peroxidase-linked whole antibody to detect glypican-1-Fc fusion protein. C and D: Western blot analysis for CR-1, Cav-1, c-src, and glypican-1 on cell lysates of COS7 cells transiently transfected with various expression vectors as described above. E: FVB/N primary mouse mammary epithelial cells and MCF-10A human mammary epithelial cells were lysed and immunoprecipitated with an anti-CR-1 rabbit polyclonal antibody. The immunoprecipitated proteins were then analyzed by Western blotting using an anti-Cav-1 rabbit polyclonal antibody and anti-CR-1 rabbit polyclonal antibody. IP, immunoprecipitation; WB, Western blot.

Techniques Used: Binding Assay, Transfection, Expressing, Immunoprecipitation, Western Blot

Colocalization of Cripto-1 and Cav-1 in COS7 cells expressing Cr-1 and Cav-1, in EpH4 Cr-1/Cav-1, EpH4 Cr-1, and FVB/N primary mammary epithelial cells. A: Immunofluorescence staining for Cr-1 and Cav-1 in COS7 cells transiently transfected with Cr-1 and Cav-1 expression vectors. B: Immunofluorescence staining for Cr-1, Cav-1, and GM-1 in EpH4 Cr-1/Cav-1 and EpH4 Cr-1 cells and immunofluorescence staining for Cr-1 and Cav-1 in primary FVB/N mouse mammary epithelial cells. C: Negative controls were obtained by replacing the primary antibody with irrelevant control isotype Ig. Fluorescence images were acquired by laser scanner confocal microscopy.
Figure Legend Snippet: Colocalization of Cripto-1 and Cav-1 in COS7 cells expressing Cr-1 and Cav-1, in EpH4 Cr-1/Cav-1, EpH4 Cr-1, and FVB/N primary mammary epithelial cells. A: Immunofluorescence staining for Cr-1 and Cav-1 in COS7 cells transiently transfected with Cr-1 and Cav-1 expression vectors. B: Immunofluorescence staining for Cr-1, Cav-1, and GM-1 in EpH4 Cr-1/Cav-1 and EpH4 Cr-1 cells and immunofluorescence staining for Cr-1 and Cav-1 in primary FVB/N mouse mammary epithelial cells. C: Negative controls were obtained by replacing the primary antibody with irrelevant control isotype Ig. Fluorescence images were acquired by laser scanner confocal microscopy.

Techniques Used: Expressing, Immunofluorescence, Staining, Transfection, Fluorescence, Confocal Microscopy

Cavtratin inhibits migration and invasion of EpH4 Cr-1 cells. Migration (A) and invasion (B) assay of EpH4 Cr-1 cells incubated in the presence of cavtratin. OD, optical density. * P
Figure Legend Snippet: Cavtratin inhibits migration and invasion of EpH4 Cr-1 cells. Migration (A) and invasion (B) assay of EpH4 Cr-1 cells incubated in the presence of cavtratin. OD, optical density. * P

Techniques Used: Migration, Incubation

Loss of Cav-1 enhances migration and invasion and induces hyperactivation of MAPK and c-src signaling molecules in primary mammary epithelial cells derived from Cav-1 null/MMTV-CR-1 double transgenic mice. A: Western blot analysis for Cav-1, CR-1, and β-actin in cell lysates derived from primary mammary epithelial cells isolated from FVB/N, Cav-1 +/− /CR-1, and Cav-1 −/− /CR-1 mice. Migration ( B) and invasion (C) assay of FVB/N, Cav-1 +/− /CR-1, and Cav-1 −/− /CR-1 primary mammary epithelial cells. Cav-1 −/− /CR-1 primary mammary epithelial cells were also treated with cavtratin (10 μmol/L) or with a control peptide (10 μmol/L). * P
Figure Legend Snippet: Loss of Cav-1 enhances migration and invasion and induces hyperactivation of MAPK and c-src signaling molecules in primary mammary epithelial cells derived from Cav-1 null/MMTV-CR-1 double transgenic mice. A: Western blot analysis for Cav-1, CR-1, and β-actin in cell lysates derived from primary mammary epithelial cells isolated from FVB/N, Cav-1 +/− /CR-1, and Cav-1 −/− /CR-1 mice. Migration ( B) and invasion (C) assay of FVB/N, Cav-1 +/− /CR-1, and Cav-1 −/− /CR-1 primary mammary epithelial cells. Cav-1 −/− /CR-1 primary mammary epithelial cells were also treated with cavtratin (10 μmol/L) or with a control peptide (10 μmol/L). * P

Techniques Used: Migration, Derivative Assay, Transgenic Assay, Mouse Assay, Western Blot, Isolation

Cav-1 inhibits Cr-1 enhanced migration, invasion, and branching of EpH4 mouse mammary epithelial cells. A: Migration or invasion assay (B) of EpH4 WT, EpH4 Cav-1, EpH4 Cr-1, and EpH4 Cr-1/Cav-1 cells. OD, optical density. * P
Figure Legend Snippet: Cav-1 inhibits Cr-1 enhanced migration, invasion, and branching of EpH4 mouse mammary epithelial cells. A: Migration or invasion assay (B) of EpH4 WT, EpH4 Cav-1, EpH4 Cr-1, and EpH4 Cr-1/Cav-1 cells. OD, optical density. * P

Techniques Used: Migration, Invasion Assay

Cav-1 expression is reduced in mammary tumors of MMTV-CR-1 transgenic mice. A: Western blot analysis for Cav-1 in tissues from one normal FVB/N or two MMTV-CR-1 virgin mice and from mammary tumors found in four MMTV-CR-1 multiparous mice (T1, T2, T3 and T4). For equal loading, the blot was reprobed with anti-β-actin antibody. B: Immunohistochemistry for Cav-1 and CR-1 in mammary tissue serial sections derived from FVB/N virgin or multiparous mice and in mammary tumor of MMTV-CR-1 mice. These sections were incubated with an anti-Cav-1 or with anti-CR-1 antibodies as described in Materials and Methods. Magnification, ×400. Negative controls are shown in the insets . WB, Western blot.
Figure Legend Snippet: Cav-1 expression is reduced in mammary tumors of MMTV-CR-1 transgenic mice. A: Western blot analysis for Cav-1 in tissues from one normal FVB/N or two MMTV-CR-1 virgin mice and from mammary tumors found in four MMTV-CR-1 multiparous mice (T1, T2, T3 and T4). For equal loading, the blot was reprobed with anti-β-actin antibody. B: Immunohistochemistry for Cav-1 and CR-1 in mammary tissue serial sections derived from FVB/N virgin or multiparous mice and in mammary tumor of MMTV-CR-1 mice. These sections were incubated with an anti-Cav-1 or with anti-CR-1 antibodies as described in Materials and Methods. Magnification, ×400. Negative controls are shown in the insets . WB, Western blot.

Techniques Used: Expressing, Transgenic Assay, Mouse Assay, Western Blot, Immunohistochemistry, Derivative Assay, Incubation

Colocalization of human CR-1 and Cav-1 in low-density sucrose fractions in 293 and COS7 cells. Western blot analysis on 5% to 40% sucrose gradient fractions isolated from 293 cells transiently transfected with expression plasmids encoding human CR-1 and Alk4-Flag (A) or from COS7 cells transiently transfected with a human CR-1 expression vector (B) . Western blot analysis was performed using anti-Flag monoclonal antibody to detect Alk4-Flag fusion protein, anti-CR-1 mouse monoclonal antibody, anti-Cav-1 mouse monoclonal antibody, and anti-β-actin mouse monoclonal antibody. The lipid raft marker GM1 was detected with horseradish peroxidase-conjugated CT-B by dot blot. Low-density sucrose fractions: 4, 5, and 6.
Figure Legend Snippet: Colocalization of human CR-1 and Cav-1 in low-density sucrose fractions in 293 and COS7 cells. Western blot analysis on 5% to 40% sucrose gradient fractions isolated from 293 cells transiently transfected with expression plasmids encoding human CR-1 and Alk4-Flag (A) or from COS7 cells transiently transfected with a human CR-1 expression vector (B) . Western blot analysis was performed using anti-Flag monoclonal antibody to detect Alk4-Flag fusion protein, anti-CR-1 mouse monoclonal antibody, anti-Cav-1 mouse monoclonal antibody, and anti-β-actin mouse monoclonal antibody. The lipid raft marker GM1 was detected with horseradish peroxidase-conjugated CT-B by dot blot. Low-density sucrose fractions: 4, 5, and 6.

Techniques Used: Western Blot, Isolation, Transfection, Expressing, Plasmid Preparation, Marker, Dot Blot

8) Product Images from "Dual regulation of hepatitis C viral RNA by cellular RNAi requires partitioning of Ago2 to lipid droplets and P-bodies"

Article Title: Dual regulation of hepatitis C viral RNA by cellular RNAi requires partitioning of Ago2 to lipid droplets and P-bodies

Journal: RNA

doi: 10.1261/rna.2523911

P-body disruption has no effect on HCV RNA replication in Huh7 cells. ( A ) Down-regulation of P-body proteins GW182 and Dcp2 had no effect on HCV RNA level in Huh7 cells as measured by subgenomic HCV replicon with firefly luciferase reporter. For comparison, silencing of Ago2 protein in the same cell line reduced HCV replicon level by ∼55%. Nontargeting (NT) siRNA was used as a negative control. NS3 siRNA was used as a positive control and inhibited HCV RNA by ∼80%. All siRNAs were transfected for 48 h. ( B ) Cell viability was not affected after 48 h post-siRNAs transfection (measured via Alamar Blue). All values are average of two sets of triplicate ± SD. ( C , D ) P-bodies are seen dispersed in Huh7 cells with subgenomic HCV replicon after 48 h down-regulation of GW182 protein. In control experiment with NT siRNA, P-bodies remain intact. ( E , F ) Ago2 foci are seen disrupted in the Huh7 cells after 48 h down-regulation of Ago2, while in the cells treated with NT siRNA, distribution of Ago2 into foci remained unaffected. Scale bar corresponds to 20 μm. ( G ) Western blot data show visible down-regulation of Ago2 expression after 48 h as identified by monoclonal 4F9 anti-Ago2. γ-Tubulin results are from the same reprobed membrane.
Figure Legend Snippet: P-body disruption has no effect on HCV RNA replication in Huh7 cells. ( A ) Down-regulation of P-body proteins GW182 and Dcp2 had no effect on HCV RNA level in Huh7 cells as measured by subgenomic HCV replicon with firefly luciferase reporter. For comparison, silencing of Ago2 protein in the same cell line reduced HCV replicon level by ∼55%. Nontargeting (NT) siRNA was used as a negative control. NS3 siRNA was used as a positive control and inhibited HCV RNA by ∼80%. All siRNAs were transfected for 48 h. ( B ) Cell viability was not affected after 48 h post-siRNAs transfection (measured via Alamar Blue). All values are average of two sets of triplicate ± SD. ( C , D ) P-bodies are seen dispersed in Huh7 cells with subgenomic HCV replicon after 48 h down-regulation of GW182 protein. In control experiment with NT siRNA, P-bodies remain intact. ( E , F ) Ago2 foci are seen disrupted in the Huh7 cells after 48 h down-regulation of Ago2, while in the cells treated with NT siRNA, distribution of Ago2 into foci remained unaffected. Scale bar corresponds to 20 μm. ( G ) Western blot data show visible down-regulation of Ago2 expression after 48 h as identified by monoclonal 4F9 anti-Ago2. γ-Tubulin results are from the same reprobed membrane.

Techniques Used: Luciferase, Negative Control, Positive Control, Transfection, Western Blot, Expressing

P-body disruption impairs NS3 siRNA inhibition of HCV RNA replication. ( A ) NS3 siRNA inhibition of subgenomic HCV replicon is compromised respectively by ∼25% in the cells with silenced GW182 (column 5) and by ∼15% in the cells with silenced Dcp2 (column 6). All values are normalized to HCV RNA level in the cells transfected with NT siRNA (column 4). HCV replicon levels measured in “no siRNA” (column 1), NT siRNA (24 h, column 2), and “NS3 siRNA only” (24 h, column 3) Huh7 cells are provided as controls. GW182, Dcp2, and NT siRNAs were transected for 48 h and subsequently, the cells were cotransfected with NS3 siRNA for additional 24 h. ( B ) Cell viability level via Alamar Blue measured after treatment with corresponding siRNAs as identified for plot A . Error bars correspond to a standard mean deviation (SD) in three independent experiments, each done in triplicate.
Figure Legend Snippet: P-body disruption impairs NS3 siRNA inhibition of HCV RNA replication. ( A ) NS3 siRNA inhibition of subgenomic HCV replicon is compromised respectively by ∼25% in the cells with silenced GW182 (column 5) and by ∼15% in the cells with silenced Dcp2 (column 6). All values are normalized to HCV RNA level in the cells transfected with NT siRNA (column 4). HCV replicon levels measured in “no siRNA” (column 1), NT siRNA (24 h, column 2), and “NS3 siRNA only” (24 h, column 3) Huh7 cells are provided as controls. GW182, Dcp2, and NT siRNAs were transected for 48 h and subsequently, the cells were cotransfected with NS3 siRNA for additional 24 h. ( B ) Cell viability level via Alamar Blue measured after treatment with corresponding siRNAs as identified for plot A . Error bars correspond to a standard mean deviation (SD) in three independent experiments, each done in triplicate.

Techniques Used: Inhibition, Transfection

Intracellular localization and activity of NS3 siRNA in Huh7 HCV cells at different intervals post-transfection. ( A ) In 2 h post-transfection, a fluorescently labeled guide strand of NS3 siRNA (blue) and Ago2 (green) are seen in the colocalizing spots in the cytoplasm. Small Dcp1a (red) spots can be found adjacent (enlargements 1 – 3 ). ( B – D ) In the course of the next 72 h, siRNA and Ago2 spots dispersed into fragmented pattern in the perinuclear area, with increase of Dcp1a punctuate staining at siRNAs sites ( B ) 24 h; ( C ) 48 h; ( D ) 72 h (enlargements 1,2 ). ( E ) Inhibition activity of labeled NS3 siRNA was not compromised by labeling. Nontargeting siRNA was used as an unspecific control. ( F ) Cell viability (measured via Alamar Blue) remained unaffected after transfection and prolonged incubation with labeled NS3 siRNA. Scale bars correspond to 20 μm in full images and 5 μm in enlargements.
Figure Legend Snippet: Intracellular localization and activity of NS3 siRNA in Huh7 HCV cells at different intervals post-transfection. ( A ) In 2 h post-transfection, a fluorescently labeled guide strand of NS3 siRNA (blue) and Ago2 (green) are seen in the colocalizing spots in the cytoplasm. Small Dcp1a (red) spots can be found adjacent (enlargements 1 – 3 ). ( B – D ) In the course of the next 72 h, siRNA and Ago2 spots dispersed into fragmented pattern in the perinuclear area, with increase of Dcp1a punctuate staining at siRNAs sites ( B ) 24 h; ( C ) 48 h; ( D ) 72 h (enlargements 1,2 ). ( E ) Inhibition activity of labeled NS3 siRNA was not compromised by labeling. Nontargeting siRNA was used as an unspecific control. ( F ) Cell viability (measured via Alamar Blue) remained unaffected after transfection and prolonged incubation with labeled NS3 siRNA. Scale bars correspond to 20 μm in full images and 5 μm in enlargements.

Techniques Used: Activity Assay, Transfection, Labeling, Staining, Inhibition, Incubation

9) Product Images from "Development of Transgenic Cloned Pig Models of Skin Inflammation by DNA Transposon-Directed Ectopic Expression of Human ?1 and ?2 Integrin"

Article Title: Development of Transgenic Cloned Pig Models of Skin Inflammation by DNA Transposon-Directed Ectopic Expression of Human ?1 and ?2 Integrin

Journal: PLoS ONE

doi: 10.1371/journal.pone.0036658

Erk1/2 phosphorylation in TPA-stimulated hITGB1-transgenic keratinocytes but not in hITGA2-transgenic keratinocytes. ( A ) Naïve and hITGB1- or hITGA2–expressing HaCaT cells were grown to subconfluency on uncoated or collagen I-coated plates. After two days of incubation in serum-depleted medium, one subset was stimulated with 100 ng/mL TPA for 10 min, after which all subsets were stained with a phosphor-Erk1/2 specific mouse mAb (E10). Increased levels of pERk1/2, indicative of increased Erk/MAPK activation, could be detected in stimulated β1 integrin-expressing HaCaT cells, whereas an inhibition upon TPA stimulation was apparent in α2 integrin-expressing cells. Asterisks (*) indicate statistical significance relative to naïve HaCaT cells. ( B ) TPA-induced activation of Erk1/2 phosphorylation in transgenic keratinocytes. Cultured hITGB1-transgenic keratinocytes were treated as described under ( A ). In case of pig #2408, a marked difference in phosphorylated Erk1/2 levels was detected by comparing TPA-stimulated and un-stimulated cells. Data are presented as mean values ± standard deviations.
Figure Legend Snippet: Erk1/2 phosphorylation in TPA-stimulated hITGB1-transgenic keratinocytes but not in hITGA2-transgenic keratinocytes. ( A ) Naïve and hITGB1- or hITGA2–expressing HaCaT cells were grown to subconfluency on uncoated or collagen I-coated plates. After two days of incubation in serum-depleted medium, one subset was stimulated with 100 ng/mL TPA for 10 min, after which all subsets were stained with a phosphor-Erk1/2 specific mouse mAb (E10). Increased levels of pERk1/2, indicative of increased Erk/MAPK activation, could be detected in stimulated β1 integrin-expressing HaCaT cells, whereas an inhibition upon TPA stimulation was apparent in α2 integrin-expressing cells. Asterisks (*) indicate statistical significance relative to naïve HaCaT cells. ( B ) TPA-induced activation of Erk1/2 phosphorylation in transgenic keratinocytes. Cultured hITGB1-transgenic keratinocytes were treated as described under ( A ). In case of pig #2408, a marked difference in phosphorylated Erk1/2 levels was detected by comparing TPA-stimulated and un-stimulated cells. Data are presented as mean values ± standard deviations.

Techniques Used: Transgenic Assay, Expressing, Incubation, Staining, Activation Assay, Inhibition, Cell Culture

Functional analysis of bicistronic SB transposon vectors. ( A ) Schematic representation of bicistronic SB-transposon vectors carrying the hITGB1 gene driven by CMV and INV promoters, respectively. LIR and RIR indicate the SB transposon left and right inverted repeats, respectively; SV40 pA and BGH pA, indicate the Simian virus 40 and bovine growth hormone polyadenylation sites, respectively. ( B ) Stable transfection rate in NIH3T3 cells co-transfected with equimolar amounts of pT2/SV40-neo or pT2/CMV-hITGB1. SV40-neo in conjunction with pCMV-mSB or pCMV-SB100X. pUC19 was included as stuffer in some transfections to ensure that equal amounts of DNA were transfected. G418-resistant colonies were counted after 14 days selection. ( C ) Western blot analysis using a mouse anti-human β1 integrin mAb (610467) on cellular protein extracts from naïve NIH3T3 cells or NIH3T3 cells stably transfected with pT2/CMV-hITGB1. SV40-neo (top panel). Top band is mature β1 integrin, lower band pre-β1 integrin. Detection of ACTB with an anti-β-actin mAb served as loading control (lower panel). ( D ) Transposase titration assay in Göttingen primary fibroblasts co-transfected with 0–100 ng pCMV-SB100X and 1.9 µg pT2/CMV-hITGB1. SV40-neo. Resistant colonies were counted after 14 days of selection with G418. ( E ) Quantitative RT-PCR on total mRNA extracted from naïve or pT2/CMV-hITGB1. SV40-neo stably transfected Göttingen primary fibroblasts. Human β1 integrin specific exon-exon primers were utilized to detect transcripts derived from the integrated transposon. The mRNA level was normalized to the level of endogenous β-actin mRNA. ( F ) Transposition assay in HaCaT cells co-transfected with 1.9 µg pT2/SV40-neo or pT2/INV-hITGB1. SV40-neo in conjunction with 100 ng pCMV-mSB or pCMV-SB100X. Resistant colonies were counted following 14 days of G418 selection. ( G ) Immunostaining of permeabilized naïve (a) or pT2/INV-hITGB1. SV40-neo-transfected (b) HaCaT cells. A mouse anti-human β1 integrin mAb (P5D2) was used to identify β1 integrin protein derived from the transposon expression cassette. Data are presented as mean values ± standard deviations.
Figure Legend Snippet: Functional analysis of bicistronic SB transposon vectors. ( A ) Schematic representation of bicistronic SB-transposon vectors carrying the hITGB1 gene driven by CMV and INV promoters, respectively. LIR and RIR indicate the SB transposon left and right inverted repeats, respectively; SV40 pA and BGH pA, indicate the Simian virus 40 and bovine growth hormone polyadenylation sites, respectively. ( B ) Stable transfection rate in NIH3T3 cells co-transfected with equimolar amounts of pT2/SV40-neo or pT2/CMV-hITGB1. SV40-neo in conjunction with pCMV-mSB or pCMV-SB100X. pUC19 was included as stuffer in some transfections to ensure that equal amounts of DNA were transfected. G418-resistant colonies were counted after 14 days selection. ( C ) Western blot analysis using a mouse anti-human β1 integrin mAb (610467) on cellular protein extracts from naïve NIH3T3 cells or NIH3T3 cells stably transfected with pT2/CMV-hITGB1. SV40-neo (top panel). Top band is mature β1 integrin, lower band pre-β1 integrin. Detection of ACTB with an anti-β-actin mAb served as loading control (lower panel). ( D ) Transposase titration assay in Göttingen primary fibroblasts co-transfected with 0–100 ng pCMV-SB100X and 1.9 µg pT2/CMV-hITGB1. SV40-neo. Resistant colonies were counted after 14 days of selection with G418. ( E ) Quantitative RT-PCR on total mRNA extracted from naïve or pT2/CMV-hITGB1. SV40-neo stably transfected Göttingen primary fibroblasts. Human β1 integrin specific exon-exon primers were utilized to detect transcripts derived from the integrated transposon. The mRNA level was normalized to the level of endogenous β-actin mRNA. ( F ) Transposition assay in HaCaT cells co-transfected with 1.9 µg pT2/SV40-neo or pT2/INV-hITGB1. SV40-neo in conjunction with 100 ng pCMV-mSB or pCMV-SB100X. Resistant colonies were counted following 14 days of G418 selection. ( G ) Immunostaining of permeabilized naïve (a) or pT2/INV-hITGB1. SV40-neo-transfected (b) HaCaT cells. A mouse anti-human β1 integrin mAb (P5D2) was used to identify β1 integrin protein derived from the transposon expression cassette. Data are presented as mean values ± standard deviations.

Techniques Used: Functional Assay, Stable Transfection, Transfection, Selection, Western Blot, Titration, Quantitative RT-PCR, Derivative Assay, Immunostaining, Expressing

Generation of hITGA2-transgenic pigs with ectopic expression of human α2 integrin. ( A ) Schematic figure of bicistronic SB transposon vectors with expression of human α2 integrin driven by the CMV and INV promoters and the neo selection marker gene driven by the SV40 promoter. LIR and RIR indicate SB transposon left and right inverted repeats, respectively; SV40 pA and BGH pA indicate Simian virus 40 and bovine growth hormone polyadenylation sites, respectively. ( B ) Immunostaining of permeabilized naïve (a) or pT2/INV-hITGA2.SV40-neo-transfected (b) HaCaT cells with a mouse anti-human α2 integrin mAb (P1E6). Background staining of endogenous α2 integrin is evident in naïve cells, whereas transfected cells show intense pancellular staining. ( C ) PCR analysis on genomic DNA extracted from cultured hITGA2-transgenic pig fibroblasts. The presence of Neo and hITGA2 was confirmed for all transgenic pigs with specific primer-pairs (upper panels). Passive integration of SB100X could be verified in all the cloned pigs by the use of primers specific for the SB100X sequence (lower panel). NT, non-transgenic control; MG366, cloned pig with known insertion of the SB100X gene; PC, plasmid control; M, 100 bp marker. ( D ) Southern blot analyses performed with 11 µg genomic DNA extracted from hITGA2-transgenic pig fibroblasts showed that all pigs, except 367, were genetically identical. DNA was digested with EcoRV and PvuI (left panel) or with EcoRV and SalI (right panel) and probed with a 32 P-labelled Neo probe. DNA from NT spiked with pT2/INV-hITGA2.SV40-neo was utilized in copy number controls with 1, 5 and 10 copies, respectively (refer to figure 2C for lengths of restriction fragments). NT, non-transgenic control; M, 1 kb marker. ( E ) qRT-PCR analysis of α2 integrin mRNA expression levels in hITGA2-transgenic pig keratinocytes, normalized to endogenous β-actin. Expression of human α2 integrin mRNA was evident in all transgenic pig keratinocytes. Representative data are presented as mean values ± standard deviations. ( F ) Representative wide-field fluorescence microscopy images of permeabilized and unstained or immunostained (with anti-human α2 integrin mAb (P1E6)) frozen cutaneous sections from the non-transgenic pig #2990 (a–b) and analyzed hITGA2-transgenic pigs (c–e). A clear staining for human α2 integrin protein was present only in transgenic keratinocytes. ( G ) Flow cytometry analysis on hITGA2-transgenic keratinocytes stained with an anti-human α2 integrin mAb (P1E6) without permeabilization (light gray bars) or with saponin permeabilization (dark gray bars). Also shown are representative histograms comparing fluorescence intensities from pig 3854 and 352 with and without permeabilization.
Figure Legend Snippet: Generation of hITGA2-transgenic pigs with ectopic expression of human α2 integrin. ( A ) Schematic figure of bicistronic SB transposon vectors with expression of human α2 integrin driven by the CMV and INV promoters and the neo selection marker gene driven by the SV40 promoter. LIR and RIR indicate SB transposon left and right inverted repeats, respectively; SV40 pA and BGH pA indicate Simian virus 40 and bovine growth hormone polyadenylation sites, respectively. ( B ) Immunostaining of permeabilized naïve (a) or pT2/INV-hITGA2.SV40-neo-transfected (b) HaCaT cells with a mouse anti-human α2 integrin mAb (P1E6). Background staining of endogenous α2 integrin is evident in naïve cells, whereas transfected cells show intense pancellular staining. ( C ) PCR analysis on genomic DNA extracted from cultured hITGA2-transgenic pig fibroblasts. The presence of Neo and hITGA2 was confirmed for all transgenic pigs with specific primer-pairs (upper panels). Passive integration of SB100X could be verified in all the cloned pigs by the use of primers specific for the SB100X sequence (lower panel). NT, non-transgenic control; MG366, cloned pig with known insertion of the SB100X gene; PC, plasmid control; M, 100 bp marker. ( D ) Southern blot analyses performed with 11 µg genomic DNA extracted from hITGA2-transgenic pig fibroblasts showed that all pigs, except 367, were genetically identical. DNA was digested with EcoRV and PvuI (left panel) or with EcoRV and SalI (right panel) and probed with a 32 P-labelled Neo probe. DNA from NT spiked with pT2/INV-hITGA2.SV40-neo was utilized in copy number controls with 1, 5 and 10 copies, respectively (refer to figure 2C for lengths of restriction fragments). NT, non-transgenic control; M, 1 kb marker. ( E ) qRT-PCR analysis of α2 integrin mRNA expression levels in hITGA2-transgenic pig keratinocytes, normalized to endogenous β-actin. Expression of human α2 integrin mRNA was evident in all transgenic pig keratinocytes. Representative data are presented as mean values ± standard deviations. ( F ) Representative wide-field fluorescence microscopy images of permeabilized and unstained or immunostained (with anti-human α2 integrin mAb (P1E6)) frozen cutaneous sections from the non-transgenic pig #2990 (a–b) and analyzed hITGA2-transgenic pigs (c–e). A clear staining for human α2 integrin protein was present only in transgenic keratinocytes. ( G ) Flow cytometry analysis on hITGA2-transgenic keratinocytes stained with an anti-human α2 integrin mAb (P1E6) without permeabilization (light gray bars) or with saponin permeabilization (dark gray bars). Also shown are representative histograms comparing fluorescence intensities from pig 3854 and 352 with and without permeabilization.

Techniques Used: Transgenic Assay, Expressing, Selection, Marker, Immunostaining, Transfection, Staining, Polymerase Chain Reaction, Cell Culture, Clone Assay, Sequencing, Plasmid Preparation, Southern Blot, Quantitative RT-PCR, Fluorescence, Microscopy, Flow Cytometry, Cytometry

10) Product Images from "MG132, a proteasome inhibitor, induces human pulmonary fibroblast cell death via increasing ROS levels and GSH depletion"

Article Title: MG132, a proteasome inhibitor, induces human pulmonary fibroblast cell death via increasing ROS levels and GSH depletion

Journal: Oncology Reports

doi: 10.3892/or.2012.1642

Effects of antioxidant-related siRNAs on cell death, ROS levels and GSH depletion in MG132-treated HPF cells. HPF cells (3–40% confluence) were transfected with either non-target control siRNA or each antioxidant-related siRNA. Two days later, cells were treated with 30 μM MG132 for additional 24 h. (A) Annexin V-FITC and PI cells were measured with a FACStar flow cytometer. The number (%) in each figure indicates Annexin-V-FITC positive cells regardless of PI negative and positive cells. (B and C) Graphs indicate DCF (ROS) levels (%) (B) and DHE (O 2 • ) levels (%) (C) compared with MG132-untreated control siRNA cells. (D) Graph shows the percent of (−) CMF (GSH-depleted) cells.
Figure Legend Snippet: Effects of antioxidant-related siRNAs on cell death, ROS levels and GSH depletion in MG132-treated HPF cells. HPF cells (3–40% confluence) were transfected with either non-target control siRNA or each antioxidant-related siRNA. Two days later, cells were treated with 30 μM MG132 for additional 24 h. (A) Annexin V-FITC and PI cells were measured with a FACStar flow cytometer. The number (%) in each figure indicates Annexin-V-FITC positive cells regardless of PI negative and positive cells. (B and C) Graphs indicate DCF (ROS) levels (%) (B) and DHE (O 2 • ) levels (%) (C) compared with MG132-untreated control siRNA cells. (D) Graph shows the percent of (−) CMF (GSH-depleted) cells.

Techniques Used: Transfection, Flow Cytometry, Cytometry

Effects of NAC, vitamin C or BSO on cell growth, cell death and MMP (ΔΨ m ) in MG132-treated HPF cells. Exponentially growing cells were treated with 10 μM MG132 for 24 h following a 1 h of pre-incubation with 2 mM NAC, 0.4 mM vitamin C or 10 μM BSO. (A) The graph shows cell growth changes in HPF cells as assessed by the MTT assay. (B and C) Annexin-V-FITC cells and MMP (ΔΨ m ) loss cells were measured with a FACStar flow cytometer. Graphs show the percent of Annexin V-positive staining cells (B) and rhodamine 123-negative [MMP (ΔΨ m ) loss] cells (C). (D) Samples of protein extracts (40 μg) were resolved by SDS-PAGE gel, transferred onto PVDF membranes and immunoblotted with the indicated antibodies against ubiquitin and β-actin. * P
Figure Legend Snippet: Effects of NAC, vitamin C or BSO on cell growth, cell death and MMP (ΔΨ m ) in MG132-treated HPF cells. Exponentially growing cells were treated with 10 μM MG132 for 24 h following a 1 h of pre-incubation with 2 mM NAC, 0.4 mM vitamin C or 10 μM BSO. (A) The graph shows cell growth changes in HPF cells as assessed by the MTT assay. (B and C) Annexin-V-FITC cells and MMP (ΔΨ m ) loss cells were measured with a FACStar flow cytometer. Graphs show the percent of Annexin V-positive staining cells (B) and rhodamine 123-negative [MMP (ΔΨ m ) loss] cells (C). (D) Samples of protein extracts (40 μg) were resolved by SDS-PAGE gel, transferred onto PVDF membranes and immunoblotted with the indicated antibodies against ubiquitin and β-actin. * P

Techniques Used: Incubation, MTT Assay, Flow Cytometry, Cytometry, Staining, SDS Page

11) Product Images from "Low Resolution Solution Structure of HAMLET and the Importance of Its Alpha-Domains in Tumoricidal Activity"

Article Title: Low Resolution Solution Structure of HAMLET and the Importance of Its Alpha-Domains in Tumoricidal Activity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0053051

Internalization of small peptides and induction of ion fluxes. (A) Internalization of biotinylated peptides (red) by A549 lung carcinoma cells counterstained with WGA (green) and Hoechst (blue) and examined by confocal microscopy. Peptides 1,10 and 11 were internalized in the absence of oleate and peptides 10 and 11 also in the presence of oleate. (B) K + and (C) Ca 2+ fluxes in tumor cells triggered by petides 1, 10 and 11 were measured by fluorescence spectrometry. Peptides 10 and 11 triggered Ca 2+ fluxes, in the presence and absence of oleate and peptide 6 a weaker Ca 2+ flux with oleate. (D) Insignificant Na + fluxes.
Figure Legend Snippet: Internalization of small peptides and induction of ion fluxes. (A) Internalization of biotinylated peptides (red) by A549 lung carcinoma cells counterstained with WGA (green) and Hoechst (blue) and examined by confocal microscopy. Peptides 1,10 and 11 were internalized in the absence of oleate and peptides 10 and 11 also in the presence of oleate. (B) K + and (C) Ca 2+ fluxes in tumor cells triggered by petides 1, 10 and 11 were measured by fluorescence spectrometry. Peptides 10 and 11 triggered Ca 2+ fluxes, in the presence and absence of oleate and peptide 6 a weaker Ca 2+ flux with oleate. (D) Insignificant Na + fluxes.

Techniques Used: Whole Genome Amplification, Confocal Microscopy, Fluorescence

Internalization of peptides into tumor cells and changes in tumor cell morphology. (A) Internalization of peptides. A549 lung carcinoma cells cultured on glass slides, were incubated with peptide-oleate mixtures for 1 hour, fixed and stained with AlexaFluor568-streptavidin, counterstained with WGA and examined by confocal microscopy. Alpha1 and alpha2 peptides, mixed with oleate, were internalized as shown by the red fluorescence. The beta peptide was not internalized. Scale bar 20 µm. (B) Morphological changes in A549 lung carcinoma cells treated with HAMLET, alpha1 peptide+oleate, alpha2 peptide+oleate and beta peptide+oleate recorded by holography imaging. Cells treated with HAMLET started to round up after 30 minutes and after 60 minutes, many cells had detached. Alpha1 peptide+oleate mixture triggers similar morphological changes as that by HAMLET. Alpha2 peptide+oleate mixture triggers similar morphological changes. Beta peptide+oleate mixture did not change cell morphology.
Figure Legend Snippet: Internalization of peptides into tumor cells and changes in tumor cell morphology. (A) Internalization of peptides. A549 lung carcinoma cells cultured on glass slides, were incubated with peptide-oleate mixtures for 1 hour, fixed and stained with AlexaFluor568-streptavidin, counterstained with WGA and examined by confocal microscopy. Alpha1 and alpha2 peptides, mixed with oleate, were internalized as shown by the red fluorescence. The beta peptide was not internalized. Scale bar 20 µm. (B) Morphological changes in A549 lung carcinoma cells treated with HAMLET, alpha1 peptide+oleate, alpha2 peptide+oleate and beta peptide+oleate recorded by holography imaging. Cells treated with HAMLET started to round up after 30 minutes and after 60 minutes, many cells had detached. Alpha1 peptide+oleate mixture triggers similar morphological changes as that by HAMLET. Alpha2 peptide+oleate mixture triggers similar morphological changes. Beta peptide+oleate mixture did not change cell morphology.

Techniques Used: Cell Culture, Incubation, Staining, Whole Genome Amplification, Confocal Microscopy, Fluorescence, Imaging

Ion fluxes and tumor cell death. (A) Peptides trigger ion fluxes in tumor cells. The free intracellular concentration of Na + , K + and Ca 2+ were measured by fluorescence spectrometry using CoroNa Green, FluxOR and Fluo-4, respectively. HAMLET, alpha1 peptide+oleate and alpha2 peptide+oleate mixtures trigger rapid fluxes of all three ions. Intracellular potassium ion concentrations were reduced due to ion efflux, while those of sodium and calcium were increased. Mean of at least two experiments. P values are explained in the text. (B) Peptide-oleate mixtures kill tumor cells. A549 lung carcinoma cells and Jurkat leukemia cells were incubated with HAMLET, oleate or peptide-oleate mixtures for 3 hours. Cell death was quantified as ATP levels and PrestoBlue, in % of control.
Figure Legend Snippet: Ion fluxes and tumor cell death. (A) Peptides trigger ion fluxes in tumor cells. The free intracellular concentration of Na + , K + and Ca 2+ were measured by fluorescence spectrometry using CoroNa Green, FluxOR and Fluo-4, respectively. HAMLET, alpha1 peptide+oleate and alpha2 peptide+oleate mixtures trigger rapid fluxes of all three ions. Intracellular potassium ion concentrations were reduced due to ion efflux, while those of sodium and calcium were increased. Mean of at least two experiments. P values are explained in the text. (B) Peptide-oleate mixtures kill tumor cells. A549 lung carcinoma cells and Jurkat leukemia cells were incubated with HAMLET, oleate or peptide-oleate mixtures for 3 hours. Cell death was quantified as ATP levels and PrestoBlue, in % of control.

Techniques Used: Concentration Assay, Fluorescence, Incubation

12) Product Images from "Small molecule inhibitors of WNT/?-catenin signaling block IL-1?- and TNF?-induced cartilage degradation"

Article Title: Small molecule inhibitors of WNT/?-catenin signaling block IL-1?- and TNF?-induced cartilage degradation

Journal: Arthritis Research & Therapy

doi: 10.1186/ar4273

Small molecule inhibitors of WNT/β-catenin signaling effectively block TCF/Lef-mediated activity of β-catenin . (A) Small molecules dose-dependently inhibit transcription factor/lymphoid enhancer-binding factor (TCF/LEF) reporter activity in HEK-293t cells, induced by the glycogen synthase kinase 3β (GSK3β) inhibitor 6-bromoindirubin 3'-oxime (BIO) (1.0 µM). Data represent the means of three independent experiments with 95% confidence intervals (CIs). (B) Metabolic activity, measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in KS483-4C3 cells, was not affected by small molecules at lower concentrations; however, at 1.0 µM (except for CGP049090) and 3.0 µM, metabolic activity was significantly decreased. Data represent the means of three independent experiments with 95% CI. (C) Treatment with 50 mM LiCl induced nuclear translocation of β-catenin. Small molecules by themselves had no effect on cellular localization of β-catenin, whereas PKF118-310 and PKF115-584 blocked LiCl-induced translocation of β-catenin to the nucleus. CGP049090 did not affect nuclear accumulation of β-catenin after LiCl treatment. A representative example of three independent experiments is shown. Scale bar represents 10 µm * P
Figure Legend Snippet: Small molecule inhibitors of WNT/β-catenin signaling effectively block TCF/Lef-mediated activity of β-catenin . (A) Small molecules dose-dependently inhibit transcription factor/lymphoid enhancer-binding factor (TCF/LEF) reporter activity in HEK-293t cells, induced by the glycogen synthase kinase 3β (GSK3β) inhibitor 6-bromoindirubin 3'-oxime (BIO) (1.0 µM). Data represent the means of three independent experiments with 95% confidence intervals (CIs). (B) Metabolic activity, measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in KS483-4C3 cells, was not affected by small molecules at lower concentrations; however, at 1.0 µM (except for CGP049090) and 3.0 µM, metabolic activity was significantly decreased. Data represent the means of three independent experiments with 95% CI. (C) Treatment with 50 mM LiCl induced nuclear translocation of β-catenin. Small molecules by themselves had no effect on cellular localization of β-catenin, whereas PKF118-310 and PKF115-584 blocked LiCl-induced translocation of β-catenin to the nucleus. CGP049090 did not affect nuclear accumulation of β-catenin after LiCl treatment. A representative example of three independent experiments is shown. Scale bar represents 10 µm * P

Techniques Used: Blocking Assay, Activity Assay, Binding Assay, Translocation Assay

13) Product Images from "Rapid Fabricating Technique for Multi-Layered Human Hepatic Cell Sheets by Forceful Contraction of the Fibroblast Monolayer"

Article Title: Rapid Fabricating Technique for Multi-Layered Human Hepatic Cell Sheets by Forceful Contraction of the Fibroblast Monolayer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0070970

Schematic diagrams of the fabrication process used for the human hepatic cell (HepaRG) sheets. The processes used to fabricate the HepaRG cell-only sheet (A) and TIG-118/HepaRG cell sheet (B).
Figure Legend Snippet: Schematic diagrams of the fabrication process used for the human hepatic cell (HepaRG) sheets. The processes used to fabricate the HepaRG cell-only sheet (A) and TIG-118/HepaRG cell sheet (B).

Techniques Used:

Cell morphologies of HepaRG cells and TIG-118/HepaRG cells on TRCD. Phase-contrast (A–D) and fluorescent (E, F) micrographs of the HepaRG cells (A, B) and TIG-118/HepaRG cells (C–F) on a TRCD. After one day (A, C, E) and three days (B, D, F) of culturing HepaRG cells. Green (CellTracker Green CMFDA): TIG-118 cells, Red (CellTracker Orange CMRA): HepaRG cells. The bars represent 200 µm (A–D) and 50 µm (E, F).
Figure Legend Snippet: Cell morphologies of HepaRG cells and TIG-118/HepaRG cells on TRCD. Phase-contrast (A–D) and fluorescent (E, F) micrographs of the HepaRG cells (A, B) and TIG-118/HepaRG cells (C–F) on a TRCD. After one day (A, C, E) and three days (B, D, F) of culturing HepaRG cells. Green (CellTracker Green CMFDA): TIG-118 cells, Red (CellTracker Orange CMRA): HepaRG cells. The bars represent 200 µm (A–D) and 50 µm (E, F).

Techniques Used:

Live and dead stained fluorescent images. (A–C) HepaRG cells and (D–F) TIG-118/HepaRG cells. (A, D) After four days of culturing HepaRG cells on the TRCD and (B, E) after two hours and (C, F) 24 hours of reculturing cell sheets on glass-based dishes. Green (Calcein-AM): viable cells, red (PI): dead cells. The bar represents 200 µm.
Figure Legend Snippet: Live and dead stained fluorescent images. (A–C) HepaRG cells and (D–F) TIG-118/HepaRG cells. (A, D) After four days of culturing HepaRG cells on the TRCD and (B, E) after two hours and (C, F) 24 hours of reculturing cell sheets on glass-based dishes. Green (Calcein-AM): viable cells, red (PI): dead cells. The bar represents 200 µm.

Techniques Used: Staining

Liver-specific functions of HepaRG cells and TIG-118/HepaRG cells on TRCD. Human albumin (A) and A1AT (B) synthesis rates.
Figure Legend Snippet: Liver-specific functions of HepaRG cells and TIG-118/HepaRG cells on TRCD. Human albumin (A) and A1AT (B) synthesis rates.

Techniques Used:

Ultrastructures of the TIG-118/HepaRG cell sheets observed by TEM. (A) Low magnification and (B–D) high magnification images of the cell-cell adhesion between HepaRG cell and TIG-118 cell (B), HepaRG cells (C) and TIG-118 cells (D). HR, HepaRG cell; TIG, TIG-118 cell; N, nucleus; BC, bile canaliculi; TG, tight junctions; GJ, gap junctions. The bars represent 5 µm (A) and 500 nm (B–D).
Figure Legend Snippet: Ultrastructures of the TIG-118/HepaRG cell sheets observed by TEM. (A) Low magnification and (B–D) high magnification images of the cell-cell adhesion between HepaRG cell and TIG-118 cell (B), HepaRG cells (C) and TIG-118 cells (D). HR, HepaRG cell; TIG, TIG-118 cell; N, nucleus; BC, bile canaliculi; TG, tight junctions; GJ, gap junctions. The bars represent 5 µm (A) and 500 nm (B–D).

Techniques Used: Transmission Electron Microscopy

Cell morphologies of HepaRG cell and TIG-118/HepaRG cell sheets. Exterior photographs (above) and phase-contrast micrographs (below) after incubation at 20°C. (A, B) HepaRG cells and (C, D) TIG-118/HepaRG cells. After one day (24 hours) (A, C) and four days (B, D) of culturing HepaRG cells. The bars represent 100 µm.
Figure Legend Snippet: Cell morphologies of HepaRG cell and TIG-118/HepaRG cell sheets. Exterior photographs (above) and phase-contrast micrographs (below) after incubation at 20°C. (A, B) HepaRG cells and (C, D) TIG-118/HepaRG cells. After one day (24 hours) (A, C) and four days (B, D) of culturing HepaRG cells. The bars represent 100 µm.

Techniques Used: Incubation

Cell distributions and HE stained images of the cross-sections of cell sheets. Fluorescent (A, B) and HE (C–F) stained images. (A, C) HepaRG cells, (B, D, E) TIG-118/HepaRG cells and (F) TIG-118 cells. Green (CellTracker Green CMFDA): TIG-118 cells, Red (Alexa Fluor 568 Phalloidin): F-actin, Blue (DAPI): nucleus. The bars represent 50 µm.
Figure Legend Snippet: Cell distributions and HE stained images of the cross-sections of cell sheets. Fluorescent (A, B) and HE (C–F) stained images. (A, C) HepaRG cells, (B, D, E) TIG-118/HepaRG cells and (F) TIG-118 cells. Green (CellTracker Green CMFDA): TIG-118 cells, Red (Alexa Fluor 568 Phalloidin): F-actin, Blue (DAPI): nucleus. The bars represent 50 µm.

Techniques Used: Staining

Cell distributions of TIG-118/HepaRG cells on TRCD. Cross-section views (A, B) and the cell population (C, D) of the TIG-118/HepaRG cells. After one day (A, C) and three days (B, D) of culturing HepaRG cells. The bars represent 50 µm.
Figure Legend Snippet: Cell distributions of TIG-118/HepaRG cells on TRCD. Cross-section views (A, B) and the cell population (C, D) of the TIG-118/HepaRG cells. After one day (A, C) and three days (B, D) of culturing HepaRG cells. The bars represent 50 µm.

Techniques Used:

14) Product Images from "Diesel Exhaust Particles Activate the Matrix-Metalloproteinase-1 Gene in Human Bronchial Epithelia in a ?-Arrestin-Dependent Manner via Activation of RAS"

Article Title: Diesel Exhaust Particles Activate the Matrix-Metalloproteinase-1 Gene in Human Bronchial Epithelia in a ?-Arrestin-Dependent Manner via Activation of RAS

Journal: Environmental Health Perspectives

doi: 10.1289/ehp.0800311

Phospho-ERK1/2 is trafficked to the nucleus as an early signaling event of the DEP MMP-1 response. ( A ) Time course of nuclear phospho-ERK1/2 after stimulation of BEAS-2B cells with DEPs (100 μg/mL). Nuclear phospho-ERK was detected by immunofluorescence and its abundance was evaluated by densitometry of the nucleus (cell numbers indicated beneath each coordinate). ( B ) Representative micrographs after DEP exposure of nuclear phospho-ERK immunofluorescence. Arrows indicate DEPs in direct contact with a cell. Western blots below show increased abundance of phospho-ERK in whole-cell lysate. ( C ) Nuclear phospho-ERK1/2 at 0 min and 30 min. A significant increase for DEP stimulation (100 μg/mL) was absent in controls (media, 0.2% DMSO). Chemical inhibition of MEK and RAF eliminated generation of a nuclear signal for phospho-ERK (MEK: UO126, PD98059, both used at 10 μM; RAF: 1 μM LBT613 and 10 μM AAL881). Eighty cells were analyzed per condition after DEP stimulation, and 40 before stimulation. ( D ) Knockdown of β-arrestins also eliminated generation of a nuclear signal for phospho-ERK. The graph (left) shows quantitation of control siRNA versus anti–pan-arrestin siRNA. The confocal micrographs (right) show representative findings. Eighty cells were analyzed for each condition after DEP stimulation, and 40 before stimulation. ( E ) Western blot as shown in ( C ) plus the corresponding Western blot for whole-cell phospho-ERK. Cell lysates sampled at 60 min. *** p
Figure Legend Snippet: Phospho-ERK1/2 is trafficked to the nucleus as an early signaling event of the DEP MMP-1 response. ( A ) Time course of nuclear phospho-ERK1/2 after stimulation of BEAS-2B cells with DEPs (100 μg/mL). Nuclear phospho-ERK was detected by immunofluorescence and its abundance was evaluated by densitometry of the nucleus (cell numbers indicated beneath each coordinate). ( B ) Representative micrographs after DEP exposure of nuclear phospho-ERK immunofluorescence. Arrows indicate DEPs in direct contact with a cell. Western blots below show increased abundance of phospho-ERK in whole-cell lysate. ( C ) Nuclear phospho-ERK1/2 at 0 min and 30 min. A significant increase for DEP stimulation (100 μg/mL) was absent in controls (media, 0.2% DMSO). Chemical inhibition of MEK and RAF eliminated generation of a nuclear signal for phospho-ERK (MEK: UO126, PD98059, both used at 10 μM; RAF: 1 μM LBT613 and 10 μM AAL881). Eighty cells were analyzed per condition after DEP stimulation, and 40 before stimulation. ( D ) Knockdown of β-arrestins also eliminated generation of a nuclear signal for phospho-ERK. The graph (left) shows quantitation of control siRNA versus anti–pan-arrestin siRNA. The confocal micrographs (right) show representative findings. Eighty cells were analyzed for each condition after DEP stimulation, and 40 before stimulation. ( E ) Western blot as shown in ( C ) plus the corresponding Western blot for whole-cell phospho-ERK. Cell lysates sampled at 60 min. *** p

Techniques Used: Immunofluorescence, Western Blot, Inhibition, Quantitation Assay

MAP kinase signaling via the RAS-RAF-MEK-ERK1/2 pathway is necessary for the DEP MMP-1 response in HBE. (A–C) MEK inhibition. ( A and B ) Dependence of the DEP MMP-1 response on MEK (and ERK1/2): transcriptional activation of MMP-1 at 2 hr ( A ; left) and 24 hr ( A ; right) in relative units (RU), and secretion of MMP-1 at the same time points ( B ). Both responses depend on MAP kinase signaling by MEK-ERK1/2, evidenced by the effect of UO126 and PD98059 (both 10 μM). (A and B; right) For transcription, differences reach statistical significance for the −1607GG MMP-1 polymorphism (A; right) For secretion of MMP-1, differences caused by MEK inhibitors are statistically signifi-cant. DEP stimulation was with 100 μg/mL; experiments conducted in triplicate with two independent experiments. ( C ) Effect of MEK inhibitors UO126 and PD98059 on primary HBE cells. Note the complete prevention of MMP-1 secretion. Experiment carried out in quadruplicate, with 100 μg/mL DEPs. ( D–F ) RAF inhibition with AAL881 and LBT613 down-regulated transcription of MMP-1 fLUC reporter genes. ( D ) Down-regulation strongest for the −1607GG polymorphism. ( E ) Secretion of MMP-1 protein from BEAS-2B cells. ( F ) Secretion of MMP-1 protein from primary airway epithelia. Experiments conducted in triplicate, with at least two independent experiments, using 100 μg/mL DEPs. ( G and H ) RAS inhibition [transient transfection of a dominant-negative (dom-neg) RAS] down-regulated the DEP MMP-1 response. ( G ) Transcriptional activation of MMP-1 and secretion of MMP-1 after 24 hr measured using MMP-1 –fLUC assays (4.4-kb MMP-1 promoter, –1607G and –1607GG). Cells transfected with the –1607G polymorphism showed no effect; further reduction of transcription for the –1607GG polymorphism was statistically significant, yet incomplete. ( H ) Transcriptional activation of MMP-1 and secretion of MMP-1 after 24 hr measured using MMP-1 ELISA. Transfection of dominant-negative RAS without DEP stimulation had no effect on MMP-1 transcription. Dominant-negative RAS virtually eliminated MMP-1. Experiment conducted in triplicate with two sets of independent experiments. * p
Figure Legend Snippet: MAP kinase signaling via the RAS-RAF-MEK-ERK1/2 pathway is necessary for the DEP MMP-1 response in HBE. (A–C) MEK inhibition. ( A and B ) Dependence of the DEP MMP-1 response on MEK (and ERK1/2): transcriptional activation of MMP-1 at 2 hr ( A ; left) and 24 hr ( A ; right) in relative units (RU), and secretion of MMP-1 at the same time points ( B ). Both responses depend on MAP kinase signaling by MEK-ERK1/2, evidenced by the effect of UO126 and PD98059 (both 10 μM). (A and B; right) For transcription, differences reach statistical significance for the −1607GG MMP-1 polymorphism (A; right) For secretion of MMP-1, differences caused by MEK inhibitors are statistically signifi-cant. DEP stimulation was with 100 μg/mL; experiments conducted in triplicate with two independent experiments. ( C ) Effect of MEK inhibitors UO126 and PD98059 on primary HBE cells. Note the complete prevention of MMP-1 secretion. Experiment carried out in quadruplicate, with 100 μg/mL DEPs. ( D–F ) RAF inhibition with AAL881 and LBT613 down-regulated transcription of MMP-1 fLUC reporter genes. ( D ) Down-regulation strongest for the −1607GG polymorphism. ( E ) Secretion of MMP-1 protein from BEAS-2B cells. ( F ) Secretion of MMP-1 protein from primary airway epithelia. Experiments conducted in triplicate, with at least two independent experiments, using 100 μg/mL DEPs. ( G and H ) RAS inhibition [transient transfection of a dominant-negative (dom-neg) RAS] down-regulated the DEP MMP-1 response. ( G ) Transcriptional activation of MMP-1 and secretion of MMP-1 after 24 hr measured using MMP-1 –fLUC assays (4.4-kb MMP-1 promoter, –1607G and –1607GG). Cells transfected with the –1607G polymorphism showed no effect; further reduction of transcription for the –1607GG polymorphism was statistically significant, yet incomplete. ( H ) Transcriptional activation of MMP-1 and secretion of MMP-1 after 24 hr measured using MMP-1 ELISA. Transfection of dominant-negative RAS without DEP stimulation had no effect on MMP-1 transcription. Dominant-negative RAS virtually eliminated MMP-1. Experiment conducted in triplicate with two sets of independent experiments. * p

Techniques Used: Inhibition, Activation Assay, Transfection, Dominant Negative Mutation, Enzyme-linked Immunosorbent Assay

DEP MMP-1 response increases in an allele-specific manner for the human–1607G(G) MMP-1 promoter polymorphism. ( A ) In BEAS-2B cells exposed to DEPs (100 μg/mL), MMP-1 mRNA formation is increased (vs. P90 nanoparticle control) as evidenced by relative abundance of the MMP-1 transcript, determined by Taqman real-time qRT-PCR. Statistically significant up-regulation of MMP-1 mRNA; experiment conducted in triplicate. ( B ) A 4.4-kb MMP-1 promoter reporter gene construct was transfected into BEAS-2B cells. Schematic illustrates the promoter with transcription factor binding sites (left). The large arrow denotes the –1607G(G) human polymorphism, which generates an ETS transcription factor binding site for GG; two smaller arrows denote additional upstream ETS binding sites. Diagram at right illustrates MMP-1 –fLUC reporter gene activity (normalized for Renilla ), in relative units (RU). Note that in response to DEPs (100 μg/mL; 24-hr incubation; triplicate assays; data based on three or more experiments), fLUC activity was strikingly increased for –1607GG (2G). ( C ) DNA sequencing of the MMP-1 promoter, encompassing –1607, from BEAS-2B cells. Like approximately 50% of all humans, BEAS-2B cells have a heterozygous genotype (–1607GG; –1607G). In ( A ), * p
Figure Legend Snippet: DEP MMP-1 response increases in an allele-specific manner for the human–1607G(G) MMP-1 promoter polymorphism. ( A ) In BEAS-2B cells exposed to DEPs (100 μg/mL), MMP-1 mRNA formation is increased (vs. P90 nanoparticle control) as evidenced by relative abundance of the MMP-1 transcript, determined by Taqman real-time qRT-PCR. Statistically significant up-regulation of MMP-1 mRNA; experiment conducted in triplicate. ( B ) A 4.4-kb MMP-1 promoter reporter gene construct was transfected into BEAS-2B cells. Schematic illustrates the promoter with transcription factor binding sites (left). The large arrow denotes the –1607G(G) human polymorphism, which generates an ETS transcription factor binding site for GG; two smaller arrows denote additional upstream ETS binding sites. Diagram at right illustrates MMP-1 –fLUC reporter gene activity (normalized for Renilla ), in relative units (RU). Note that in response to DEPs (100 μg/mL; 24-hr incubation; triplicate assays; data based on three or more experiments), fLUC activity was strikingly increased for –1607GG (2G). ( C ) DNA sequencing of the MMP-1 promoter, encompassing –1607, from BEAS-2B cells. Like approximately 50% of all humans, BEAS-2B cells have a heterozygous genotype (–1607GG; –1607G). In ( A ), * p

Techniques Used: Quantitative RT-PCR, Construct, Transfection, Binding Assay, Activity Assay, Incubation, DNA Sequencing

Active MMP-1 is secreted from HBE in response to DEPs. ( A ) Results of MMP and cytokine screen of DEP-stimulated BEAS-2B cell supernatant: secretory responses of several MMPs, TIMPs, and IL-6 (positive control). Cells were stimulated in triplicate (50 μg/mL DEPs; supernatants pooled after 6 hr). ( B ) After exposure to increasing concentrations of DEPs, secretion of MMP-1 by BEAS-2B cells followed a time course and a dose–response pattern by ELISA. P90 nanoparticles served as control; experiment conducted six times. ( C ) In primary HBE, DEP (100 μg/mL) exposure evoked robust MMP-1 secretion by ELISA. Note also the high amount of unstimulated MMP-1 secretion. P90 nanoparticles served as control; experiment conducted in triplicate. ( D ) MMP-1 immunofluorescence (BEAS-2B, primary HBE cells) identifies cell-bound MMP-1 immunoreactivity (green). In BEAS-2B cells, stimulation with P90 nanoparticles does not surpass background; DEP stimulation (100 μg/mL) leads to robust up-regulation, uniformly staining all cells. Specificity of the DEP MMP-1 response could also be observed in primary HBE cells, yet here a more heterogeneous MMP-1 immunoreactivity is apparent. 4’-6’-diamidino-2-phenylindole (DAPI) nuclear stain illustrates increased cell density (blue insert in the lower right-hand micrograph). Scale bars = 20 μm for MMP-1 immunolabeling, 80 μm for the DAPI. * p
Figure Legend Snippet: Active MMP-1 is secreted from HBE in response to DEPs. ( A ) Results of MMP and cytokine screen of DEP-stimulated BEAS-2B cell supernatant: secretory responses of several MMPs, TIMPs, and IL-6 (positive control). Cells were stimulated in triplicate (50 μg/mL DEPs; supernatants pooled after 6 hr). ( B ) After exposure to increasing concentrations of DEPs, secretion of MMP-1 by BEAS-2B cells followed a time course and a dose–response pattern by ELISA. P90 nanoparticles served as control; experiment conducted six times. ( C ) In primary HBE, DEP (100 μg/mL) exposure evoked robust MMP-1 secretion by ELISA. Note also the high amount of unstimulated MMP-1 secretion. P90 nanoparticles served as control; experiment conducted in triplicate. ( D ) MMP-1 immunofluorescence (BEAS-2B, primary HBE cells) identifies cell-bound MMP-1 immunoreactivity (green). In BEAS-2B cells, stimulation with P90 nanoparticles does not surpass background; DEP stimulation (100 μg/mL) leads to robust up-regulation, uniformly staining all cells. Specificity of the DEP MMP-1 response could also be observed in primary HBE cells, yet here a more heterogeneous MMP-1 immunoreactivity is apparent. 4’-6’-diamidino-2-phenylindole (DAPI) nuclear stain illustrates increased cell density (blue insert in the lower right-hand micrograph). Scale bars = 20 μm for MMP-1 immunolabeling, 80 μm for the DAPI. * p

Techniques Used: Positive Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Immunolabeling

15) Product Images from "Diversity and evolution of phycobilisomes in marine Synechococcus spp.: a comparative genomics study"

Article Title: Diversity and evolution of phycobilisomes in marine Synechococcus spp.: a comparative genomics study

Journal: Genome Biology

doi: 10.1186/gb-2007-8-12-r259

Proposed models of PBS structure for the different Synechococcus pigment types and subtypes. PBS cores are generally composed of three cylinders, but in some chromatic adapters possessing an extended L CM , it is likely composed of two additional half cylinders (see, for example, [58]). In pigment type 1, rods are composed of C-PC only; in pigment type 2, rods are composed of either C-PC, or R-PCIII and a PEI-like phycobiliprotein; in pigment type 3, rods comprise R-PC and two PE types (PEI and PEII). Cells of the latter pigment type bind PEB and PUB at a low (3a), medium (3b), high (3c) or variable (3d or type IV chromatic adapter) ratio. Colored stars indicate the pigment type of each strain (see Figure 1 for color code).
Figure Legend Snippet: Proposed models of PBS structure for the different Synechococcus pigment types and subtypes. PBS cores are generally composed of three cylinders, but in some chromatic adapters possessing an extended L CM , it is likely composed of two additional half cylinders (see, for example, [58]). In pigment type 1, rods are composed of C-PC only; in pigment type 2, rods are composed of either C-PC, or R-PCIII and a PEI-like phycobiliprotein; in pigment type 3, rods comprise R-PC and two PE types (PEI and PEII). Cells of the latter pigment type bind PEB and PUB at a low (3a), medium (3b), high (3c) or variable (3d or type IV chromatic adapter) ratio. Colored stars indicate the pigment type of each strain (see Figure 1 for color code).

Techniques Used:

Absorption (continuous line) and fluorescence (dotted line) properties of isolated PBP complexes. (a) C-PC (as in Synechococcus spp. RS9917, WH5701 and WH8018); (b) PEI-A* (as in Synechococcus spp. WH8018 and WH7805); (c) PEI-A (as in Synechococcus spp. WH7803, Almo03 and RS9912); (d) PEI-B (as in Synechococcus spp. WH8102, CC9605 and Oli31).
Figure Legend Snippet: Absorption (continuous line) and fluorescence (dotted line) properties of isolated PBP complexes. (a) C-PC (as in Synechococcus spp. RS9917, WH5701 and WH8018); (b) PEI-A* (as in Synechococcus spp. WH8018 and WH7805); (c) PEI-A (as in Synechococcus spp. WH7803, Almo03 and RS9912); (d) PEI-B (as in Synechococcus spp. WH8102, CC9605 and Oli31).

Techniques Used: Fluorescence, Isolation

Absorption (continuous line) and fluorescence (dotted line) properties of the isolated PEII complexes. (a) PEII-A (as in Synechococcus sp. WH7803); (b) PEII-B (as in Synechococcus sp. RCC307); (c) PEII-C (as in Synechococcus spp. CC9605 and WH8102). Type IV chromatic adapters have a PEII-B under white or green light and a PEII-C under blue light [34].
Figure Legend Snippet: Absorption (continuous line) and fluorescence (dotted line) properties of the isolated PEII complexes. (a) PEII-A (as in Synechococcus sp. WH7803); (b) PEII-B (as in Synechococcus sp. RCC307); (c) PEII-C (as in Synechococcus spp. CC9605 and WH8102). Type IV chromatic adapters have a PEII-B under white or green light and a PEII-C under blue light [34].

Techniques Used: Fluorescence, Isolation

Comparison of PBS rod gene regions of the different pigment types of marine Synechococcus . Rectangles above and below the lines have a length proportional to the size of ORFs and correspond to the forward and the reverse strand, respectively. In several genomes, the sense of the rod region was inversed so that the regions all appear in the same direction. For the group formed by the chromatic adapters and RCC307, a few variations can be found with regard to the region shown here, which corresponds to BL107. First, the lyase-encoding gene(s) located near the 3'-end can either be a rpcE-F operon or rpcG , a pecEF -like fusion gene (see text). Second, the gene organization at the 5'-end can vary: unk1 is located elsewhere in the genome of RCC307 and the gene following unk2 is either the lyase gene cpcT in RS9916 and RCC307, unk3 in BL107 and CC9902, or none of these in CC9311. Colored stars indicate the pigment type of each strain (see Figure 1 for color code).
Figure Legend Snippet: Comparison of PBS rod gene regions of the different pigment types of marine Synechococcus . Rectangles above and below the lines have a length proportional to the size of ORFs and correspond to the forward and the reverse strand, respectively. In several genomes, the sense of the rod region was inversed so that the regions all appear in the same direction. For the group formed by the chromatic adapters and RCC307, a few variations can be found with regard to the region shown here, which corresponds to BL107. First, the lyase-encoding gene(s) located near the 3'-end can either be a rpcE-F operon or rpcG , a pecEF -like fusion gene (see text). Second, the gene organization at the 5'-end can vary: unk1 is located elsewhere in the genome of RCC307 and the gene following unk2 is either the lyase gene cpcT in RS9916 and RCC307, unk3 in BL107 and CC9902, or none of these in CC9311. Colored stars indicate the pigment type of each strain (see Figure 1 for color code).

Techniques Used:

ML trees made with concatenated amino acid sequences of (a) all 51 ribosomal proteins (6,754 amino acid positions), (b) the AP proteins ApcA-B-C-D-F (710 amino acid positions), (c) the PC proteins CpcA-B or RpcA-B (332 amino acid positions), (d) the PEI proteins CpeA-B-Y-Z (943 amino acid positions) and (e) the PEII proteins MpeA-B-Y and Unk7-8-9 (1,007 amino acid positions). The first four trees are rooted with corresponding proteins from the primitive, freshwater cyanobacterium Gloeobacter violaceus , taken as an outgroup. The PEII tree is unrooted since these proteins are specific for marine Synechococcus spp. Numbers at internal branches correspond to bootstrap values for 1,000 replicate trees obtained with ML/NJ/MP methods. Colored stars indicate the pigment type of each strain (see Figure 1 for color code).
Figure Legend Snippet: ML trees made with concatenated amino acid sequences of (a) all 51 ribosomal proteins (6,754 amino acid positions), (b) the AP proteins ApcA-B-C-D-F (710 amino acid positions), (c) the PC proteins CpcA-B or RpcA-B (332 amino acid positions), (d) the PEI proteins CpeA-B-Y-Z (943 amino acid positions) and (e) the PEII proteins MpeA-B-Y and Unk7-8-9 (1,007 amino acid positions). The first four trees are rooted with corresponding proteins from the primitive, freshwater cyanobacterium Gloeobacter violaceus , taken as an outgroup. The PEII tree is unrooted since these proteins are specific for marine Synechococcus spp. Numbers at internal branches correspond to bootstrap values for 1,000 replicate trees obtained with ML/NJ/MP methods. Colored stars indicate the pigment type of each strain (see Figure 1 for color code).

Techniques Used:

The diversity of pigment types among marine Synechococcus spp. (a) Photograph of representative cultured strains of the major pigment types (1-3) and subtypes (3a-c) of marine Synechococcus grown under low white light and (b) corresponding absorption properties of whole cells. Pigment subtype 3d corresponds to type IV chromatic adapters, which are able to modify their PBS pigmentation from subtype 3b when grown under white or green light to subtype 3c when grown under blue light. The different colors of stars in panel A are a code for the different pigment types.
Figure Legend Snippet: The diversity of pigment types among marine Synechococcus spp. (a) Photograph of representative cultured strains of the major pigment types (1-3) and subtypes (3a-c) of marine Synechococcus grown under low white light and (b) corresponding absorption properties of whole cells. Pigment subtype 3d corresponds to type IV chromatic adapters, which are able to modify their PBS pigmentation from subtype 3b when grown under white or green light to subtype 3c when grown under blue light. The different colors of stars in panel A are a code for the different pigment types.

Techniques Used: Cell Culture

16) Product Images from "The SARS Coronavirus E Protein Interacts with PALS1 and Alters Tight Junction Formation and Epithelial Morphogenesis"

Article Title: The SARS Coronavirus E Protein Interacts with PALS1 and Alters Tight Junction Formation and Epithelial Morphogenesis

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E10-04-0338

MDCKII cyst morphogenesis is altered by expression of SARS-E protein. Single MDCKII, eGFP-PALS1 cells were embedded into cell culture medium supplemented with 4% GelTrex matrix and incubated at 37°C for five days until cysts developed. Mature cysts were fixed, permeabilized, and stained with antibodies against ZO-1 (a marker of tight junctions), the apical protein GP-135 and hemagglutinin (HA) tag at the amino terminus of HA-E (wt/ΔPBM) proteins, as indicated. Images were acquired with an inverted confocal microscope (Zeiss LSM 510 Axiovert 200M). (A) Control cells stably expressing eGFP-PALS1 formed cysts with a single lumen. (B) In contrast, transfection with SARS-E (wt; upper panels) or ΔPBM (lower panels) resulted in the formation of cysts with multiple lumens, as shown in these examples. Occasionally, a low fraction of E (wt) could be detected with eGFP-PALS1 at the apical site of the cell (white arrow). Bar, 10 μm. (C) Qualitative analysis of cyst formation by MDCKII cells ectopically expressing the indicated constructs. The number of single lumen cysts with preserved polarity is expressed as percentage of total count of cysts scored. Results are shown as means ± SEM of the specified number of replicates from three independent experiments. For each cell line, we have counted total of 870–885 cysts. ***p
Figure Legend Snippet: MDCKII cyst morphogenesis is altered by expression of SARS-E protein. Single MDCKII, eGFP-PALS1 cells were embedded into cell culture medium supplemented with 4% GelTrex matrix and incubated at 37°C for five days until cysts developed. Mature cysts were fixed, permeabilized, and stained with antibodies against ZO-1 (a marker of tight junctions), the apical protein GP-135 and hemagglutinin (HA) tag at the amino terminus of HA-E (wt/ΔPBM) proteins, as indicated. Images were acquired with an inverted confocal microscope (Zeiss LSM 510 Axiovert 200M). (A) Control cells stably expressing eGFP-PALS1 formed cysts with a single lumen. (B) In contrast, transfection with SARS-E (wt; upper panels) or ΔPBM (lower panels) resulted in the formation of cysts with multiple lumens, as shown in these examples. Occasionally, a low fraction of E (wt) could be detected with eGFP-PALS1 at the apical site of the cell (white arrow). Bar, 10 μm. (C) Qualitative analysis of cyst formation by MDCKII cells ectopically expressing the indicated constructs. The number of single lumen cysts with preserved polarity is expressed as percentage of total count of cysts scored. Results are shown as means ± SEM of the specified number of replicates from three independent experiments. For each cell line, we have counted total of 870–885 cysts. ***p

Techniques Used: Expressing, Cell Culture, Incubation, Staining, Marker, Microscopy, Stable Transfection, Transfection, Construct

17) Product Images from "Galactosylated poly(ethylene glycol)-b-poly (l-lactide-co-?-malic acid) block copolymer micelles for targeted drug delivery: preparation and in vitro characterization"

Article Title: Galactosylated poly(ethylene glycol)-b-poly (l-lactide-co-?-malic acid) block copolymer micelles for targeted drug delivery: preparation and in vitro characterization

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S14280

Confocal laser scanning microscopic images of HepG2 cells (5 × 10 4 cells/mL) after 12 hours of in vitro exposure to ( A ) free doxorubicin, ( B ) doxorubicin-loaded PEG-b-PLMA micelles, and ( C ) doxorubicin-loaded Gal-PEG-b-PLMA micelles. Abbreviations: PEG-b-PLMA, methoxy poly(ethylene glycol)/poly (l-lactide-co-β-malic acid); Gal-PEG-b-PLMA, galactosylated methoxy poly(ethylene glycol)/poly(llactide-co-β-malic acid).
Figure Legend Snippet: Confocal laser scanning microscopic images of HepG2 cells (5 × 10 4 cells/mL) after 12 hours of in vitro exposure to ( A ) free doxorubicin, ( B ) doxorubicin-loaded PEG-b-PLMA micelles, and ( C ) doxorubicin-loaded Gal-PEG-b-PLMA micelles. Abbreviations: PEG-b-PLMA, methoxy poly(ethylene glycol)/poly (l-lactide-co-β-malic acid); Gal-PEG-b-PLMA, galactosylated methoxy poly(ethylene glycol)/poly(llactide-co-β-malic acid).

Techniques Used: In Vitro

Size distributions of ( A ) empty, ( B ) doxorubicin-loaded Gal-PEG-b-PLMA micelles, and ( C ) their mean sizes measured by dynamic light scattering method. Abbreviation: Gal-PEG-b-PLMA, galactosylated methoxy poly(ethylene glycol)/poly(l-lactide-co-β-malic acid) block copolymer.
Figure Legend Snippet: Size distributions of ( A ) empty, ( B ) doxorubicin-loaded Gal-PEG-b-PLMA micelles, and ( C ) their mean sizes measured by dynamic light scattering method. Abbreviation: Gal-PEG-b-PLMA, galactosylated methoxy poly(ethylene glycol)/poly(l-lactide-co-β-malic acid) block copolymer.

Techniques Used: Blocking Assay

In vitro cytotoxicity of free doxorubicin, doxorubicin-loaded PEG-b-PLMA micelles and doxorubicin-loaded Gal-PEG-b-PLMA micelles in HepG2 cells. Data are shown as mean ± standard deviation ( n = 3). Abbreviations: PEG-b-PLMA, methoxy poly(ethylene glycol)/poly(l-lactide-co-β-malic acid); Gal-PEG-b-PLMA, galactosylated methoxy poly(ethylene glycol)/poly (l-lactide-co-β-malic acid).
Figure Legend Snippet: In vitro cytotoxicity of free doxorubicin, doxorubicin-loaded PEG-b-PLMA micelles and doxorubicin-loaded Gal-PEG-b-PLMA micelles in HepG2 cells. Data are shown as mean ± standard deviation ( n = 3). Abbreviations: PEG-b-PLMA, methoxy poly(ethylene glycol)/poly(l-lactide-co-β-malic acid); Gal-PEG-b-PLMA, galactosylated methoxy poly(ethylene glycol)/poly (l-lactide-co-β-malic acid).

Techniques Used: In Vitro, Standard Deviation

In vitro release behavior of doxorubicin from PEG-b-PLMA micelles at pH 4.5, 5.5, and 7.4. Each point and error bar represents mean ± standard deviation (n ± 3). Abbreviation: PEG-b-PLMA, methoxy poly(ethylene glycol)/poly(l-lactide-co-β-malic acid).
Figure Legend Snippet: In vitro release behavior of doxorubicin from PEG-b-PLMA micelles at pH 4.5, 5.5, and 7.4. Each point and error bar represents mean ± standard deviation (n ± 3). Abbreviation: PEG-b-PLMA, methoxy poly(ethylene glycol)/poly(l-lactide-co-β-malic acid).

Techniques Used: In Vitro, Standard Deviation

Transmission electron microscopic images of ( A) empty and ( B) doxorubicin loaded Gal-PEG-b-PLMA micelles. Abbreviation: Gal-PEG-b-PLMA, galactosylated methoxy poly (ethylene glycol)/poly (l-lactide-co-β-malic acid) block copolymer.
Figure Legend Snippet: Transmission electron microscopic images of ( A) empty and ( B) doxorubicin loaded Gal-PEG-b-PLMA micelles. Abbreviation: Gal-PEG-b-PLMA, galactosylated methoxy poly (ethylene glycol)/poly (l-lactide-co-β-malic acid) block copolymer.

Techniques Used: Transmission Assay, Blocking Assay

The cell cycle profiles of HepG2 cells before and after doxorubicin treatment for 12 hours: A ) control group, B ) free doxorubicin group, C ) doxorubicin-loaded PEG-b-PLMA micelle group, and D ) doxorubicin-loaded Gal-PEG-b-PLMA micelle group. The data represent mean ± standard deviation where n = 3. Abbreviations: PEG-b-PLMA, methoxy poly(ethylene glycol)/poly(l-lactide-co-β-malic acid); Gal-PEG-b-PLMA, galactosylated methoxy poly(ethylene glycol)/poly (l-lactide-co-β-malic acid).
Figure Legend Snippet: The cell cycle profiles of HepG2 cells before and after doxorubicin treatment for 12 hours: A ) control group, B ) free doxorubicin group, C ) doxorubicin-loaded PEG-b-PLMA micelle group, and D ) doxorubicin-loaded Gal-PEG-b-PLMA micelle group. The data represent mean ± standard deviation where n = 3. Abbreviations: PEG-b-PLMA, methoxy poly(ethylene glycol)/poly(l-lactide-co-β-malic acid); Gal-PEG-b-PLMA, galactosylated methoxy poly(ethylene glycol)/poly (l-lactide-co-β-malic acid).

Techniques Used: Standard Deviation

18) Product Images from "Antrodin C Inhibits Epithelial-to-Mesenchymal Transition and Metastasis of Breast Cancer Cells via Suppression of Smad2/3 and β-Catenin Signaling Pathways"

Article Title: Antrodin C Inhibits Epithelial-to-Mesenchymal Transition and Metastasis of Breast Cancer Cells via Suppression of Smad2/3 and β-Catenin Signaling Pathways

Journal: PLoS ONE

doi: 10.1371/journal.pone.0117111

Schematic representation represents the anti-metastatic potential of ADC. TGF-β ligand binding with TGF-β receptor II (TGF-βRII) recruit TGF-βRI into a tetrameric receptor complex resulting in transphosphorylation and activation of TGF-βRI, which then phosphorylates Smad2 or Smad3. Phosphorylated Smad2/3 associate with Smad4 and translocate into the nucleus, where they activate transcription of target genes including snail, slug, and Twist. These genes regulate EMT through suppression of epithelial markers and induction of mesenchymal markers. Pretreatment with ADC inhibits TGF-β-induced EMT by inhibiting phosphorylation and transcriptional activation of Smad2/3. In addition, TGF-β activates β-catenin signaling cascade through the suppression of GSK3β, a negative regulator of β-catenin. The free form of β-catenin translocate into the nucleus and transcribe number of migration marker genes including MMP-2, MMP-9, and uPA. Pretreatment with ADC blocks TGF-β1-induced migration and invasion of MCF-7 cells through the suppression of transcriptional activation of β-catenin followed by down-regulation of MMP-2, MMP-9, and uPA. In addition, previous reports demonstrated that MMPs expression also regulated by snail, transcription factor regulates EMT. There is a possible that reduction in snail expression by may influence MMPs activity. Moreover, Twist was reported to be one of the down-stream target of β-catenin. Therefore, we believe that there may be a cross-talk existing between ADC-induced down-regulation of β-catenin and Twist.
Figure Legend Snippet: Schematic representation represents the anti-metastatic potential of ADC. TGF-β ligand binding with TGF-β receptor II (TGF-βRII) recruit TGF-βRI into a tetrameric receptor complex resulting in transphosphorylation and activation of TGF-βRI, which then phosphorylates Smad2 or Smad3. Phosphorylated Smad2/3 associate with Smad4 and translocate into the nucleus, where they activate transcription of target genes including snail, slug, and Twist. These genes regulate EMT through suppression of epithelial markers and induction of mesenchymal markers. Pretreatment with ADC inhibits TGF-β-induced EMT by inhibiting phosphorylation and transcriptional activation of Smad2/3. In addition, TGF-β activates β-catenin signaling cascade through the suppression of GSK3β, a negative regulator of β-catenin. The free form of β-catenin translocate into the nucleus and transcribe number of migration marker genes including MMP-2, MMP-9, and uPA. Pretreatment with ADC blocks TGF-β1-induced migration and invasion of MCF-7 cells through the suppression of transcriptional activation of β-catenin followed by down-regulation of MMP-2, MMP-9, and uPA. In addition, previous reports demonstrated that MMPs expression also regulated by snail, transcription factor regulates EMT. There is a possible that reduction in snail expression by may influence MMPs activity. Moreover, Twist was reported to be one of the down-stream target of β-catenin. Therefore, we believe that there may be a cross-talk existing between ADC-induced down-regulation of β-catenin and Twist.

Techniques Used: Ligand Binding Assay, Activation Assay, Migration, Marker, Expressing, Activity Assay

ADC inhibits TGF-β1-induced transcriptional activity of Smad2/Smad3. ( A ) MCF-7 cells were pre-treated with ADC (5–20 μM) for 2 h prior to stimulation with TGF-β1 (20 ng/mL) for 24 h. Western blot analysis was performed to examine the protein expression levels of Snail, Slug, and TWIST with specific antibodies. β-actin was used as an internal loading control. ( B ) Cells were transfected with pGL3-SBE-4-Luc reporter construct, and then pre-treated with ADC (5–20 μM) prior to stimulation with TGF-β1 for 3 h. The luciferase activity was expressed as a relative value compared to that of the untreated cells which was set to 1-fold. ( C ) Western blot was performed to measure the total and phosphorylated levels of Smad2 and Smad3 proteins. ( D ) TGF-β1-induced nuclear translocation of phosphorylated Smad2 and Smad3 were examined by immunofluorescence analysis with confocal microscope. Cells were pre-treated with ADC (20 μM) for 2 h, and then incubated with TGF-β1 for 1 h. After treatment, cells were fixed, permiabilized, and incubated with Phos-Smad2 or Phos-Smad3 primary antibodies for overnight followed by FITC and TRITC secondary antibodies, respectively for 1 h. The cellular DNA was stained with DAPI (1 μg/mL) and images were captured by confocal microscope (magnification 200). Bars, 20 μm. The data reported as mean ± SD of three independent experiments. Θ P
Figure Legend Snippet: ADC inhibits TGF-β1-induced transcriptional activity of Smad2/Smad3. ( A ) MCF-7 cells were pre-treated with ADC (5–20 μM) for 2 h prior to stimulation with TGF-β1 (20 ng/mL) for 24 h. Western blot analysis was performed to examine the protein expression levels of Snail, Slug, and TWIST with specific antibodies. β-actin was used as an internal loading control. ( B ) Cells were transfected with pGL3-SBE-4-Luc reporter construct, and then pre-treated with ADC (5–20 μM) prior to stimulation with TGF-β1 for 3 h. The luciferase activity was expressed as a relative value compared to that of the untreated cells which was set to 1-fold. ( C ) Western blot was performed to measure the total and phosphorylated levels of Smad2 and Smad3 proteins. ( D ) TGF-β1-induced nuclear translocation of phosphorylated Smad2 and Smad3 were examined by immunofluorescence analysis with confocal microscope. Cells were pre-treated with ADC (20 μM) for 2 h, and then incubated with TGF-β1 for 1 h. After treatment, cells were fixed, permiabilized, and incubated with Phos-Smad2 or Phos-Smad3 primary antibodies for overnight followed by FITC and TRITC secondary antibodies, respectively for 1 h. The cellular DNA was stained with DAPI (1 μg/mL) and images were captured by confocal microscope (magnification 200). Bars, 20 μm. The data reported as mean ± SD of three independent experiments. Θ P

Techniques Used: Activity Assay, Western Blot, Expressing, Transfection, Construct, Luciferase, Translocation Assay, Immunofluorescence, Microscopy, Incubation, Staining

ADC down-regulates TGF-β1-induced MMPs activity in breast cancer cells. ( A ) MCF-7 cells were pre-treated with ADC (5–20 μM) for 2 h, and then stimulated with TGF-β1 (20 ng/mL) for 48 h. Gelatin zymography was performed with conditioned media collected from MCF-7 cells. ( B ) Protein expression levels of MT1-MMP, MMP-2, MMP-9, and uPA were determined by western blot analysis with specific antibodies. The house-keeping protein β-actin served as an internal loading control. Arrows denote pro- and cleaved forms of MMP-2. The data reported as mean ± SD of three independent experiments. Θ P
Figure Legend Snippet: ADC down-regulates TGF-β1-induced MMPs activity in breast cancer cells. ( A ) MCF-7 cells were pre-treated with ADC (5–20 μM) for 2 h, and then stimulated with TGF-β1 (20 ng/mL) for 48 h. Gelatin zymography was performed with conditioned media collected from MCF-7 cells. ( B ) Protein expression levels of MT1-MMP, MMP-2, MMP-9, and uPA were determined by western blot analysis with specific antibodies. The house-keeping protein β-actin served as an internal loading control. Arrows denote pro- and cleaved forms of MMP-2. The data reported as mean ± SD of three independent experiments. Θ P

Techniques Used: Activity Assay, Zymography, Expressing, Western Blot

Effect of ADC on MCF-7 cell viability. ( A ) MCF-7 cells were incubated with increasing concentrations of ADC (5–40 μM) for 48 h. ( B ) Cells were pre-treated with ADC (5–20 μM) for 2 h, and then incubated with TGF-β1 for 48 h. Cell viability was determined by MTT colorimetric assay. The percentage of cell viability was cauculated by the absorption of control cells (0.1% DMSO) as 100%. The data reported as mean ± SD of three independent experiments. Θ P
Figure Legend Snippet: Effect of ADC on MCF-7 cell viability. ( A ) MCF-7 cells were incubated with increasing concentrations of ADC (5–40 μM) for 48 h. ( B ) Cells were pre-treated with ADC (5–20 μM) for 2 h, and then incubated with TGF-β1 for 48 h. Cell viability was determined by MTT colorimetric assay. The percentage of cell viability was cauculated by the absorption of control cells (0.1% DMSO) as 100%. The data reported as mean ± SD of three independent experiments. Θ P

Techniques Used: Incubation, MTT Assay, Colorimetric Assay

ADC suppressed TGF-β1-induced transcriptional activation of β-catenin breast cancer cells. ( A ) MCF-7 cells were co-transfected with TOP-flash or FOP-flash or pCMV-β-Gal harboring luciferase reporter construct. After transfection, cells were pre-treated with ADC (5–20 μM) for 2 h and then stimulated with TGF-β1 for 3 h. Luciferase activity was determined and normalized with β-gal activity. The histogram shows the relative luciferase activity (fold increase). ( B ) Cells were pre-treated with ADC (5–20 μM) for 2 h and then stimulated with TGF-β1 for 2 h. β-catenin expression in cytoplasam and the nucleus were determined by western blot analysis using specific cytosolic and nuclear extracts. β-actin and histone were served as an internal control for cytosolic and nuclear fractions, respectively. ( C ) The nuclear localization of β-catenin in MCF-7 cells were determined by immunofluorescence staining. MCF-7 cells were seeded in a 8-well Tek chamber and allowed to adhere for 24 h. ADC (5–20 μM) for 2 h and then stimulated with TGF-β1 for 2 h. After treatment, cells were fixed, permiabilized, and incubated with β-catenin primary antibody overnight, followed by FITC secondary antibody for 1 h. The cellular DNA was stained with DAPI (1 μg/mL) and images were captured by confocal microscope (magnification 200). ( D ) Cells were pre-incubated with ADC (5–20 μM) for 2 h, and then stimulated with TGF-β1 for 1 h. The phosphorylated and total protein expression levels of β-catenin and GSK3β were determined by western blot analysis. The data reported as mean ± SD of three independent experiments. Θ P
Figure Legend Snippet: ADC suppressed TGF-β1-induced transcriptional activation of β-catenin breast cancer cells. ( A ) MCF-7 cells were co-transfected with TOP-flash or FOP-flash or pCMV-β-Gal harboring luciferase reporter construct. After transfection, cells were pre-treated with ADC (5–20 μM) for 2 h and then stimulated with TGF-β1 for 3 h. Luciferase activity was determined and normalized with β-gal activity. The histogram shows the relative luciferase activity (fold increase). ( B ) Cells were pre-treated with ADC (5–20 μM) for 2 h and then stimulated with TGF-β1 for 2 h. β-catenin expression in cytoplasam and the nucleus were determined by western blot analysis using specific cytosolic and nuclear extracts. β-actin and histone were served as an internal control for cytosolic and nuclear fractions, respectively. ( C ) The nuclear localization of β-catenin in MCF-7 cells were determined by immunofluorescence staining. MCF-7 cells were seeded in a 8-well Tek chamber and allowed to adhere for 24 h. ADC (5–20 μM) for 2 h and then stimulated with TGF-β1 for 2 h. After treatment, cells were fixed, permiabilized, and incubated with β-catenin primary antibody overnight, followed by FITC secondary antibody for 1 h. The cellular DNA was stained with DAPI (1 μg/mL) and images were captured by confocal microscope (magnification 200). ( D ) Cells were pre-incubated with ADC (5–20 μM) for 2 h, and then stimulated with TGF-β1 for 1 h. The phosphorylated and total protein expression levels of β-catenin and GSK3β were determined by western blot analysis. The data reported as mean ± SD of three independent experiments. Θ P

Techniques Used: Activation Assay, Transfection, Luciferase, Construct, Activity Assay, Expressing, Western Blot, Immunofluorescence, Staining, Incubation, Microscopy

ADC blocks TGF-β1-induced EMT in breast cancer cells. MCF-7 cells were pre-treated with ADC (5–20 μM) for 2 h prior to stimulation with TGF-β1 (20 ng/mL) for 48 h. ( A ) Morphological changes especially cell scattering was examined by phase-contrast microscope. Photomicrography shown here are from one of the three independent experiments. ( B ) ADC inhibits TGF-β1-induced actin cytoskeleton reorganization in MCF-7 cells. ADC and TGF-β1 treated cells were fixed, permiabilized, and stained with FITC-phalloidin to visuvalize the F-actin cytoskeleton reorganization. The images are representative of three independent experiments. Bars, 20 μm. ( C ) Protein samples were isolated from control, ADC, and TGF-β treated cells for the detection of E-cadherin, occluding, vimentin, N-cadherin, and β-actin proteins. β-actin was used as an internal control. Western blot data presented are representative of those obtained in at least three independent experiments.
Figure Legend Snippet: ADC blocks TGF-β1-induced EMT in breast cancer cells. MCF-7 cells were pre-treated with ADC (5–20 μM) for 2 h prior to stimulation with TGF-β1 (20 ng/mL) for 48 h. ( A ) Morphological changes especially cell scattering was examined by phase-contrast microscope. Photomicrography shown here are from one of the three independent experiments. ( B ) ADC inhibits TGF-β1-induced actin cytoskeleton reorganization in MCF-7 cells. ADC and TGF-β1 treated cells were fixed, permiabilized, and stained with FITC-phalloidin to visuvalize the F-actin cytoskeleton reorganization. The images are representative of three independent experiments. Bars, 20 μm. ( C ) Protein samples were isolated from control, ADC, and TGF-β treated cells for the detection of E-cadherin, occluding, vimentin, N-cadherin, and β-actin proteins. β-actin was used as an internal control. Western blot data presented are representative of those obtained in at least three independent experiments.

Techniques Used: Microscopy, Staining, Isolation, Western Blot

ADC inhibits TGF-β1-induced breast cancer cell migration and invasion. (A) Cell migration was determined by wound healing assay, the confluent MCF-7 monolayer was pre-treated with ADC (5–20 μM) for 2 h, cells were scratched by 200 μL pipet tips, and washed to remove the debris followed by addition of freshmedium containing 1% FBS and TGF-β1 (20 ng/mL). Cells were then incubated for 48 h. Photographs were taken at 0 h, and 48 h using inverted microscope with 10 × magnification. TGF-β1-induced cell motility was determined by measuring the area of wound closure as shown by histogram. The closure area at 48 h was compared with 0 h in the same samples. (B) For the invasion assay, the pre-treated cells were seed into the upper chamber of 24-well transwell chamber containing DMEM with 1% FBS. The lower chamber was filled with complete serum media. The cells were allowed to invade for 48 h. Invading cells were then fixed, and stained with Giemsa stain solution and counted in 5 random fields. The average invaded cells in each group was presented by histogram. The data reported as mean ± SD of three independent experiments. Θ P
Figure Legend Snippet: ADC inhibits TGF-β1-induced breast cancer cell migration and invasion. (A) Cell migration was determined by wound healing assay, the confluent MCF-7 monolayer was pre-treated with ADC (5–20 μM) for 2 h, cells were scratched by 200 μL pipet tips, and washed to remove the debris followed by addition of freshmedium containing 1% FBS and TGF-β1 (20 ng/mL). Cells were then incubated for 48 h. Photographs were taken at 0 h, and 48 h using inverted microscope with 10 × magnification. TGF-β1-induced cell motility was determined by measuring the area of wound closure as shown by histogram. The closure area at 48 h was compared with 0 h in the same samples. (B) For the invasion assay, the pre-treated cells were seed into the upper chamber of 24-well transwell chamber containing DMEM with 1% FBS. The lower chamber was filled with complete serum media. The cells were allowed to invade for 48 h. Invading cells were then fixed, and stained with Giemsa stain solution and counted in 5 random fields. The average invaded cells in each group was presented by histogram. The data reported as mean ± SD of three independent experiments. Θ P

Techniques Used: Migration, Wound Healing Assay, Incubation, Inverted Microscopy, Invasion Assay, Staining, Giemsa Stain

19) Product Images from "Systematic Evaluation of the Metabolic to Mitogenic Potency Ratio for B10-Substituted Insulin Analogues"

Article Title: Systematic Evaluation of the Metabolic to Mitogenic Potency Ratio for B10-Substituted Insulin Analogues

Journal: PLoS ONE

doi: 10.1371/journal.pone.0029198

Representative dose-response profiles for mitogenic potency determination. Human insulin (•) or insulin analogue (B10D (♦), B10E (▪), or B10A (▾)) stimulated incorporation of [ 3 H]-thymidine into DNA is shown in ( A ) L6-hIR cells and ( B ) HMECs. Data points are means ± SEM (n = 3).
Figure Legend Snippet: Representative dose-response profiles for mitogenic potency determination. Human insulin (•) or insulin analogue (B10D (♦), B10E (▪), or B10A (▾)) stimulated incorporation of [ 3 H]-thymidine into DNA is shown in ( A ) L6-hIR cells and ( B ) HMECs. Data points are means ± SEM (n = 3).

Techniques Used:

20) Product Images from "Bioguided Fractionation Shows Cassia alata Extract to Inhibit Staphylococcus epidermidis and Pseudomonas aeruginosa Growth and Biofilm Formation"

Article Title: Bioguided Fractionation Shows Cassia alata Extract to Inhibit Staphylococcus epidermidis and Pseudomonas aeruginosa Growth and Biofilm Formation

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2012/867103

(A) Growth of CaRP-treated P. aeruginosa ATCC27853, CRT: control; (B) Inhibition of biofilm formation of P. aeruginosa * P
Figure Legend Snippet: (A) Growth of CaRP-treated P. aeruginosa ATCC27853, CRT: control; (B) Inhibition of biofilm formation of P. aeruginosa * P

Techniques Used: Inhibition

21) Product Images from "Microbial inhibition of oral epithelial wound recovery: potential role for quorum sensing molecules?"

Article Title: Microbial inhibition of oral epithelial wound recovery: potential role for quorum sensing molecules?

Journal: AMB Express

doi: 10.1186/s13568-015-0116-5

a Wound healing capacity of TR146 epithelial cells in presence of the conditioned medium of K. oxytoca -exposed TR146 cells (mean + SD; *p
Figure Legend Snippet: a Wound healing capacity of TR146 epithelial cells in presence of the conditioned medium of K. oxytoca -exposed TR146 cells (mean + SD; *p

Techniques Used:

a Concentrations of glucose, l - and d -lactate found in the basal cell culture medium after 24 h of co-culture of the TR146 cells with different oral microbial species (mean ± SD; *p
Figure Legend Snippet: a Concentrations of glucose, l - and d -lactate found in the basal cell culture medium after 24 h of co-culture of the TR146 cells with different oral microbial species (mean ± SD; *p

Techniques Used: Cell Culture, Co-Culture Assay

a Wound healing capacity of TR146 epithelial cells confronted with K. oxytoca alone or mixed with S. salivarius , S. oralis or S. mitis (mean + SD; *p
Figure Legend Snippet: a Wound healing capacity of TR146 epithelial cells confronted with K. oxytoca alone or mixed with S. salivarius , S. oralis or S. mitis (mean + SD; *p

Techniques Used:

a Wound healing capacity of TR146 cells treated with different concentrations of tilivalline (mean + SD; *p
Figure Legend Snippet: a Wound healing capacity of TR146 cells treated with different concentrations of tilivalline (mean + SD; *p

Techniques Used:

a Wound healing capacity of TR146 epithelial cells confronted with inserts containing 5.5–6 log CFU of monocultures of different oral species. The relative area of the wound after 24 h is plotted (mean + SD; *p
Figure Legend Snippet: a Wound healing capacity of TR146 epithelial cells confronted with inserts containing 5.5–6 log CFU of monocultures of different oral species. The relative area of the wound after 24 h is plotted (mean + SD; *p

Techniques Used:

Wound healing capacity of TR146 cells treated with different concentrations of N -(3-oxododecanoyl)- l -homoserine lactone (AHL) (mean + SD; *p
Figure Legend Snippet: Wound healing capacity of TR146 cells treated with different concentrations of N -(3-oxododecanoyl)- l -homoserine lactone (AHL) (mean + SD; *p

Techniques Used:

22) Product Images from "The Connexin46 Mutant, Cx46T19M, Causes Loss of Gap Junction Function and Alters Hemi-channel Gating"

Article Title: The Connexin46 Mutant, Cx46T19M, Causes Loss of Gap Junction Function and Alters Hemi-channel Gating

Journal: The Journal of Membrane Biology

doi: 10.1007/s00232-014-9752-y

Representative families of current traces recorded from single HeLa cells transfected with vector alone ( a ), wild-type rat Cx46 ( b ), or T19M ( c ). Families of current traces were recorded in response to a series of voltage clamp steps between −60 and 50 mV in increments of 10 mV from a holding potential of −60 mV. Dashed line indicates zero current level. d Average steady-state I–V relationships for vector alone ( open squares , n = 5), wild-type ( solid circles , n = 4), and T19M ( open triangles , n = 3)
Figure Legend Snippet: Representative families of current traces recorded from single HeLa cells transfected with vector alone ( a ), wild-type rat Cx46 ( b ), or T19M ( c ). Families of current traces were recorded in response to a series of voltage clamp steps between −60 and 50 mV in increments of 10 mV from a holding potential of −60 mV. Dashed line indicates zero current level. d Average steady-state I–V relationships for vector alone ( open squares , n = 5), wild-type ( solid circles , n = 4), and T19M ( open triangles , n = 3)

Techniques Used: Transfection, Plasmid Preparation

Effects of T19M mutation on human Cx46. Photomicrographs showing the distribution of human wild-type Cx46 ( a ) and T19M ( b ) in transfected HeLa cells. Bar 30 µm. c Bar graph summarizes the DAPI uptake data for vector alone, human wild-type Cx46 and T19M obtained in transfected HeLa cells when exposed to extracellular solutions containing 1 mM Ca 2+ , 1 mM Mg 2+ ( gray bars ) or 1 mM Ca 2+ , 1 mM Mg 2+ , 0.2 mM La 3+ ( black bars ). Data are graphed as mean ± SEM. * p
Figure Legend Snippet: Effects of T19M mutation on human Cx46. Photomicrographs showing the distribution of human wild-type Cx46 ( a ) and T19M ( b ) in transfected HeLa cells. Bar 30 µm. c Bar graph summarizes the DAPI uptake data for vector alone, human wild-type Cx46 and T19M obtained in transfected HeLa cells when exposed to extracellular solutions containing 1 mM Ca 2+ , 1 mM Mg 2+ ( gray bars ) or 1 mM Ca 2+ , 1 mM Mg 2+ , 0.2 mM La 3+ ( black bars ). Data are graphed as mean ± SEM. * p

Techniques Used: Mutagenesis, Transfection, Plasmid Preparation

T19M hemi-channels show alterations in voltage gating. Ensemble averaged current traces recorded from cells expressing wild-type rat Cx46 ( a ) or T19M ( b ) in response to a 2-s voltage clamp step to 80 mV followed by a hyperpolarizing step to −60 mV. The holding potential was −60 mV. Dashed line indicates zero current level. c Averaged peak tail currents at −60 mV. The number of cells tested is indicated within parentheses . d Bar graph summarizes the t 1/2 ’s of deactivation of peak tail currents for wild-type rat Cx46 ( hatched bars ) and T19M ( black bars ) at −80, −60, and −40 mV. Data are graphed as mean ± SEM. * p
Figure Legend Snippet: T19M hemi-channels show alterations in voltage gating. Ensemble averaged current traces recorded from cells expressing wild-type rat Cx46 ( a ) or T19M ( b ) in response to a 2-s voltage clamp step to 80 mV followed by a hyperpolarizing step to −60 mV. The holding potential was −60 mV. Dashed line indicates zero current level. c Averaged peak tail currents at −60 mV. The number of cells tested is indicated within parentheses . d Bar graph summarizes the t 1/2 ’s of deactivation of peak tail currents for wild-type rat Cx46 ( hatched bars ) and T19M ( black bars ) at −80, −60, and −40 mV. Data are graphed as mean ± SEM. * p

Techniques Used: Expressing

Effect of lanthanum ions. Currents before ( a ) and after the application of La 3+ ( b ; 200 µM) recorded from a HeLa cell expressing T19M. Families of current traces were recorded in response to a series of voltage clamp steps between −60 and 70 mV in increments of 10 mV from a holding potential of −60 mV. Dashed line indicates zero current level. c I–V relations obtained from the data shown in ( a , b ). The current was measured at the end of the 1-s pulse and plotted as a function of voltage. The concentrations of divalent cations in the bath solution were reduced to zero added Ca 2+ and 0.5 mM Mg 2+ to augment the size of the hemi-channel currents. d Bar graph summarizes the input conductance measured at −60 mV in HeLa cells expressing wild-type Cx46, T19M, or vector ( alone ) when exposed to extracellular solutions containing 0 mM Ca 2+ , 0.5 mM Mg 2+ ( black bars ) or 0 mM Ca 2+ , 0.5 mM Mg 2+ , 0.2 mM La 3+ ( gray bars ). The number of cells analyzed is indicated within parentheses
Figure Legend Snippet: Effect of lanthanum ions. Currents before ( a ) and after the application of La 3+ ( b ; 200 µM) recorded from a HeLa cell expressing T19M. Families of current traces were recorded in response to a series of voltage clamp steps between −60 and 70 mV in increments of 10 mV from a holding potential of −60 mV. Dashed line indicates zero current level. c I–V relations obtained from the data shown in ( a , b ). The current was measured at the end of the 1-s pulse and plotted as a function of voltage. The concentrations of divalent cations in the bath solution were reduced to zero added Ca 2+ and 0.5 mM Mg 2+ to augment the size of the hemi-channel currents. d Bar graph summarizes the input conductance measured at −60 mV in HeLa cells expressing wild-type Cx46, T19M, or vector ( alone ) when exposed to extracellular solutions containing 0 mM Ca 2+ , 0.5 mM Mg 2+ ( black bars ) or 0 mM Ca 2+ , 0.5 mM Mg 2+ , 0.2 mM La 3+ ( gray bars ). The number of cells analyzed is indicated within parentheses

Techniques Used: Expressing, Plasmid Preparation

T19M does not induce gap junctional coupling when expressed by itself, and it acts as a loss-of-function mutation without dominant-negative inhibition when co-expressed with wild-type lens connexins. Bar graphs show mean gap junctional conductances in pairs of oocytes expressing different combinations of wild-type and mutant lens connexins as determined using the double two-electrode voltage clamp technique. a Rat Cx46 or T19M were expressed alone or in combination with each other. b Mouse Cx50 was expressed alone or in combination with either rat Cx46 or T19M. AS indicates oocytes that were injected with no cRNA (i.e., Xenopus Cx38 antisense oligonucleotide alone). The number of pairs tested is indicated within parentheses . * p
Figure Legend Snippet: T19M does not induce gap junctional coupling when expressed by itself, and it acts as a loss-of-function mutation without dominant-negative inhibition when co-expressed with wild-type lens connexins. Bar graphs show mean gap junctional conductances in pairs of oocytes expressing different combinations of wild-type and mutant lens connexins as determined using the double two-electrode voltage clamp technique. a Rat Cx46 or T19M were expressed alone or in combination with each other. b Mouse Cx50 was expressed alone or in combination with either rat Cx46 or T19M. AS indicates oocytes that were injected with no cRNA (i.e., Xenopus Cx38 antisense oligonucleotide alone). The number of pairs tested is indicated within parentheses . * p

Techniques Used: Mutagenesis, Dominant Negative Mutation, Inhibition, Expressing, Injection

T19M is inefficient at forming gap junction plaques. Photomicrographs show the distribution of wild-type rat Cx46 ( a ) and T19M ( b – d ) at the indicated times following transient transfection of HeLa cells. Bar 30 μM
Figure Legend Snippet: T19M is inefficient at forming gap junction plaques. Photomicrographs show the distribution of wild-type rat Cx46 ( a ) and T19M ( b – d ) at the indicated times following transient transfection of HeLa cells. Bar 30 μM

Techniques Used: Transfection

The rate of DAPI uptake is increased by lowering divalent cations and inhibited by La 3+ . Average time course of DAPI uptake by transfected HeLa cells in control solution (1 mM Ca 2+ , 1 mM Mg 2+ ), in external solutions with no added divalent cations and in control solution plus 200 µM La 3+ . a Wild-type rat Cx46 ( closed circles ); T19M ( open triangles ). To measure changes in the rate of dye uptake over time, the mean DAPI fluorescence intensity per pixel from ROI’s located in the nuclei of Zaza-green positive cells were normalized to mean DAPI fluorescence intensity of the ROI’s at 60 min, averaged and plotted as a function of time. The cells were initially bathed in control solution (containing 1 mM Ca 2+ , 1 mM Mg 2+ ). Then, the cells were exposed to a solution containing no added divalent cations followed by reperfusion with control solution containing 200 μM La 3+ . All the solutions contained 4 μM DAPI. b Bar graph shows the rates of DAPI uptake in cells expressing wild-type Cx46, T19M, or vector alone in the presence of 1 mM Ca 2+ , 1 mM Mg 2+ ( gray bar ); or 1 mM Ca 2+ , 1 mM Mg 2+ , 0.2 mM La 3+ ( black bar ). Data are presented as the mean ± SEM. * p
Figure Legend Snippet: The rate of DAPI uptake is increased by lowering divalent cations and inhibited by La 3+ . Average time course of DAPI uptake by transfected HeLa cells in control solution (1 mM Ca 2+ , 1 mM Mg 2+ ), in external solutions with no added divalent cations and in control solution plus 200 µM La 3+ . a Wild-type rat Cx46 ( closed circles ); T19M ( open triangles ). To measure changes in the rate of dye uptake over time, the mean DAPI fluorescence intensity per pixel from ROI’s located in the nuclei of Zaza-green positive cells were normalized to mean DAPI fluorescence intensity of the ROI’s at 60 min, averaged and plotted as a function of time. The cells were initially bathed in control solution (containing 1 mM Ca 2+ , 1 mM Mg 2+ ). Then, the cells were exposed to a solution containing no added divalent cations followed by reperfusion with control solution containing 200 μM La 3+ . All the solutions contained 4 μM DAPI. b Bar graph shows the rates of DAPI uptake in cells expressing wild-type Cx46, T19M, or vector alone in the presence of 1 mM Ca 2+ , 1 mM Mg 2+ ( gray bar ); or 1 mM Ca 2+ , 1 mM Mg 2+ , 0.2 mM La 3+ ( black bar ). Data are presented as the mean ± SEM. * p

Techniques Used: Transfection, Fluorescence, Expressing, Plasmid Preparation

T19M causes increased uptake of connexon-permeant dyes. Photomicrographs show examples of HeLa cells that were transfected with wild-type rat Cx46 ( a – c ) or T19M ( d–i ) (using the vector PBI-CMV3 which also drives expression of Zaza green) and incubated a day later with DAPI in Na gluconate Ringer’s solution containing 0 mM Ca 2+ ( a – f ) or 5 mM Ca +2 ( g – i ) for 20 min. Phase contrast images ( a , d , g ). Zaza-green fluorescence ( b , e , h ). DAPI fluorescence ( c , f , i ). After a 20-min incubation in control solution containing 0 mM Ca 2+ , cells expressing T19M showed DAPI uptake ( e , f ) that was mostly inhibited by 5 mM Ca 2+ ( h , i ). Bar graph summarizes the quantification of the DAPI uptake data ( j ). Data are graphed as mean ± SEM. The number of cells tested is indicated within parentheses . * p
Figure Legend Snippet: T19M causes increased uptake of connexon-permeant dyes. Photomicrographs show examples of HeLa cells that were transfected with wild-type rat Cx46 ( a – c ) or T19M ( d–i ) (using the vector PBI-CMV3 which also drives expression of Zaza green) and incubated a day later with DAPI in Na gluconate Ringer’s solution containing 0 mM Ca 2+ ( a – f ) or 5 mM Ca +2 ( g – i ) for 20 min. Phase contrast images ( a , d , g ). Zaza-green fluorescence ( b , e , h ). DAPI fluorescence ( c , f , i ). After a 20-min incubation in control solution containing 0 mM Ca 2+ , cells expressing T19M showed DAPI uptake ( e , f ) that was mostly inhibited by 5 mM Ca 2+ ( h , i ). Bar graph summarizes the quantification of the DAPI uptake data ( j ). Data are graphed as mean ± SEM. The number of cells tested is indicated within parentheses . * p

Techniques Used: Transfection, Plasmid Preparation, Expressing, Incubation, Fluorescence

23) Product Images from "Induction of release and up-regulated gene expression of interleukin (IL)-8 in A549 cells by serine proteinases"

Article Title: Induction of release and up-regulated gene expression of interleukin (IL)-8 in A549 cells by serine proteinases

Journal: BMC Cell Biology

doi: 10.1186/1471-2121-7-22

Effect of SFLLR-NH 2 , an agonist peptide of PAR-1 and its reverse peptide RLLFS-NH 2 on IL-8 release from A549 cells. Cells were incubated ( A ) with various concentrations of SFLLR-NH 2 (open bar) or RLLFS-NH 2 (hatched bar) at 37°C for 16 h or ( B ) with 100 μM of SFLLR-NH 2 and RLLFS-NH 2 for 2 h, 8 h and 16 h. Values shown are Mean ± SE for five separate experiments performed in duplicate. * P
Figure Legend Snippet: Effect of SFLLR-NH 2 , an agonist peptide of PAR-1 and its reverse peptide RLLFS-NH 2 on IL-8 release from A549 cells. Cells were incubated ( A ) with various concentrations of SFLLR-NH 2 (open bar) or RLLFS-NH 2 (hatched bar) at 37°C for 16 h or ( B ) with 100 μM of SFLLR-NH 2 and RLLFS-NH 2 for 2 h, 8 h and 16 h. Values shown are Mean ± SE for five separate experiments performed in duplicate. * P

Techniques Used: Incubation

Effect of serine proteinases and agonist peptides of PARs on the expression of IL-8 mRNA in A549 cells. Total cellular RNA was isolated, reverse transcribed to cDNA, and the cDNA was used for real-time PCR. Cells were treated with the testing compounds at 37°C for 2 (open bars), 8 (left hatched bars) and 16 h (right hatched bars), respectively. The data were normalized to the housekeeping gene (β-actin gene) and were expressed as mean ± SE fold of control for three separate experiments performed in duplicate. * p
Figure Legend Snippet: Effect of serine proteinases and agonist peptides of PARs on the expression of IL-8 mRNA in A549 cells. Total cellular RNA was isolated, reverse transcribed to cDNA, and the cDNA was used for real-time PCR. Cells were treated with the testing compounds at 37°C for 2 (open bars), 8 (left hatched bars) and 16 h (right hatched bars), respectively. The data were normalized to the housekeeping gene (β-actin gene) and were expressed as mean ± SE fold of control for three separate experiments performed in duplicate. * p

Techniques Used: Expressing, Isolation, Real-time Polymerase Chain Reaction

Effect of GYPGQV-NH 2 , a PAR-4 agonist peptide and its reverse peptide VQGPYG-NH 2 on release of IL-8 from A549 cells. Cells were incubated with various concentrations of GYPGQV-NH 2 or VQGPYG-NH 2 at 37°C for 16 h (A), or with 10 μM of GYPGQV-NH 2 for 2 h, 8 h and 16 h (B). Values shown are mean ± SE for 5 separate experiments. * P
Figure Legend Snippet: Effect of GYPGQV-NH 2 , a PAR-4 agonist peptide and its reverse peptide VQGPYG-NH 2 on release of IL-8 from A549 cells. Cells were incubated with various concentrations of GYPGQV-NH 2 or VQGPYG-NH 2 at 37°C for 16 h (A), or with 10 μM of GYPGQV-NH 2 for 2 h, 8 h and 16 h (B). Values shown are mean ± SE for 5 separate experiments. * P

Techniques Used: Incubation

Effect of trypsin on the release of IL-8 from A549 cells. Cells were incubated with various concentrations of trypsin at 37°C for 2 h, 8 h or 16 h. Values shown are mean ± SE for 5 separate experiments. * P
Figure Legend Snippet: Effect of trypsin on the release of IL-8 from A549 cells. Cells were incubated with various concentrations of trypsin at 37°C for 2 h, 8 h or 16 h. Values shown are mean ± SE for 5 separate experiments. * P

Techniques Used: Incubation

Effect of tryptase and elastase on the release of IL-8 from A549 cells. Cells were incubated with various concentrations of tryptase or elastase at 37°C for 16 h (A), or with 2 μg/ml tryptase or 0.3 μg/ml elastase for 2 h, 8 h and 16 h (B). Values shown are mean ± SE for 5 separate experiments. * P
Figure Legend Snippet: Effect of tryptase and elastase on the release of IL-8 from A549 cells. Cells were incubated with various concentrations of tryptase or elastase at 37°C for 16 h (A), or with 2 μg/ml tryptase or 0.3 μg/ml elastase for 2 h, 8 h and 16 h (B). Values shown are mean ± SE for 5 separate experiments. * P

Techniques Used: Incubation

Analysis of expression of PARs on A549 cells by flow cytometry ( A ), Immunofluorescent microscopy ( B ) and RT-PCR ( C ). For PAR-1 and PAR-2 labeling, cells were incubated with PE-conjugated mouse anti-human PAR-1 monoclonal antibody and FITC-conjugated mouse anti-human PAR-2 monoclonal antibody for 30 min on ice. For PAR-3 and PAR-4 labeling, cells were incubated with rabbit anti-human PAR-3 or PAR-4 polyclonal antibodies for 30 min on ice followed by addition of FITC-conjugated goat anti-rabbit polyclonal antibodies. To detect cytoplasmic PARs, A549 cells were permeabilized with 0.2% Triton X-100 for 5 min at room temperature before analysis. Immunofluorescent microscopy was performed with a laser scanning confocal microscope. For RT-PCR analysis of expression of mRNAs of PARs in A549 cells, the gene products of PARs were separated in 1.5% agarose gels, stained with SYBR Green I Nucleic Acid Gel Stain and photographed under UV light. Lane1-6 represented DNA marker, PAR-1 (500 bp), PAR-2 (461 bp), PAR-3 (403 bp), PAR-4 (542 bp) and β-actin (148 bp), respectively.
Figure Legend Snippet: Analysis of expression of PARs on A549 cells by flow cytometry ( A ), Immunofluorescent microscopy ( B ) and RT-PCR ( C ). For PAR-1 and PAR-2 labeling, cells were incubated with PE-conjugated mouse anti-human PAR-1 monoclonal antibody and FITC-conjugated mouse anti-human PAR-2 monoclonal antibody for 30 min on ice. For PAR-3 and PAR-4 labeling, cells were incubated with rabbit anti-human PAR-3 or PAR-4 polyclonal antibodies for 30 min on ice followed by addition of FITC-conjugated goat anti-rabbit polyclonal antibodies. To detect cytoplasmic PARs, A549 cells were permeabilized with 0.2% Triton X-100 for 5 min at room temperature before analysis. Immunofluorescent microscopy was performed with a laser scanning confocal microscope. For RT-PCR analysis of expression of mRNAs of PARs in A549 cells, the gene products of PARs were separated in 1.5% agarose gels, stained with SYBR Green I Nucleic Acid Gel Stain and photographed under UV light. Lane1-6 represented DNA marker, PAR-1 (500 bp), PAR-2 (461 bp), PAR-3 (403 bp), PAR-4 (542 bp) and β-actin (148 bp), respectively.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Microscopy, Reverse Transcription Polymerase Chain Reaction, Labeling, Incubation, Staining, SYBR Green Assay, Marker

Effect of PAR-2 agonist peptides tc-LIGRLO-NH 2 and SLIGKV-NH 2 and their reverse peptides, tc-OLRGIL and VKGILS-NH 2 on IL-8 release from A549 cells. Cells were incubated with various concentrations of tc-LIGRLO-NH 2 (open triangle), tc-OLRGIL (open reverse triangle), SLIGKV-NH 2 (open circle) or VKGILS-NH 2 (open square) at 37°C for (A) 2 h, (B) 8 h and (C) 16 h. Values shown are Mean ± SE for five separate experiments performed in duplicate. * P
Figure Legend Snippet: Effect of PAR-2 agonist peptides tc-LIGRLO-NH 2 and SLIGKV-NH 2 and their reverse peptides, tc-OLRGIL and VKGILS-NH 2 on IL-8 release from A549 cells. Cells were incubated with various concentrations of tc-LIGRLO-NH 2 (open triangle), tc-OLRGIL (open reverse triangle), SLIGKV-NH 2 (open circle) or VKGILS-NH 2 (open square) at 37°C for (A) 2 h, (B) 8 h and (C) 16 h. Values shown are Mean ± SE for five separate experiments performed in duplicate. * P

Techniques Used: Incubation

Effect of thrombin on the release of IL-8 from A549 cells. Cells were incubated ( A ) with various concentrations of thrombin at 37°C for 16 h, or ( B ) with 10 U/ml of thrombin for 2 h, 8 h and 16 h. Values shown are mean ± SE for 5 separate experiments. * P
Figure Legend Snippet: Effect of thrombin on the release of IL-8 from A549 cells. Cells were incubated ( A ) with various concentrations of thrombin at 37°C for 16 h, or ( B ) with 10 U/ml of thrombin for 2 h, 8 h and 16 h. Values shown are mean ± SE for 5 separate experiments. * P

Techniques Used: Incubation

24) Product Images from "The safety profile of Bald’s eyesalve for the treatment of bacterial infections"

Article Title: The safety profile of Bald’s eyesalve for the treatment of bacterial infections

Journal: bioRxiv

doi: 10.1101/2020.04.23.041749

Lactate dehydrogenase assay of THP-1 cells treated with eyesalve. THP-1 cells were treated with three batches of eyesalve (B7, B8 and B9) in the undiluted and diluted (1/10) forms. The controls include cells only (untreated), Neosporin (Neo), a safe antibiotic for wound infections and Optrex™ chloramphenicol (chl) treated cells (n = 4 replicates). The preface “dil” represents cells treated with a 1 in 10 dilution of either the chloramphenicol or the different eyesalve batches.
Figure Legend Snippet: Lactate dehydrogenase assay of THP-1 cells treated with eyesalve. THP-1 cells were treated with three batches of eyesalve (B7, B8 and B9) in the undiluted and diluted (1/10) forms. The controls include cells only (untreated), Neosporin (Neo), a safe antibiotic for wound infections and Optrex™ chloramphenicol (chl) treated cells (n = 4 replicates). The preface “dil” represents cells treated with a 1 in 10 dilution of either the chloramphenicol or the different eyesalve batches.

Techniques Used: Lactate Dehydrogenase Assay

25) Product Images from "Oncogenic mutation or overexpression of oncogenic KRAS or BRAF is not sufficient to confer oncogene addiction"

Article Title: Oncogenic mutation or overexpression of oncogenic KRAS or BRAF is not sufficient to confer oncogene addiction

Journal: bioRxiv

doi: 10.1101/2020.05.26.117911

Evaluation of oncogenic KRAS or BRAF addiction in MCF-10A cells overexpressing KRAS G12V or BRAF V600E. (A and B) Cell growth assays following siRNA-mediated negative control, KRAS, or BRAF ablation in parental MCF-10A, KRAS G12V OE, and BRAF V600E OE late cell lines grown in the partial growth medium. Three days after transfection with siRNA, relative cell densities were determined by crystal violet staining. Representative 96-well plates are shown (A). The mean relative cell number is shown with the SD (n = 4) (B). (C) Cell growth assays in empty vector-introduced MCF-10A, KRAS G12V OE, and BRAF V600E OE late cell lines grown in the starvation medium for four days with the indicated siRNA. Relative cell densities were determined by crystal violet staining. Representative 96-well plates are shown (lower). Mean relative cell number are shown with the SD (empty vector control, n = 3; KRAS G12V OE, n =8; BRAFV600E OE, n = 8) (upper). (D and E) Cell growth assays following siRNA-mediated negative control, KRAS, or BRAF ablation in the KRAS G12V OE cell line grown in the starvation medium. Four days after transfection with siRNA, relative cell densities were determined by crystal violet staining. Representative 96-well plates are shown (D). The mean relative cell number is shown with the SD (n = 8) (E).
Figure Legend Snippet: Evaluation of oncogenic KRAS or BRAF addiction in MCF-10A cells overexpressing KRAS G12V or BRAF V600E. (A and B) Cell growth assays following siRNA-mediated negative control, KRAS, or BRAF ablation in parental MCF-10A, KRAS G12V OE, and BRAF V600E OE late cell lines grown in the partial growth medium. Three days after transfection with siRNA, relative cell densities were determined by crystal violet staining. Representative 96-well plates are shown (A). The mean relative cell number is shown with the SD (n = 4) (B). (C) Cell growth assays in empty vector-introduced MCF-10A, KRAS G12V OE, and BRAF V600E OE late cell lines grown in the starvation medium for four days with the indicated siRNA. Relative cell densities were determined by crystal violet staining. Representative 96-well plates are shown (lower). Mean relative cell number are shown with the SD (empty vector control, n = 3; KRAS G12V OE, n =8; BRAFV600E OE, n = 8) (upper). (D and E) Cell growth assays following siRNA-mediated negative control, KRAS, or BRAF ablation in the KRAS G12V OE cell line grown in the starvation medium. Four days after transfection with siRNA, relative cell densities were determined by crystal violet staining. Representative 96-well plates are shown (D). The mean relative cell number is shown with the SD (n = 8) (E).

Techniques Used: Negative Control, Transfection, Staining, Plasmid Preparation

Evaluation of oncogenic KRAS or BRAF addiction in MCF-10A cells harboring a single allele mutation of KRAS G12V or BRAF V600E. (A and B) Cell growth assays following siRNA-mediated negative control, KRAS, or BRAF ablation in the A549, H358, and A375 cell lines. Four days after transfection with siRNA, relative cell densities were determined by crystal violet staining. Representative 96-well plates are shown (A). The mean relative cell number is shown with the SD (n = 2) (B). (C and D) Cell growth assays following siRNA-mediated negative control, KRAS, or BRAF ablation in the parental MCF-10A, KRAS G12V/+, and BRAF V600E/+ cell lines grown in the partial growth medium. Three days after transfection with siRNA, relative cell densities were determined by crystal violet staining. Representative 96-well plates are shown (C). The mean relative cell number is shown with the SD (Parental, n = 4; KRAS G12V/+, n = 4; BRAF V600E/+, n=3) (D). (E) Cell growth assays in the parental MCF-10A, KRAS G12V/+, and BRAF V600E/+ cell lines grown in starvation medium for four days with the indicated siRNA. Relative cell densities were determined by crystal violet staining. Representative 96-well plates are shown (lower). Mean relative cell numbers are shown with the SD (Parental, n = 3; KRAS G12V/+, n = 9; BRAF V600E/+, n=8) (upper). (F and G) Cell growth assays following siRNA-mediated negative control, KRAS, or BRAF ablation in the KRAS G12V/+ and BRAF V600E/+ cell lines grown in starvation medium. Four days after transfection with siRNA, relative cell densities were determined by crystal violet staining. Representative 96-well plates are shown (F). The mean relative cell number is shown with the SD (n = 8) (G).
Figure Legend Snippet: Evaluation of oncogenic KRAS or BRAF addiction in MCF-10A cells harboring a single allele mutation of KRAS G12V or BRAF V600E. (A and B) Cell growth assays following siRNA-mediated negative control, KRAS, or BRAF ablation in the A549, H358, and A375 cell lines. Four days after transfection with siRNA, relative cell densities were determined by crystal violet staining. Representative 96-well plates are shown (A). The mean relative cell number is shown with the SD (n = 2) (B). (C and D) Cell growth assays following siRNA-mediated negative control, KRAS, or BRAF ablation in the parental MCF-10A, KRAS G12V/+, and BRAF V600E/+ cell lines grown in the partial growth medium. Three days after transfection with siRNA, relative cell densities were determined by crystal violet staining. Representative 96-well plates are shown (C). The mean relative cell number is shown with the SD (Parental, n = 4; KRAS G12V/+, n = 4; BRAF V600E/+, n=3) (D). (E) Cell growth assays in the parental MCF-10A, KRAS G12V/+, and BRAF V600E/+ cell lines grown in starvation medium for four days with the indicated siRNA. Relative cell densities were determined by crystal violet staining. Representative 96-well plates are shown (lower). Mean relative cell numbers are shown with the SD (Parental, n = 3; KRAS G12V/+, n = 9; BRAF V600E/+, n=8) (upper). (F and G) Cell growth assays following siRNA-mediated negative control, KRAS, or BRAF ablation in the KRAS G12V/+ and BRAF V600E/+ cell lines grown in starvation medium. Four days after transfection with siRNA, relative cell densities were determined by crystal violet staining. Representative 96-well plates are shown (F). The mean relative cell number is shown with the SD (n = 8) (G).

Techniques Used: Mutagenesis, Negative Control, Transfection, Staining

26) Product Images from "Transient “rest” induces functional reinvigoration and epigenetic remodeling in exhausted CAR-T cells"

Article Title: Transient “rest” induces functional reinvigoration and epigenetic remodeling in exhausted CAR-T cells

Journal: bioRxiv

doi: 10.1101/2020.01.26.920496

A GD2-targeting CAR fused to an ecDHFR destabilizing domain exhibits rapid ON/OFF kinetics and drug-dependent, analog control of expression and function in vivo . Related to Figure 1 . A) CAR-T cells were stained with 1A7 anti-CAR idiotype antibody and analyzed via flow cytometry. Dose-dependent control (left) and ON/OFF kinetics (middle, right) were demonstrated via trimethoprim (TMP). CAR MFI was used to generate non-linear dose-response curves. Error bars represent mean ± SEM of 3 individual donors. B) 1×10 6 Nalm6-GD2 leukemia cells were engrafted in mice. On days 5-7 post-engraftment, mice were dosed with vehicle (water, CAR OFF) or 200mg/kg trimethoprim (TMP, CAR ON) once daily. On day 7 post-engraftment, 1×10 7 GD2.28 ζ .ecDHFR CAR-T cells were infused. 24 hours after CAR-T cell infusion, peripheral blood and spleens were harvested for flow cytometry analyses. Contour plots demonstrate a TMP-dependent increase in co-expression of CAR and the activation marker CD69 in CAR ON mice compared CAR OFF mice. Representative mouse from n=7 total mice from 2 independent experiments. C) 1×10 6 Nalm6-GD2 leukemia cells were engrafted in mice at day 0 and 2×10 6 GD2.28 ζ .ecDHFR CAR-T cells expanded in vitro in the absence of TMP for 15 days were infused IV on day 7 post-engraftment. Mice were dosed 6 days per week with vehicle (CAR OFF) or 200mg/kg TMP (CAR ON). Bioluminescence imaging of the tumor indicates TMP-dependent anti-tumor activity in CAR ON mice compared to CAR OFF mice (n=5 mice/group). Representative experiment (also shown in Figure 1F-G ) from 3 individual experiments.
Figure Legend Snippet: A GD2-targeting CAR fused to an ecDHFR destabilizing domain exhibits rapid ON/OFF kinetics and drug-dependent, analog control of expression and function in vivo . Related to Figure 1 . A) CAR-T cells were stained with 1A7 anti-CAR idiotype antibody and analyzed via flow cytometry. Dose-dependent control (left) and ON/OFF kinetics (middle, right) were demonstrated via trimethoprim (TMP). CAR MFI was used to generate non-linear dose-response curves. Error bars represent mean ± SEM of 3 individual donors. B) 1×10 6 Nalm6-GD2 leukemia cells were engrafted in mice. On days 5-7 post-engraftment, mice were dosed with vehicle (water, CAR OFF) or 200mg/kg trimethoprim (TMP, CAR ON) once daily. On day 7 post-engraftment, 1×10 7 GD2.28 ζ .ecDHFR CAR-T cells were infused. 24 hours after CAR-T cell infusion, peripheral blood and spleens were harvested for flow cytometry analyses. Contour plots demonstrate a TMP-dependent increase in co-expression of CAR and the activation marker CD69 in CAR ON mice compared CAR OFF mice. Representative mouse from n=7 total mice from 2 independent experiments. C) 1×10 6 Nalm6-GD2 leukemia cells were engrafted in mice at day 0 and 2×10 6 GD2.28 ζ .ecDHFR CAR-T cells expanded in vitro in the absence of TMP for 15 days were infused IV on day 7 post-engraftment. Mice were dosed 6 days per week with vehicle (CAR OFF) or 200mg/kg TMP (CAR ON). Bioluminescence imaging of the tumor indicates TMP-dependent anti-tumor activity in CAR ON mice compared to CAR OFF mice (n=5 mice/group). Representative experiment (also shown in Figure 1F-G ) from 3 individual experiments.

Techniques Used: Expressing, In Vivo, Staining, Flow Cytometry, Mouse Assay, Activation Assay, Marker, In Vitro, Imaging, Activity Assay

Transient rest reinvigorates exhausted CAR-T cells and improves anti-tumor function. A) CAR expression was normalized across all HA.28z.FKBP-expressing cells ( Figure 4A ) by adding shield-1 to cultures approximately 16 hours prior to in vitro tumor challenge. B) D15 incucyte assay shows that transient rest and α PD-1 enhances cytotoxicity in response to 143B-GL osteosarcoma (1:8 E:T, normalized to t=0) compared to exhausted Always ON controls. Error bars represent mean ± SD of 3 triplicate wells from one representative donor (n=3 individual donors). C) D15 co-culture assay with Nalm6-GD2 leukemia demonstrates that transient rest, but not α PD-1, augments IL-2 and IFN γ secretion. Error bars represent mean ± SEM of 4-6 individual donors. D-E) Intracellular cytokine staining and flow cytometry analyses of CD8+ CAR+ T cells demonstrate that rest reduces the frequency of non-responsive cells and increases the frequency of polyfunctional cells. SPICE analysis from 1 representative donor was conducted in (D). Error bars in (E) represent mean ± SEM of 3 individual donors. F-G) CAR-T cells were crosslinked with immobilized 1A7 anti-CAR idiotype antibody for 24 hours. (F) Non-linear dose-response curves demonstrate that T cell rest, but not PD-1 blockade, augments IL-2 and IFN γ secretion in response to both low and high idiotype densities. Error bars represent mean ± SD of 3 triplicate wells from one representative donor (n=3 individual donors). (G) IL-2 secretion in response to low density (1.25 μ g/mL) anti-CAR idiotype was normalized to secretion levels from Always OFF cells. Error bars represent mean ± SEM of 3 individual donors. H-I) 1×10 6 Nalm6-GD2 leukemia cells were engrafted in mice on day 0 and 1-2×10 6 HA.28 ζ .ecDHFR CAR-T cells expanded for 15 days in vitro (as depicted in Figure 4A ) and were infused IV on day 7 post-engraftment. Mice were dosed with 200mg/kg TMP 6 days/week. (I) Bioluminescent imaging on day 30 post-engraftment demonstrates augmented control of tumor growth in Always OFF and rested conditions compared to Always ON cells. Error bars represent mean ± SEM of 3-5 mice from 1 representative experiment (n=3 independent experiments). Statistics were calculated using one or two-way ANOVA and Dunnett’s multiple comparisons test. *, p
Figure Legend Snippet: Transient rest reinvigorates exhausted CAR-T cells and improves anti-tumor function. A) CAR expression was normalized across all HA.28z.FKBP-expressing cells ( Figure 4A ) by adding shield-1 to cultures approximately 16 hours prior to in vitro tumor challenge. B) D15 incucyte assay shows that transient rest and α PD-1 enhances cytotoxicity in response to 143B-GL osteosarcoma (1:8 E:T, normalized to t=0) compared to exhausted Always ON controls. Error bars represent mean ± SD of 3 triplicate wells from one representative donor (n=3 individual donors). C) D15 co-culture assay with Nalm6-GD2 leukemia demonstrates that transient rest, but not α PD-1, augments IL-2 and IFN γ secretion. Error bars represent mean ± SEM of 4-6 individual donors. D-E) Intracellular cytokine staining and flow cytometry analyses of CD8+ CAR+ T cells demonstrate that rest reduces the frequency of non-responsive cells and increases the frequency of polyfunctional cells. SPICE analysis from 1 representative donor was conducted in (D). Error bars in (E) represent mean ± SEM of 3 individual donors. F-G) CAR-T cells were crosslinked with immobilized 1A7 anti-CAR idiotype antibody for 24 hours. (F) Non-linear dose-response curves demonstrate that T cell rest, but not PD-1 blockade, augments IL-2 and IFN γ secretion in response to both low and high idiotype densities. Error bars represent mean ± SD of 3 triplicate wells from one representative donor (n=3 individual donors). (G) IL-2 secretion in response to low density (1.25 μ g/mL) anti-CAR idiotype was normalized to secretion levels from Always OFF cells. Error bars represent mean ± SEM of 3 individual donors. H-I) 1×10 6 Nalm6-GD2 leukemia cells were engrafted in mice on day 0 and 1-2×10 6 HA.28 ζ .ecDHFR CAR-T cells expanded for 15 days in vitro (as depicted in Figure 4A ) and were infused IV on day 7 post-engraftment. Mice were dosed with 200mg/kg TMP 6 days/week. (I) Bioluminescent imaging on day 30 post-engraftment demonstrates augmented control of tumor growth in Always OFF and rested conditions compared to Always ON cells. Error bars represent mean ± SEM of 3-5 mice from 1 representative experiment (n=3 independent experiments). Statistics were calculated using one or two-way ANOVA and Dunnett’s multiple comparisons test. *, p

Techniques Used: Expressing, In Vitro, Co-culture Assay, Staining, Flow Cytometry, Mouse Assay, Imaging

27) Product Images from "Mitf links neuronal activity and long-term homeostatic intrinsic plasticity"

Article Title: Mitf links neuronal activity and long-term homeostatic intrinsic plasticity

Journal: bioRxiv

doi: 10.1101/507640

Activity-dependent increase in Kcnd3 expression requires MITF enhancer activity. a. RNA in situ hybridization of c-Fos (red) and Kcnd3 (green) performed on Mitf mi-vga9/mi-vga9 OB following AA. b . Kcnd3 dots per cell in Mitf mi-vga9/mi-vga9 OB following AA. N=3-5 per time point. c . ChIPseq peaks of MITF, H3K27ac and H3K4me1 on KCND3 gene in 501mel cells. The MITF peaks are labeled A (orange) and B (yellow). Yellow shows overlapping peak of MITF binding in Strub and Laurette datasets (B) whereas Orange indicates MITF peaks that overlap with H3K27ac and H3K4me1 peaks (A). d. Sequence of the basic region of wild-type (MITF-M) and transcriptionally inactive MITF with four argenines mutated to alanines (MITF-M B4RA). e. Transcription activation assays performed in HEK293T cells co-transfected with constructs containing the hTYR, hKCND3_pA, and hKCND3_pB regulatory regions fused to luciferase, together with empty vector or wild-type or transcriptionally inactive (B4RA) MITF constructs. N=3. f . Transcription activation assays performed in N2A cells co-transfected with constructs containing the hTYR, hKCND3_pA, and hKCND3_pB regulatory regions fused to luciferase, together with empty vector or wild-type or transcriptionally inactive (B4RA) MITF constructs. N=3. The values on the graphs are mean ± SEM. DAPI nuclear staining is shown in blue. Scale bars are 50µm. P-values were calculated using two-way ANOVA ( b, e, f ) *P
Figure Legend Snippet: Activity-dependent increase in Kcnd3 expression requires MITF enhancer activity. a. RNA in situ hybridization of c-Fos (red) and Kcnd3 (green) performed on Mitf mi-vga9/mi-vga9 OB following AA. b . Kcnd3 dots per cell in Mitf mi-vga9/mi-vga9 OB following AA. N=3-5 per time point. c . ChIPseq peaks of MITF, H3K27ac and H3K4me1 on KCND3 gene in 501mel cells. The MITF peaks are labeled A (orange) and B (yellow). Yellow shows overlapping peak of MITF binding in Strub and Laurette datasets (B) whereas Orange indicates MITF peaks that overlap with H3K27ac and H3K4me1 peaks (A). d. Sequence of the basic region of wild-type (MITF-M) and transcriptionally inactive MITF with four argenines mutated to alanines (MITF-M B4RA). e. Transcription activation assays performed in HEK293T cells co-transfected with constructs containing the hTYR, hKCND3_pA, and hKCND3_pB regulatory regions fused to luciferase, together with empty vector or wild-type or transcriptionally inactive (B4RA) MITF constructs. N=3. f . Transcription activation assays performed in N2A cells co-transfected with constructs containing the hTYR, hKCND3_pA, and hKCND3_pB regulatory regions fused to luciferase, together with empty vector or wild-type or transcriptionally inactive (B4RA) MITF constructs. N=3. The values on the graphs are mean ± SEM. DAPI nuclear staining is shown in blue. Scale bars are 50µm. P-values were calculated using two-way ANOVA ( b, e, f ) *P

Techniques Used: Activity Assay, Expressing, RNA In Situ Hybridization, Labeling, Binding Assay, Sequencing, Activation Assay, Transfection, Construct, Luciferase, Plasmid Preparation, Staining

28) Product Images from "The cellular basis of protease-activated receptor 2–evoked mechanical and affective pain"

Article Title: The cellular basis of protease-activated receptor 2–evoked mechanical and affective pain

Journal: JCI Insight

doi: 10.1172/jci.insight.137393

F2rl1 is expressed by a small subset of sensory neurons. DRG neurons ( A – E ) and hind paw skin ( F ) from F2rl1 flox Pirt +/+ and F2rl1 flox Pirt Cre mice were dissected and prepared for RNAscope in situ hybridization. White arrows indicate cells positive for F2rl1 mRNA. ( A ) Representative original magnification ×20 images of Calca (green), P2rx3 (red), and F2rl1 (white) mRNA signal in the DRG of F2rl1 flox Pirt +/+ and F2rl1 flox Pirt Cre mice. These images show that the F2rl1 flox Pirt Cre mice do not express F2rl1 mRNA in sensory neurons while the F2rl1 flox Pirt +/+ mice do. Scale bar: 50 μm. ( B ) Original magnification ×40 overlay image showing RNAscope signal at the cellular level. Scale bar: 10 μm. ( C ) Percentage of Calca + and P2rx3 + neurons that coexpress F2rl1 . Around 2% of Calca + neurons express F2rl1 mRNA while around 6% of P2rx3 + neurons express F2rl1 mRNA. ( D ) Histogram illustrating the diameter of neurons expressing F2rl1 . F2rl1 + neurons are small- to medium-diameter neurons (16–46 μm). ( E ) Pie chart illustration of the percentage of F2rl1 + cells that colocalize with Calca + and P2rx3 + neurons. About 3%–4% of DRG neurons are F2rl1 + , of which almost all are P2rx3 + neurons. ( F ) Representative hind paw skin images of F2rl1 (white) and DAPI (blue) signal from a F2rl1 flox Pirt +/+ mouse and a F2rl1 flox Pirt Cre mouse. The last image shows hind paw skin from a F2rl1 flox Pirt +/+ mouse stained with a negative probe control (bacterial dapB ). These images show the specificity of conditional knockout of F2rl1 expression is restricted to only sensory neurons and not skin cells. Scale bar: 20 μm.
Figure Legend Snippet: F2rl1 is expressed by a small subset of sensory neurons. DRG neurons ( A – E ) and hind paw skin ( F ) from F2rl1 flox Pirt +/+ and F2rl1 flox Pirt Cre mice were dissected and prepared for RNAscope in situ hybridization. White arrows indicate cells positive for F2rl1 mRNA. ( A ) Representative original magnification ×20 images of Calca (green), P2rx3 (red), and F2rl1 (white) mRNA signal in the DRG of F2rl1 flox Pirt +/+ and F2rl1 flox Pirt Cre mice. These images show that the F2rl1 flox Pirt Cre mice do not express F2rl1 mRNA in sensory neurons while the F2rl1 flox Pirt +/+ mice do. Scale bar: 50 μm. ( B ) Original magnification ×40 overlay image showing RNAscope signal at the cellular level. Scale bar: 10 μm. ( C ) Percentage of Calca + and P2rx3 + neurons that coexpress F2rl1 . Around 2% of Calca + neurons express F2rl1 mRNA while around 6% of P2rx3 + neurons express F2rl1 mRNA. ( D ) Histogram illustrating the diameter of neurons expressing F2rl1 . F2rl1 + neurons are small- to medium-diameter neurons (16–46 μm). ( E ) Pie chart illustration of the percentage of F2rl1 + cells that colocalize with Calca + and P2rx3 + neurons. About 3%–4% of DRG neurons are F2rl1 + , of which almost all are P2rx3 + neurons. ( F ) Representative hind paw skin images of F2rl1 (white) and DAPI (blue) signal from a F2rl1 flox Pirt +/+ mouse and a F2rl1 flox Pirt Cre mouse. The last image shows hind paw skin from a F2rl1 flox Pirt +/+ mouse stained with a negative probe control (bacterial dapB ). These images show the specificity of conditional knockout of F2rl1 expression is restricted to only sensory neurons and not skin cells. Scale bar: 20 μm.

Techniques Used: Mouse Assay, In Situ Hybridization, Expressing, Staining, Knock-Out

2AT-evoked increased phosphorylated ERK signal intensity is specific for F2rl1 -expressing neurons. Primary mouse DRG cultures were prepared for RNAscope in situ hybridization and immunocytochemistry. ( A ) Representative original magnification ×40 images of P2rx3 and F2rl1 mRNA and Neurofilament 200 (NF200) protein signal of cultured DRG neurons from WT mice. White arrows indicate a neuron positive for F2rl1 mRNA signal. Smaller image panels display zoomed-in images of the indicated single neuron. Scale bar: 20 μm. Zoomed-in image scale bar: 2 μm. ( B ) Pie chart illustrating the percentage distribution of neuronal F2rl1 and P2rx3 expression in vitro. About 3%–4% of primary cultured DRG neurons are F2rl1 + , almost all of which are also P2rx3 + . ( C ) Representative images of F2rl1 mRNA signal and phosphorylated ERK (p-ERK) immunolabeling in cultured DRG neurons from WT mice after treatment with vehicle or 2AT (1 μM) for 10 minutes. Scale bar: 2 μm. F2rl1 + DRG neurons treated with 2AT show increased p-ERK signal when compared with vehicle treatment. ( D ) Signal intensity of p-ERK increased markedly in F2rl1 + neurons after treatment with 2AT (1 μM) when compared with the vehicle treatment group. No significant difference in p-ERK signal intensity is seen between vehicle- and 2AT-treated groups in the F2rl1 – neurons. p-ERK signal was quantified through mean gray intensity value and normalized to the average p-ERK signal intensity value for the vehicle treatment groups. n = 15 and n = 16 for F2rl1 + neurons treated with vehicle or 2AT, respectively. n = 88 for both vehicle and 2AT treatment groups in F2rl1 – neurons. Data represent mean ± SEM. One-way ANOVA with Bonferroni’s multiple comparisons ( D ) *** P
Figure Legend Snippet: 2AT-evoked increased phosphorylated ERK signal intensity is specific for F2rl1 -expressing neurons. Primary mouse DRG cultures were prepared for RNAscope in situ hybridization and immunocytochemistry. ( A ) Representative original magnification ×40 images of P2rx3 and F2rl1 mRNA and Neurofilament 200 (NF200) protein signal of cultured DRG neurons from WT mice. White arrows indicate a neuron positive for F2rl1 mRNA signal. Smaller image panels display zoomed-in images of the indicated single neuron. Scale bar: 20 μm. Zoomed-in image scale bar: 2 μm. ( B ) Pie chart illustrating the percentage distribution of neuronal F2rl1 and P2rx3 expression in vitro. About 3%–4% of primary cultured DRG neurons are F2rl1 + , almost all of which are also P2rx3 + . ( C ) Representative images of F2rl1 mRNA signal and phosphorylated ERK (p-ERK) immunolabeling in cultured DRG neurons from WT mice after treatment with vehicle or 2AT (1 μM) for 10 minutes. Scale bar: 2 μm. F2rl1 + DRG neurons treated with 2AT show increased p-ERK signal when compared with vehicle treatment. ( D ) Signal intensity of p-ERK increased markedly in F2rl1 + neurons after treatment with 2AT (1 μM) when compared with the vehicle treatment group. No significant difference in p-ERK signal intensity is seen between vehicle- and 2AT-treated groups in the F2rl1 – neurons. p-ERK signal was quantified through mean gray intensity value and normalized to the average p-ERK signal intensity value for the vehicle treatment groups. n = 15 and n = 16 for F2rl1 + neurons treated with vehicle or 2AT, respectively. n = 88 for both vehicle and 2AT treatment groups in F2rl1 – neurons. Data represent mean ± SEM. One-way ANOVA with Bonferroni’s multiple comparisons ( D ) *** P

Techniques Used: Expressing, In Situ Hybridization, Immunocytochemistry, Cell Culture, Mouse Assay, In Vitro, Immunolabeling

29) Product Images from "Expression of Heat Shock Protein 27 in Prostate Cancer Cell Lines According to the Extent of Malignancy and Doxazosin Treatment"

Article Title: Expression of Heat Shock Protein 27 in Prostate Cancer Cell Lines According to the Extent of Malignancy and Doxazosin Treatment

Journal: The World Journal of Men's Health

doi: 10.5534/wjmh.2013.31.3.247

Heat shock protein 27 immunofluorescence staining according to the cell type (RWPE-1, LNCaP, PC-3, and TSU-Pr1). The staining for TSU-Pr1 was more intense than that of the other cell lines. The intensity of staining was RWPE-1
Figure Legend Snippet: Heat shock protein 27 immunofluorescence staining according to the cell type (RWPE-1, LNCaP, PC-3, and TSU-Pr1). The staining for TSU-Pr1 was more intense than that of the other cell lines. The intensity of staining was RWPE-1

Techniques Used: Immunofluorescence, Staining

HSP27 reverse transcription polymerase chain reaction results according to the cell type (RWPE-1, LNCaP, PC-3, and TSU-Pr1). The density of HSP27 mRNA expression was shown to be higher than the low grade malignant cell line. Values are mean±standard error of mean. HSP27: heat shock protein 27, GAPDH: glyceraldehyde-3-phosphate dehydrogenase. a p
Figure Legend Snippet: HSP27 reverse transcription polymerase chain reaction results according to the cell type (RWPE-1, LNCaP, PC-3, and TSU-Pr1). The density of HSP27 mRNA expression was shown to be higher than the low grade malignant cell line. Values are mean±standard error of mean. HSP27: heat shock protein 27, GAPDH: glyceraldehyde-3-phosphate dehydrogenase. a p

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

30) Product Images from "Drug testing and flow cytometry analysis on a large number of uniform sized tumor spheroids using a microfluidic device"

Article Title: Drug testing and flow cytometry analysis on a large number of uniform sized tumor spheroids using a microfluidic device

Journal: Scientific Reports

doi: 10.1038/srep21061

Size distribution analysis of the HepG2 tumor spheroids located in up-, middle- and down-stream of the microfluidic device and different devices with cell culture chamber dimensions (width × length) of ( A ) 200 × 200 μm 2 and ( B ) 300 × 300 μm 2 , and the height is 250 μm. Data are expressed as the mean ± standard deviation (16 and 9 spheroids are analyzed in each location within a device with 200 × 200 μm 2 and 300 × 300 μm 2 cell culture chambers, respectively).
Figure Legend Snippet: Size distribution analysis of the HepG2 tumor spheroids located in up-, middle- and down-stream of the microfluidic device and different devices with cell culture chamber dimensions (width × length) of ( A ) 200 × 200 μm 2 and ( B ) 300 × 300 μm 2 , and the height is 250 μm. Data are expressed as the mean ± standard deviation (16 and 9 spheroids are analyzed in each location within a device with 200 × 200 μm 2 and 300 × 300 μm 2 cell culture chambers, respectively).

Techniques Used: Cell Culture, Standard Deviation

Bright field microscopic images of the HepG2 tumor spheroids cultured in the microfluidic devices with cell culture chamber dimensions (width × length) of ( A ) 200 × 200 μm 2 and ( B ) 300 × 300 μm 2 , and the height is 250 μm for total 3 days and treated with drugs for 48 hours. ( C ) Quantitative characterization of the diameters of the HepG2 tumor spheroids before and after the drug treatments by imaging analysis on the bright field microscopic images.
Figure Legend Snippet: Bright field microscopic images of the HepG2 tumor spheroids cultured in the microfluidic devices with cell culture chamber dimensions (width × length) of ( A ) 200 × 200 μm 2 and ( B ) 300 × 300 μm 2 , and the height is 250 μm for total 3 days and treated with drugs for 48 hours. ( C ) Quantitative characterization of the diameters of the HepG2 tumor spheroids before and after the drug treatments by imaging analysis on the bright field microscopic images.

Techniques Used: Cell Culture, Imaging

Left: Flow-cytometry analysis of dissociated HepG2 tumor spheroids with forward-scattering light (FSC), side-scattering light (SSC), and gated areas. Right: Viability assay using Calcein-AM and 7-AAD (right panel) on dissociated HepG2 tumor spheroids after 3-day culture in the microfluidic devices with cell culture chamber dimensions (width × length) of ( A ) 200 × 200 μm 2 and ( B ) 300 × 300 μm 2 , and the height is 250 μm.
Figure Legend Snippet: Left: Flow-cytometry analysis of dissociated HepG2 tumor spheroids with forward-scattering light (FSC), side-scattering light (SSC), and gated areas. Right: Viability assay using Calcein-AM and 7-AAD (right panel) on dissociated HepG2 tumor spheroids after 3-day culture in the microfluidic devices with cell culture chamber dimensions (width × length) of ( A ) 200 × 200 μm 2 and ( B ) 300 × 300 μm 2 , and the height is 250 μm.

Techniques Used: Flow Cytometry, Cytometry, Viability Assay, Cell Culture

Density plots of flow cytometry analysis on APC-Annexin V and 7-AAD stained HepG2 cells (apoptosis and necrosis assay) cultured in 2D Petri-dish and dissociated from different sized tumor spheroids after 48-hour drug treatments.
Figure Legend Snippet: Density plots of flow cytometry analysis on APC-Annexin V and 7-AAD stained HepG2 cells (apoptosis and necrosis assay) cultured in 2D Petri-dish and dissociated from different sized tumor spheroids after 48-hour drug treatments.

Techniques Used: Flow Cytometry, Cytometry, Staining, Cell Culture

Density plots of flow cytometry analysis on APC-Annexin V and 7-AAD stained HepG2 cells (apoptosis and necrosis assay) cultured in 2D Petri-dish and dissociated from tumor spheroids formed in 300 × 300 μm 2 (width × length) cell culture chambers after 48-hour treatments of drug combinations.
Figure Legend Snippet: Density plots of flow cytometry analysis on APC-Annexin V and 7-AAD stained HepG2 cells (apoptosis and necrosis assay) cultured in 2D Petri-dish and dissociated from tumor spheroids formed in 300 × 300 μm 2 (width × length) cell culture chambers after 48-hour treatments of drug combinations.

Techniques Used: Flow Cytometry, Cytometry, Staining, Cell Culture

31) Product Images from "Interaction of RNA with a C-terminal fragment of the amyotrophic lateral sclerosis-associated TDP43 reduces cytotoxicity"

Article Title: Interaction of RNA with a C-terminal fragment of the amyotrophic lateral sclerosis-associated TDP43 reduces cytotoxicity

Journal: Scientific Reports

doi: 10.1038/srep19230

Structural characterization of inclusion bodies (IBs) containing TDP43 carboxyl-terminal fragments (CTFs). ( a , b ) Western blotting of GFP-tagged TDP43 and TDP43 CTFs after fractionation into 0.1% SDS soluble (s) or insoluble (p) parts. ( c ) The fluorescence recovery curve of GFP-TDP25 in IBs in the cytoplasm with and without the proteasome inhibitor MG-132 (magenta and green, respectively). The dashed gray line indicates the values of zero and 1.0 of relative fluorescence intensity (RFI). Inset values show the maximum recovery rate.
Figure Legend Snippet: Structural characterization of inclusion bodies (IBs) containing TDP43 carboxyl-terminal fragments (CTFs). ( a , b ) Western blotting of GFP-tagged TDP43 and TDP43 CTFs after fractionation into 0.1% SDS soluble (s) or insoluble (p) parts. ( c ) The fluorescence recovery curve of GFP-TDP25 in IBs in the cytoplasm with and without the proteasome inhibitor MG-132 (magenta and green, respectively). The dashed gray line indicates the values of zero and 1.0 of relative fluorescence intensity (RFI). Inset values show the maximum recovery rate.

Techniques Used: Western Blot, Fractionation, Fluorescence

Distinct properties of cytoplasmic and nuclear inclusion bodies (IBs) containing a TDP43 carboxyl-terminal fragment (CTF). ( a , c ) Confocal fluorescence microscopy of GFP-tagged TDP43 and TDP43 CTFs during a normal state ( a ), during proteasome inhibition ( c ). The white arrow and arrowhead indicate IBs in the cytoplasm and nucleus, respectively. Scale bar = 5 μm. ( b ) Confocal immunofluorescence microscopy of GFP-tagged TDP35 or TDP25 with an anti-phosphorylated Ser409/410 in TDP43 antibody. Arrowhead indicate IBs in the cytoplasm. Scale bar = 5 μm. ( d ) Quantification and comparison of cytoplasmic IB-positive cells (percentages). The numbers on the bar graph show mean values. The error bars denote mean and SD (n = 3). Light and dark gray mean without (−) and with (+) the proteasome inhibitor MG-132, respectively. ( e ) Airyscan confocal super-resolution microscopy images of TDP43-GFP, GFP-NLS, and GFP-NLS-TDP25. The white arrowhead indicates the position of the nucleolus. Scale bar = 5 μm.
Figure Legend Snippet: Distinct properties of cytoplasmic and nuclear inclusion bodies (IBs) containing a TDP43 carboxyl-terminal fragment (CTF). ( a , c ) Confocal fluorescence microscopy of GFP-tagged TDP43 and TDP43 CTFs during a normal state ( a ), during proteasome inhibition ( c ). The white arrow and arrowhead indicate IBs in the cytoplasm and nucleus, respectively. Scale bar = 5 μm. ( b ) Confocal immunofluorescence microscopy of GFP-tagged TDP35 or TDP25 with an anti-phosphorylated Ser409/410 in TDP43 antibody. Arrowhead indicate IBs in the cytoplasm. Scale bar = 5 μm. ( d ) Quantification and comparison of cytoplasmic IB-positive cells (percentages). The numbers on the bar graph show mean values. The error bars denote mean and SD (n = 3). Light and dark gray mean without (−) and with (+) the proteasome inhibitor MG-132, respectively. ( e ) Airyscan confocal super-resolution microscopy images of TDP43-GFP, GFP-NLS, and GFP-NLS-TDP25. The white arrowhead indicates the position of the nucleolus. Scale bar = 5 μm.

Techniques Used: Fluorescence, Microscopy, Inhibition, Immunofluorescence

Comparison of the death rate among the Neuro2A cells expressing TDP43 and the carboxyl-terminal fragments (CTFs) with and without treatment with a proteasome inhibitor. Dead cell rate of Neuro2A cells expressing proteins tagged with GFP ( a ) or FLAG ( b ). The error bars indicate mean and SD (n = 3). Significance was assessed by Student’s t test. Significance compared to GFP as a negative control without proteasome inhibition is marked as † p
Figure Legend Snippet: Comparison of the death rate among the Neuro2A cells expressing TDP43 and the carboxyl-terminal fragments (CTFs) with and without treatment with a proteasome inhibitor. Dead cell rate of Neuro2A cells expressing proteins tagged with GFP ( a ) or FLAG ( b ). The error bars indicate mean and SD (n = 3). Significance was assessed by Student’s t test. Significance compared to GFP as a negative control without proteasome inhibition is marked as † p

Techniques Used: Expressing, Negative Control, Inhibition

32) Product Images from "EGF-induced adipose tissue mesothelial cells undergo functional vascular smooth muscle differentiation"

Article Title: EGF-induced adipose tissue mesothelial cells undergo functional vascular smooth muscle differentiation

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.271

Generation of spreading ATMC-derived VSM-like cell spheroids for their use in contraction studies. ( a ) Schematic representation of steps performed to generate ATMC-derived VSM-like cell spheroids. Right, phase contrast pictures of spheroid taken after 0 and 24 h of their transfer onto plastic adherent surface into BMe+50EGF. Scale bar is 200 μ m. ( b ) Immunofluorescence characterization of SMCs markers (SM22 α and α SMA) expression in spheroids fixed after 24 h of spreading onto plastic surface
Figure Legend Snippet: Generation of spreading ATMC-derived VSM-like cell spheroids for their use in contraction studies. ( a ) Schematic representation of steps performed to generate ATMC-derived VSM-like cell spheroids. Right, phase contrast pictures of spheroid taken after 0 and 24 h of their transfer onto plastic adherent surface into BMe+50EGF. Scale bar is 200 μ m. ( b ) Immunofluorescence characterization of SMCs markers (SM22 α and α SMA) expression in spheroids fixed after 24 h of spreading onto plastic surface

Techniques Used: Derivative Assay, Immunofluorescence, Expressing

33) Product Images from "Epirubicin-Adsorbed Nanodiamonds Kill Chemoresistant Hepatic Cancer Stem Cells"

Article Title: Epirubicin-Adsorbed Nanodiamonds Kill Chemoresistant Hepatic Cancer Stem Cells

Journal: ACS Nano

doi: 10.1021/nn503491e

Evaluation of EPND efficacy in vitro . (a) Dose response curves of LT2-MYC cells after exposure to PBS, nanodiamond (ND) and a range of Epirubicin (Epi) and EPND concentrations. IC 50 of Epi =16 nM, IC 50 of EPND = 450 nM. Data are represented as mean ± SD. (b) Live cell number counting during 5 days of PBS, ND, Epi (100 nM) and EPND (100 nM) treatment. Data are represented as mean ± SD, *, p
Figure Legend Snippet: Evaluation of EPND efficacy in vitro . (a) Dose response curves of LT2-MYC cells after exposure to PBS, nanodiamond (ND) and a range of Epirubicin (Epi) and EPND concentrations. IC 50 of Epi =16 nM, IC 50 of EPND = 450 nM. Data are represented as mean ± SD. (b) Live cell number counting during 5 days of PBS, ND, Epi (100 nM) and EPND (100 nM) treatment. Data are represented as mean ± SD, *, p

Techniques Used: In Vitro

Enhanced efficacy of EPND against side population cells in vitro . (a) Representative images of flow cytometry analysis of side-population (SP) in LT2-MYC cell line after 48 h of Epirubicin (Epi) (100 nM and 200 nM) or EPND (100 nM and 200 nM) treatment using Hoechst dye 33342 staining, PBS and nanodiamond (ND) as control. SP gate was determined by absence of cell population after addition of 500 μM verapamil in PBS control. The percentages of SP cells in the total live cells gated are shown. (b) Quantitative analysis of SP cells in LT2-MYC cell line after 48 h of Epi, EPND, PBS and ND treatment ( n = 3). Data are represented as mean ± SD, *, p
Figure Legend Snippet: Enhanced efficacy of EPND against side population cells in vitro . (a) Representative images of flow cytometry analysis of side-population (SP) in LT2-MYC cell line after 48 h of Epirubicin (Epi) (100 nM and 200 nM) or EPND (100 nM and 200 nM) treatment using Hoechst dye 33342 staining, PBS and nanodiamond (ND) as control. SP gate was determined by absence of cell population after addition of 500 μM verapamil in PBS control. The percentages of SP cells in the total live cells gated are shown. (b) Quantitative analysis of SP cells in LT2-MYC cell line after 48 h of Epi, EPND, PBS and ND treatment ( n = 3). Data are represented as mean ± SD, *, p

Techniques Used: In Vitro, Flow Cytometry, Cytometry, Staining

EPND uptake is mediated by multiple endocytic pathways. (a) Representative images of cellular EPND uptake after dynasore treatment by fluorescent microscopy. LT2-MYC cells treated with or without dynasore (100 μM) for 30 min followed by 30 min treatment with 35 μM EPND (upper panels) or PBS control (bottom panels). Blue and red fluorescent signals represent diamidino-2-phenylindole (DAPI) staining and Epirubicin (Epi) respectively. Scale bar (white), 50 μm. (b) Quantification of percent cellular EPND uptake normalized to DAPI signal ( n = 3 per treatment condition). Data are represented as mean ± SD; *, p
Figure Legend Snippet: EPND uptake is mediated by multiple endocytic pathways. (a) Representative images of cellular EPND uptake after dynasore treatment by fluorescent microscopy. LT2-MYC cells treated with or without dynasore (100 μM) for 30 min followed by 30 min treatment with 35 μM EPND (upper panels) or PBS control (bottom panels). Blue and red fluorescent signals represent diamidino-2-phenylindole (DAPI) staining and Epirubicin (Epi) respectively. Scale bar (white), 50 μm. (b) Quantification of percent cellular EPND uptake normalized to DAPI signal ( n = 3 per treatment condition). Data are represented as mean ± SD; *, p

Techniques Used: Microscopy, Staining

EPND is less toxic than Epirubicin. (a) Bodyweight analysis of MYC-tumor bearing mice injected with PBS, nanodiamond (ND), Epirubicin (Epi) (5 mg/kg) and EPND (5 mg/kg) ( n = 5) every 3 days. Black arrow denotes injection day. Data are presented as mean ± SD. (b) Bodyweight analysis of healthy FVB/N mice injected with PBS, ND, Epi (20 mg/kg) and EPND (20 mg/kg) ( n = 5) every 3 days. Black arrow denotes injection day. Data are presented as mean ± SD; **, p
Figure Legend Snippet: EPND is less toxic than Epirubicin. (a) Bodyweight analysis of MYC-tumor bearing mice injected with PBS, nanodiamond (ND), Epirubicin (Epi) (5 mg/kg) and EPND (5 mg/kg) ( n = 5) every 3 days. Black arrow denotes injection day. Data are presented as mean ± SD. (b) Bodyweight analysis of healthy FVB/N mice injected with PBS, ND, Epi (20 mg/kg) and EPND (20 mg/kg) ( n = 5) every 3 days. Black arrow denotes injection day. Data are presented as mean ± SD; **, p

Techniques Used: Mouse Assay, Injection

EPND can inhibit tumor-initiation in murine hepatic tumor allografts. (a) Representative image of FVB/N mouse seeded in hindquarters with cells from PBS, nanodiamond (ND), Epirubicin (Epi) or EPND treated MYC-driven tumors 58 days after allograft transplantation. Two mice in each panel were under same treatment conditions. Black arrows indicate treatment condition and number. (b) Quantitative analysis of percentage allograft tumor formation in FVB/N mice injected with cells from drug treated MYC-driven tumors ( n = 10 per treatment condition). Data are represented as mean. N.D. denotes nondetectable tumor. (c) Quantitative analysis of allograft tumor volumes in FVB/N mice injected with cells from drug treated MYC-driven tumor ( n = 10 per treatment condition). Data are represented as mean ± SD. N.D. denotes nondetectable tumor.
Figure Legend Snippet: EPND can inhibit tumor-initiation in murine hepatic tumor allografts. (a) Representative image of FVB/N mouse seeded in hindquarters with cells from PBS, nanodiamond (ND), Epirubicin (Epi) or EPND treated MYC-driven tumors 58 days after allograft transplantation. Two mice in each panel were under same treatment conditions. Black arrows indicate treatment condition and number. (b) Quantitative analysis of percentage allograft tumor formation in FVB/N mice injected with cells from drug treated MYC-driven tumors ( n = 10 per treatment condition). Data are represented as mean. N.D. denotes nondetectable tumor. (c) Quantitative analysis of allograft tumor volumes in FVB/N mice injected with cells from drug treated MYC-driven tumor ( n = 10 per treatment condition). Data are represented as mean ± SD. N.D. denotes nondetectable tumor.

Techniques Used: Transplantation Assay, Mouse Assay, Injection

Cellular drug retention in hepatic tumor cell line of EPND and Epirubicin. (a) Representative images of cellular drug retention in LT2-MYC cells by fluorescent microscopy. LT2-MYC cells treated with Epirubicin (Epi) (35 μM), EPND (35 μM) or Liposomal–Epirubicin (Lipo-Epi) (35 μM) for 1 h before 12 h efflux. Blue and red fluorescent signals represent diamidino-2-phenylindole (DAPI) staining and Epirubicin, respectively. Scale bar (white), 50 μm. (b) Cellular retention is quantified by percent of drug retention normalized to DAPI signal ( n = 3). Data are represented as mean ± SD; *, p
Figure Legend Snippet: Cellular drug retention in hepatic tumor cell line of EPND and Epirubicin. (a) Representative images of cellular drug retention in LT2-MYC cells by fluorescent microscopy. LT2-MYC cells treated with Epirubicin (Epi) (35 μM), EPND (35 μM) or Liposomal–Epirubicin (Lipo-Epi) (35 μM) for 1 h before 12 h efflux. Blue and red fluorescent signals represent diamidino-2-phenylindole (DAPI) staining and Epirubicin, respectively. Scale bar (white), 50 μm. (b) Cellular retention is quantified by percent of drug retention normalized to DAPI signal ( n = 3). Data are represented as mean ± SD; *, p

Techniques Used: Microscopy, Staining

Characterization of nanodiamond, Epirubicin, EPND complex and release profile of Epirubicin. (a) Schematic model showing surface and chemical structure of nanodiamond (ND) and Epirubicin (Epi), synthesis and aggregation of EPND. ND represented in truncated octahedron structure with different surface charge denoted with color. ND surface functional group indicated, including benzene ring, carboxyl group and hydrogen group. Molecular skeleton representing carbon, oxygen and nitrogen atoms in Epi molecule was shown in red. Synthesis of EPND was performed under basic condition of 2.5 mM NaOH through physical adsorption between Epi and ND. Aggregation around 90 nm was formed after EPND synthesis. (b) FTIR spectra of ND (black line), Epi (red line), and EPND (blue line) indicating surface functional groups. Black arrows indicating surface functional groups of O–H stretch signals between 3300 and 3500 cm –1 and C=C stretch signals between 1400 and 1600 cm –1 presenting aromatic rings of anthracyclines. (c) Size distribution of ND (10.9 ± 3.6 nm) and EPND (89.2 ± 3.3 nm) after Epirubicin loading analyzed via dynamic light scattering (DLS) analysis. Data are represented as mean ± SD. (d) Zeta-potential of ND (48.6 ± 3.3) and EPND (19.6 ± 1.1) indicating surface charge. Data are represented as mean ± SD; ***, p
Figure Legend Snippet: Characterization of nanodiamond, Epirubicin, EPND complex and release profile of Epirubicin. (a) Schematic model showing surface and chemical structure of nanodiamond (ND) and Epirubicin (Epi), synthesis and aggregation of EPND. ND represented in truncated octahedron structure with different surface charge denoted with color. ND surface functional group indicated, including benzene ring, carboxyl group and hydrogen group. Molecular skeleton representing carbon, oxygen and nitrogen atoms in Epi molecule was shown in red. Synthesis of EPND was performed under basic condition of 2.5 mM NaOH through physical adsorption between Epi and ND. Aggregation around 90 nm was formed after EPND synthesis. (b) FTIR spectra of ND (black line), Epi (red line), and EPND (blue line) indicating surface functional groups. Black arrows indicating surface functional groups of O–H stretch signals between 3300 and 3500 cm –1 and C=C stretch signals between 1400 and 1600 cm –1 presenting aromatic rings of anthracyclines. (c) Size distribution of ND (10.9 ± 3.6 nm) and EPND (89.2 ± 3.3 nm) after Epirubicin loading analyzed via dynamic light scattering (DLS) analysis. Data are represented as mean ± SD. (d) Zeta-potential of ND (48.6 ± 3.3) and EPND (19.6 ± 1.1) indicating surface charge. Data are represented as mean ± SD; ***, p

Techniques Used: Functional Assay, Adsorption

Nanodiamond delivery of Epirubicin can effectively target side population cells in murine liver tumor models. (a) Schematic diagram showing the murine model for MYC-driven tumor induction, long-term drug treatment and experimental workflow. MYC-driven tumor was induced in 6-week old female FVB/N mice by hydrodynamic transfection. Six weeks after transfection, mice were treated with PBS ( n = 4), nanodiamond (ND) (1 mg/kg, n = 5), Epirubicin (Epi) (5 mg/kg, n = 5) or EPND (5 mg/kg, n = 5) via tail vein (i.v.) injection every 3 d. Tumors were isolated for further analysis 3 d after the final drug treatment. (b) Representative images of flow cytometry analysis of side population (SP) cells in MYC-driven tumor tissue after Epi, EPND 63 and ND treatment using Hoechst dye 33342 staining, PBS as control. SP gate was determined by absence of cell population after addition of 100 μM verapamil in PBS control. The percentages of SP cells in the total live cells gated are shown. (c) Quantitative analysis of SP cells in MYC-driven tumor tissue after Epi, EPND, ND and PBS treatment ( n = 3). Data are represented as mean ± SD; *, p
Figure Legend Snippet: Nanodiamond delivery of Epirubicin can effectively target side population cells in murine liver tumor models. (a) Schematic diagram showing the murine model for MYC-driven tumor induction, long-term drug treatment and experimental workflow. MYC-driven tumor was induced in 6-week old female FVB/N mice by hydrodynamic transfection. Six weeks after transfection, mice were treated with PBS ( n = 4), nanodiamond (ND) (1 mg/kg, n = 5), Epirubicin (Epi) (5 mg/kg, n = 5) or EPND (5 mg/kg, n = 5) via tail vein (i.v.) injection every 3 d. Tumors were isolated for further analysis 3 d after the final drug treatment. (b) Representative images of flow cytometry analysis of side population (SP) cells in MYC-driven tumor tissue after Epi, EPND 63 and ND treatment using Hoechst dye 33342 staining, PBS as control. SP gate was determined by absence of cell population after addition of 100 μM verapamil in PBS control. The percentages of SP cells in the total live cells gated are shown. (c) Quantitative analysis of SP cells in MYC-driven tumor tissue after Epi, EPND, ND and PBS treatment ( n = 3). Data are represented as mean ± SD; *, p

Techniques Used: Mouse Assay, Transfection, Injection, Isolation, Flow Cytometry, Cytometry, Staining

34) Product Images from "Epigenetically maintained SW13+ and SW13- subtypes have different oncogenic potential and convert with HDAC1 inhibition"

Article Title: Epigenetically maintained SW13+ and SW13- subtypes have different oncogenic potential and convert with HDAC1 inhibition

Journal: BMC Cancer

doi: 10.1186/s12885-016-2353-7

Inhibitors of HDAC1 increase BRM and VIM expression in a time-dependent manner. Total RNA was isolated from untreated SW13- cells (0 h), and SW13- cells treated with 0.15 μM MGCD0103, 0.51 μM MS-275, or 2 nM FK228 for 24, 48, and 72 h. Fold change in ( a ) BRM and ( b ) VIM mRNA expression from 0 h was determined by real-time PCR analysis using the 2 -ΔΔCt method normalized to GAPDH. Data are presented as mean ± SEM. Superscripts indicate statistical significance, p
Figure Legend Snippet: Inhibitors of HDAC1 increase BRM and VIM expression in a time-dependent manner. Total RNA was isolated from untreated SW13- cells (0 h), and SW13- cells treated with 0.15 μM MGCD0103, 0.51 μM MS-275, or 2 nM FK228 for 24, 48, and 72 h. Fold change in ( a ) BRM and ( b ) VIM mRNA expression from 0 h was determined by real-time PCR analysis using the 2 -ΔΔCt method normalized to GAPDH. Data are presented as mean ± SEM. Superscripts indicate statistical significance, p

Techniques Used: Expressing, Isolation, Mass Spectrometry, Real-time Polymerase Chain Reaction

Validation of select genes related to epigenetic chromatin modification and remodeling by qPCR. Relative expression of a total of bromodomain containing 2 (BRD2), HDAC7, HDAC9, HDAC10, lysine (K)-specific demethylase 5c (KDM5C), and methyl-CpG binding domain protein 2 (MBD2) was examined in stable SW13- and SW13+ subtypes and SW13- cells which had been treated for 24 h with either 0.51 μM MS-275 or 2 nM FK228. Fold-regulation was determined by the 2 -ΔΔCT method using GAPDH as the invariant control. Data are presented as mean ± SEM. *Denotes statistical difference from SW13-, p
Figure Legend Snippet: Validation of select genes related to epigenetic chromatin modification and remodeling by qPCR. Relative expression of a total of bromodomain containing 2 (BRD2), HDAC7, HDAC9, HDAC10, lysine (K)-specific demethylase 5c (KDM5C), and methyl-CpG binding domain protein 2 (MBD2) was examined in stable SW13- and SW13+ subtypes and SW13- cells which had been treated for 24 h with either 0.51 μM MS-275 or 2 nM FK228. Fold-regulation was determined by the 2 -ΔΔCT method using GAPDH as the invariant control. Data are presented as mean ± SEM. *Denotes statistical difference from SW13-, p

Techniques Used: Modification, Real-time Polymerase Chain Reaction, Expressing, Binding Assay, Mass Spectrometry

HDAC1 inhibitors reduce cell proliferation and promote restoration of VIM and BRM protein to varying degrees. a SW13- and SW13+ cells as well as SW13- cells treated with either 0.51 μM MS-275 or 2 nM FK228 for 24 h were labeled with EdU for 24 h to determine proliferation rates. b Representative images of two independent experiments performed in triplicate are shown. c Nuclear protein was isolated from SW13- and SW13+ cells, as well as from SW13- cells which had been treated with either 0.51 μM MS-275 or 2 nM FK228 for 24 h and the expression of SWI/SNF protein components were examined by western blot. Total histone H3 was used as a loading control. Data are presented as mean ± SEM. Superscripts indicate statistical significance, p
Figure Legend Snippet: HDAC1 inhibitors reduce cell proliferation and promote restoration of VIM and BRM protein to varying degrees. a SW13- and SW13+ cells as well as SW13- cells treated with either 0.51 μM MS-275 or 2 nM FK228 for 24 h were labeled with EdU for 24 h to determine proliferation rates. b Representative images of two independent experiments performed in triplicate are shown. c Nuclear protein was isolated from SW13- and SW13+ cells, as well as from SW13- cells which had been treated with either 0.51 μM MS-275 or 2 nM FK228 for 24 h and the expression of SWI/SNF protein components were examined by western blot. Total histone H3 was used as a loading control. Data are presented as mean ± SEM. Superscripts indicate statistical significance, p

Techniques Used: Mass Spectrometry, Labeling, Isolation, Expressing, Western Blot

Histone acetylation in stable SW13- and SW13+ subtypes and following treatment with the HDAC inhibitors MS-275 and FK228. Pure histones were isolated from SW13- and SW13+ cells, as well as from SW13- cells which had been treated with either 0.51 μM MS-275 or 2 nM FK228 for 24 h. Histones were separated on tris-tricine gels and transferred to a PVDF membrane for blotting and assessment of histone modifications. Total histone H3 was used as a loading control. Data are presented as mean ± SEM. Superscripts indicate statistical significance, p
Figure Legend Snippet: Histone acetylation in stable SW13- and SW13+ subtypes and following treatment with the HDAC inhibitors MS-275 and FK228. Pure histones were isolated from SW13- and SW13+ cells, as well as from SW13- cells which had been treated with either 0.51 μM MS-275 or 2 nM FK228 for 24 h. Histones were separated on tris-tricine gels and transferred to a PVDF membrane for blotting and assessment of histone modifications. Total histone H3 was used as a loading control. Data are presented as mean ± SEM. Superscripts indicate statistical significance, p

Techniques Used: Mass Spectrometry, Isolation

SW13- and SW13+ subtype characterization: differences in morphology, actin organization, vimentin expression, and VIM and BRM levels. a Subtypes have distinct morphology, actin organization, and levels of vimentin expression. Left-hand panel: light microscopy photographs; Middle panel: visualization of actin filaments with fluorescent phalloidin; Right-hand panel: expression of vimentin by immunofluorescence. Images were taken using a 40× oil-immersion objective lens. b qPCR reveals VIM and BRM mRNA expression is ~ 8 -fold higher in the SW13+ cells compared to the SW13- cells. Data are presented as mean ± SEM. *Denotes statistical difference between subtypes, p
Figure Legend Snippet: SW13- and SW13+ subtype characterization: differences in morphology, actin organization, vimentin expression, and VIM and BRM levels. a Subtypes have distinct morphology, actin organization, and levels of vimentin expression. Left-hand panel: light microscopy photographs; Middle panel: visualization of actin filaments with fluorescent phalloidin; Right-hand panel: expression of vimentin by immunofluorescence. Images were taken using a 40× oil-immersion objective lens. b qPCR reveals VIM and BRM mRNA expression is ~ 8 -fold higher in the SW13+ cells compared to the SW13- cells. Data are presented as mean ± SEM. *Denotes statistical difference between subtypes, p

Techniques Used: Expressing, Light Microscopy, Immunofluorescence, Real-time Polymerase Chain Reaction

SW13- cells are more proliferative and have higher rates of anchorage independent growth than SW13+. SW13- and SW13+ cells were seeded in 6-well plates at 1 × 10 4 cells per well and counted using a hemocytometer and trypan exclusion every 24 h for 7 days. a The growth rate of SW13- cells is significantly higher than SW13+ cells. b SW13- cells have higher rates of proliferation as determined by the higher number of EdU positive cells ( green ) per total number of cells indicated by Hoechst 33342 staining ( blue ). Images were taken using a 10× objective lens and quantitated ( f ) using ImageJ software. c Soft agar colony formation assays revealed SW13- cells exhibit increased rates of anchorage independent growth as indicated by ( d ) increased colony number and ( e ) increased colony size. Data are presented as mean ± SEM. *Denotes statistical difference between subtypes, p
Figure Legend Snippet: SW13- cells are more proliferative and have higher rates of anchorage independent growth than SW13+. SW13- and SW13+ cells were seeded in 6-well plates at 1 × 10 4 cells per well and counted using a hemocytometer and trypan exclusion every 24 h for 7 days. a The growth rate of SW13- cells is significantly higher than SW13+ cells. b SW13- cells have higher rates of proliferation as determined by the higher number of EdU positive cells ( green ) per total number of cells indicated by Hoechst 33342 staining ( blue ). Images were taken using a 10× objective lens and quantitated ( f ) using ImageJ software. c Soft agar colony formation assays revealed SW13- cells exhibit increased rates of anchorage independent growth as indicated by ( d ) increased colony number and ( e ) increased colony size. Data are presented as mean ± SEM. *Denotes statistical difference between subtypes, p

Techniques Used: Staining, Software

The ability of selected HDAC inhibitors to induce the switching from the SW13- to the SW13+ subtype. a Induction of vimentin expression was assayed at 24, 48, and 72 h using fluorescence intensity of vimentin normalized to DAPI staining at each time point ( left columns ). Dose response was measured by fold induction in BRM mRNA using qPCR for two doses of each inhibitor ( right columns ). b Representative immunofluorescence images which illustrate the range of vimentin induction in panel ( a ). Scale ranges from no detectable vimentin immunofluorescence signal (−) to + representing
Figure Legend Snippet: The ability of selected HDAC inhibitors to induce the switching from the SW13- to the SW13+ subtype. a Induction of vimentin expression was assayed at 24, 48, and 72 h using fluorescence intensity of vimentin normalized to DAPI staining at each time point ( left columns ). Dose response was measured by fold induction in BRM mRNA using qPCR for two doses of each inhibitor ( right columns ). b Representative immunofluorescence images which illustrate the range of vimentin induction in panel ( a ). Scale ranges from no detectable vimentin immunofluorescence signal (−) to + representing

Techniques Used: Expressing, Fluorescence, Staining, Real-time Polymerase Chain Reaction, Immunofluorescence

SW13+ cells display properties associated with increased rates of metastasis to a greater extent than do SW13- cells. a and c Transwell migration assays indicate SW13+ cells have increased rates of chemotaxis compared to the SW13- cells at 24 and 48 h. Images were taken using a 4× objective lens ( b and d ) SW13+ cells exhibited higher levels of MMP2/9 (collagenase) activity as measured by increased fluorescence after incubation with a fluorescein-quenched gelatin substrate (DQ gelatin). Addition of a broad-spectrum MMP inhibitor reduced the fluorescence signal in both subtypes. Images were taken using a 40× objective lens. Data are presented as mean ± SEM. *Denotes statistical difference between subtypes, p
Figure Legend Snippet: SW13+ cells display properties associated with increased rates of metastasis to a greater extent than do SW13- cells. a and c Transwell migration assays indicate SW13+ cells have increased rates of chemotaxis compared to the SW13- cells at 24 and 48 h. Images were taken using a 4× objective lens ( b and d ) SW13+ cells exhibited higher levels of MMP2/9 (collagenase) activity as measured by increased fluorescence after incubation with a fluorescein-quenched gelatin substrate (DQ gelatin). Addition of a broad-spectrum MMP inhibitor reduced the fluorescence signal in both subtypes. Images were taken using a 40× objective lens. Data are presented as mean ± SEM. *Denotes statistical difference between subtypes, p

Techniques Used: Migration, Chemotaxis Assay, Activity Assay, Fluorescence, Incubation

35) Product Images from "Creation of an engineered APC system to explore and optimize the presentation of immunodominant peptides of major allergens"

Article Title: Creation of an engineered APC system to explore and optimize the presentation of immunodominant peptides of major allergens

Journal: Scientific Reports

doi: 10.1038/srep31580

Generation and characterization of allergen-specific T cell stimulator cells and allergen-specific T cell reporter cells. ( a ) Scheme illustrating the generation of K562 cell-based engineered APC (left) and expression analysis of surface molecules on these cells by flow cytometry (right). Open histograms: K562 cells; filled histograms: respective engineered APC. ( b ) Scheme illustrating the generation of Jurkat-based allergen-specific T cell reporter cells (left) and expression analysis of the allergen-specific TCRs using antibodies specific for the Vß-chains of the respective transgenic TCR (right). Open histograms: Staining of allergen-specific reporter cells with isotype control antibody; filled histograms: staining with antibodies specific for the transgenic TCR-Vß chain. ( c ) Activation of Je6 NF-κB reporters with allergen-specific stimulator cells harboring the appropriate restriction element in absence (open histograms) or presence (grey histograms) of allergenic peptides Art v 1 23–36 or Bet v 1 142–153 (left). T cell reporter specific for Art v 1 and Bet v 1 were cocultured with eAPC expressing the indicated molecules in presence or absence of allergenic peptides. Mean gMFI ± SD of triplicates is shown and experiment is representative for three independently performed experiments. ( d ) Reporter gene-expression and formation of immunological synapses visualized by cell imaging using chamber slides. K562 stimulator cells expressing HLA-DRB1 (transduced with mCherry for microscopic visualization) are cocultivated with Je6 NF-κB-eGFP-reporter cell line without and with allergenic peptide addition (upper and lower panel, respectively).
Figure Legend Snippet: Generation and characterization of allergen-specific T cell stimulator cells and allergen-specific T cell reporter cells. ( a ) Scheme illustrating the generation of K562 cell-based engineered APC (left) and expression analysis of surface molecules on these cells by flow cytometry (right). Open histograms: K562 cells; filled histograms: respective engineered APC. ( b ) Scheme illustrating the generation of Jurkat-based allergen-specific T cell reporter cells (left) and expression analysis of the allergen-specific TCRs using antibodies specific for the Vß-chains of the respective transgenic TCR (right). Open histograms: Staining of allergen-specific reporter cells with isotype control antibody; filled histograms: staining with antibodies specific for the transgenic TCR-Vß chain. ( c ) Activation of Je6 NF-κB reporters with allergen-specific stimulator cells harboring the appropriate restriction element in absence (open histograms) or presence (grey histograms) of allergenic peptides Art v 1 23–36 or Bet v 1 142–153 (left). T cell reporter specific for Art v 1 and Bet v 1 were cocultured with eAPC expressing the indicated molecules in presence or absence of allergenic peptides. Mean gMFI ± SD of triplicates is shown and experiment is representative for three independently performed experiments. ( d ) Reporter gene-expression and formation of immunological synapses visualized by cell imaging using chamber slides. K562 stimulator cells expressing HLA-DRB1 (transduced with mCherry for microscopic visualization) are cocultivated with Je6 NF-κB-eGFP-reporter cell line without and with allergenic peptide addition (upper and lower panel, respectively).

Techniques Used: Expressing, Flow Cytometry, Cytometry, Transgenic Assay, Staining, Activation Assay, Imaging, Transduction

36) Product Images from "The Role of Parathyroid Hormone-Related Protein (PTHrP) in Osteoblast Response to Microgravity: Mechanistic Implications for Osteoporosis Development"

Article Title: The Role of Parathyroid Hormone-Related Protein (PTHrP) in Osteoblast Response to Microgravity: Mechanistic Implications for Osteoporosis Development

Journal: PLoS ONE

doi: 10.1371/journal.pone.0160034

Allelic effect of endogenous PTHrP levels in simulated microgravity, and compensation by exogenous PTHrP 1–36 (6-day experiments). (A) Microgravity simulation apparatus (RCCS) with one rotating culture unit (HARV). (B) MicroHex carriers with attached trabecular osteoblasts in culture. (C) Cell-induced MicroHex aggregate in culture (occurs for both trabecular and calvarial osteoblasts). (D) Trabecular osteoblast viability after 6 days on MicroHex carriers in normal gravity (1 g ) or in simulated microgravity (0 g ) (triplicate 6-day experiments). White bars: untreated; black bars: 2-h daily treatment with PTHrP 1-36 10 -8 M. ** p
Figure Legend Snippet: Allelic effect of endogenous PTHrP levels in simulated microgravity, and compensation by exogenous PTHrP 1–36 (6-day experiments). (A) Microgravity simulation apparatus (RCCS) with one rotating culture unit (HARV). (B) MicroHex carriers with attached trabecular osteoblasts in culture. (C) Cell-induced MicroHex aggregate in culture (occurs for both trabecular and calvarial osteoblasts). (D) Trabecular osteoblast viability after 6 days on MicroHex carriers in normal gravity (1 g ) or in simulated microgravity (0 g ) (triplicate 6-day experiments). White bars: untreated; black bars: 2-h daily treatment with PTHrP 1-36 10 -8 M. ** p

Techniques Used:

37) Product Images from "An Antibody to De-N-Acetyl Sialic Acid Containing-Polysialic Acid Identifies an Intracellular Antigen and Induces Apoptosis in Human Cancer Cell Lines"

Article Title: An Antibody to De-N-Acetyl Sialic Acid Containing-Polysialic Acid Identifies an Intracellular Antigen and Induces Apoptosis in Human Cancer Cell Lines

Journal: PLoS ONE

doi: 10.1371/journal.pone.0027249

SEAM 3 mediates antibody dependent cytotoxicity by inducting apoptosis. (A), Antibody dependent cytotoxicity of SEAM 3 against SK-MEL-28, CHP-134, SH-SY5Y, and Jurkat cells as measured by LDH release assay. Each cell line was incubated with increasing concentrations of SEAM 3 for 16 hrs. LDH release was measured and percent cytotoxicity was determined using spontaneous release and maximal release following treatment with Triton X-100. (B), Analysis of SEAM 3 mediated apoptosis against SK-MEL-28 melanoma cells by flow cytometry. SK-MEL-28 cells were incubated with an irrelevant IgG2b mAb (5 µg/ml), DMSO, 0.1 µM Staurosporine, or 5 µg/ml SEAM 3 for 12 or 24 hours. Cells were then stained with fluorescently labeled annexin V and propidium iodide and the fraction of live (open bars), apoptotic (cross-hatched bars), and dead cells (black bars) was measured by flow cytometry.
Figure Legend Snippet: SEAM 3 mediates antibody dependent cytotoxicity by inducting apoptosis. (A), Antibody dependent cytotoxicity of SEAM 3 against SK-MEL-28, CHP-134, SH-SY5Y, and Jurkat cells as measured by LDH release assay. Each cell line was incubated with increasing concentrations of SEAM 3 for 16 hrs. LDH release was measured and percent cytotoxicity was determined using spontaneous release and maximal release following treatment with Triton X-100. (B), Analysis of SEAM 3 mediated apoptosis against SK-MEL-28 melanoma cells by flow cytometry. SK-MEL-28 cells were incubated with an irrelevant IgG2b mAb (5 µg/ml), DMSO, 0.1 µM Staurosporine, or 5 µg/ml SEAM 3 for 12 or 24 hours. Cells were then stained with fluorescently labeled annexin V and propidium iodide and the fraction of live (open bars), apoptotic (cross-hatched bars), and dead cells (black bars) was measured by flow cytometry.

Techniques Used: Lactate Dehydrogenase Assay, Incubation, Flow Cytometry, Cytometry, Staining, Labeling

Intracellular localization of de-N-acetyl PSA in melanoma and neuroblastoma cell lines. SK-MEL-28 and SH-SY5Y cells were either untreated to detect surface binding (upper panel in each set of two panels), or treated with Triton-X 100 to detect intracellular binding (lower panel) by flow cytometry. Cells were incubated with 5 µg/ml of each primary antibody, followed by incubation with 2 µg/ml Alexa Fluor 488-conjugated secondary antibody. Irrelevant murine IgG2b, IgG3, and IgG1 mAbs were used as negative controls for SEAM 3, anti-GD3, and anti-NCAM, respectively, and were used to determine baseline fluorescence. Binding was detected using Guava EasyCyte flow cytometer. Gates used to define cells positive for binding are indicated at the top of each histogram.
Figure Legend Snippet: Intracellular localization of de-N-acetyl PSA in melanoma and neuroblastoma cell lines. SK-MEL-28 and SH-SY5Y cells were either untreated to detect surface binding (upper panel in each set of two panels), or treated with Triton-X 100 to detect intracellular binding (lower panel) by flow cytometry. Cells were incubated with 5 µg/ml of each primary antibody, followed by incubation with 2 µg/ml Alexa Fluor 488-conjugated secondary antibody. Irrelevant murine IgG2b, IgG3, and IgG1 mAbs were used as negative controls for SEAM 3, anti-GD3, and anti-NCAM, respectively, and were used to determine baseline fluorescence. Binding was detected using Guava EasyCyte flow cytometer. Gates used to define cells positive for binding are indicated at the top of each histogram.

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

Immuno-fluorescence analysis of SEAM 3 binding to human SK-MEL-28 melanoma and CHP-134 neuroblastoma cells. Light micrographs of SK-MEL-28 and CHP-134 show the general shape of cells with nuclear DNA indicated in blue. Anti-NeuPSA mAb SEAM 3 binding (in red) to SK-MEL-28 and CHP-134 cells not treated or treated with Triton X-100 to permeablize the cells, was compared to anti-GD3 and –PSA in green, as indicated. Subcellular localization of S3RA in SK-MEL-28 cells with Golgi (giantin, golgin 97 and Tuba) and ER (calnexin) markers are shown in the bottom panel in yellow. Arrows indicate granular vesicular-like structures with relatively intense SEAM 3 staining. Reference bars = 20 µm.
Figure Legend Snippet: Immuno-fluorescence analysis of SEAM 3 binding to human SK-MEL-28 melanoma and CHP-134 neuroblastoma cells. Light micrographs of SK-MEL-28 and CHP-134 show the general shape of cells with nuclear DNA indicated in blue. Anti-NeuPSA mAb SEAM 3 binding (in red) to SK-MEL-28 and CHP-134 cells not treated or treated with Triton X-100 to permeablize the cells, was compared to anti-GD3 and –PSA in green, as indicated. Subcellular localization of S3RA in SK-MEL-28 cells with Golgi (giantin, golgin 97 and Tuba) and ER (calnexin) markers are shown in the bottom panel in yellow. Arrows indicate granular vesicular-like structures with relatively intense SEAM 3 staining. Reference bars = 20 µm.

Techniques Used: Fluorescence, Binding Assay, Staining

Relative quantification of PST mRNA in SK-MEL-28 cells treated with scrambled siRNA or PST-specific siRNA. SK-MEL-28 cells were transiently transfected with 50 nM siRNA for 72 hours in triplicate, then total RNA from each cell line was reverse-transcribed into cDNA and used as the template in the qRT-PCR reaction. Relative quantification was determined using the comparative CT method, normalized to GAPDH mRNA.
Figure Legend Snippet: Relative quantification of PST mRNA in SK-MEL-28 cells treated with scrambled siRNA or PST-specific siRNA. SK-MEL-28 cells were transiently transfected with 50 nM siRNA for 72 hours in triplicate, then total RNA from each cell line was reverse-transcribed into cDNA and used as the template in the qRT-PCR reaction. Relative quantification was determined using the comparative CT method, normalized to GAPDH mRNA.

Techniques Used: Transfection, Quantitative RT-PCR

38) Product Images from "Investigation of Griffithsin's Interactions with Human Cells Confirms Its Outstanding Safety and Efficacy Profile as a Microbicide Candidate"

Article Title: Investigation of Griffithsin's Interactions with Human Cells Confirms Its Outstanding Safety and Efficacy Profile as a Microbicide Candidate

Journal: PLoS ONE

doi: 10.1371/journal.pone.0022635

Effect of GRFT on cervico-vaginal cell proliferation as measured by BrdU incorporation. Cell proliferation was measured by the incorporation of BrdU in newly synthesized DNA using a colorimetric assay after treatment of Ect1/E6E7, End1/E6E7 and VK2/E6E7 with GRFT (1 or 8 µM), PKM (3.12 or 0.31 µM) or PBS. Cells without BrdU were assessed in the assay as a negative control according to the manufacturer's intructions. Values are given as mean ± standard deviation of the relative cell proliferation rate derived from BrdU incorporated as a percentage of that found in PBS.
Figure Legend Snippet: Effect of GRFT on cervico-vaginal cell proliferation as measured by BrdU incorporation. Cell proliferation was measured by the incorporation of BrdU in newly synthesized DNA using a colorimetric assay after treatment of Ect1/E6E7, End1/E6E7 and VK2/E6E7 with GRFT (1 or 8 µM), PKM (3.12 or 0.31 µM) or PBS. Cells without BrdU were assessed in the assay as a negative control according to the manufacturer's intructions. Values are given as mean ± standard deviation of the relative cell proliferation rate derived from BrdU incorporated as a percentage of that found in PBS.

Techniques Used: BrdU Incorporation Assay, Synthesized, Colorimetric Assay, Negative Control, Standard Deviation, Derivative Assay

Effect of GRFT on cervico-vaginal cell viability. Cell viability was evaluated in an MTT assay after 72 hours treatment of Ect1/E6E7 (white), End1/E6E7 (grey) and VK2/E6E7 (black) with different test compounds listed on the X-axis together with their concentrations. Values are given as mean ± standard deviation of OD570 – OD630.
Figure Legend Snippet: Effect of GRFT on cervico-vaginal cell viability. Cell viability was evaluated in an MTT assay after 72 hours treatment of Ect1/E6E7 (white), End1/E6E7 (grey) and VK2/E6E7 (black) with different test compounds listed on the X-axis together with their concentrations. Values are given as mean ± standard deviation of OD570 – OD630.

Techniques Used: MTT Assay, Standard Deviation

39) Product Images from "In Vivo Delivery of Gremlin siRNA Plasmid Reveals Therapeutic Potential against Diabetic Nephropathy by Recovering Bone Morphogenetic Protein-7"

Article Title: In Vivo Delivery of Gremlin siRNA Plasmid Reveals Therapeutic Potential against Diabetic Nephropathy by Recovering Bone Morphogenetic Protein-7

Journal: PLoS ONE

doi: 10.1371/journal.pone.0011709

Cell proliferation and apoptosis in diabetic mouse kidneys. ( A ) Detection of proliferating cell nuclear antigen (PCNA) by immunoperoxidase staining, in the kidneys of non-diabetic control mice (N), streptozotocin-induced diabetic mice treated with pBAsi mU6 Neo control plasmid (STZ) or pBAsi mU6 Neo gremlin siRNA plasmid (Gremlin siRNA). ( B and C ) PCNA positive cells in kidneys from the STZ group dramatically increase at week-1 and -2, and pBAsi mU6 Neo gremlin siRNA plasmid treatment significantly reduces PCNA positive cells both in glomeruli and tubules. Proliferating cells are barely seen in all three groups at week 12. ( D ) Co-immunostaining of diabetic kidney sections with antibodies against PCNA and Gremlin. Intensive Gremlin expression is often seen in the cells with PCNA positive signal. ( E, F ) In situ TUNEL assay. Apoptotic cells are observed predominantly in tubules in the STZ group at week-12. The number of apoptotic cells is significantly reduced by pBAsi mU6 Neo gremlin siRNA plasmid treatment. (* p
Figure Legend Snippet: Cell proliferation and apoptosis in diabetic mouse kidneys. ( A ) Detection of proliferating cell nuclear antigen (PCNA) by immunoperoxidase staining, in the kidneys of non-diabetic control mice (N), streptozotocin-induced diabetic mice treated with pBAsi mU6 Neo control plasmid (STZ) or pBAsi mU6 Neo gremlin siRNA plasmid (Gremlin siRNA). ( B and C ) PCNA positive cells in kidneys from the STZ group dramatically increase at week-1 and -2, and pBAsi mU6 Neo gremlin siRNA plasmid treatment significantly reduces PCNA positive cells both in glomeruli and tubules. Proliferating cells are barely seen in all three groups at week 12. ( D ) Co-immunostaining of diabetic kidney sections with antibodies against PCNA and Gremlin. Intensive Gremlin expression is often seen in the cells with PCNA positive signal. ( E, F ) In situ TUNEL assay. Apoptotic cells are observed predominantly in tubules in the STZ group at week-12. The number of apoptotic cells is significantly reduced by pBAsi mU6 Neo gremlin siRNA plasmid treatment. (* p

Techniques Used: Immunoperoxidase Staining, Mouse Assay, Plasmid Preparation, Immunostaining, Expressing, In Situ, TUNEL Assay

Cell proliferation in human mesangial cells cultured under high glucose conditions. Human mesangial cells were cultured in RPMI 1640 containing normal glucose (100 mg/dl D-glucose; NG) and high glucose (300 mg/dl D-glucose; HG). Cells under HG conditions were transfected with pBAsi mU6 Neo control plasmid (HG+V) or pBAsi mU6 Neo gremlin siRNA plasmid (HG+gremlin si) 12 hours before the glucose stimulation. Cell proliferation was examined by PCNA staining 12 hours after glucose stimulation. Gremlin expression is examined in human mesangial cells by Western blot ( A ); the secreted Gremlin in culture medium is observed by ELISA ( B ). The HG stimulated Gremlin expression in human mesangial cells is successfully inhibited by the transfection of pBAsi mU6 Neo gremlin siRNA plasmid. ( C ) High glucose-induced cell proliferation is inhibited in the HG+gremlin si group. (* p
Figure Legend Snippet: Cell proliferation in human mesangial cells cultured under high glucose conditions. Human mesangial cells were cultured in RPMI 1640 containing normal glucose (100 mg/dl D-glucose; NG) and high glucose (300 mg/dl D-glucose; HG). Cells under HG conditions were transfected with pBAsi mU6 Neo control plasmid (HG+V) or pBAsi mU6 Neo gremlin siRNA plasmid (HG+gremlin si) 12 hours before the glucose stimulation. Cell proliferation was examined by PCNA staining 12 hours after glucose stimulation. Gremlin expression is examined in human mesangial cells by Western blot ( A ); the secreted Gremlin in culture medium is observed by ELISA ( B ). The HG stimulated Gremlin expression in human mesangial cells is successfully inhibited by the transfection of pBAsi mU6 Neo gremlin siRNA plasmid. ( C ) High glucose-induced cell proliferation is inhibited in the HG+gremlin si group. (* p

Techniques Used: Cell Culture, Transfection, Plasmid Preparation, Staining, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

The effects of gremlin siRNA plasmid delivery on diabetic nephropathy in diabetic mice post-uninephrectomy. ( A ) Increased spot urinary protein levels and ( B ) serum creatinine in STZ-induced diabetic mice treated with pBAsi mU6 Neo control plasmid (STZ) compared with nondiabetic control animals (N). The effect is decreased by treatment of diabetic animals with pBAsi mU6 Neo gremlin siRNA plasmid (Gremlin-si). ( C, D ) Increase in glomerular and tubular diameters at week 2 and week 12 is ameliorated by treatment with gremlin siRNA plasmid. ( E, F ) Increases in cell numbers in both glomeruli and tubules at week 2 and week 12 are significantly reduced in the Gremlin siRNA group. ( G ) PAS staining of kidney tissues shows glomerular and tubular hypertrophy and mesangial matrix accumulation in the STZ group 12 weeks after STZ injection. Treatment with gremlin siRNA plasmid prevents these pathological changes. ( H ) Collagen type IV expression in the kidneys at week 12. High expression of collagen IV is seen in diabetic kidney and the treatment of gremlin siRNA plasmid significantly down-regulated the accumulation of collagen IV. (* p
Figure Legend Snippet: The effects of gremlin siRNA plasmid delivery on diabetic nephropathy in diabetic mice post-uninephrectomy. ( A ) Increased spot urinary protein levels and ( B ) serum creatinine in STZ-induced diabetic mice treated with pBAsi mU6 Neo control plasmid (STZ) compared with nondiabetic control animals (N). The effect is decreased by treatment of diabetic animals with pBAsi mU6 Neo gremlin siRNA plasmid (Gremlin-si). ( C, D ) Increase in glomerular and tubular diameters at week 2 and week 12 is ameliorated by treatment with gremlin siRNA plasmid. ( E, F ) Increases in cell numbers in both glomeruli and tubules at week 2 and week 12 are significantly reduced in the Gremlin siRNA group. ( G ) PAS staining of kidney tissues shows glomerular and tubular hypertrophy and mesangial matrix accumulation in the STZ group 12 weeks after STZ injection. Treatment with gremlin siRNA plasmid prevents these pathological changes. ( H ) Collagen type IV expression in the kidneys at week 12. High expression of collagen IV is seen in diabetic kidney and the treatment of gremlin siRNA plasmid significantly down-regulated the accumulation of collagen IV. (* p

Techniques Used: Plasmid Preparation, Mouse Assay, Staining, Injection, Expressing

Collagen type IVand TGF-βexpression and MMP-2 activity in mouse mesangial cells cultured under high glucose conditions. Mouse mesangial cells were cultured in RPMI 1640 and transfected with pBAsi mU6 Neo or pBAsi mU6 Neo gremlin siRNA plasmid as described in the methods . Culture medium was collected for measurement of collagen type IV concentration by radio-immunoassay, and cells were subjected to Western blot analysis to determine MMP-2 and TGF-βexpression levels 48 hours after glucose stimulation. ( A ) Increased collagen type IV accumulation is observed in the HG group, and gremlin siRNA plasmid transfection significantly inhibits collagen type IV secretion. ( B ) Compared to the normal glucose control group (NG), TGF-β expression is significantly increased under high glucose conditions, and the HG stimulated TGF-β expression remains the same after gremlin siRNA transfection. ( C ) Compared with the NG group, MMP-2 activity in culture medium is significantly decreased in the HG and HG+V groups, and this is prevented by transfection with gremlin siRNA plasmid. (* p
Figure Legend Snippet: Collagen type IVand TGF-βexpression and MMP-2 activity in mouse mesangial cells cultured under high glucose conditions. Mouse mesangial cells were cultured in RPMI 1640 and transfected with pBAsi mU6 Neo or pBAsi mU6 Neo gremlin siRNA plasmid as described in the methods . Culture medium was collected for measurement of collagen type IV concentration by radio-immunoassay, and cells were subjected to Western blot analysis to determine MMP-2 and TGF-βexpression levels 48 hours after glucose stimulation. ( A ) Increased collagen type IV accumulation is observed in the HG group, and gremlin siRNA plasmid transfection significantly inhibits collagen type IV secretion. ( B ) Compared to the normal glucose control group (NG), TGF-β expression is significantly increased under high glucose conditions, and the HG stimulated TGF-β expression remains the same after gremlin siRNA transfection. ( C ) Compared with the NG group, MMP-2 activity in culture medium is significantly decreased in the HG and HG+V groups, and this is prevented by transfection with gremlin siRNA plasmid. (* p

Techniques Used: Activity Assay, Cell Culture, Transfection, Plasmid Preparation, Concentration Assay, Western Blot, Expressing

BMP-7 activity in mouse mesangial cells transfected with gremlin siRNA plasmid. Mouse mesangial cells were transfected with pBAsi mU6 Neo or pBAsi mU6 Neo gremlin siRNA plasmid and stimulated with NG and HG. Cells were collected 48 hours after HG stimulation and subjected to RT-PCR and Western blot. BMP-7 mRNA level was found decreased after gremlin siRNA transfection ( A B ). The protein levels of BMP-7 and Phos-Smad-5/Smad-5 decreased after 48 hours incubation with high glucose. Transfection with gremlin siRNA plasmid significantly increased the Phos-Smad-5/Smad-5 level (* p
Figure Legend Snippet: BMP-7 activity in mouse mesangial cells transfected with gremlin siRNA plasmid. Mouse mesangial cells were transfected with pBAsi mU6 Neo or pBAsi mU6 Neo gremlin siRNA plasmid and stimulated with NG and HG. Cells were collected 48 hours after HG stimulation and subjected to RT-PCR and Western blot. BMP-7 mRNA level was found decreased after gremlin siRNA transfection ( A B ). The protein levels of BMP-7 and Phos-Smad-5/Smad-5 decreased after 48 hours incubation with high glucose. Transfection with gremlin siRNA plasmid significantly increased the Phos-Smad-5/Smad-5 level (* p

Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Incubation

Delivery of gremlin siRNA plasmid into diabetic CD-1 mice post-uninephrectomy. ( A ) Gremlin protein expression by western blotting in whole-kidney homogenates at different time points after injection of pBAsi mU6 Neo control vector or pBAsi mU6 Neo gremlin siRNA plasmid, respectively. Compared to those treated with pBAsi mU6 Neo plasmid (STZ group), animals administered pBAsi mU6 Neo gremlin siRNA plasmid (Gremlin siRNA group) show low expression of Gremlin in the kidneys. ( B ) Immunostaining of kidney sections shows the localization of Gremlin protein after the delivery of plasmids. Marked Gremlin expression is observed in both glomeruli and tubules in the STZ group, which is significantly inhibited by the delivery of gremlin siRNA plasmid. (* p
Figure Legend Snippet: Delivery of gremlin siRNA plasmid into diabetic CD-1 mice post-uninephrectomy. ( A ) Gremlin protein expression by western blotting in whole-kidney homogenates at different time points after injection of pBAsi mU6 Neo control vector or pBAsi mU6 Neo gremlin siRNA plasmid, respectively. Compared to those treated with pBAsi mU6 Neo plasmid (STZ group), animals administered pBAsi mU6 Neo gremlin siRNA plasmid (Gremlin siRNA group) show low expression of Gremlin in the kidneys. ( B ) Immunostaining of kidney sections shows the localization of Gremlin protein after the delivery of plasmids. Marked Gremlin expression is observed in both glomeruli and tubules in the STZ group, which is significantly inhibited by the delivery of gremlin siRNA plasmid. (* p

Techniques Used: Plasmid Preparation, Mouse Assay, Expressing, Western Blot, Injection, Immunostaining

40) Product Images from "Measurement of in-plane elasticity of live cell layers using a pressure sensor embedded microfluidic device"

Article Title: Measurement of in-plane elasticity of live cell layers using a pressure sensor embedded microfluidic device

Journal: Scientific Reports

doi: 10.1038/srep36425

( a ) Bright field phase images of MRC-5 cells cultured in the microfluidic device for 24 hours. ( b ) Comparison between MRC-5 cells cultured in a T25 flask and in a microfluidic device. ( c ) Cell morphologies before and after the in-plane elasticity measurements. ( d ) Fluorescence image (live/dead stain) of MRC-5 cells cultured in the device after the in-plane elasticity measurement. Live cells show green fluorescence, and dead cells show red fluorescence.
Figure Legend Snippet: ( a ) Bright field phase images of MRC-5 cells cultured in the microfluidic device for 24 hours. ( b ) Comparison between MRC-5 cells cultured in a T25 flask and in a microfluidic device. ( c ) Cell morphologies before and after the in-plane elasticity measurements. ( d ) Fluorescence image (live/dead stain) of MRC-5 cells cultured in the device after the in-plane elasticity measurement. Live cells show green fluorescence, and dead cells show red fluorescence.

Techniques Used: Cell Culture, Fluorescence, Staining

( a ) Fluorescence images of untreated and TGF-β treated MRC-5 cells (Green: α-SMA; Blue: nuclei). ( b ) Average sensitivity ratios (sensitivity with cell /sensitivity without cell ) of the embedded pressure sensors when culturing untreated and TGF-β treated MRC-5 cells in the microfluidic devices. Data are expressed as mean ± sd (n = 3). ( c ) Topography and elasticity of untreated and TGF-β treated MRC-5 cells measured using AFM.
Figure Legend Snippet: ( a ) Fluorescence images of untreated and TGF-β treated MRC-5 cells (Green: α-SMA; Blue: nuclei). ( b ) Average sensitivity ratios (sensitivity with cell /sensitivity without cell ) of the embedded pressure sensors when culturing untreated and TGF-β treated MRC-5 cells in the microfluidic devices. Data are expressed as mean ± sd (n = 3). ( c ) Topography and elasticity of untreated and TGF-β treated MRC-5 cells measured using AFM.

Techniques Used: Fluorescence

Related Articles

Transfection:

Article Title: A novel form of JARID2 is required for differentiation in lineage‐committed cells
Article Snippet: .. 2 × 105 HaCaT cells were transiently transfected with 80 pmol siRNAs using Lipofectamine RNAiMAX (Invitrogen) for 48 h. Non‐silencing control (NSC) was used as control. .. HEK‐293T were transiently transfected as 3:1 ratio of reagent to DNA using X‐tremeGENE9 DNA transfection reagent (Roche).

In Vitro:

Article Title: SWI/SNF interacts with cleavage and polyadenylation factors and facilitates pre-mRNA 3′ end processing
Article Snippet: .. Double-stranded RNAs (dsRNAs) complementary to dBRM or GFP were synthesized by in vitro transcription using the MegaScript RNAi kit (Ambion) from gene-specific PCR fragments with incorporated T7 promoters at both ends. .. The sequences of the PCR primers used for dsRNA synthesis are provided as additional text in the .

Synthesized:

Article Title: SWI/SNF interacts with cleavage and polyadenylation factors and facilitates pre-mRNA 3′ end processing
Article Snippet: .. Double-stranded RNAs (dsRNAs) complementary to dBRM or GFP were synthesized by in vitro transcription using the MegaScript RNAi kit (Ambion) from gene-specific PCR fragments with incorporated T7 promoters at both ends. .. The sequences of the PCR primers used for dsRNA synthesis are provided as additional text in the .

Construct:

Article Title: Mucosal antibody responses to vaccines targeting SIV protease cleavage sites or full-length Gag and Env proteins in Mauritian cynomolgus macaques
Article Snippet: .. To test SIV protein expression, the Gag and Env DNA vaccine constructs were each used to transfect HEK293T cells using lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). .. HEK293 T cells were cultured to 90% confluence (in the same media as used for VeroE6 described above) in 6-well plates.

Polymerase Chain Reaction:

Article Title: SWI/SNF interacts with cleavage and polyadenylation factors and facilitates pre-mRNA 3′ end processing
Article Snippet: .. Double-stranded RNAs (dsRNAs) complementary to dBRM or GFP were synthesized by in vitro transcription using the MegaScript RNAi kit (Ambion) from gene-specific PCR fragments with incorporated T7 promoters at both ends. .. The sequences of the PCR primers used for dsRNA synthesis are provided as additional text in the .

Cell Culture:

Article Title: Thyroid transcription factor 1 enhances cellular statin sensitivity via perturbing cholesterol metabolism
Article Snippet: .. BEAS-2B cells were cultured in Keratinocyte-SFM (1X) with supplement kit containing recombinant EGF and bovine pituitary extract (Life Technologies). ..

Article Title: Serum microRNA miR-206 is decreased in hyperthyroidism and mediates thyroid hormone regulation of lipid metabolism in HepG2 human hepatoblastoma cells
Article Snippet: .. HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, and 100 µg/ml streptomycin. .. For treatment with T3 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), cells were seeded in 12-well culture plates at a density of 3×105 cells/well, and maintained for 2 days incubated at 37°C in a humidified chamber containing 5% CO2 .

Expressing:

Article Title: Mucosal antibody responses to vaccines targeting SIV protease cleavage sites or full-length Gag and Env proteins in Mauritian cynomolgus macaques
Article Snippet: .. To test SIV protein expression, the Gag and Env DNA vaccine constructs were each used to transfect HEK293T cells using lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). .. HEK293 T cells were cultured to 90% confluence (in the same media as used for VeroE6 described above) in 6-well plates.

Modification:

Article Title: Serum microRNA miR-206 is decreased in hyperthyroidism and mediates thyroid hormone regulation of lipid metabolism in HepG2 human hepatoblastoma cells
Article Snippet: .. HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, and 100 µg/ml streptomycin. .. For treatment with T3 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), cells were seeded in 12-well culture plates at a density of 3×105 cells/well, and maintained for 2 days incubated at 37°C in a humidified chamber containing 5% CO2 .

Lysis:

Article Title: Serum and Ectopic Endometrium from Women with Endometriosis Modulate Macrophage M1/M2 Polarization via the Smad2/Smad3 Pathway
Article Snippet: .. The THP-1 cells were harvested and lysed in 50 μ l lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA). .. The mixture was centrifuged at 14000 rpm for 10 min. Total protein in the supernatant was quantified with a bicinchoninic acid (BCA) kit (Thermo Scientific Pierce, Rockford, IL, USA).

Recombinant:

Article Title: Thyroid transcription factor 1 enhances cellular statin sensitivity via perturbing cholesterol metabolism
Article Snippet: .. BEAS-2B cells were cultured in Keratinocyte-SFM (1X) with supplement kit containing recombinant EGF and bovine pituitary extract (Life Technologies). ..

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  • 99
    Thermo Fisher 4 well plates
    Overexpression of STI1 protects both  C. elegans  and primary hippocampal mouse neurons from Aβ-toxicity.  a  The average percent of dead cells ((# of dead cells)/(# of live + # of dead cells))× 100) in E17.5 primary hippocampal neuronal cultures from WT or TgA embryos. Cells were imaged in 4 well dishes from 8 random fields ( N  = 6 individual embryos/genotype). Dishes were treated with either no Aβ, 0.1, 0.5 or 1 μM AβOs for 48 h.  b  Likewise, WT or TgA primary hippocampal neurons were treated with 0, 0.25, 0.5 or 1 μM for 48 h, and LDH release was measured using colorimetric assay, read at 450 nm.  N  = 3–4 individual embryos for WT hippocampal cultures for each condition and  N  = 5 individual embryos per condition for TgA hippocampal cultures.  c  Quantification of percentage of cell death in hippocampal neuronal cultures treated for 48 h with 1 μM scrambled Aβ control, antibody against STI1 (1:500), AβO alone (1 μM) or dishes treated with both AβO (1 μM) and anti-STI1 (1:500). At least four individual embryos were used for each condition and genotype.  d  Percentage of body paralysis over 10 days in nematodes expressing Aβ (3–42)  (strain CL2006 (dvIs2 [pcL12 (unc-54/human Aβ peptide minigene) + pRF4 ]) in the bodywall muscle and treated with empty vector control RNAi (black triangle),  sti-1  RNAi (black circle), or  hsp-90  RNAi (black square).  e  Percentage of paralysis in worms expressing Aβ ( 3–42)  (black circle), Aβ ( 3–42)  worms overexpressing HSP90 in body wall (strain AM988 (rmIs347( unc-54p::HSP-90::RFP )) (black square), Aβ ( 3–42)  worms overexpressing STI-1 in muscle cells (strain PVH40 (PPI1972 ( unc-54p::STI-1::GFP);dvIs2 ))) (black triangle) and Aβ (3–42)  worms overexpressing both STI-1 and HSP-90 in the bodywall muscle (strain PVH71 (rmIs347( unc-54p::HSP-90::RFP );( unc-54p::STI-1::GFP);dvIs2 ) (black stars). For  C. elegans  experiments, 100 age synchronized animals were used for analyses. For panels a and b, data were analyzed using Two-Way ANOVA, with Sidak’s or Bonferroni’s post-hoc tests for multiple comparisons, respectively, comparing WT vs TgA across the different concentrations of the AβOs. For both panels c and d, groups were analyzed using Wilcoxon statistics, comparing to Aβ (3–42)  expressing organisms. * p
    4 Well Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher elisa plates
    Description and characterization of the chimeric human <t>FasL-derived</t> constructs Panel A: Schematic representation of soluble FasL (sFasL), Flag-tagged sFasL (sfFasL), polymeric Flag-tagged soluble FasL (pfFasL), polymeric TCR γ4 and δ5 Flag-tagged soluble FasL generating the TCR-pfFasL upon cotransfection, and beta2-microglobulin-fused HLA-A*02: 01 Flag-tagged soluble FasL (HLA-pfFasL). The f and p symbols represent the flag epitope and the LIF receptor-derived domain triggering the polymerisation of the FasL oligomers, respectively. Panel B: direct immunoblot of the supernatants from COS cells transfected with the empty vector (control) or the FasL constructs sFasL, sfFasL and pfFasL. Panel C: immunoprecipitation of the TCR-pfFasL chimera from transfected HEK cells, using an irrelevant IgG1 antibody, the anti-Flag (clone M2), the anti-FasL (clone 10F2), the anti-TCRγδ (clone IMU-510) or the anti-TCRδ5 (clone 12C7) antibodies. Panel D: immunoprecipitation of the HLA-pfFasL chimera from the supernatant of COS cells, with anti-Flag, anti-FasL or anti-β2microglobulin antibodies. As controls, the same experiment was performed with irrelevant IgG1 and IgG2 antibodies. Panel E: cytotoxic effect of the FasL chimeras. The indicated chimeras, as supernatants from transfected cells and quantitated using the <t>ELISA</t> for FasL, were incubated at the indicated concentrations with Jurkat cells. After 18 h, the MTT cell viability assay was performed. The anti-Flag M2 antibody at 0.5 µg/ml was added to sfFasL to render it cytotoxic.
    Elisa Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 564 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher 4 well cell culture plates
    Topography and architecture of V. cholerae biofilms. Each strain was grown in a <t>4-well</t> cell culture plate containing 500 μl FSLW. A glass cover slip was dipped into each culture well and incubated overnight statically at room temperature. The glass cover slips were stained with SYTO 9 and the images were obtained using a laser scanning confocal microscopy with an excitation and emission wavelengths of 484 and 500 nm, respectively. (A) Images of x–y sections (top panels) and x–z projections of the same biofilms (bottom panels) were analyzed with DAIME software; magnification, x200. (B) Average biofilm heights (μm) for each strain measured across five random x–z sections. (C) Total volume of biofilm (μm 3 ) for each strain calculated by x–y and x–z projections. A p-value of
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    93
    Thermo Fisher 96 well maxisorp plates nunc
    L1CAM is a mediator of organoid and tumor regeneration. a , L1CAM is required for organoid regeneration. CRC107Li organoid-derived cells were transduced with lentivirus directing the expression of either Cas9 alone or Cas9 with sgRNAs targeting L1CAM and allowed to grow under antibiotic selection for 14 d, when they were flow-sorted and seeded at a concentration of 2,000 cells per 40 μl of Matrigel in independent wells of a <t>96-well</t> plate. The number of organoids (mean ± s.e.m.) established from each population 14 d after sorting and seeding is shown. From left to right, n = 10, 13 and 11 organoid cultures per group; two-tailed Mann-Whitney U test. b , c , L1CAM knockdown inhibits regrowth of multiple patient-derived organoids. Organoids derived from four patients with metastatic CRC were transduced with lentiviruses directing the expression of doxycycline (Dox)-inducible shRNA targeting L1CAM , expanded and, where indicated, treated with doxycycline for 48 h before dissociation and seeding at a concentration of 2,000 cells per 40 μl of Matrigel. Knockdown efficiencies of two independent L1CAM -targeting shRNAs in four patient-derived organoids ( b ) and relative cell viability on day 14 as compared to day 0 (mean ± s.e.m.) after plating of organoid-derived single cells ( c ) are shown. n = 6 organoid cultures per group; two-sided Student’s t tests. d , L1CAM is required for subcutaneous tumor growth in vivo. MSK107Li organoid-derived cells (50,000) expressing a doxycycline-inducible shRNA targeting L1CAM were injected subcutaneously into each flank of immunodeficient NSG mice. Where indicated, organoids were treated with doxycycline 2 d before transplantation and mice were maintained on a doxycycline diet for the duration of the experiment. Tumor volume (mean ± s.e.m.) was measured with calipers at the indicated time points after subcutaneous inoculation. n = 10 tumors from five mice per group; two-tailed Mann-Whitney U test. e , Representative image and quantification of tumor bioluminescence measured 35 d after inoculation. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 10 tumors from five mice per group; two-sided Mann-Whitney U test. f , Day 21 steady-state MSK107Li and MSK121Li organoids were incubated in medium containing 50 μM irinotecan, and L1CAM expression was measured in residual DAPI − cells 7 d later. Top- flow cytometry plots showing distribution of the data. Bottom: bars showing median fluorescence intensity of L1CAM expression in each population. From left to right, n = 6,512, 130, 8,542 and 49 cells per group, representative of three independent experiments. g , Single cells derived from CRC107Li organoids transduced with lentivirus directing expression of the indicated shRNAs were seeded at a concentration of 2,000 cells per 40 μl grown as organoids for 21 d and then treated with doxycycline and/or irinotecan (irino) as indicated. The viability assay shows the luminescence (mean ± s.e.m.) of each population relative to the luminescence at the time that drug treatment was started (day 0); n = 5 organoid cultures per group; two-sided Mann-Whitney U test. h , Solid-phase binding assay showing dose-response curves of recombinant human L1CAM-Fc binding to plates coated with equimolar concentrations of the indicated proteins. After washing, bound L1CAM-Fc was detected with horseradish peroxidase (HRP)-conjugated anti-human IgG, HRP substrate was added and OD 450 was measured. Data are shown as the mean ± s.e.m; n = 5 wells per time point, representative of three independent experiments; two-tailed Mann-Whitney U test. i , L1CAM mediates the interaction of dissociated CRC cells with laminin isoforms. Single cells derived from MSK121Li organoids (3,000) cultured in the presence or absence of doxycycline to knock down L1CAM were seeded in wells coated with 30 nM of the indicated proteins. After 1 h of adhesion and extensive washing, the percentage of adherent cells (mean ± s.e.m.) was measured as the relative luminescence of each well immediately after plating. n = 10 organoid cultures per condition; two-tailed Mann-Whitney U tests.
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    Overexpression of STI1 protects both  C. elegans  and primary hippocampal mouse neurons from Aβ-toxicity.  a  The average percent of dead cells ((# of dead cells)/(# of live + # of dead cells))× 100) in E17.5 primary hippocampal neuronal cultures from WT or TgA embryos. Cells were imaged in 4 well dishes from 8 random fields ( N  = 6 individual embryos/genotype). Dishes were treated with either no Aβ, 0.1, 0.5 or 1 μM AβOs for 48 h.  b  Likewise, WT or TgA primary hippocampal neurons were treated with 0, 0.25, 0.5 or 1 μM for 48 h, and LDH release was measured using colorimetric assay, read at 450 nm.  N  = 3–4 individual embryos for WT hippocampal cultures for each condition and  N  = 5 individual embryos per condition for TgA hippocampal cultures.  c  Quantification of percentage of cell death in hippocampal neuronal cultures treated for 48 h with 1 μM scrambled Aβ control, antibody against STI1 (1:500), AβO alone (1 μM) or dishes treated with both AβO (1 μM) and anti-STI1 (1:500). At least four individual embryos were used for each condition and genotype.  d  Percentage of body paralysis over 10 days in nematodes expressing Aβ (3–42)  (strain CL2006 (dvIs2 [pcL12 (unc-54/human Aβ peptide minigene) + pRF4 ]) in the bodywall muscle and treated with empty vector control RNAi (black triangle),  sti-1  RNAi (black circle), or  hsp-90  RNAi (black square).  e  Percentage of paralysis in worms expressing Aβ ( 3–42)  (black circle), Aβ ( 3–42)  worms overexpressing HSP90 in body wall (strain AM988 (rmIs347( unc-54p::HSP-90::RFP )) (black square), Aβ ( 3–42)  worms overexpressing STI-1 in muscle cells (strain PVH40 (PPI1972 ( unc-54p::STI-1::GFP);dvIs2 ))) (black triangle) and Aβ (3–42)  worms overexpressing both STI-1 and HSP-90 in the bodywall muscle (strain PVH71 (rmIs347( unc-54p::HSP-90::RFP );( unc-54p::STI-1::GFP);dvIs2 ) (black stars). For  C. elegans  experiments, 100 age synchronized animals were used for analyses. For panels a and b, data were analyzed using Two-Way ANOVA, with Sidak’s or Bonferroni’s post-hoc tests for multiple comparisons, respectively, comparing WT vs TgA across the different concentrations of the AβOs. For both panels c and d, groups were analyzed using Wilcoxon statistics, comparing to Aβ (3–42)  expressing organisms. * p

    Journal: Acta Neuropathologica Communications

    Article Title: Increased levels of Stress-inducible phosphoprotein-1 accelerates amyloid-β deposition in a mouse model of Alzheimer’s disease

    doi: 10.1186/s40478-020-01013-5

    Figure Lengend Snippet: Overexpression of STI1 protects both C. elegans and primary hippocampal mouse neurons from Aβ-toxicity. a The average percent of dead cells ((# of dead cells)/(# of live + # of dead cells))× 100) in E17.5 primary hippocampal neuronal cultures from WT or TgA embryos. Cells were imaged in 4 well dishes from 8 random fields ( N  = 6 individual embryos/genotype). Dishes were treated with either no Aβ, 0.1, 0.5 or 1 μM AβOs for 48 h. b Likewise, WT or TgA primary hippocampal neurons were treated with 0, 0.25, 0.5 or 1 μM for 48 h, and LDH release was measured using colorimetric assay, read at 450 nm. N  = 3–4 individual embryos for WT hippocampal cultures for each condition and N  = 5 individual embryos per condition for TgA hippocampal cultures. c Quantification of percentage of cell death in hippocampal neuronal cultures treated for 48 h with 1 μM scrambled Aβ control, antibody against STI1 (1:500), AβO alone (1 μM) or dishes treated with both AβO (1 μM) and anti-STI1 (1:500). At least four individual embryos were used for each condition and genotype. d Percentage of body paralysis over 10 days in nematodes expressing Aβ (3–42) (strain CL2006 (dvIs2 [pcL12 (unc-54/human Aβ peptide minigene) + pRF4 ]) in the bodywall muscle and treated with empty vector control RNAi (black triangle), sti-1 RNAi (black circle), or hsp-90 RNAi (black square). e Percentage of paralysis in worms expressing Aβ ( 3–42) (black circle), Aβ ( 3–42) worms overexpressing HSP90 in body wall (strain AM988 (rmIs347( unc-54p::HSP-90::RFP )) (black square), Aβ ( 3–42) worms overexpressing STI-1 in muscle cells (strain PVH40 (PPI1972 ( unc-54p::STI-1::GFP);dvIs2 ))) (black triangle) and Aβ (3–42) worms overexpressing both STI-1 and HSP-90 in the bodywall muscle (strain PVH71 (rmIs347( unc-54p::HSP-90::RFP );( unc-54p::STI-1::GFP);dvIs2 ) (black stars). For C. elegans experiments, 100 age synchronized animals were used for analyses. For panels a and b, data were analyzed using Two-Way ANOVA, with Sidak’s or Bonferroni’s post-hoc tests for multiple comparisons, respectively, comparing WT vs TgA across the different concentrations of the AβOs. For both panels c and d, groups were analyzed using Wilcoxon statistics, comparing to Aβ (3–42) expressing organisms. * p

    Article Snippet: Approximately 4–6 × 104 cells were added to each well in 4-well plates (ThermoFisher, Cat#176740) pre-coated with Poly-L-Lysine (Catalog# P6407, Sigma).

    Techniques: Over Expression, Colorimetric Assay, Expressing, Plasmid Preparation

    Description and characterization of the chimeric human FasL-derived constructs Panel A: Schematic representation of soluble FasL (sFasL), Flag-tagged sFasL (sfFasL), polymeric Flag-tagged soluble FasL (pfFasL), polymeric TCR γ4 and δ5 Flag-tagged soluble FasL generating the TCR-pfFasL upon cotransfection, and beta2-microglobulin-fused HLA-A*02: 01 Flag-tagged soluble FasL (HLA-pfFasL). The f and p symbols represent the flag epitope and the LIF receptor-derived domain triggering the polymerisation of the FasL oligomers, respectively. Panel B: direct immunoblot of the supernatants from COS cells transfected with the empty vector (control) or the FasL constructs sFasL, sfFasL and pfFasL. Panel C: immunoprecipitation of the TCR-pfFasL chimera from transfected HEK cells, using an irrelevant IgG1 antibody, the anti-Flag (clone M2), the anti-FasL (clone 10F2), the anti-TCRγδ (clone IMU-510) or the anti-TCRδ5 (clone 12C7) antibodies. Panel D: immunoprecipitation of the HLA-pfFasL chimera from the supernatant of COS cells, with anti-Flag, anti-FasL or anti-β2microglobulin antibodies. As controls, the same experiment was performed with irrelevant IgG1 and IgG2 antibodies. Panel E: cytotoxic effect of the FasL chimeras. The indicated chimeras, as supernatants from transfected cells and quantitated using the ELISA for FasL, were incubated at the indicated concentrations with Jurkat cells. After 18 h, the MTT cell viability assay was performed. The anti-Flag M2 antibody at 0.5 µg/ml was added to sfFasL to render it cytotoxic.

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Description and characterization of the chimeric human FasL-derived constructs Panel A: Schematic representation of soluble FasL (sFasL), Flag-tagged sFasL (sfFasL), polymeric Flag-tagged soluble FasL (pfFasL), polymeric TCR γ4 and δ5 Flag-tagged soluble FasL generating the TCR-pfFasL upon cotransfection, and beta2-microglobulin-fused HLA-A*02: 01 Flag-tagged soluble FasL (HLA-pfFasL). The f and p symbols represent the flag epitope and the LIF receptor-derived domain triggering the polymerisation of the FasL oligomers, respectively. Panel B: direct immunoblot of the supernatants from COS cells transfected with the empty vector (control) or the FasL constructs sFasL, sfFasL and pfFasL. Panel C: immunoprecipitation of the TCR-pfFasL chimera from transfected HEK cells, using an irrelevant IgG1 antibody, the anti-Flag (clone M2), the anti-FasL (clone 10F2), the anti-TCRγδ (clone IMU-510) or the anti-TCRδ5 (clone 12C7) antibodies. Panel D: immunoprecipitation of the HLA-pfFasL chimera from the supernatant of COS cells, with anti-Flag, anti-FasL or anti-β2microglobulin antibodies. As controls, the same experiment was performed with irrelevant IgG1 and IgG2 antibodies. Panel E: cytotoxic effect of the FasL chimeras. The indicated chimeras, as supernatants from transfected cells and quantitated using the ELISA for FasL, were incubated at the indicated concentrations with Jurkat cells. After 18 h, the MTT cell viability assay was performed. The anti-Flag M2 antibody at 0.5 µg/ml was added to sfFasL to render it cytotoxic.

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Derivative Assay, Construct, Cotransfection, FLAG-tag, Transfection, Plasmid Preparation, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Incubation, MTT Assay, Viability Assay

    Direct association of sFasL to the pfFasL-containing chimeric proteins during co-expression. Panel A: Identical amounts of pfFasL (1 µg, according to the Flag ELISA) produced in the presence of the indicated ratios of added sFasL plasmid (left panels) was immunoprecipitated with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (3 µg according to the FasL ELISA, right panel). Panel B: Densitometric detection and quantification of the pfFasL (grey bars) and the sFasL (black bars) fractions, following transfection of the pfFasL plasmid in the presence of the indicated proportion of the sFasL plasmid. The measures were normalized to the condition lacking sFasL. Mean+/- sd from three experiments. Panel C: The TCR-pfFasL chimera (2 µg, according to an ELISA specific for the TCR-pFasL molecule using anti-TCRδ5 (clone 12C7) and anti-FasL (clone 10F2) as capture and tracing antibodies, respectively), produced in the absence or the presence of the sFasL plasmid at the indicated ratio, was immunoprecipitated with the anti-TCRδ5 antibody, then separated by 10% SDS-PAGE under reducing conditions and revealed by immunoblotting with the anti-FasL antibody. As a control, the immunoprecipitation experiment was performed with 2 µg of sFasL protein. Panel D: COS supernatants containing pfFasL (4 µg/ml according to the Flag ELISA) produced alone, was mixed with culture medium or sFasL (15 µg/ml) produced separately in a total volume of 1 ml, and incubated for 24 h at 37°C. Then the recombinant proteins were immunoprecipitated (left panels) with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (15 µg according to the FasL ELISA, right panel).

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Direct association of sFasL to the pfFasL-containing chimeric proteins during co-expression. Panel A: Identical amounts of pfFasL (1 µg, according to the Flag ELISA) produced in the presence of the indicated ratios of added sFasL plasmid (left panels) was immunoprecipitated with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (3 µg according to the FasL ELISA, right panel). Panel B: Densitometric detection and quantification of the pfFasL (grey bars) and the sFasL (black bars) fractions, following transfection of the pfFasL plasmid in the presence of the indicated proportion of the sFasL plasmid. The measures were normalized to the condition lacking sFasL. Mean+/- sd from three experiments. Panel C: The TCR-pfFasL chimera (2 µg, according to an ELISA specific for the TCR-pFasL molecule using anti-TCRδ5 (clone 12C7) and anti-FasL (clone 10F2) as capture and tracing antibodies, respectively), produced in the absence or the presence of the sFasL plasmid at the indicated ratio, was immunoprecipitated with the anti-TCRδ5 antibody, then separated by 10% SDS-PAGE under reducing conditions and revealed by immunoblotting with the anti-FasL antibody. As a control, the immunoprecipitation experiment was performed with 2 µg of sFasL protein. Panel D: COS supernatants containing pfFasL (4 µg/ml according to the Flag ELISA) produced alone, was mixed with culture medium or sFasL (15 µg/ml) produced separately in a total volume of 1 ml, and incubated for 24 h at 37°C. Then the recombinant proteins were immunoprecipitated (left panels) with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (15 µg according to the FasL ELISA, right panel).

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Produced, Plasmid Preparation, Immunoprecipitation, SDS Page, Transfection, Incubation, Recombinant

    Effect of sFasL on the supernatant production of the Flag-tagged FasL constructs. Panels A to D : An increasing amount expressed in percentage, of the sFasL encoding plasmid, was co-transfected with a fixed amount of the plasmids encoding sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D). The secreted proteins were quantified in culture supernatants using an ELISA specific for FasL (shaded histograms, right-hand scale) and for Flag-tagged FasL (curves, left-hand scale). For the Flag ELISA, the measured concentrations were normalized according to the condition lacking sFasL. Are presented the mean +/- sd of four independent transfection experiments. * 0.02≤p≤0.05; ** p≤0.02. Panel E : direct anti-FasL immunoblot analysis of identical volumes of the cell culture supernatant containing pfFasL produced alone and with 50% of the sFasL plasmid, after SDS-PAGE separation under reducing conditions.

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Effect of sFasL on the supernatant production of the Flag-tagged FasL constructs. Panels A to D : An increasing amount expressed in percentage, of the sFasL encoding plasmid, was co-transfected with a fixed amount of the plasmids encoding sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D). The secreted proteins were quantified in culture supernatants using an ELISA specific for FasL (shaded histograms, right-hand scale) and for Flag-tagged FasL (curves, left-hand scale). For the Flag ELISA, the measured concentrations were normalized according to the condition lacking sFasL. Are presented the mean +/- sd of four independent transfection experiments. * 0.02≤p≤0.05; ** p≤0.02. Panel E : direct anti-FasL immunoblot analysis of identical volumes of the cell culture supernatant containing pfFasL produced alone and with 50% of the sFasL plasmid, after SDS-PAGE separation under reducing conditions.

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Construct, Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Produced, SDS Page

    Effect of sFasL on cell targeting of the FasL-containing chimeras. Panel A : Schematic description of the experimental model used. The chimera is enriched at the surface of the CD32-expressing L-cells via its HLA targeting module and an anti-HLA monoclonal antibody. Panel B: murine Fas (continuous line), human CD32 (dashed line) and IgG1 isotype-matched control (shaded histogram) staining of the CD32+ L-cell transfectant. Living cells were gated on the basis of the morphological parameters. Panel C : Fas sensitivity of the CD32+ L-cell transfectant to the indicated concentrations of the anti-Fas JO-2 antibody (circles), the HLA-pfFasL chimera expressed alone (triangle) or in the presence of 25% of the sFasL plasmid (squares), in the MTT viability assay. Panel D : The CD32+ L-cells were incubated with the HLA-pfFasL chimera produced in the presence (black bars) or in the absence (white bars) of 25% of the sFasL plasmid, together with the indicated irrelevant IgG1 isotype-matched, anti-beta-2 microglobulin or anti-Flag antibodies. The concentrations of the chimera that triggered 15% of cell death and were at 15 and 0.3 ng/ml in the absence and presence of sFasL, as estimated using the ELISA specific for the Flag-tagged FasL. Cytotoxicity was measured with the propidium iodide assay and normalized to the effect of the chimera in the absence of antibody. Are presented the mean +/- sd of three independent experiments. Panel E: reversal in the presence of the blocking anti-FasL and anti-CD32 antibodies, of the cytotoxic effect of the immune complexes between the anti-Flag antibody and HLA-pfFasL co-expressed with sFasL. Are presented the mean +/- sd of three independent experiments. ns : non significant ; ** p≤0.02.

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Effect of sFasL on cell targeting of the FasL-containing chimeras. Panel A : Schematic description of the experimental model used. The chimera is enriched at the surface of the CD32-expressing L-cells via its HLA targeting module and an anti-HLA monoclonal antibody. Panel B: murine Fas (continuous line), human CD32 (dashed line) and IgG1 isotype-matched control (shaded histogram) staining of the CD32+ L-cell transfectant. Living cells were gated on the basis of the morphological parameters. Panel C : Fas sensitivity of the CD32+ L-cell transfectant to the indicated concentrations of the anti-Fas JO-2 antibody (circles), the HLA-pfFasL chimera expressed alone (triangle) or in the presence of 25% of the sFasL plasmid (squares), in the MTT viability assay. Panel D : The CD32+ L-cells were incubated with the HLA-pfFasL chimera produced in the presence (black bars) or in the absence (white bars) of 25% of the sFasL plasmid, together with the indicated irrelevant IgG1 isotype-matched, anti-beta-2 microglobulin or anti-Flag antibodies. The concentrations of the chimera that triggered 15% of cell death and were at 15 and 0.3 ng/ml in the absence and presence of sFasL, as estimated using the ELISA specific for the Flag-tagged FasL. Cytotoxicity was measured with the propidium iodide assay and normalized to the effect of the chimera in the absence of antibody. Are presented the mean +/- sd of three independent experiments. Panel E: reversal in the presence of the blocking anti-FasL and anti-CD32 antibodies, of the cytotoxic effect of the immune complexes between the anti-Flag antibody and HLA-pfFasL co-expressed with sFasL. Are presented the mean +/- sd of three independent experiments. ns : non significant ; ** p≤0.02.

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Expressing, Staining, Transfection, Plasmid Preparation, MTT Assay, Viability Assay, Incubation, Produced, Enzyme-linked Immunosorbent Assay, Blocking Assay

    Effect of sFasL on the cytotoxic activity of the Flag-tagged FasL chimeras. The FasL-derived proteins sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D) were expressed alone or upon co-transfection with the indicated percentage of the plasmid encoding sFasL. A fixed concentration triggering 25 to 40% of cell death (1.9 ng/ml for sfFasL, 0.6 ng/ml for pfFasL, 0.7 ng/ml for HLA-pfFasL and 2.2 ng/ml for TCR-pfFasL), for the FasL-derived protein quantitated with the ELISA specific for Flag-tagged FasL, was incubated with the Fas-sensitive Jurkat cells. For the sfFasL construct, the filled squares and the empty squares depict the cytotoxicity of sfFasL in the presence and absence of the cross-linking anti-Flag antibody at 0.5 µg/ml), respectively. Cytotoxicity was estimated by a measure of the remaining viable cells using the MTT assay. Are presented the mean +/- sd of four independent transfection experiments. * 0.01≤p≤0.05; ** p≤0.01.

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Effect of sFasL on the cytotoxic activity of the Flag-tagged FasL chimeras. The FasL-derived proteins sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D) were expressed alone or upon co-transfection with the indicated percentage of the plasmid encoding sFasL. A fixed concentration triggering 25 to 40% of cell death (1.9 ng/ml for sfFasL, 0.6 ng/ml for pfFasL, 0.7 ng/ml for HLA-pfFasL and 2.2 ng/ml for TCR-pfFasL), for the FasL-derived protein quantitated with the ELISA specific for Flag-tagged FasL, was incubated with the Fas-sensitive Jurkat cells. For the sfFasL construct, the filled squares and the empty squares depict the cytotoxicity of sfFasL in the presence and absence of the cross-linking anti-Flag antibody at 0.5 µg/ml), respectively. Cytotoxicity was estimated by a measure of the remaining viable cells using the MTT assay. Are presented the mean +/- sd of four independent transfection experiments. * 0.01≤p≤0.05; ** p≤0.01.

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Activity Assay, Derivative Assay, Cotransfection, Plasmid Preparation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Construct, MTT Assay, Transfection

    Topography and architecture of V. cholerae biofilms. Each strain was grown in a 4-well cell culture plate containing 500 μl FSLW. A glass cover slip was dipped into each culture well and incubated overnight statically at room temperature. The glass cover slips were stained with SYTO 9 and the images were obtained using a laser scanning confocal microscopy with an excitation and emission wavelengths of 484 and 500 nm, respectively. (A) Images of x–y sections (top panels) and x–z projections of the same biofilms (bottom panels) were analyzed with DAIME software; magnification, x200. (B) Average biofilm heights (μm) for each strain measured across five random x–z sections. (C) Total volume of biofilm (μm 3 ) for each strain calculated by x–y and x–z projections. A p-value of

    Journal: PLoS ONE

    Article Title: Vibrio cholerae Persisted in Microcosm for 700 Days Inhibits Motility but Promotes Biofilm Formation in Nutrient-Poor Lake Water Microcosms

    doi: 10.1371/journal.pone.0092883

    Figure Lengend Snippet: Topography and architecture of V. cholerae biofilms. Each strain was grown in a 4-well cell culture plate containing 500 μl FSLW. A glass cover slip was dipped into each culture well and incubated overnight statically at room temperature. The glass cover slips were stained with SYTO 9 and the images were obtained using a laser scanning confocal microscopy with an excitation and emission wavelengths of 484 and 500 nm, respectively. (A) Images of x–y sections (top panels) and x–z projections of the same biofilms (bottom panels) were analyzed with DAIME software; magnification, x200. (B) Average biofilm heights (μm) for each strain measured across five random x–z sections. (C) Total volume of biofilm (μm 3 ) for each strain calculated by x–y and x–z projections. A p-value of

    Article Snippet: 108 cfu/ml) in 4-well cell culture plates (Thermo Scientific Nunc, Pittsburgh, PA) containing 500 μl FSLW.

    Techniques: Cell Culture, Incubation, Staining, Confocal Microscopy, Software

    L1CAM is a mediator of organoid and tumor regeneration. a , L1CAM is required for organoid regeneration. CRC107Li organoid-derived cells were transduced with lentivirus directing the expression of either Cas9 alone or Cas9 with sgRNAs targeting L1CAM and allowed to grow under antibiotic selection for 14 d, when they were flow-sorted and seeded at a concentration of 2,000 cells per 40 μl of Matrigel in independent wells of a 96-well plate. The number of organoids (mean ± s.e.m.) established from each population 14 d after sorting and seeding is shown. From left to right, n = 10, 13 and 11 organoid cultures per group; two-tailed Mann-Whitney U test. b , c , L1CAM knockdown inhibits regrowth of multiple patient-derived organoids. Organoids derived from four patients with metastatic CRC were transduced with lentiviruses directing the expression of doxycycline (Dox)-inducible shRNA targeting L1CAM , expanded and, where indicated, treated with doxycycline for 48 h before dissociation and seeding at a concentration of 2,000 cells per 40 μl of Matrigel. Knockdown efficiencies of two independent L1CAM -targeting shRNAs in four patient-derived organoids ( b ) and relative cell viability on day 14 as compared to day 0 (mean ± s.e.m.) after plating of organoid-derived single cells ( c ) are shown. n = 6 organoid cultures per group; two-sided Student’s t tests. d , L1CAM is required for subcutaneous tumor growth in vivo. MSK107Li organoid-derived cells (50,000) expressing a doxycycline-inducible shRNA targeting L1CAM were injected subcutaneously into each flank of immunodeficient NSG mice. Where indicated, organoids were treated with doxycycline 2 d before transplantation and mice were maintained on a doxycycline diet for the duration of the experiment. Tumor volume (mean ± s.e.m.) was measured with calipers at the indicated time points after subcutaneous inoculation. n = 10 tumors from five mice per group; two-tailed Mann-Whitney U test. e , Representative image and quantification of tumor bioluminescence measured 35 d after inoculation. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 10 tumors from five mice per group; two-sided Mann-Whitney U test. f , Day 21 steady-state MSK107Li and MSK121Li organoids were incubated in medium containing 50 μM irinotecan, and L1CAM expression was measured in residual DAPI − cells 7 d later. Top- flow cytometry plots showing distribution of the data. Bottom: bars showing median fluorescence intensity of L1CAM expression in each population. From left to right, n = 6,512, 130, 8,542 and 49 cells per group, representative of three independent experiments. g , Single cells derived from CRC107Li organoids transduced with lentivirus directing expression of the indicated shRNAs were seeded at a concentration of 2,000 cells per 40 μl grown as organoids for 21 d and then treated with doxycycline and/or irinotecan (irino) as indicated. The viability assay shows the luminescence (mean ± s.e.m.) of each population relative to the luminescence at the time that drug treatment was started (day 0); n = 5 organoid cultures per group; two-sided Mann-Whitney U test. h , Solid-phase binding assay showing dose-response curves of recombinant human L1CAM-Fc binding to plates coated with equimolar concentrations of the indicated proteins. After washing, bound L1CAM-Fc was detected with horseradish peroxidase (HRP)-conjugated anti-human IgG, HRP substrate was added and OD 450 was measured. Data are shown as the mean ± s.e.m; n = 5 wells per time point, representative of three independent experiments; two-tailed Mann-Whitney U test. i , L1CAM mediates the interaction of dissociated CRC cells with laminin isoforms. Single cells derived from MSK121Li organoids (3,000) cultured in the presence or absence of doxycycline to knock down L1CAM were seeded in wells coated with 30 nM of the indicated proteins. After 1 h of adhesion and extensive washing, the percentage of adherent cells (mean ± s.e.m.) was measured as the relative luminescence of each well immediately after plating. n = 10 organoid cultures per condition; two-tailed Mann-Whitney U tests.

    Journal: Nature cancer

    Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

    doi: 10.1038/s43018-019-0006-x

    Figure Lengend Snippet: L1CAM is a mediator of organoid and tumor regeneration. a , L1CAM is required for organoid regeneration. CRC107Li organoid-derived cells were transduced with lentivirus directing the expression of either Cas9 alone or Cas9 with sgRNAs targeting L1CAM and allowed to grow under antibiotic selection for 14 d, when they were flow-sorted and seeded at a concentration of 2,000 cells per 40 μl of Matrigel in independent wells of a 96-well plate. The number of organoids (mean ± s.e.m.) established from each population 14 d after sorting and seeding is shown. From left to right, n = 10, 13 and 11 organoid cultures per group; two-tailed Mann-Whitney U test. b , c , L1CAM knockdown inhibits regrowth of multiple patient-derived organoids. Organoids derived from four patients with metastatic CRC were transduced with lentiviruses directing the expression of doxycycline (Dox)-inducible shRNA targeting L1CAM , expanded and, where indicated, treated with doxycycline for 48 h before dissociation and seeding at a concentration of 2,000 cells per 40 μl of Matrigel. Knockdown efficiencies of two independent L1CAM -targeting shRNAs in four patient-derived organoids ( b ) and relative cell viability on day 14 as compared to day 0 (mean ± s.e.m.) after plating of organoid-derived single cells ( c ) are shown. n = 6 organoid cultures per group; two-sided Student’s t tests. d , L1CAM is required for subcutaneous tumor growth in vivo. MSK107Li organoid-derived cells (50,000) expressing a doxycycline-inducible shRNA targeting L1CAM were injected subcutaneously into each flank of immunodeficient NSG mice. Where indicated, organoids were treated with doxycycline 2 d before transplantation and mice were maintained on a doxycycline diet for the duration of the experiment. Tumor volume (mean ± s.e.m.) was measured with calipers at the indicated time points after subcutaneous inoculation. n = 10 tumors from five mice per group; two-tailed Mann-Whitney U test. e , Representative image and quantification of tumor bioluminescence measured 35 d after inoculation. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 10 tumors from five mice per group; two-sided Mann-Whitney U test. f , Day 21 steady-state MSK107Li and MSK121Li organoids were incubated in medium containing 50 μM irinotecan, and L1CAM expression was measured in residual DAPI − cells 7 d later. Top- flow cytometry plots showing distribution of the data. Bottom: bars showing median fluorescence intensity of L1CAM expression in each population. From left to right, n = 6,512, 130, 8,542 and 49 cells per group, representative of three independent experiments. g , Single cells derived from CRC107Li organoids transduced with lentivirus directing expression of the indicated shRNAs were seeded at a concentration of 2,000 cells per 40 μl grown as organoids for 21 d and then treated with doxycycline and/or irinotecan (irino) as indicated. The viability assay shows the luminescence (mean ± s.e.m.) of each population relative to the luminescence at the time that drug treatment was started (day 0); n = 5 organoid cultures per group; two-sided Mann-Whitney U test. h , Solid-phase binding assay showing dose-response curves of recombinant human L1CAM-Fc binding to plates coated with equimolar concentrations of the indicated proteins. After washing, bound L1CAM-Fc was detected with horseradish peroxidase (HRP)-conjugated anti-human IgG, HRP substrate was added and OD 450 was measured. Data are shown as the mean ± s.e.m; n = 5 wells per time point, representative of three independent experiments; two-tailed Mann-Whitney U test. i , L1CAM mediates the interaction of dissociated CRC cells with laminin isoforms. Single cells derived from MSK121Li organoids (3,000) cultured in the presence or absence of doxycycline to knock down L1CAM were seeded in wells coated with 30 nM of the indicated proteins. After 1 h of adhesion and extensive washing, the percentage of adherent cells (mean ± s.e.m.) was measured as the relative luminescence of each well immediately after plating. n = 10 organoid cultures per condition; two-tailed Mann-Whitney U tests.

    Article Snippet: 96-well Maxisorp plates (Nunc) were coated overnight at 4 °C with 30 nM recombinant protein, washed with PBS and blocked with protein-free blocking buffer (TBS, Thermo Fisher Scientific) for 2 h at room temperature.

    Techniques: Derivative Assay, Transduction, Expressing, Selection, Concentration Assay, Two Tailed Test, MANN-WHITNEY, shRNA, In Vivo, Injection, Mouse Assay, Transplantation Assay, Incubation, Flow Cytometry, Fluorescence, Viability Assay, Binding Assay, Recombinant, Cell Culture