hoechst 33342  (Thermo Fisher)


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    Structured Review

    Thermo Fisher hoechst 33342
    Co-localisation during the life cycle (A) MyoB-GFP and (B) MyoE-GFP localised by super-resolution microscopy of ookinetes. Shown are DIC image, GFP (green) with higher magnification inset, 13.1(red) and DAPI (blue), and merged image. (C) Co-localisation of MyoB-GFP or MyoE-GFP E with MyoA-mCherry or ISP1-mCherry in ookinetes using live cell imaging. Shown are: DIC image, GFP (green), mCherry (red) and merged image including <t>Hoechst</t> 33342 (blue). (D) Co-localisation of GFP-myosins J and F with mCherry-MyoA in oocysts. Shown are: DIC image, GFP (green), mCherry (red) and merged image including Hoechst 33342 (blue). Scale bar = 5 μm.
    Hoechst 33342, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hoechst 33342 - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Systematic analysis of Plasmodium myosins reveals differential expression, localization and function in invasive and proliferative parasite stages"

    Article Title: Systematic analysis of Plasmodium myosins reveals differential expression, localization and function in invasive and proliferative parasite stages

    Journal: bioRxiv

    doi: 10.1101/671578

    Co-localisation during the life cycle (A) MyoB-GFP and (B) MyoE-GFP localised by super-resolution microscopy of ookinetes. Shown are DIC image, GFP (green) with higher magnification inset, 13.1(red) and DAPI (blue), and merged image. (C) Co-localisation of MyoB-GFP or MyoE-GFP E with MyoA-mCherry or ISP1-mCherry in ookinetes using live cell imaging. Shown are: DIC image, GFP (green), mCherry (red) and merged image including Hoechst 33342 (blue). (D) Co-localisation of GFP-myosins J and F with mCherry-MyoA in oocysts. Shown are: DIC image, GFP (green), mCherry (red) and merged image including Hoechst 33342 (blue). Scale bar = 5 μm.
    Figure Legend Snippet: Co-localisation during the life cycle (A) MyoB-GFP and (B) MyoE-GFP localised by super-resolution microscopy of ookinetes. Shown are DIC image, GFP (green) with higher magnification inset, 13.1(red) and DAPI (blue), and merged image. (C) Co-localisation of MyoB-GFP or MyoE-GFP E with MyoA-mCherry or ISP1-mCherry in ookinetes using live cell imaging. Shown are: DIC image, GFP (green), mCherry (red) and merged image including Hoechst 33342 (blue). (D) Co-localisation of GFP-myosins J and F with mCherry-MyoA in oocysts. Shown are: DIC image, GFP (green), mCherry (red) and merged image including Hoechst 33342 (blue). Scale bar = 5 μm.

    Techniques Used: Microscopy, Live Cell Imaging

    Expression and localisation of Plasmodium myosins throughout the life cycle (A) There are six Plasmodium myosins. Two class XIV myosins have a ‘head’ and ‘neck’ region but no tail. The remaining class XIV and class XXII and VI myosins have a ‘tail’, which in the case of MyoF contains WD40 repeat domains. (B) Plot of normalised transcript expression levels for each myosin gene throughout the Plasmodium life cycle. RNA was prepared from asexual blood stages (AS), blood stage schizonts (Sch), non-activated gametocytes (NAG), activated gametocytes (AG), ookinetes (Ook) and sporozoites (Spr). Two genes, arginine-tRNA synthetase and hsp70 , were used as controls for normalisation. Each point is the mean of three biological replicates ± SEM. (C) Summary of GFP-tagged myosin expression throughout the life cycle, in merozoites (Mero), schizonts (Sch), non-activated gametocytes (NAG), activated gametocytes (AG), zygotes (Zyg), retort-forms (Reto), ookinetes (Ook), oocysts (Ooc) and sporozoites (Spor). Expression of (D) MyoA-GFP, (E) MyoB-GFP and (F) MyoE-GFP in schizonts, ookinetes and sporozoites using live cell imaging. MyoB and MyoE with higher magnification inset. Expression of (G) MyoF-GFP in ookinetes and oocysts, (H) MyoJ-GFP in an oocyst and residual oocyst body following sporozoite egression and (I) MyoK-GFP in a gametocyte and early (stage IV) retort/ookinete using live cell imaging. Shown are DIC image, Hoechst 33342 (blue); GFP (green); Merge: blue, green and 13.1 (red), a cy3-conjugated antibody recognising P28 (on activated female gametocytes, zygotes and ookinetes only). Size marker = 5 μm.
    Figure Legend Snippet: Expression and localisation of Plasmodium myosins throughout the life cycle (A) There are six Plasmodium myosins. Two class XIV myosins have a ‘head’ and ‘neck’ region but no tail. The remaining class XIV and class XXII and VI myosins have a ‘tail’, which in the case of MyoF contains WD40 repeat domains. (B) Plot of normalised transcript expression levels for each myosin gene throughout the Plasmodium life cycle. RNA was prepared from asexual blood stages (AS), blood stage schizonts (Sch), non-activated gametocytes (NAG), activated gametocytes (AG), ookinetes (Ook) and sporozoites (Spr). Two genes, arginine-tRNA synthetase and hsp70 , were used as controls for normalisation. Each point is the mean of three biological replicates ± SEM. (C) Summary of GFP-tagged myosin expression throughout the life cycle, in merozoites (Mero), schizonts (Sch), non-activated gametocytes (NAG), activated gametocytes (AG), zygotes (Zyg), retort-forms (Reto), ookinetes (Ook), oocysts (Ooc) and sporozoites (Spor). Expression of (D) MyoA-GFP, (E) MyoB-GFP and (F) MyoE-GFP in schizonts, ookinetes and sporozoites using live cell imaging. MyoB and MyoE with higher magnification inset. Expression of (G) MyoF-GFP in ookinetes and oocysts, (H) MyoJ-GFP in an oocyst and residual oocyst body following sporozoite egression and (I) MyoK-GFP in a gametocyte and early (stage IV) retort/ookinete using live cell imaging. Shown are DIC image, Hoechst 33342 (blue); GFP (green); Merge: blue, green and 13.1 (red), a cy3-conjugated antibody recognising P28 (on activated female gametocytes, zygotes and ookinetes only). Size marker = 5 μm.

    Techniques Used: Expressing, SPR Assay, Live Cell Imaging, Marker

    2) Product Images from "A manual multiplex immunofluorescence method for investigating neurodegenerative diseases"

    Article Title: A manual multiplex immunofluorescence method for investigating neurodegenerative diseases

    Journal: bioRxiv

    doi: 10.1101/533547

    MC1 (orange) across five rounds of staining, stripping, and re-staining using βME-based stripping along with TSA-developed NeuN (pink) and Hoechst 33342 (blue). (A) Stripping controls which were stained in each round up until the final elution for that section where stripping was confirmed by confirming the lack of signal after development with AF546-conjugated secondary. (B) Signal from MC1 (orange) following staining, stripping, and re-staining across five rounds. NeuN (pink) was only developed in round 1 with AF647-TSA. Hoechst 33342 was stained in each round, as it is in the Prolong mounting media. Signal from MC1 appears across each of the rounds in the same locations. However a decrease in signal intensity is noted by Round 5.. Scale bar: 10 μm
    Figure Legend Snippet: MC1 (orange) across five rounds of staining, stripping, and re-staining using βME-based stripping along with TSA-developed NeuN (pink) and Hoechst 33342 (blue). (A) Stripping controls which were stained in each round up until the final elution for that section where stripping was confirmed by confirming the lack of signal after development with AF546-conjugated secondary. (B) Signal from MC1 (orange) following staining, stripping, and re-staining across five rounds. NeuN (pink) was only developed in round 1 with AF647-TSA. Hoechst 33342 was stained in each round, as it is in the Prolong mounting media. Signal from MC1 appears across each of the rounds in the same locations. However a decrease in signal intensity is noted by Round 5.. Scale bar: 10 μm

    Techniques Used: Staining, Stripping Membranes

    3) Product Images from "Continuous Exposure to 1.7 GHz LTE Electromagnetic Fields Increases Intracellular Reactive Oxygen Species to Decrease Human Cell Proliferation and Induce Senescence"

    Article Title: Continuous Exposure to 1.7 GHz LTE Electromagnetic Fields Increases Intracellular Reactive Oxygen Species to Decrease Human Cell Proliferation and Induce Senescence

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-65732-4

    Continuous exposure to 1.7 GHz LTE RF-EMF decreased cell proliferation by inducing intracellular ROS in ASCs and Huh7 cells. ( A–D ) ASCs and Huh7 cells pre-treated or not with 100 μM NAC were exposed to 1.7 GHz RF-EMF for 72 h at 2 SAR, while the sham control cells were incubated for 72 h without RF-EMF exposure. After the exposure, ( A,C ) the cells were collected and counted with a cell counter (Nexcelom Bioscience). Huh7 cells ( B ) and ASCs ( D ) were stained with carboxy-H 2 DCFDA. Cells treat with TBHP were used as a positive control for intracellular ROS generation. ( E,F ) ASCs and Huh7 cells were exposed to 1.7 GHz RF-EMF for 72 h at 1 SAR or 2 SAR, and were stained with MitoSOX. ( B,D–F ) Nuclei were stained with Hoechst 33342. Images were taken with an Axioplan2 fluorescence microscope (Zeiss) under a 200× objective. Scale bar, 25 μm. All experiments consisted of three independent replicates.
    Figure Legend Snippet: Continuous exposure to 1.7 GHz LTE RF-EMF decreased cell proliferation by inducing intracellular ROS in ASCs and Huh7 cells. ( A–D ) ASCs and Huh7 cells pre-treated or not with 100 μM NAC were exposed to 1.7 GHz RF-EMF for 72 h at 2 SAR, while the sham control cells were incubated for 72 h without RF-EMF exposure. After the exposure, ( A,C ) the cells were collected and counted with a cell counter (Nexcelom Bioscience). Huh7 cells ( B ) and ASCs ( D ) were stained with carboxy-H 2 DCFDA. Cells treat with TBHP were used as a positive control for intracellular ROS generation. ( E,F ) ASCs and Huh7 cells were exposed to 1.7 GHz RF-EMF for 72 h at 1 SAR or 2 SAR, and were stained with MitoSOX. ( B,D–F ) Nuclei were stained with Hoechst 33342. Images were taken with an Axioplan2 fluorescence microscope (Zeiss) under a 200× objective. Scale bar, 25 μm. All experiments consisted of three independent replicates.

    Techniques Used: Incubation, Staining, Positive Control, Fluorescence, Microscopy

    4) Product Images from "TDP-43, a protein central to amyotrophic lateral sclerosis, is destabilized by tankyrase-1 and -2"

    Article Title: TDP-43, a protein central to amyotrophic lateral sclerosis, is destabilized by tankyrase-1 and -2

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.245811

    Deletion of the TBD promotes MG132-induced nuclear ubiquitination of TDP-43. (A) COS-7 cells expressing TDP-43-WT-YFP and TDP-43-ΔTBD-YFP were treated with vehicle or MG132 for the indicated time periods. Cells were fixed and immunolabeled for the cytoplasmic protein G3BP1 (yellow) and ubiquitin (magenta), and counterstained with Hoechst 33342 (blue). Both TDP-43-YFP proteins formed nuclear foci upon proteasome inhibition (green). Nuclear foci of TDP-43-ΔTBD were co-labeled with ubiquitin by 2 h of MG132 treatment, whereas TDP-43-WT was only co-labeled with ubiquitin by 4 h of MG132 treatment. Nuclei are outlined in white dashed lines. (B) Cells were quantified for the presence of nuclear TDP-43-YFP foci and for nuclear TDP-43-YFP foci that co-labeled with ubiquitin. Both TDP-43 variant proteins (WT and ΔTBD) formed nuclear foci in response to MG132 treatment. Cells were quantified for the presence of nuclear TDP-43-YFP foci that co-labeled with ubiquitin. A significant increase in nuclear TDP-43-ΔTBD foci co-labeled with ubiquitin was detected at an earlier time point compared with TDP-43-WT (2 h versus 4 h). Mean±s.e.m. of three independent experiments. Two-way ANOVA (left graph, P
    Figure Legend Snippet: Deletion of the TBD promotes MG132-induced nuclear ubiquitination of TDP-43. (A) COS-7 cells expressing TDP-43-WT-YFP and TDP-43-ΔTBD-YFP were treated with vehicle or MG132 for the indicated time periods. Cells were fixed and immunolabeled for the cytoplasmic protein G3BP1 (yellow) and ubiquitin (magenta), and counterstained with Hoechst 33342 (blue). Both TDP-43-YFP proteins formed nuclear foci upon proteasome inhibition (green). Nuclear foci of TDP-43-ΔTBD were co-labeled with ubiquitin by 2 h of MG132 treatment, whereas TDP-43-WT was only co-labeled with ubiquitin by 4 h of MG132 treatment. Nuclei are outlined in white dashed lines. (B) Cells were quantified for the presence of nuclear TDP-43-YFP foci and for nuclear TDP-43-YFP foci that co-labeled with ubiquitin. Both TDP-43 variant proteins (WT and ΔTBD) formed nuclear foci in response to MG132 treatment. Cells were quantified for the presence of nuclear TDP-43-YFP foci that co-labeled with ubiquitin. A significant increase in nuclear TDP-43-ΔTBD foci co-labeled with ubiquitin was detected at an earlier time point compared with TDP-43-WT (2 h versus 4 h). Mean±s.e.m. of three independent experiments. Two-way ANOVA (left graph, P

    Techniques Used: Expressing, Immunolabeling, Inhibition, Labeling, Variant Assay

    Tnks-1/2 inhibitor G007-LK reduces TDP-43-associated loss of rat primary cortical neurons. (A) Cortical neurons isolated from Sprague Dawley embryos (E16-E18) were seeded in 24-well plate format at a density of 100,000 neurons. After 15-18 days in vitro (DIV) neurons were virally infected with either HSV-LacZ or HSV-TDP-43 and treated with DMSO or G007-LK. Neurons were fixed and immunostained at 7 days post infection (DPI). See Fig. S2 for expanded images. (B) Neurons were immunolabeled for the neuronal marker tubulin β-III chain (green) and counterstained with Hoechst 33342 (blue). Arrows indicate neurons. (C) Viral infection with HSV-TDP-43 at 5 moi resulted in significant loss in cortical neurons compared with the HSV-LacZ control. Co-treatment with G007-LK (at 1 and 10 µM) significantly suppressed TDP-43-associated neuronal loss. Graph shows individual data points and the mean±s.d. from the same pregnant female analyzed by one-way ANOVA ( P
    Figure Legend Snippet: Tnks-1/2 inhibitor G007-LK reduces TDP-43-associated loss of rat primary cortical neurons. (A) Cortical neurons isolated from Sprague Dawley embryos (E16-E18) were seeded in 24-well plate format at a density of 100,000 neurons. After 15-18 days in vitro (DIV) neurons were virally infected with either HSV-LacZ or HSV-TDP-43 and treated with DMSO or G007-LK. Neurons were fixed and immunostained at 7 days post infection (DPI). See Fig. S2 for expanded images. (B) Neurons were immunolabeled for the neuronal marker tubulin β-III chain (green) and counterstained with Hoechst 33342 (blue). Arrows indicate neurons. (C) Viral infection with HSV-TDP-43 at 5 moi resulted in significant loss in cortical neurons compared with the HSV-LacZ control. Co-treatment with G007-LK (at 1 and 10 µM) significantly suppressed TDP-43-associated neuronal loss. Graph shows individual data points and the mean±s.d. from the same pregnant female analyzed by one-way ANOVA ( P

    Techniques Used: Isolation, In Vitro, Infection, Immunolabeling, Marker

    MG132-treatment led to the formation of nuclear TDP-43 foci that co-label with ubiquitin.  (A) TDP-43-WT-GFP diffusely localizes to the nucleus whereas TDP-43-ΔNLS/PBM-GFP is diffusely cytoplasmic. Addition of an exogenous NLS from either TDP-43 (-ΔNLS/PBM TDP-43 ) or hnRNPA1 (-ΔNLS/PBM A1 ) localizes TDP-43-ΔNLS/PBM-GFP to the nucleus. COS-7 cells were treated with DMSO for 3 h, fixed and counterstained with Hoechst 33342. (B) Treatment with 10 µM MG132 (3 h) led to the formation of nuclear TDP-43-WT-GFP foci that co-labeled with ubiquitin, whereas TDP-43-ΔNLS/PBM-GFP in the cytoplasm remained diffuse. TDP-43-ΔNLS/PBM re-localized to the nucleus by an exogenous NLS, from either TDP-43 (-ΔNLS/PBM TDP-43 ) or hnRNPA1 (-ΔNLS/PBM A1 ), formed MG132-induced nuclear foci that co-labeled with ubiquitin. These data suggest that mutation in the NLS/PBM does not inhibit foci formation. COS-7 cells were immunolabeled for ubiquitin, fixed and counterstained with Hoechst 33342. (C) TDP-43-GFP localized to the nucleus (-WT, -ΔNLS/PBM TDP-43  and -ΔNLS/PBM A1 ) forms foci that co-label with ubiquitin upon treatment with 10 µM MG132 (3 h). Cells were quantified for the presence of nuclear TDP-43-GFP foci and the presence of nuclear TDP-43-GFP foci that co-labeled with ubiquitin. Mean±s.e.m. of three independent experiments. Two-way-ANOVA (both graphs,  P
    Figure Legend Snippet: MG132-treatment led to the formation of nuclear TDP-43 foci that co-label with ubiquitin. (A) TDP-43-WT-GFP diffusely localizes to the nucleus whereas TDP-43-ΔNLS/PBM-GFP is diffusely cytoplasmic. Addition of an exogenous NLS from either TDP-43 (-ΔNLS/PBM TDP-43 ) or hnRNPA1 (-ΔNLS/PBM A1 ) localizes TDP-43-ΔNLS/PBM-GFP to the nucleus. COS-7 cells were treated with DMSO for 3 h, fixed and counterstained with Hoechst 33342. (B) Treatment with 10 µM MG132 (3 h) led to the formation of nuclear TDP-43-WT-GFP foci that co-labeled with ubiquitin, whereas TDP-43-ΔNLS/PBM-GFP in the cytoplasm remained diffuse. TDP-43-ΔNLS/PBM re-localized to the nucleus by an exogenous NLS, from either TDP-43 (-ΔNLS/PBM TDP-43 ) or hnRNPA1 (-ΔNLS/PBM A1 ), formed MG132-induced nuclear foci that co-labeled with ubiquitin. These data suggest that mutation in the NLS/PBM does not inhibit foci formation. COS-7 cells were immunolabeled for ubiquitin, fixed and counterstained with Hoechst 33342. (C) TDP-43-GFP localized to the nucleus (-WT, -ΔNLS/PBM TDP-43 and -ΔNLS/PBM A1 ) forms foci that co-label with ubiquitin upon treatment with 10 µM MG132 (3 h). Cells were quantified for the presence of nuclear TDP-43-GFP foci and the presence of nuclear TDP-43-GFP foci that co-labeled with ubiquitin. Mean±s.e.m. of three independent experiments. Two-way-ANOVA (both graphs, P

    Techniques Used: Labeling, Mutagenesis, Immunolabeling

    Tnks-1/2 inhibition promotes nuclear localization of TDP-43. (A) Treatment with Tnks-1/2 inhibitor G007-LK (10 µM) reduced cytoplasmic accumulation of TDP-43-WT-GFP. Cells were immunostained for the cytoplasmic protein G3BP1 (magenta) and counterstained with Hoechst 33342 (blue). Inner dashed line marks the nuclear boundary and outer solid line marks the cytoplasmic boundary. (B) Treatment of cells with Tnks-1/2 inhibitors G007-LK and IWR1-endo led to a significant reduction in the percentage of cells with cytoplasmic TDP-43-GFP. The percentage of cells with cytoplasmic TDP-43-GFP showing a diffuse GFP signal or GFP-positive puncta was quantified. Mean±s.e.m. of three independent experiments. One-way ANOVA ( P =0.0032) and a Tukey's tests. (C) Treatment with the Tnks-1/2 inhibitor XAV939 led to a significant reduction in the percentage of cells with cytoplasmic TDP-43-GFP. Mean±s.e.m. of three independent experiments. One-way ANOVA ( P
    Figure Legend Snippet: Tnks-1/2 inhibition promotes nuclear localization of TDP-43. (A) Treatment with Tnks-1/2 inhibitor G007-LK (10 µM) reduced cytoplasmic accumulation of TDP-43-WT-GFP. Cells were immunostained for the cytoplasmic protein G3BP1 (magenta) and counterstained with Hoechst 33342 (blue). Inner dashed line marks the nuclear boundary and outer solid line marks the cytoplasmic boundary. (B) Treatment of cells with Tnks-1/2 inhibitors G007-LK and IWR1-endo led to a significant reduction in the percentage of cells with cytoplasmic TDP-43-GFP. The percentage of cells with cytoplasmic TDP-43-GFP showing a diffuse GFP signal or GFP-positive puncta was quantified. Mean±s.e.m. of three independent experiments. One-way ANOVA ( P =0.0032) and a Tukey's tests. (C) Treatment with the Tnks-1/2 inhibitor XAV939 led to a significant reduction in the percentage of cells with cytoplasmic TDP-43-GFP. Mean±s.e.m. of three independent experiments. One-way ANOVA ( P

    Techniques Used: Inhibition

    5) Product Images from "Secretion of a mammalian chondroitinase ABC aids glial integration at PNS/CNS boundaries"

    Article Title: Secretion of a mammalian chondroitinase ABC aids glial integration at PNS/CNS boundaries

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-67526-0

    mChABC can be transduced, expressed, and secreted by Schwann cells. Schwann cells were control, bChABC treated, or transduced with LV-plasmid control, LV-mChABC, LV-fGFP, or LV-mChABC + LV-fGFP ( a – d ) Images show ( a ) LV-plasmid control and ( b ) LV-mChABC + LV-fGFP transduced cells immunostained for Hoechst-33342 (blue); GFP (green) and p75 (red), scale bar = 40 μm. ( c ) Transduction did not alter rate of Schwann cell division (N = 4, one-way ANOVA F(5,18) = 0.528, p = 0.753). ( s ) The same transduction efficiencies were achieved for LV-fGFP and LV-mChABC + LV-fGFP cells (N = 30, one-way ANOVA F(5,174) = 6.932, p
    Figure Legend Snippet: mChABC can be transduced, expressed, and secreted by Schwann cells. Schwann cells were control, bChABC treated, or transduced with LV-plasmid control, LV-mChABC, LV-fGFP, or LV-mChABC + LV-fGFP ( a – d ) Images show ( a ) LV-plasmid control and ( b ) LV-mChABC + LV-fGFP transduced cells immunostained for Hoechst-33342 (blue); GFP (green) and p75 (red), scale bar = 40 μm. ( c ) Transduction did not alter rate of Schwann cell division (N = 4, one-way ANOVA F(5,18) = 0.528, p = 0.753). ( s ) The same transduction efficiencies were achieved for LV-fGFP and LV-mChABC + LV-fGFP cells (N = 30, one-way ANOVA F(5,174) = 6.932, p

    Techniques Used: Transduction, Plasmid Preparation

    6) Product Images from "An Alginate-Based Hydrogel with a High Angiogenic Capacity and a High Osteogenic Potential"

    Article Title: An Alginate-Based Hydrogel with a High Angiogenic Capacity and a High Osteogenic Potential

    Journal: BioResearch Open Access

    doi: 10.1089/biores.2020.0010

    Viability of cells cultured on the alginate-based hydrogel for 24 or 96 h. The alginate-based hydrogel was seeded with MSCs alone (A, D) , ECs alone (B, E), or co-cultured MSCs and ECs (C, F) . The hydrogel was then cultured for 24 or 96 h. Representative fluorescence images of MSCs previously labeled with cell tracker green (A, D) , ECs (B, E) , or a co-culture of MSCs + ECs (C F) seeded on alginate-based hydrogel after 24 h (A–C) or 96 h (D–F) . The cell nuclei were stained with Hoechst 33342 (blue). Red fluorescence (after staining with ethidium homodimer-1) indicated the loss of plasma membrane integrity (i.e., dead cells). ECs, endothelial cells; MSCs, mesenchymal stem cells.
    Figure Legend Snippet: Viability of cells cultured on the alginate-based hydrogel for 24 or 96 h. The alginate-based hydrogel was seeded with MSCs alone (A, D) , ECs alone (B, E), or co-cultured MSCs and ECs (C, F) . The hydrogel was then cultured for 24 or 96 h. Representative fluorescence images of MSCs previously labeled with cell tracker green (A, D) , ECs (B, E) , or a co-culture of MSCs + ECs (C F) seeded on alginate-based hydrogel after 24 h (A–C) or 96 h (D–F) . The cell nuclei were stained with Hoechst 33342 (blue). Red fluorescence (after staining with ethidium homodimer-1) indicated the loss of plasma membrane integrity (i.e., dead cells). ECs, endothelial cells; MSCs, mesenchymal stem cells.

    Techniques Used: Cell Culture, Fluorescence, Labeling, Co-Culture Assay, Staining

    7) Product Images from "In vitro analysis of putative cancer stem cell populations and chemosensitivity in the SW480 and SW620 colon cancer metastasis model"

    Article Title: In vitro analysis of putative cancer stem cell populations and chemosensitivity in the SW480 and SW620 colon cancer metastasis model

    Journal: Oncology Letters

    doi: 10.3892/ol.2018.8431

    Sensitivity of adherent SW620 and SW480 cells to anti-cancer drugs. (A) Sensitivity of SW480 and SW620 cells exposed to increasing dosage of 5-fluorouracil (0–300 µM), oxaliplatin (0–200 µM), geldanamycin (0–1,000 nM) and novobiocin (0–500 µM) analysed by WST-1 after 72 h (represented as the mean ± standard deviation for triplicate independent analysis). EC 50 : The concentration at which 50% of cells remain viable. The P-value was determined for the EC 50 : Values, comparing between the cell lines for each drug, using an unpaired t-test. (B) Western blot analysis of Hsp90 levels in replicate SW480 and SW620 whole cell lysates. (C) Distribution of Hsp90 in SW480 and SW620 cells by fluorescent microscopy (nucleus stained with Hoechst 33342). Images were taken using a Zeiss AxioVert.A1 Fluorescence LED inverted microscope (scale bars, 0.02 mm). ***P
    Figure Legend Snippet: Sensitivity of adherent SW620 and SW480 cells to anti-cancer drugs. (A) Sensitivity of SW480 and SW620 cells exposed to increasing dosage of 5-fluorouracil (0–300 µM), oxaliplatin (0–200 µM), geldanamycin (0–1,000 nM) and novobiocin (0–500 µM) analysed by WST-1 after 72 h (represented as the mean ± standard deviation for triplicate independent analysis). EC 50 : The concentration at which 50% of cells remain viable. The P-value was determined for the EC 50 : Values, comparing between the cell lines for each drug, using an unpaired t-test. (B) Western blot analysis of Hsp90 levels in replicate SW480 and SW620 whole cell lysates. (C) Distribution of Hsp90 in SW480 and SW620 cells by fluorescent microscopy (nucleus stained with Hoechst 33342). Images were taken using a Zeiss AxioVert.A1 Fluorescence LED inverted microscope (scale bars, 0.02 mm). ***P

    Techniques Used: Standard Deviation, Concentration Assay, Western Blot, Microscopy, Staining, Fluorescence, Inverted Microscopy

    Putative cancer stem cell identification by Hoechst 33342 dye exclusion in SW480 and SW620 cells. The proportion of cells representing the SP in the SW480 and SW620 cell lines was determined using Hoechst 33342 dye efflux. (A) Representative dot plots indicating the SP within SW480 and SW620 cells stained with 5 µg/ml Hoechst 33342. The SP was detected by the loss in the fluorescent population (H blue-/ H red− ) upon treatment with the ABCG2 inhibitor, verapamil (50 µM). (B) Putative cancer stem cell populations representative of the average percentage SP observed (mean ± standard deviation) from triplicate independent experiments measured as: SP = (% cells H blue−/ H red− without verapamil) - (% cells H blue−/ H red− with verapamil). No significant differences were identified when comparing the SP of the SW480 and SW620 cell lines (t-test with Welch's correction). SP, side population.
    Figure Legend Snippet: Putative cancer stem cell identification by Hoechst 33342 dye exclusion in SW480 and SW620 cells. The proportion of cells representing the SP in the SW480 and SW620 cell lines was determined using Hoechst 33342 dye efflux. (A) Representative dot plots indicating the SP within SW480 and SW620 cells stained with 5 µg/ml Hoechst 33342. The SP was detected by the loss in the fluorescent population (H blue-/ H red− ) upon treatment with the ABCG2 inhibitor, verapamil (50 µM). (B) Putative cancer stem cell populations representative of the average percentage SP observed (mean ± standard deviation) from triplicate independent experiments measured as: SP = (% cells H blue−/ H red− without verapamil) - (% cells H blue−/ H red− with verapamil). No significant differences were identified when comparing the SP of the SW480 and SW620 cell lines (t-test with Welch's correction). SP, side population.

    Techniques Used: Staining, Standard Deviation, T-Test

    8) Product Images from "DICER regulates the expression of major satellite repeat transcripts and meiotic chromosome segregation during spermatogenesis"

    Article Title: DICER regulates the expression of major satellite repeat transcripts and meiotic chromosome segregation during spermatogenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa460

    Meiotic chromosome segregation is defective in Dicer1 knockout germ cells. ( A ) Nuclei of early round spermatids at stage I-II appear unevenly sized in Dicer1 cKO testes compared to control (CTRL). Nuclei were stained by DAPI (grey). Basal lamina is indicated with a grey line. The border between pachytene spermatocyte (PSpc) and round spermatid (RS) layers is indicated with a dashed white line. ES, elongating spermatids. Some examples of abnormally large spermatid nuclei are indicated with white arrows. Scale bar: 20 μm. ( B ) A representative Hoechst 33342 live-cell fluorescent DNA staining and flow cytometric analysis of DNA ploidy for testicular cells isolated from CTRL and Dicer1 cKO mice. Dicer1 cKO testes display the expected 1C, 2C and 4C populations but specifically lack the elongating spermatid population. 1C-R, round spermatids; 1C–E, elongating spermatids; 2C, diploid cells; 4C, tetraploid cells; FSC, forward scatter light. See also Supplementary Figure S6 . ( C , D ) An in-depth analysis of the 1C-R population reveals that the number of round spermatids having a higher DNA content is significantly higher in Dicer1 cKO than in CTRL, n = 3, SEM, ** P
    Figure Legend Snippet: Meiotic chromosome segregation is defective in Dicer1 knockout germ cells. ( A ) Nuclei of early round spermatids at stage I-II appear unevenly sized in Dicer1 cKO testes compared to control (CTRL). Nuclei were stained by DAPI (grey). Basal lamina is indicated with a grey line. The border between pachytene spermatocyte (PSpc) and round spermatid (RS) layers is indicated with a dashed white line. ES, elongating spermatids. Some examples of abnormally large spermatid nuclei are indicated with white arrows. Scale bar: 20 μm. ( B ) A representative Hoechst 33342 live-cell fluorescent DNA staining and flow cytometric analysis of DNA ploidy for testicular cells isolated from CTRL and Dicer1 cKO mice. Dicer1 cKO testes display the expected 1C, 2C and 4C populations but specifically lack the elongating spermatid population. 1C-R, round spermatids; 1C–E, elongating spermatids; 2C, diploid cells; 4C, tetraploid cells; FSC, forward scatter light. See also Supplementary Figure S6 . ( C , D ) An in-depth analysis of the 1C-R population reveals that the number of round spermatids having a higher DNA content is significantly higher in Dicer1 cKO than in CTRL, n = 3, SEM, ** P

    Techniques Used: Knock-Out, Staining, Isolation, Mouse Assay

    9) Product Images from "Ultrasound-assisted multicomponent synthesis of 4H-pyrans in water and DNA binding studies"

    Article Title: Ultrasound-assisted multicomponent synthesis of 4H-pyrans in water and DNA binding studies

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-68076-1

    Titration experiment of a solution of ctDNA (200 µM) and Hoechst 33342 (2.5 µM) in Tris/HCl (0.1 M, pH 7.2) with increasing concentrations of compound 3n (0–150 µM). The plot is normalized to the maximum intensity of the initial experiment.
    Figure Legend Snippet: Titration experiment of a solution of ctDNA (200 µM) and Hoechst 33342 (2.5 µM) in Tris/HCl (0.1 M, pH 7.2) with increasing concentrations of compound 3n (0–150 µM). The plot is normalized to the maximum intensity of the initial experiment.

    Techniques Used: Titration

    Titration experiment of a solution of ctDNA (200 µM) and Hoechst 33342 (2.5 µM) in Tris/HCl (0.1 M, pH 7.2) with increasing concentrations of compound 3m (0–150 µM). The plot is normalized to the maximum intensity of the initial experiment.
    Figure Legend Snippet: Titration experiment of a solution of ctDNA (200 µM) and Hoechst 33342 (2.5 µM) in Tris/HCl (0.1 M, pH 7.2) with increasing concentrations of compound 3m (0–150 µM). The plot is normalized to the maximum intensity of the initial experiment.

    Techniques Used: Titration

    10) Product Images from "Akt and Src mediate the photocrosslinked fibroin-induced neural differentiation."

    Article Title: Akt and Src mediate the photocrosslinked fibroin-induced neural differentiation.

    Journal: Neuroreport

    doi: 10.1097/WNR.0000000000001482

    Spreading and differentiation of SH-SY5Y cells on different substrates. (a) Cells were stained with phalloidin-Alexa 488 (actin) and Hoechst 33342 (nuclei). Scale bar, 25 µm. (b) Cells were stained with antibodies to βIII-tubulin and NCAM and counterstained with Hoechst 33342 at days 3 and 12 of culture. Bar, 25 µm. * P
    Figure Legend Snippet: Spreading and differentiation of SH-SY5Y cells on different substrates. (a) Cells were stained with phalloidin-Alexa 488 (actin) and Hoechst 33342 (nuclei). Scale bar, 25 µm. (b) Cells were stained with antibodies to βIII-tubulin and NCAM and counterstained with Hoechst 33342 at days 3 and 12 of culture. Bar, 25 µm. * P

    Techniques Used: Staining

    11) Product Images from "Vacancies on 2D transition metal dichalcogenides elicit ferroptotic cell death"

    Article Title: Vacancies on 2D transition metal dichalcogenides elicit ferroptotic cell death

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17300-7

    Biomarkers of ferroptosis in TMD-exposed cells. a Representative images of Fe 2+ in cells by confocal microscope. BEAS-2B cells treated by 100 μM Fe(NH 4 ) 2 (SO 4 ) 2 , 200 μg/mL WS 2 and MoS 2 were stained by FeRhoNox (red) to visualize the cellular distribution of Fe 2+ . Hoechst 33342 and WGA were used to stain nuclei (blue) and cell membrane (green), respectively (scale bar: 10 μm). Shown are the representative images from three independent cell samples. b Effects of Fe 2+ chelators on WS 2 and MoS 2 induced cytotoxicity ( n = 3 biologically independent cell samples). BEAS-2B cells pretreated by 2 mM DFP or 0.4 mM DFX were exposed to 200 μg/mL WS 2 and MoS 2 and examine cell viability after 48 h incubation. Data are presented as mean values ± SD. *** p
    Figure Legend Snippet: Biomarkers of ferroptosis in TMD-exposed cells. a Representative images of Fe 2+ in cells by confocal microscope. BEAS-2B cells treated by 100 μM Fe(NH 4 ) 2 (SO 4 ) 2 , 200 μg/mL WS 2 and MoS 2 were stained by FeRhoNox (red) to visualize the cellular distribution of Fe 2+ . Hoechst 33342 and WGA were used to stain nuclei (blue) and cell membrane (green), respectively (scale bar: 10 μm). Shown are the representative images from three independent cell samples. b Effects of Fe 2+ chelators on WS 2 and MoS 2 induced cytotoxicity ( n = 3 biologically independent cell samples). BEAS-2B cells pretreated by 2 mM DFP or 0.4 mM DFX were exposed to 200 μg/mL WS 2 and MoS 2 and examine cell viability after 48 h incubation. Data are presented as mean values ± SD. *** p

    Techniques Used: Microscopy, Staining, Whole Genome Amplification, Incubation

    12) Product Images from "Deep Learning of Cancer Stem Cell Morphology Using Conditional Generative Adversarial Networks"

    Article Title: Deep Learning of Cancer Stem Cell Morphology Using Conditional Generative Adversarial Networks

    Journal: Biomolecules

    doi: 10.3390/biom10060931

    Deep learning of miPS-T47Dcm cell morphology in tumor tissue. ( a ) Primary subcutaneous tumors; arrowhead indicates tumor tissue. ( b ) Tumor tissue section visualized with phase contrast, Hoechst 33342, and GFP fluorescence using objection lens 20×. P: phase contrast; H: Hoechst 33342; G: GFP. Bars = 100 μm. An area in overlay (P, H, G) is shown in detail. ( c , e ) Effect of training steps on loss functions. ( d , f ) Output examples by AI models. Test phase contrast images were subjected to AI models for depicting fluorescence images. Input and target are the pair image for the depicted image evaluation. The AI models trained with the set of ( c , d ) phase contrast and GFP images, and ( e , f ) Hoechst 33342 overlaid-phase contrast and GFP images. Bars = 100 µm.
    Figure Legend Snippet: Deep learning of miPS-T47Dcm cell morphology in tumor tissue. ( a ) Primary subcutaneous tumors; arrowhead indicates tumor tissue. ( b ) Tumor tissue section visualized with phase contrast, Hoechst 33342, and GFP fluorescence using objection lens 20×. P: phase contrast; H: Hoechst 33342; G: GFP. Bars = 100 μm. An area in overlay (P, H, G) is shown in detail. ( c , e ) Effect of training steps on loss functions. ( d , f ) Output examples by AI models. Test phase contrast images were subjected to AI models for depicting fluorescence images. Input and target are the pair image for the depicted image evaluation. The AI models trained with the set of ( c , d ) phase contrast and GFP images, and ( e , f ) Hoechst 33342 overlaid-phase contrast and GFP images. Bars = 100 µm.

    Techniques Used: Fluorescence

    Comparison between depicted CSC image in tissue by AI models and original GFP fluorescence. Images of Hoechst 33342 overlaid-phase contrast (+) or not overlaid-phase contrast (-) were used for training AI for AI models. The AI output images and each true target image were compared using the values of ( a ) recall, ( b ) precision, ( c ) specificity, ( d ) F-measure, and ( e ) image correlation coefficient. Closed circles indicate maximum values. Mean ± S.D., n = 684. *** p
    Figure Legend Snippet: Comparison between depicted CSC image in tissue by AI models and original GFP fluorescence. Images of Hoechst 33342 overlaid-phase contrast (+) or not overlaid-phase contrast (-) were used for training AI for AI models. The AI output images and each true target image were compared using the values of ( a ) recall, ( b ) precision, ( c ) specificity, ( d ) F-measure, and ( e ) image correlation coefficient. Closed circles indicate maximum values. Mean ± S.D., n = 684. *** p

    Techniques Used: Fluorescence

    13) Product Images from "A Stable Thoracic Hox Code and Epimorphosis Characterize Posterior Regeneration in Capitella teleta"

    Article Title: A Stable Thoracic Hox Code and Epimorphosis Characterize Posterior Regeneration in Capitella teleta

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0149724

    Blastema formation, cell division patterns and nerve cord dynamics during posterior regeneration of C . teleta . All panels show posterior ends of amputated juveniles in ventral view, with anterior to the left. Amputations were conducted at the boundary of segment 10 and 11. White dotted lines indicate approximate position of amputation, and all tissue to the right of dotted lines is regenerated tissue. The panels in each row are from a single individual. The regenerative process is described as progressing through different stages (left of rows), and specific stain, chemical or antibody is indicated at the top of columns. (A-G) Hoechst 33342 staining showing nuclei; (A’-G’) EdU incorporation marking dividing cells; (A”-G”) anti-acetylated α-tubulin labeling neurites. Scale bars in A”-G” are 50 μm. White circle in A”, F” and G” shows cilia of the hindgut. White arrowheads show mature ganglia in A, A”, B, B”, G and G”. Open arrowheads show peripheral nerves in A” and G”. White arrows in A”, F” and G” mark the Y-shaped neurites which extend into the pygidium. Blue arrows in E” mark nerve tracts. Blue arrows in F” mark medial axons that branch from the ventral nerve cord and red arrows mark axons that branch from lateral nerves. White arrows in E’ indicate bilateral clusters of EdU-positive cells. Chaetae are autofluorescent and are visible in A’, B’, C’ and D’.
    Figure Legend Snippet: Blastema formation, cell division patterns and nerve cord dynamics during posterior regeneration of C . teleta . All panels show posterior ends of amputated juveniles in ventral view, with anterior to the left. Amputations were conducted at the boundary of segment 10 and 11. White dotted lines indicate approximate position of amputation, and all tissue to the right of dotted lines is regenerated tissue. The panels in each row are from a single individual. The regenerative process is described as progressing through different stages (left of rows), and specific stain, chemical or antibody is indicated at the top of columns. (A-G) Hoechst 33342 staining showing nuclei; (A’-G’) EdU incorporation marking dividing cells; (A”-G”) anti-acetylated α-tubulin labeling neurites. Scale bars in A”-G” are 50 μm. White circle in A”, F” and G” shows cilia of the hindgut. White arrowheads show mature ganglia in A, A”, B, B”, G and G”. Open arrowheads show peripheral nerves in A” and G”. White arrows in A”, F” and G” mark the Y-shaped neurites which extend into the pygidium. Blue arrows in E” mark nerve tracts. Blue arrows in F” mark medial axons that branch from the ventral nerve cord and red arrows mark axons that branch from lateral nerves. White arrows in E’ indicate bilateral clusters of EdU-positive cells. Chaetae are autofluorescent and are visible in A’, B’, C’ and D’.

    Techniques Used: Staining, Labeling

    14) Product Images from "RNAi screen reveals a role of SPHK2 in dengue virus–mediated apoptosis in hepatic cell lines"

    Article Title: RNAi screen reveals a role of SPHK2 in dengue virus–mediated apoptosis in hepatic cell lines

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0188121

    Alteration of SPHK2 protein expression and subcellular localization in DENV-infected Huh7 cells and HepG2 cells. Huh7 cells, HepG2 cells and A549 cells were infected with DENV at the MOI of 1, MOI of 5 and MOI of 1 for 24 hours, respectively. SPHK2 and DENV E proteins were det ected by IFA and represented as green and red fluorescence, respectively. Hoechst 33342 was used to stain the nucleus. Mock cells (upper panel) and DENV-infected cells (lower panel) are (A) Huh7 cells (B) HepG2 cells and (C) A549 cells, respectively.
    Figure Legend Snippet: Alteration of SPHK2 protein expression and subcellular localization in DENV-infected Huh7 cells and HepG2 cells. Huh7 cells, HepG2 cells and A549 cells were infected with DENV at the MOI of 1, MOI of 5 and MOI of 1 for 24 hours, respectively. SPHK2 and DENV E proteins were det ected by IFA and represented as green and red fluorescence, respectively. Hoechst 33342 was used to stain the nucleus. Mock cells (upper panel) and DENV-infected cells (lower panel) are (A) Huh7 cells (B) HepG2 cells and (C) A549 cells, respectively.

    Techniques Used: Expressing, Infection, Immunofluorescence, Fluorescence, Staining

    15) Product Images from "nev (cyfip2) IS REQUIRED FOR RETINAL LAMINATION AND AXON GUIDANCE IN THE ZEBRAFISH RETINOTECTAL SYSTEM"

    Article Title: nev (cyfip2) IS REQUIRED FOR RETINAL LAMINATION AND AXON GUIDANCE IN THE ZEBRAFISH RETINOTECTAL SYSTEM

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2010.05.512

    cyfip2 acts both cell autonomously and cell nonautonomously in lamination. Representative coronal sections through a nev eye with WT donor cells (A–B‴), a WT eye with nev donor cells (C–D‴), and a nev eye with nev donor cells (E–F‴). (A, C, E) Hoechst 33342 stain (blue), biotin-dextran-positive (BDA+) donor cells (red), and isl2b:GFP + donor RGCs (green). Insets are shown magnified below. (B–B‴) WT donor cells in a nev host are misplaced in the IPL, showing nev can act cell nonautonomously. Arrows show a BDA+ donor cell next to a BDA- cell in the IPL. (D–D‴) nev donor cells in a WT host are misplaced in the IPL, showing that nev can also act cell autonomously. Arrows show a BDA+/ isl2b:GFP + cell in the IPL. (F–F‴) Control transplants show nev donor cells in a nev host that are misplaced in the IPL. Arrow shows a BDA+/ isl2b:GFP + cell misplaced in the IPL. Scale bar=50μm.
    Figure Legend Snippet: cyfip2 acts both cell autonomously and cell nonautonomously in lamination. Representative coronal sections through a nev eye with WT donor cells (A–B‴), a WT eye with nev donor cells (C–D‴), and a nev eye with nev donor cells (E–F‴). (A, C, E) Hoechst 33342 stain (blue), biotin-dextran-positive (BDA+) donor cells (red), and isl2b:GFP + donor RGCs (green). Insets are shown magnified below. (B–B‴) WT donor cells in a nev host are misplaced in the IPL, showing nev can act cell nonautonomously. Arrows show a BDA+ donor cell next to a BDA- cell in the IPL. (D–D‴) nev donor cells in a WT host are misplaced in the IPL, showing that nev can also act cell autonomously. Arrows show a BDA+/ isl2b:GFP + cell in the IPL. (F–F‴) Control transplants show nev donor cells in a nev host that are misplaced in the IPL. Arrow shows a BDA+/ isl2b:GFP + cell misplaced in the IPL. Scale bar=50μm.

    Techniques Used: Staining, Activated Clotting Time Assay

    Retinal lamination is disrupted in nev . Coronal sections through a WT eye (A–A‴) and a nev eye (B–B‴) at 5dpf. (A and B) Hoechst 33342 stain to visualize lamination. The retinal ganglion cell (RGC) layer, inner plexiform layer (IPL), inner nuclear layer (INL), and outer nuclear layer (ONL) are all clearly visible at this stage. Arrowheads in B show displaced cells in the IPL of nev . Boxed regions are magnified in A′–A‴ and B′–B‴ and show nuclei labeled with Hoechst 33342 (magenta; A′–A‴, B′–B‴), RGCs labeled with isl2b:GFP (green; A″, B″), and a subset of amacrine cells labeled with anti-parvalbumin (green; A‴, B‴). In nev, RGCs and amacrine cells are intermingled in the IPL. Arrowheads in B″ and B‴ show displaced RGCs and amacrine cells in the IPL, respectively. ON, optic nerve. Scale bar= 50μm.
    Figure Legend Snippet: Retinal lamination is disrupted in nev . Coronal sections through a WT eye (A–A‴) and a nev eye (B–B‴) at 5dpf. (A and B) Hoechst 33342 stain to visualize lamination. The retinal ganglion cell (RGC) layer, inner plexiform layer (IPL), inner nuclear layer (INL), and outer nuclear layer (ONL) are all clearly visible at this stage. Arrowheads in B show displaced cells in the IPL of nev . Boxed regions are magnified in A′–A‴ and B′–B‴ and show nuclei labeled with Hoechst 33342 (magenta; A′–A‴, B′–B‴), RGCs labeled with isl2b:GFP (green; A″, B″), and a subset of amacrine cells labeled with anti-parvalbumin (green; A‴, B‴). In nev, RGCs and amacrine cells are intermingled in the IPL. Arrowheads in B″ and B‴ show displaced RGCs and amacrine cells in the IPL, respectively. ON, optic nerve. Scale bar= 50μm.

    Techniques Used: Staining, Labeling

    16) Product Images from "Analysis of the Interaction between Globular Head Modules of Human C1q and Its Candidate Receptor gC1qR"

    Article Title: Analysis of the Interaction between Globular Head Modules of Human C1q and Its Candidate Receptor gC1qR

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2016.00567

    Interaction of ghA, ghB, and ghC with monocyte/macrophages . PMBCs (1 × 10 6 ) were seeded on 13 mm coverslips and incubated in complete RPMI 1640 medium for 2 weeks at 37°C in 5% CO 2  incubator. Cells were treated with 10 μg of each globular head module and incubated with serum-free RPMI 1640 medium for 1 h at 37°C. After washing with PBS, cells were fixed with 4% PFA, permeabilzed with Triton X-100, and probed with anti-gC1qR polyclonal antibody and anti-MBP monoclonal antibody to reveal gC1qR and bound globular head modules, respectively. Cells were washed and treated with Alexa Fluor 488 conjugated secondary goat anti-rabbit antibody and Alexa Fluor 647 conjugated secondary donkey anti-mouse antibody and the nucleus was stained with Hoechst 33342. Cells were then examined under Leica fluorescence microscope with 40× magnification. In the merged images gC1qR is green; globular heads are red; and nucleus is blue. Arrows point to bound globular heads with colocalization of globular heads and gC1qR seen in orange in the merged images. Scale bars: 10 μm.
    Figure Legend Snippet: Interaction of ghA, ghB, and ghC with monocyte/macrophages . PMBCs (1 × 10 6 ) were seeded on 13 mm coverslips and incubated in complete RPMI 1640 medium for 2 weeks at 37°C in 5% CO 2 incubator. Cells were treated with 10 μg of each globular head module and incubated with serum-free RPMI 1640 medium for 1 h at 37°C. After washing with PBS, cells were fixed with 4% PFA, permeabilzed with Triton X-100, and probed with anti-gC1qR polyclonal antibody and anti-MBP monoclonal antibody to reveal gC1qR and bound globular head modules, respectively. Cells were washed and treated with Alexa Fluor 488 conjugated secondary goat anti-rabbit antibody and Alexa Fluor 647 conjugated secondary donkey anti-mouse antibody and the nucleus was stained with Hoechst 33342. Cells were then examined under Leica fluorescence microscope with 40× magnification. In the merged images gC1qR is green; globular heads are red; and nucleus is blue. Arrows point to bound globular heads with colocalization of globular heads and gC1qR seen in orange in the merged images. Scale bars: 10 μm.

    Techniques Used: Incubation, Staining, Fluorescence, Microscopy

    17) Product Images from "The PI3K and MAPK/p38 pathways control stress granule assembly in a hierarchical manner"

    Article Title: The PI3K and MAPK/p38 pathways control stress granule assembly in a hierarchical manner

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800257

    p38 promotes mTORC1 activation and stress granule formation when PI3K is inactive. (A)  p38 mediates mTORC1 activation when PI3K is inactive. MCF-7 cells were serum-starved and treated with arsenite in the presence of carrier (DMSO) or wortmannin (100 nM, PI3K inhibitor). In addition, the cells were treated with carrier (DMSO) versus LY2228820 (1 μM, p38 inhibitor). MK2-pT334, Akt-pT308, Akt-pS473, p70-S6K-pT389, and eIF2α-pS51 were monitored by immunoblot. Data represent five biological replicates.  (B)  Quantification of data shown in (A) when PI3K is active. Akt-pS473 and p70-S6K-pT389 were compared between carrier (DMSO) and LY2228820-treated cells using a two-way ANOVA followed by a Bonferroni multiple comparison test across five biological replicates. Data represent the mean ± SEM. The  P -values for the Bonferroni multiple comparison tests are shown. *** P  ≤ 0.001.  (C)  Quantification of data shown in (A) when PI3K is inactive. Akt-pS473 and p70-S6K-pT389 were compared between wortmannin- and wortmannin + LY2228820–treated cells using a two-way ANOVA followed by a Bonferroni multiple comparison test across five biological replicates. Data represent the mean ± SEM. The  P -values for the Bonferroni multiple comparison tests are shown. * P  ≤ 0.05; *** P  ≤ 0.001.  (D)  p38 drives mTORC1 activity when PI3K is inactive. Quantification of data shown in   Fig S14A . 4E-BP1-pT37/46 relative intensity was normalized separately for conditions without or with wortmannin. Significance of 4E-BP1-pT37/46 inhibition by LY2228820 was tested using a two-tailed  t  test across five biological replicates. Data represent the mean ± SEM. * P  ≤ 0.05.  (E)  Prediction on the extent of mTORC1 inhibition upon LY2228820 treatment when PI3K is active or inactive. Prediction was performed with model V. The red lines depict the time points measured experimentally (  Fig 5A–C ).  (F)  When PI3K activity declines, p38 drives mTORC1 activity. Quantification of data shown in   Fig S13 L. MCF-7 cells were serum-starved and treated with arsenite for 60 min in the presence of different concentrations of wortmannin (as indicated, PI3K inhibitor) in carrier (DMSO) versus LY2228820 (1 μM, p38 inhibitor)-treated cells. p70-S6K-pT389 relative intensity was normalized separately for each wortmannin concentration. Significance of p70-S6K-pT389 inhibition by LY2228820 was tested using a two-tailed  t  test across five biological replicates. Data represent the mean ± SEM. * P  ≤ 0.05.  (G)  p38 drives mTORC1 activity in several cell lines, as PI3K activity declines. Quantification of data shown in   Fig S14D–G . MCF-7, CAL51, LN18, HEK293T, and HeLa cells were serum-starved and exposed to arsenite for 60 min in combination with wortmannin (100 nM, PI3K inhibitor) and/or LY2228820 (1 mM, p38 inhibitor). Data represent 3–4 biological replicates (see   Fig S14D–G ). 4E-BP1-pT37/46 relative intensity was normalized separately for conditions without or with wortmannin. Significance of 4E-BP1-pT37/46 inhibition by LY2228820 was tested using a two-tailed  t  test across three biological replicates. Data represent the mean ± SEM. * P  ≤ 0.05; ** P  ≤ 0.01.  (H)  Stress granule numbers upon PI3K and p38 inhibition. MCF-7 cells were serum-starved and treated with arsenite for 30 min in the presence of carrier (DMSO), wortmannin (100 nM, PI3K inhibitor), LY2228820 (1 μM, p38 inhibitor), or wortmannin + LY2228820. Stress granules were visualized by immunofluorescence staining of G3BP1. Nuclei were visualized with Hoechst 33342. Data represent four biological replicates. White square indicates region of insert and blue arrow highlights stress granules; scale bar 10 μm.  (I)  Quantification of data shown in (H). The number of stress granules (SGs) per cell (normalized to the arsenite condition) across four biological replicates. Stress granule formation between carrier and LY2228820 as well as wortmannin- and wortmannin + LY2228820–treated cells was compared using a two-tailed  t  test across four biological replicates. Data represent the mean ± SEM. * P  ≤ 0.01. ns, not significant.
    Figure Legend Snippet: p38 promotes mTORC1 activation and stress granule formation when PI3K is inactive. (A) p38 mediates mTORC1 activation when PI3K is inactive. MCF-7 cells were serum-starved and treated with arsenite in the presence of carrier (DMSO) or wortmannin (100 nM, PI3K inhibitor). In addition, the cells were treated with carrier (DMSO) versus LY2228820 (1 μM, p38 inhibitor). MK2-pT334, Akt-pT308, Akt-pS473, p70-S6K-pT389, and eIF2α-pS51 were monitored by immunoblot. Data represent five biological replicates. (B) Quantification of data shown in (A) when PI3K is active. Akt-pS473 and p70-S6K-pT389 were compared between carrier (DMSO) and LY2228820-treated cells using a two-way ANOVA followed by a Bonferroni multiple comparison test across five biological replicates. Data represent the mean ± SEM. The P -values for the Bonferroni multiple comparison tests are shown. *** P ≤ 0.001. (C) Quantification of data shown in (A) when PI3K is inactive. Akt-pS473 and p70-S6K-pT389 were compared between wortmannin- and wortmannin + LY2228820–treated cells using a two-way ANOVA followed by a Bonferroni multiple comparison test across five biological replicates. Data represent the mean ± SEM. The P -values for the Bonferroni multiple comparison tests are shown. * P ≤ 0.05; *** P ≤ 0.001. (D) p38 drives mTORC1 activity when PI3K is inactive. Quantification of data shown in Fig S14A . 4E-BP1-pT37/46 relative intensity was normalized separately for conditions without or with wortmannin. Significance of 4E-BP1-pT37/46 inhibition by LY2228820 was tested using a two-tailed t test across five biological replicates. Data represent the mean ± SEM. * P ≤ 0.05. (E) Prediction on the extent of mTORC1 inhibition upon LY2228820 treatment when PI3K is active or inactive. Prediction was performed with model V. The red lines depict the time points measured experimentally ( Fig 5A–C ). (F) When PI3K activity declines, p38 drives mTORC1 activity. Quantification of data shown in Fig S13 L. MCF-7 cells were serum-starved and treated with arsenite for 60 min in the presence of different concentrations of wortmannin (as indicated, PI3K inhibitor) in carrier (DMSO) versus LY2228820 (1 μM, p38 inhibitor)-treated cells. p70-S6K-pT389 relative intensity was normalized separately for each wortmannin concentration. Significance of p70-S6K-pT389 inhibition by LY2228820 was tested using a two-tailed t test across five biological replicates. Data represent the mean ± SEM. * P ≤ 0.05. (G) p38 drives mTORC1 activity in several cell lines, as PI3K activity declines. Quantification of data shown in Fig S14D–G . MCF-7, CAL51, LN18, HEK293T, and HeLa cells were serum-starved and exposed to arsenite for 60 min in combination with wortmannin (100 nM, PI3K inhibitor) and/or LY2228820 (1 mM, p38 inhibitor). Data represent 3–4 biological replicates (see Fig S14D–G ). 4E-BP1-pT37/46 relative intensity was normalized separately for conditions without or with wortmannin. Significance of 4E-BP1-pT37/46 inhibition by LY2228820 was tested using a two-tailed t test across three biological replicates. Data represent the mean ± SEM. * P ≤ 0.05; ** P ≤ 0.01. (H) Stress granule numbers upon PI3K and p38 inhibition. MCF-7 cells were serum-starved and treated with arsenite for 30 min in the presence of carrier (DMSO), wortmannin (100 nM, PI3K inhibitor), LY2228820 (1 μM, p38 inhibitor), or wortmannin + LY2228820. Stress granules were visualized by immunofluorescence staining of G3BP1. Nuclei were visualized with Hoechst 33342. Data represent four biological replicates. White square indicates region of insert and blue arrow highlights stress granules; scale bar 10 μm. (I) Quantification of data shown in (H). The number of stress granules (SGs) per cell (normalized to the arsenite condition) across four biological replicates. Stress granule formation between carrier and LY2228820 as well as wortmannin- and wortmannin + LY2228820–treated cells was compared using a two-tailed t test across four biological replicates. Data represent the mean ± SEM. * P ≤ 0.01. ns, not significant.

    Techniques Used: Activation Assay, Activity Assay, Inhibition, Two Tailed Test, Concentration Assay, Immunofluorescence, Staining

    Stress activates mTORC1 to promote stress granule formation. (A)  Arsenite stress enhances phosphorylation of eIF2α-S51 and mTORC1 substrates. MCF-7 cells were serum-starved and treated with arsenite. p70-S6K-pT389, 4EBP1-pT37/46, and eIF2α-pS51 were monitored by immunoblot. Data represent six biological replicates.  (B)  Quantification of data shown in (A). eIF2α-pS51, p70-S6K-pT389, and 4E-BP1-pT37/46 levels were compared between control and arsenite-treated cells using a two-tailed  t  test across six biological replicates. Data represent the mean ± SEM. ** P  ≤ 0.01; *** P  ≤ 0.001.  (C)  Arsenite induces stress granules. MCF-7 cells were serum-starved and exposed to arsenite for 30 min. Stress granules were visualized by immunofluorescence staining of G3BP1. Data represent three biological replicates. Nuclei were visualized using Hoechst 33342. Scale bar: 10 μm.  (D)  mTOR mediates induction of p70-S6K-pT389 and 4EBP1-pT37/46 by stress. MCF-7 cells were serum-starved and treated with arsenite in the presence of carrier (DMSO), everolimus (100 nM, mTORC1 inhibitor), or AZD8055 (100 nM, mTOR inhibitor). p70-S6K-pT389, 4E-BP1-pT37/46, G3BP1, and eIF2α-pS51 were monitored by immunoblot. Data represent four biological replicates.  (E)  Quantification of data shown in (D). p70-S6K-pT389 and 4E-BP1-pT37/46 levels were compared between the different treatments using a two-tailed  t  test across four biological replicates. Data represent the mean ± SEM. ns, not significant; * P  ≤ 0.05; ** P  ≤ 0.01.  (F)  mTOR inhibition reduces stress granule numbers. MCF-7 cells were serum-starved and treated with arsenite for 30 min in the presence of carrier (DMSO), everolimus (100 nM, mTORC1 inhibitor), or AZD8055 (100 nM, mTOR inhibitor). Stress granules were visualized by immunofluorescence staining of G3BP1. Nuclei were visualized with Hoechst 33342. Data represent three biological replicates. White squares indicate region of insert and blue arrows highlight stress granules; scale bar 10 μm.  (G)  Quantification of data shown in (F): number of stress granules (SGs) per cell (normalized to the arsenite condition) across three biological replicates. Stress granule numbers were compared between the carrier and everolimus, or carrier and AZD8055-treated cells, using a two-tailed  t  test across three biological replicates. Data represent the mean ± SEM. * P  ≤ 0.05.
    Figure Legend Snippet: Stress activates mTORC1 to promote stress granule formation. (A) Arsenite stress enhances phosphorylation of eIF2α-S51 and mTORC1 substrates. MCF-7 cells were serum-starved and treated with arsenite. p70-S6K-pT389, 4EBP1-pT37/46, and eIF2α-pS51 were monitored by immunoblot. Data represent six biological replicates. (B) Quantification of data shown in (A). eIF2α-pS51, p70-S6K-pT389, and 4E-BP1-pT37/46 levels were compared between control and arsenite-treated cells using a two-tailed t test across six biological replicates. Data represent the mean ± SEM. ** P ≤ 0.01; *** P ≤ 0.001. (C) Arsenite induces stress granules. MCF-7 cells were serum-starved and exposed to arsenite for 30 min. Stress granules were visualized by immunofluorescence staining of G3BP1. Data represent three biological replicates. Nuclei were visualized using Hoechst 33342. Scale bar: 10 μm. (D) mTOR mediates induction of p70-S6K-pT389 and 4EBP1-pT37/46 by stress. MCF-7 cells were serum-starved and treated with arsenite in the presence of carrier (DMSO), everolimus (100 nM, mTORC1 inhibitor), or AZD8055 (100 nM, mTOR inhibitor). p70-S6K-pT389, 4E-BP1-pT37/46, G3BP1, and eIF2α-pS51 were monitored by immunoblot. Data represent four biological replicates. (E) Quantification of data shown in (D). p70-S6K-pT389 and 4E-BP1-pT37/46 levels were compared between the different treatments using a two-tailed t test across four biological replicates. Data represent the mean ± SEM. ns, not significant; * P ≤ 0.05; ** P ≤ 0.01. (F) mTOR inhibition reduces stress granule numbers. MCF-7 cells were serum-starved and treated with arsenite for 30 min in the presence of carrier (DMSO), everolimus (100 nM, mTORC1 inhibitor), or AZD8055 (100 nM, mTOR inhibitor). Stress granules were visualized by immunofluorescence staining of G3BP1. Nuclei were visualized with Hoechst 33342. Data represent three biological replicates. White squares indicate region of insert and blue arrows highlight stress granules; scale bar 10 μm. (G) Quantification of data shown in (F): number of stress granules (SGs) per cell (normalized to the arsenite condition) across three biological replicates. Stress granule numbers were compared between the carrier and everolimus, or carrier and AZD8055-treated cells, using a two-tailed t test across three biological replicates. Data represent the mean ± SEM. * P ≤ 0.05.

    Techniques Used: Two Tailed Test, Immunofluorescence, Staining, Inhibition

    PI3K enhances stress granule formation through mTORC1 activation. (A)  mTORC1 is activated by arsenite in a PI3K-dependent manner. MCF-7 cells were serum-starved and treated with arsenite in the presence of carrier (DMSO), wortmannin (100 nM, PI3K inhibitor), or GDC-0941 (1 μM, PI3K inhibitor). Akt-pT308, TSC2-pT1462, p70-S6K-pT389, and eIF2α-pS51 were monitored by immunoblot. Data represent three biological replicates.  (B)  Quantification of data shown in (A). Akt-pT308, TSC2-pT1462, and p70-S6K-pT389 levels were compared between carrier and wortmannin as well as carrier and GDC-0941-treated cells using a two-tailed  t  test across three biological replicates. Data represent the mean ± SEM. * P  ≤ 0.05; ** P  ≤ 0.01; *** P  ≤ 0.001.  (C)  PDK1 mediates stress activation of mTORC1. MCF-7 cells were serum-starved and treated with arsenite in the presence of carrier (DMSO) or GSK2334470 (1 μM, PDK1 inhibitor). Akt-pT308, p70-S6K-pT389, and eIF2α-pS51 were monitored by immunoblot. Data represent three biological replicates.  (D)  Quantification of data shown in (C). Akt-pT308 and p70-S6K-pT389 levels were compared between carrier and GSK2334470-treated cells using a two-way ANOVA followed by a Bonferroni multiple comparison test across three biological replicates. Data represent the mean ± SEM.  P -values for the Bonferroni multiple comparison tests are shown above the columns. * P  ≤ 0.05; *** P  ≤ 0.001.  (E)  Stress activation of mTORC1 is IRS1 independent. MCF-7 cells treated with non-targeting scramble siRNA (siControl) or with two different siRNA sequences targeting IRS1 (siIRS1 #1 and #2) were serum-starved and treated with arsenite. Akt-pT308, p70-S6K-pT389, and eIF2α-pS51 were monitored by immunoblot. Data represent four biological replicates.  (F)  Quantification of data shown in (E). IRS1, Akt-pT308, and p70-S6K-pT389 levels were compared between siControl, siIRS1 #1–, and siIRS2 #2–treated cells using a two-way ANOVA followed by a Bonferroni multiple comparison test across four biological replicates. Data represent the mean ± SEM.  P -values for the Bonferroni multiple comparison tests are depicted above the corresponding time point. ns, not significant; * P  ≤ 0.05; ** P  ≤ 0.01; *** P  ≤ 0.001.  (G)  Stress activates RAS. MCF-7 cells were serum-starved and treated with arsenite. RAS activity was measured using GST-coupled RAF-RAS–binding domain pull down experiments. Data represent three biological replicates.  (H)  Quantification of data shown in (G). RAS-GTP levels were compared over an arsenite time course using a one-way ANOVA followed by a Bonferroni multiple comparison test across three biological replicates. Data represent the mean ± SEM. The significances for the Bonferroni multiple comparison tests between time points is shown above the column, the  P -value for the ANOVA is  P  = 0.0318. * P  ≤ 0.05.  (I)  PI3K inhibition reduces stress granule numbers. MCF-7 cells were serum-starved and treated with arsenite for 30 min in the presence of carrier (DMSO) or wortmannin (100 nM, PI3K inhibitor). Stress granules were visualized by immunofluorescence staining of G3BP1. Nuclei were visualized with Hoechst 33342. Data represent four biological replicates. White square indicates region of insert and blue arrow highlights stress granules; scale bar 10 μm.  (J)  Quantification of data shown in (I): number of stress granules (SGs) per cell (normalized to the arsenite condition) across four biological replicates. A two-tailed  t  test across four biological replicates was applied. Data represent the mean ± SEM. ** P  ≤ 0.01; *** P  ≤ 0.001.
    Figure Legend Snippet: PI3K enhances stress granule formation through mTORC1 activation. (A) mTORC1 is activated by arsenite in a PI3K-dependent manner. MCF-7 cells were serum-starved and treated with arsenite in the presence of carrier (DMSO), wortmannin (100 nM, PI3K inhibitor), or GDC-0941 (1 μM, PI3K inhibitor). Akt-pT308, TSC2-pT1462, p70-S6K-pT389, and eIF2α-pS51 were monitored by immunoblot. Data represent three biological replicates. (B) Quantification of data shown in (A). Akt-pT308, TSC2-pT1462, and p70-S6K-pT389 levels were compared between carrier and wortmannin as well as carrier and GDC-0941-treated cells using a two-tailed t test across three biological replicates. Data represent the mean ± SEM. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. (C) PDK1 mediates stress activation of mTORC1. MCF-7 cells were serum-starved and treated with arsenite in the presence of carrier (DMSO) or GSK2334470 (1 μM, PDK1 inhibitor). Akt-pT308, p70-S6K-pT389, and eIF2α-pS51 were monitored by immunoblot. Data represent three biological replicates. (D) Quantification of data shown in (C). Akt-pT308 and p70-S6K-pT389 levels were compared between carrier and GSK2334470-treated cells using a two-way ANOVA followed by a Bonferroni multiple comparison test across three biological replicates. Data represent the mean ± SEM. P -values for the Bonferroni multiple comparison tests are shown above the columns. * P ≤ 0.05; *** P ≤ 0.001. (E) Stress activation of mTORC1 is IRS1 independent. MCF-7 cells treated with non-targeting scramble siRNA (siControl) or with two different siRNA sequences targeting IRS1 (siIRS1 #1 and #2) were serum-starved and treated with arsenite. Akt-pT308, p70-S6K-pT389, and eIF2α-pS51 were monitored by immunoblot. Data represent four biological replicates. (F) Quantification of data shown in (E). IRS1, Akt-pT308, and p70-S6K-pT389 levels were compared between siControl, siIRS1 #1–, and siIRS2 #2–treated cells using a two-way ANOVA followed by a Bonferroni multiple comparison test across four biological replicates. Data represent the mean ± SEM. P -values for the Bonferroni multiple comparison tests are depicted above the corresponding time point. ns, not significant; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. (G) Stress activates RAS. MCF-7 cells were serum-starved and treated with arsenite. RAS activity was measured using GST-coupled RAF-RAS–binding domain pull down experiments. Data represent three biological replicates. (H) Quantification of data shown in (G). RAS-GTP levels were compared over an arsenite time course using a one-way ANOVA followed by a Bonferroni multiple comparison test across three biological replicates. Data represent the mean ± SEM. The significances for the Bonferroni multiple comparison tests between time points is shown above the column, the P -value for the ANOVA is P = 0.0318. * P ≤ 0.05. (I) PI3K inhibition reduces stress granule numbers. MCF-7 cells were serum-starved and treated with arsenite for 30 min in the presence of carrier (DMSO) or wortmannin (100 nM, PI3K inhibitor). Stress granules were visualized by immunofluorescence staining of G3BP1. Nuclei were visualized with Hoechst 33342. Data represent four biological replicates. White square indicates region of insert and blue arrow highlights stress granules; scale bar 10 μm. (J) Quantification of data shown in (I): number of stress granules (SGs) per cell (normalized to the arsenite condition) across four biological replicates. A two-tailed t test across four biological replicates was applied. Data represent the mean ± SEM. ** P ≤ 0.01; *** P ≤ 0.001.

    Techniques Used: Activation Assay, Two Tailed Test, Activity Assay, Binding Assay, Inhibition, Immunofluorescence, Staining

    18) Product Images from "d(−) Lactic Acid-Induced Adhesion of Bovine Neutrophils onto Endothelial Cells Is Dependent on Neutrophils Extracellular Traps Formation and CD11b Expression"

    Article Title: d(−) Lactic Acid-Induced Adhesion of Bovine Neutrophils onto Endothelial Cells Is Dependent on Neutrophils Extracellular Traps Formation and CD11b Expression

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00975

    d (−) lactic acid-induced neutrophil extracellular trap (NET) formation. (A,B) Immunofluorescent images of bovine neutrophils treated with 5 mM d (−) lactic acid for 30 min using anti-H 4 citrullinated 3 (A) and CD11b (B) markers, with picogreen and Hoechst 33342 as DNA staining probes, respectively; representative images of five independent experiments, scale bars: 60 µm for (A) and 50 µm for (B) . (C) Fold of control [(number of NETs/number of neutrophils) × 100]. (D) Images from scanning electronic microscopy of neutrophils treated with d (−) lactic acid, scale bar: 5 µm, representative images from three independent experiments. (E) Extracellular DNA quantifications of supernatants from neutrophils treated with different concentrations of d (−) lactic acid (0.1–5.0 mM), ionomycin (positive control) or vehicle using picogreen to stain DNA, n = 5. ** P
    Figure Legend Snippet: d (−) lactic acid-induced neutrophil extracellular trap (NET) formation. (A,B) Immunofluorescent images of bovine neutrophils treated with 5 mM d (−) lactic acid for 30 min using anti-H 4 citrullinated 3 (A) and CD11b (B) markers, with picogreen and Hoechst 33342 as DNA staining probes, respectively; representative images of five independent experiments, scale bars: 60 µm for (A) and 50 µm for (B) . (C) Fold of control [(number of NETs/number of neutrophils) × 100]. (D) Images from scanning electronic microscopy of neutrophils treated with d (−) lactic acid, scale bar: 5 µm, representative images from three independent experiments. (E) Extracellular DNA quantifications of supernatants from neutrophils treated with different concentrations of d (−) lactic acid (0.1–5.0 mM), ionomycin (positive control) or vehicle using picogreen to stain DNA, n = 5. ** P

    Techniques Used: Staining, Microscopy, Positive Control

    19) Product Images from "CRISPR/Cas9-mediated gene knockout reveals a guardian role of NF-κB/RelA in maintaining the homeostasis of human vascular cells"

    Article Title: CRISPR/Cas9-mediated gene knockout reveals a guardian role of NF-κB/RelA in maintaining the homeostasis of human vascular cells

    Journal: Protein & Cell

    doi: 10.1007/s13238-018-0560-5

    Derivation of RelA −/− VECs, VSMCs, and MSCs from RelA −/− ESCs . (A) Flow cytometric analysis of WT and  RelA −/−  VECs with VEC-specific markers CD34 and CD201. IgG-FITC and IgG-PE were used as isotype controls. (B) Flow cytometric analysis of WT and  RelA −/−  VSMCs with VSMC-specific marker, CD140b. IgG-APC was used as an isotype control. (C) Flow cytometric analysis of WT and  RelA −/−  MSCs with MSC-specific markers, CD73, CD90 and CD105. IgG-FITC, IgG-PE and IgG-APC were used as isotype controls. (D) Immunostaining of WT and  RelA −/−  VECs with VEC-specific markers, vWF and CD31. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (E) Immunostaining of WT and  RelA −/−  VSMCs with VSMC-specific markers, SM22 and Calponin. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (F) Western blot analysis of RelA protein in WT and  RelA −/−  VECs, VSMCs and MSCs, respectively. β-Actin was used as a loading control. (G) Immunostaining of RelA in WT and  RelA −/−  VECs, VSMCs and MSCs under basal condition. DNA was labeled by Hoechst 33342. Scale bar, 10 μm
    Figure Legend Snippet: Derivation of RelA −/− VECs, VSMCs, and MSCs from RelA −/− ESCs . (A) Flow cytometric analysis of WT and RelA −/− VECs with VEC-specific markers CD34 and CD201. IgG-FITC and IgG-PE were used as isotype controls. (B) Flow cytometric analysis of WT and RelA −/− VSMCs with VSMC-specific marker, CD140b. IgG-APC was used as an isotype control. (C) Flow cytometric analysis of WT and RelA −/− MSCs with MSC-specific markers, CD73, CD90 and CD105. IgG-FITC, IgG-PE and IgG-APC were used as isotype controls. (D) Immunostaining of WT and RelA −/− VECs with VEC-specific markers, vWF and CD31. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (E) Immunostaining of WT and RelA −/− VSMCs with VSMC-specific markers, SM22 and Calponin. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (F) Western blot analysis of RelA protein in WT and RelA −/− VECs, VSMCs and MSCs, respectively. β-Actin was used as a loading control. (G) Immunostaining of RelA in WT and RelA −/− VECs, VSMCs and MSCs under basal condition. DNA was labeled by Hoechst 33342. Scale bar, 10 μm

    Techniques Used: Flow Cytometry, Marker, Immunostaining, Labeling, Western Blot

    Transcriptomic analysis revealed the effect of IκBα deficiency on RelA signaling . (A) Schemic diagram of  IκB α knockout strategy via CRISPR/Cas9 in human ESCs. A neomycin-resistant cassette (Neo) was included for positive selection. (B) Genomic PCR verification of the deletion of  IκB α exon 1 in  IκB α −/−  ESCs. Water was used as a negative control (NC). (C) Western blot analysis showing IκBα protein levels in WT and  IκB α −/−  ESCs. β-Actin was used as a loading control. (D) Transcriptional signal of IκBα in WT and  IκB α −/−  in VECs, VSMCs and MSCs. Transcriptional signals were normalized by RPKM at bin size 10 bp. (E) Venn diagrams showing the overlap between upregulated genes in  IκB α −/−  vascular cells and downregulated genes in  RelA −/−  vascular cells compared to WT vascular cells under basal condition. (F) Heatmaps revealing the transcriptional patterns of genes upregulated only in  IκB α −/−  vascular cells (pink), downregulated only in  RelA −/−  vascular cells (green), and genes overlapped (blue) under basal condition. (G) Venn diagrams showing the overalp between upregulated genes in  IκB α −/−  vascular cells and downregulated genes in  RelA −/−  vascular cells compared to WT vascular cells upon TNFα treatment. (H) Heatmaps revealing the transcriptional patterns of genes upregulated only in  IκB α −/−  vascular cells (pink), downregulated only in  RelA −/−  vascular cells (green) and genes overlapped (blue) upon TNFα treatment. (I) Immunostaining of RelA in WT and  IκB α −/−  MSCs under basal and TNFα-treated conditions. DNA was labeled by Hoechst 33342. Scale bar, 10 μm
    Figure Legend Snippet: Transcriptomic analysis revealed the effect of IκBα deficiency on RelA signaling . (A) Schemic diagram of IκB α knockout strategy via CRISPR/Cas9 in human ESCs. A neomycin-resistant cassette (Neo) was included for positive selection. (B) Genomic PCR verification of the deletion of IκB α exon 1 in IκB α −/− ESCs. Water was used as a negative control (NC). (C) Western blot analysis showing IκBα protein levels in WT and IκB α −/− ESCs. β-Actin was used as a loading control. (D) Transcriptional signal of IκBα in WT and IκB α −/− in VECs, VSMCs and MSCs. Transcriptional signals were normalized by RPKM at bin size 10 bp. (E) Venn diagrams showing the overlap between upregulated genes in IκB α −/− vascular cells and downregulated genes in RelA −/− vascular cells compared to WT vascular cells under basal condition. (F) Heatmaps revealing the transcriptional patterns of genes upregulated only in IκB α −/− vascular cells (pink), downregulated only in RelA −/− vascular cells (green), and genes overlapped (blue) under basal condition. (G) Venn diagrams showing the overalp between upregulated genes in IκB α −/− vascular cells and downregulated genes in RelA −/− vascular cells compared to WT vascular cells upon TNFα treatment. (H) Heatmaps revealing the transcriptional patterns of genes upregulated only in IκB α −/− vascular cells (pink), downregulated only in RelA −/− vascular cells (green) and genes overlapped (blue) upon TNFα treatment. (I) Immunostaining of RelA in WT and IκB α −/− MSCs under basal and TNFα-treated conditions. DNA was labeled by Hoechst 33342. Scale bar, 10 μm

    Techniques Used: Knock-Out, CRISPR, Selection, Polymerase Chain Reaction, Negative Control, Western Blot, Immunostaining, Labeling

    RelA deficiency affected vascular cell homeostasis . (A) Immunostaining and flow cytometry analysis of the Dil-Ac-LDL uptake capacity in WT and  RelA −/−  VECs. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (B) Representative micrographs of matrigel tubes formed by WT and  RelA −/−  VECs  in vitro  ( n  = 3). Scale bar, 3 mm. (C) Oil red O staining of WT and  RelA −/−  adipocytes derived from MSCs, respectively. The quantification of adipocytes was measured by absorbance at 510 nm ( n  = 4). ***  P
    Figure Legend Snippet: RelA deficiency affected vascular cell homeostasis . (A) Immunostaining and flow cytometry analysis of the Dil-Ac-LDL uptake capacity in WT and RelA −/− VECs. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (B) Representative micrographs of matrigel tubes formed by WT and RelA −/− VECs in vitro ( n  = 3). Scale bar, 3 mm. (C) Oil red O staining of WT and RelA −/− adipocytes derived from MSCs, respectively. The quantification of adipocytes was measured by absorbance at 510 nm ( n  = 4). *** P

    Techniques Used: Immunostaining, Flow Cytometry, Cytometry, Labeling, In Vitro, Staining, Derivative Assay

    Generation and characterization of RelA −/− human ESCs . (A) Schemic diagram of  RelA  knockout strategy via CRISPR/Cas9 in human ESCs. A neomycin-resistant cassette (Neo) was included for positive selection. (B) Genomic PCR verification of  RelA  exon 1 knockout in  RelA −/−  ESCs. Water was used as a negative control (NC). (C) Western blot analysis of RelA protein levels in WT and  RelA −/−  ESCs. β-Actin was used as a loading control. (D) Representative colony morphology and immunostaining of pluripotency markers in WT and  RelA −/−  ESCs. Scale bar, 30 μm. (E) Measurement of the mRNA expression levels of pluripotency markers by semi-quantitative PCR in WT and  RelA −/−  ESCs.  18S  was used as a loading control. (F) Teratoma analysis of WT and  RelA −/−  ESCs with three germ layer markers. Markers were stained in red; DNA was labeled in blue by Hoechst 33342. Scale bar, 100 μm. (G) Karyotype analysis of WT and  RelA −/−  ESCs. (H) Ki67 immunostaining in WT and  RelA −/−  ESCs. Ki67 was stained in red; DNA was labeled by Hoechst 33342. Scale bar, 30 μm
    Figure Legend Snippet: Generation and characterization of RelA −/− human ESCs . (A) Schemic diagram of RelA knockout strategy via CRISPR/Cas9 in human ESCs. A neomycin-resistant cassette (Neo) was included for positive selection. (B) Genomic PCR verification of RelA exon 1 knockout in RelA −/− ESCs. Water was used as a negative control (NC). (C) Western blot analysis of RelA protein levels in WT and RelA −/− ESCs. β-Actin was used as a loading control. (D) Representative colony morphology and immunostaining of pluripotency markers in WT and RelA −/− ESCs. Scale bar, 30 μm. (E) Measurement of the mRNA expression levels of pluripotency markers by semi-quantitative PCR in WT and RelA −/− ESCs. 18S was used as a loading control. (F) Teratoma analysis of WT and RelA −/− ESCs with three germ layer markers. Markers were stained in red; DNA was labeled in blue by Hoechst 33342. Scale bar, 100 μm. (G) Karyotype analysis of WT and RelA −/− ESCs. (H) Ki67 immunostaining in WT and RelA −/− ESCs. Ki67 was stained in red; DNA was labeled by Hoechst 33342. Scale bar, 30 μm

    Techniques Used: Knock-Out, CRISPR, Selection, Polymerase Chain Reaction, Negative Control, Western Blot, Immunostaining, Expressing, Real-time Polymerase Chain Reaction, Staining, Labeling

    20) Product Images from "Severe Early-Onset Obesity Due to Bioinactive Leptin Caused by a p.N103K Mutation in the Leptin Gene"

    Article Title: Severe Early-Onset Obesity Due to Bioinactive Leptin Caused by a p.N103K Mutation in the Leptin Gene

    Journal: The Journal of Clinical Endocrinology and Metabolism

    doi: 10.1210/jc.2015-2263

    Bioinactivity due to a p.N103K mutation in leptin. A, Body weight curves of the patients (boy, blue line; girl, red line) and the healthy, normal-weight sister (black line) compared to body weight percentiles for boys (blue dotted lines) and girls (red dotted lines). The triangle indicates the start of treatment. B, Pedigree indicating the mutation status of the parents and their three children. The BMI is given in kilograms per square meter, along with the corresponding age in years. C, HEK293 cells were transfected with empty vector (Ctrl) or vector encoding wild-type (Wt) or p.N103K (N103K) leptin. After 48 hours, media supernatants (Sup) were collected, cell lysates (Lys) were prepared, and leptin immunoreactivity was examined by Western blot analysis. β-Actin served as a loading control. One representative experiment out of three performed is shown. D, HEK293 cells were transfected with either empty vector (Ev) or vector encoding the human leptin receptor (hLR). Cells were treated with media supernatants from HEK293 cells transfected with empty vector (Ctrl) or vector encoding wild-type (Wt) or p.N103K (N103K) leptin for 15 minutes. Concentrations of wild-type and p.N103K leptin were adjusted to 30 ng/mL. Cell lysates were prepared and subjected to Western blot analysis using pStat3 and Stat3 antibodies. α-Tubulin served as a loading control. One representative experiment out of three performed is shown. E, HEK293 cells were transfected with vector encoding the human leptin receptor. Cells were treated with media supernatants from HEK293 cells transfected with vector encoding mCherry-labeled wild-type (Wt) or p.N103K (N103K) leptin for 45 minutes. Concentrations of wild-type and p.N103K leptin were adjusted to 30 ng/mL. The cells were fixed, the nuclei were stained with Hoechst 33342 (Hoechst), and the cells were analyzed by fluorescence microscopy. Blue, nuclei; red, mCherry-labeled leptin. One representative experiment out of three performed is shown.
    Figure Legend Snippet: Bioinactivity due to a p.N103K mutation in leptin. A, Body weight curves of the patients (boy, blue line; girl, red line) and the healthy, normal-weight sister (black line) compared to body weight percentiles for boys (blue dotted lines) and girls (red dotted lines). The triangle indicates the start of treatment. B, Pedigree indicating the mutation status of the parents and their three children. The BMI is given in kilograms per square meter, along with the corresponding age in years. C, HEK293 cells were transfected with empty vector (Ctrl) or vector encoding wild-type (Wt) or p.N103K (N103K) leptin. After 48 hours, media supernatants (Sup) were collected, cell lysates (Lys) were prepared, and leptin immunoreactivity was examined by Western blot analysis. β-Actin served as a loading control. One representative experiment out of three performed is shown. D, HEK293 cells were transfected with either empty vector (Ev) or vector encoding the human leptin receptor (hLR). Cells were treated with media supernatants from HEK293 cells transfected with empty vector (Ctrl) or vector encoding wild-type (Wt) or p.N103K (N103K) leptin for 15 minutes. Concentrations of wild-type and p.N103K leptin were adjusted to 30 ng/mL. Cell lysates were prepared and subjected to Western blot analysis using pStat3 and Stat3 antibodies. α-Tubulin served as a loading control. One representative experiment out of three performed is shown. E, HEK293 cells were transfected with vector encoding the human leptin receptor. Cells were treated with media supernatants from HEK293 cells transfected with vector encoding mCherry-labeled wild-type (Wt) or p.N103K (N103K) leptin for 45 minutes. Concentrations of wild-type and p.N103K leptin were adjusted to 30 ng/mL. The cells were fixed, the nuclei were stained with Hoechst 33342 (Hoechst), and the cells were analyzed by fluorescence microscopy. Blue, nuclei; red, mCherry-labeled leptin. One representative experiment out of three performed is shown.

    Techniques Used: Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Labeling, Staining, Fluorescence, Microscopy

    21) Product Images from "Prestin Contributes to Membrane Compartmentalization and Is Required for Normal Innervation of Outer Hair Cells"

    Article Title: Prestin Contributes to Membrane Compartmentalization and Is Required for Normal Innervation of Outer Hair Cells

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2018.00211

    Representative immunofluorescent images of the organ of Corti from prestin WT (A) and KO (B) mice. (C) An enlarged OHC from an apical turn of prestin-KO cochlea (P21, male). Radial cochlear sections from WT (P138, male) and prestin-KO (P37, male) mice were stained with anti-KCNQ4 (green). Phalloidin-Alexa 546 (red), and Hoechst 33342 (blue) for actin and nuclei, respectively, were also used. White arrow indicates KCNQ4 signal on the LM of OHC in prestin-KO mouse. IHC, inner hair cells; DCs, Dieters’ cells. Scale bars, 10 μm.
    Figure Legend Snippet: Representative immunofluorescent images of the organ of Corti from prestin WT (A) and KO (B) mice. (C) An enlarged OHC from an apical turn of prestin-KO cochlea (P21, male). Radial cochlear sections from WT (P138, male) and prestin-KO (P37, male) mice were stained with anti-KCNQ4 (green). Phalloidin-Alexa 546 (red), and Hoechst 33342 (blue) for actin and nuclei, respectively, were also used. White arrow indicates KCNQ4 signal on the LM of OHC in prestin-KO mouse. IHC, inner hair cells; DCs, Dieters’ cells. Scale bars, 10 μm.

    Techniques Used: Mouse Assay, Staining, Immunohistochemistry

    Abnormal localization of MOC synapses in OHCs from prestin-KO mice. Representative images are shown for each panel. (A) 3D reconstructions of z-stack images of cochlear whole mounts from the middle turn of 129/B6 WT (P55, female) and prestin-KO (P26, male) mice, showing three rows of OHCs oriented such that the cuticular plate is facing upward and the first OHC row is toward the front. In WT, MOC terminals are found at the bottom of the OHCs, while in the prestin-KO, MOC terminals are disorganized and extend upward to the cuticular plate. Green: anti-synaptophysin for synaptic vesicles. Red: phalloidin-Alexa 546 for actin. Blue: Hoechst 33342 for nuclei. (B) 3D reconstructions of z-stack images of cochlear whole mounts from the middle turn of 499-prestin-KI (P21, female) mouse, showing three rows of OHCs. MOC terminals (stained with anti-synaptophysin) were found at the bottom of the OHCs. (C) 3D reconstruction of z-stack images of a cochlear whole mount from the apical turn of a prestin-KO (P22, male), stained with anti-VAChT (green), phalloidin-Alexa 546 (actin) and Hoechst 33342 (nuclei; OHC nuclei were marked with white dotted circles). MOC terminals above OHC nucleus (white arrows) are often present in addition to the subnuclear terminals at the bottom (yellow arrow). Scale bar, 10 μm. (D) TEM image of prestin-KO OHC with two MOC terminals at the side (E) and the bottom (F) of the cell. Boxed regions correspond to the images in (E,F) , respectively. 1200×, scale bar, 1 μm. (E,F) Enlarged images of supranuclear synapse along the LM of OHCs (E) and subnuclear synapse (F) . Both terminals in (E,F) contain numerous small synaptic vesicles. 10,000×, scale bars, 100 nm.
    Figure Legend Snippet: Abnormal localization of MOC synapses in OHCs from prestin-KO mice. Representative images are shown for each panel. (A) 3D reconstructions of z-stack images of cochlear whole mounts from the middle turn of 129/B6 WT (P55, female) and prestin-KO (P26, male) mice, showing three rows of OHCs oriented such that the cuticular plate is facing upward and the first OHC row is toward the front. In WT, MOC terminals are found at the bottom of the OHCs, while in the prestin-KO, MOC terminals are disorganized and extend upward to the cuticular plate. Green: anti-synaptophysin for synaptic vesicles. Red: phalloidin-Alexa 546 for actin. Blue: Hoechst 33342 for nuclei. (B) 3D reconstructions of z-stack images of cochlear whole mounts from the middle turn of 499-prestin-KI (P21, female) mouse, showing three rows of OHCs. MOC terminals (stained with anti-synaptophysin) were found at the bottom of the OHCs. (C) 3D reconstruction of z-stack images of a cochlear whole mount from the apical turn of a prestin-KO (P22, male), stained with anti-VAChT (green), phalloidin-Alexa 546 (actin) and Hoechst 33342 (nuclei; OHC nuclei were marked with white dotted circles). MOC terminals above OHC nucleus (white arrows) are often present in addition to the subnuclear terminals at the bottom (yellow arrow). Scale bar, 10 μm. (D) TEM image of prestin-KO OHC with two MOC terminals at the side (E) and the bottom (F) of the cell. Boxed regions correspond to the images in (E,F) , respectively. 1200×, scale bar, 1 μm. (E,F) Enlarged images of supranuclear synapse along the LM of OHCs (E) and subnuclear synapse (F) . Both terminals in (E,F) contain numerous small synaptic vesicles. 10,000×, scale bars, 100 nm.

    Techniques Used: Mouse Assay, Staining, Transmission Electron Microscopy

    22) Product Images from "PLK1 (polo like kinase 1) inhibits MTOR complex 1 and promotes autophagy"

    Article Title: PLK1 (polo like kinase 1) inhibits MTOR complex 1 and promotes autophagy

    Journal: Autophagy

    doi: 10.1080/15548627.2016.1263781

    PLK1 resides with MTORC1 at lysosomes, and overexpression of active PLK1 decreases lysosomal association of the PLK1-MTORC1 complex. (A) Immunofluorescence analysis of HeLa cells that were cultured in full medium and stained with LAMP2 and MTOR antibodies. White regions in merged image (right) of LAMP2 (green) and MTOR (magenta) indicate colocalization. Nuclei were stained with Hoechst 33342. Scale bar 20 µm. Representative images are shown for n = 3 independent experiments. (B) Immunofluorescence analysis of HeLa cells that were cultured in full medium and stained with PLK1 and MTOR antibodies. White regions in merged image (right) of PLK1 (green) and MTOR (magenta) indicate colocalization. Nuclei were stained with Hoechst 33342. Scale bar 20 µm. Representative images are shown for n = 3 independent experiments. (C) Immunofluorescence analysis of HeLa cells that were synchronized in prometaphase with nocodazole for 16 h and released for 30 min in full medium. Cells were stained with PLK1 antibody. Nuclei were stained with Hoechst 33342. Scale bar: 10 µm. Representative images of cells in metaphase (left) and anaphase (right) are shown for n = 3 independent experiments. (D) Analysis of input sample taken before fractionation in sucrose gradient (E). The mitotic cell lysate was collected from HeLa sh PLK1  knockdown cultures without mitotic shake-off. Samples were analyzed by immunoblotting. Data shown are representative of n = 2 independent experiments. (E) HeLa cells were starved for 1 h for amino acids and growth factors and stimulated with amino acids and insulin for 35 min. Samples were separated in a 10 to 40% sucrose gradient and analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments. (F) Quantification of data shown in (E) for n = 3 independent experiments. The percentage of PLK1 in either the lysosomal or the nuclear fraction is displayed. Data are represented as mean ± SEM. (G) HeLa cells were cultured in full medium. Immunoprecipitation (IP) was performed with PLK1 and control (mock) antibodies. Samples were analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments. (H) HeLa cells overexpressing wild type MYC-PLK1 (WT) or empty vector were cultured in full medium. Immunoprecipitation (IP) was performed with PLK1 and control (mock) antibodies. Samples were analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments. (I) HeLa cells overexpressing MYC-PLK1 (WT) or kinase-defective, dominant negative MYC-PLK1 K82R  were cultured in full medium. Immunoprecipitation (IP) was performed with PLK1 and control (mock) antibodies. Samples were analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments. (J) HeLa cells overexpressing MYC-PLK1 (WT) or kinase-defective, dominant negative MYC-PLK1 K82R  were starved for 1 h for amino acids and growth factors, and stimulated with amino acids and insulin for 35 min. Cells were then starved for amino acids for 10 min as indicated, and samples were analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments.
    Figure Legend Snippet: PLK1 resides with MTORC1 at lysosomes, and overexpression of active PLK1 decreases lysosomal association of the PLK1-MTORC1 complex. (A) Immunofluorescence analysis of HeLa cells that were cultured in full medium and stained with LAMP2 and MTOR antibodies. White regions in merged image (right) of LAMP2 (green) and MTOR (magenta) indicate colocalization. Nuclei were stained with Hoechst 33342. Scale bar 20 µm. Representative images are shown for n = 3 independent experiments. (B) Immunofluorescence analysis of HeLa cells that were cultured in full medium and stained with PLK1 and MTOR antibodies. White regions in merged image (right) of PLK1 (green) and MTOR (magenta) indicate colocalization. Nuclei were stained with Hoechst 33342. Scale bar 20 µm. Representative images are shown for n = 3 independent experiments. (C) Immunofluorescence analysis of HeLa cells that were synchronized in prometaphase with nocodazole for 16 h and released for 30 min in full medium. Cells were stained with PLK1 antibody. Nuclei were stained with Hoechst 33342. Scale bar: 10 µm. Representative images of cells in metaphase (left) and anaphase (right) are shown for n = 3 independent experiments. (D) Analysis of input sample taken before fractionation in sucrose gradient (E). The mitotic cell lysate was collected from HeLa sh PLK1 knockdown cultures without mitotic shake-off. Samples were analyzed by immunoblotting. Data shown are representative of n = 2 independent experiments. (E) HeLa cells were starved for 1 h for amino acids and growth factors and stimulated with amino acids and insulin for 35 min. Samples were separated in a 10 to 40% sucrose gradient and analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments. (F) Quantification of data shown in (E) for n = 3 independent experiments. The percentage of PLK1 in either the lysosomal or the nuclear fraction is displayed. Data are represented as mean ± SEM. (G) HeLa cells were cultured in full medium. Immunoprecipitation (IP) was performed with PLK1 and control (mock) antibodies. Samples were analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments. (H) HeLa cells overexpressing wild type MYC-PLK1 (WT) or empty vector were cultured in full medium. Immunoprecipitation (IP) was performed with PLK1 and control (mock) antibodies. Samples were analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments. (I) HeLa cells overexpressing MYC-PLK1 (WT) or kinase-defective, dominant negative MYC-PLK1 K82R were cultured in full medium. Immunoprecipitation (IP) was performed with PLK1 and control (mock) antibodies. Samples were analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments. (J) HeLa cells overexpressing MYC-PLK1 (WT) or kinase-defective, dominant negative MYC-PLK1 K82R were starved for 1 h for amino acids and growth factors, and stimulated with amino acids and insulin for 35 min. Cells were then starved for amino acids for 10 min as indicated, and samples were analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments.

    Techniques Used: Over Expression, Immunofluorescence, Cell Culture, Staining, Fractionation, Immunoprecipitation, Plasmid Preparation, Dominant Negative Mutation

    PLK1 inhibition impairs autophagy in nonmitotic cells and in  C. elegans  dauers. (A) HeLa cells were transfected with mRFP-GFP-LC3 tandem reporter, followed by  PLK1  siRNA transfection on the next day. Cells were either kept in full medium, or starved for serum and amino acids for 16 h. Mitotic cells were removed by shake-off before fixation of cells 24 h after siRNA transfection. Representative images are shown for each condition. Scale bar 10 µm. Data shown are representative of n = 2 independent experiments. (B) Quantification of experiment shown in (A). The numbers of green puncta (autophagosomes) and red puncta (autolysosomes plus autophagosomes) were counted for nonmitotic cells. Data shown are represented as mean ± SEM for n = 30 cells for control knockdown, full medium, n = 43 cells for si PLK1 , full medium, n = 35 cells for control knockdown, starvation condition, and n = 26 for si PLK1  starvation condition. A nonparametric 2-tailed Student  t  test was applied; *,  P  ≤ 0.05; ***,  P  ≤ 0.001. (C) Immunofluorescence analysis of HeLa cells that were starved for 1 h for amino acids and growth factors, stimulated with amino acids and insulin for 35 min, followed by 30 min amino acid starvation. All media were supplemented with bafilomycin A 1 . Staining was performed with SQSTM1 antibody and Hoechst 33342. Shown are maximum intensity projections. Scale bar 20 µm. Representative images are shown for n = 3 independent experiments. (D) Quantification of experiment shown in (C). The total area of SQSTM1-positive foci was calculated and normalized to the number of nuclei. n = 126 cells for control condition and n = 105 cells for BI2536 treated condition. Data are represented as mean ± SEM, and are representative of n = 3 independent experiments. A nonparametric 2-tailed Student  t  test was applied; *,  P  ≤ 0.05. (E   F)  daf-2(e1370)  animals expressing GFP::LGG-1 were fed bacteria expressing control,  atg-18  or  plk-1  dsRNA from hatching, and raised at the nonpermissible temperature (25°C) to induce dauers. (E) Micrographs of ∼8 to 10 dauer animals lined up next to each other were taken 6 d after the temperature shift. Scale bar 200 µm. (F) GFP::LGG-1 fluorescence (mean ± s.d. of ∼8–10 animals, ** P
    Figure Legend Snippet: PLK1 inhibition impairs autophagy in nonmitotic cells and in C. elegans dauers. (A) HeLa cells were transfected with mRFP-GFP-LC3 tandem reporter, followed by PLK1 siRNA transfection on the next day. Cells were either kept in full medium, or starved for serum and amino acids for 16 h. Mitotic cells were removed by shake-off before fixation of cells 24 h after siRNA transfection. Representative images are shown for each condition. Scale bar 10 µm. Data shown are representative of n = 2 independent experiments. (B) Quantification of experiment shown in (A). The numbers of green puncta (autophagosomes) and red puncta (autolysosomes plus autophagosomes) were counted for nonmitotic cells. Data shown are represented as mean ± SEM for n = 30 cells for control knockdown, full medium, n = 43 cells for si PLK1 , full medium, n = 35 cells for control knockdown, starvation condition, and n = 26 for si PLK1 starvation condition. A nonparametric 2-tailed Student t test was applied; *, P ≤ 0.05; ***, P ≤ 0.001. (C) Immunofluorescence analysis of HeLa cells that were starved for 1 h for amino acids and growth factors, stimulated with amino acids and insulin for 35 min, followed by 30 min amino acid starvation. All media were supplemented with bafilomycin A 1 . Staining was performed with SQSTM1 antibody and Hoechst 33342. Shown are maximum intensity projections. Scale bar 20 µm. Representative images are shown for n = 3 independent experiments. (D) Quantification of experiment shown in (C). The total area of SQSTM1-positive foci was calculated and normalized to the number of nuclei. n = 126 cells for control condition and n = 105 cells for BI2536 treated condition. Data are represented as mean ± SEM, and are representative of n = 3 independent experiments. A nonparametric 2-tailed Student t test was applied; *, P ≤ 0.05. (E F) daf-2(e1370) animals expressing GFP::LGG-1 were fed bacteria expressing control, atg-18 or plk-1 dsRNA from hatching, and raised at the nonpermissible temperature (25°C) to induce dauers. (E) Micrographs of ∼8 to 10 dauer animals lined up next to each other were taken 6 d after the temperature shift. Scale bar 200 µm. (F) GFP::LGG-1 fluorescence (mean ± s.d. of ∼8–10 animals, ** P

    Techniques Used: Inhibition, Transfection, Immunofluorescence, Staining, Expressing, Fluorescence

    Starvation enhances PLK1-MTOR binding and cytoplasmic PLK1-MTOR colocalization. (A) HeLa cells were cultured in full medium and treated for 30 min with Torin1 or carrier, respectively. Immunoprecipitation (IP) was performed with PLK1 and control (mock) antibodies. Samples were analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments. (B) Quantification of IP samples shown in (A). Ratio of MTOR:PLK1 was calculated for n = 3 independent experiments. Data are normalized to 1 for full medium condition and represented as mean ± SEM. A nonparametric 2-tailed Student  t  test was applied; ns, nonsignificant. (C) HeLa cells were either cultured in full medium or starved for amino acids and growth factors for 16 h. Cells were then treated with BI2536 or carrier, as indicated. Immunoprecipitation (IP) was performed with PLK1 and control (mock) antibodies. Samples were analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments. (D) Quantification of data shown in (C). Fold change of MTOR:PLK1 ratio in starved versus control cells was calculated across n = 3 independent experiments, for carrier or BI2536 treated cells, as indicated. Data are normalized to lane 1 (C), and represented as mean ± SEM. A nonparametric 2-tailed Student  t  test was applied; ns, nonsignificant. (E) HeLa cells were starved for 1 h for amino acids and growth factors, and stimulated with amino acids and insulin for 35 min. Afterwards, for starvation, amino acids were withdrawn for 30 min. Immunoprecipitation (IP) was performed with PLK1 and control (mock) antibodies. Samples were analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments. (F) Quantification of data shown in (E). Ratio of MTOR:PLK1 was calculated for n = 3 independent experiments. Data are normalized to 1 for amino acids and insulin condition, and represented as mean ± SEM. A nonparametric 2-tailed Student  t  test was applied; ***,  P  ≤ 0.001. (G) Immunofluorescence analysis of HeLa cells that were starved for 1 h for amino acids and growth factors, stimulated with amino acids and insulin for 35 min, followed by 30 min of amino acid starvation, as indicated. Staining was performed with PLK1 and MTOR antibodies. White regions in merged image (right) of PLK1 (green) and MTOR (magenta) staining indicate colocalization; insert 1 shows a region with lysosomal MTOR pattern; insert 2 shows a cytoplasmic region without lysosomal MTOR pattern. Nuclei were stained with Hoechst 33342. Scale bar: 20 µm. Representative images are shown for n = 3 independent experiments. (H) Analysis of PLK1-MTOR colocalization by the Pearson correlation coefficient for experiment shown in (G). Ten representative cells were quantified for each condition. Data are represented as mean ± SEM and are representative of n = 3 independent experiments. A nonparametric 2-tailed Student  t  test was applied; *,  P  ≤ 0.05. (E, F, G, H) aa, amino acids; ins, insulin.
    Figure Legend Snippet: Starvation enhances PLK1-MTOR binding and cytoplasmic PLK1-MTOR colocalization. (A) HeLa cells were cultured in full medium and treated for 30 min with Torin1 or carrier, respectively. Immunoprecipitation (IP) was performed with PLK1 and control (mock) antibodies. Samples were analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments. (B) Quantification of IP samples shown in (A). Ratio of MTOR:PLK1 was calculated for n = 3 independent experiments. Data are normalized to 1 for full medium condition and represented as mean ± SEM. A nonparametric 2-tailed Student t test was applied; ns, nonsignificant. (C) HeLa cells were either cultured in full medium or starved for amino acids and growth factors for 16 h. Cells were then treated with BI2536 or carrier, as indicated. Immunoprecipitation (IP) was performed with PLK1 and control (mock) antibodies. Samples were analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments. (D) Quantification of data shown in (C). Fold change of MTOR:PLK1 ratio in starved versus control cells was calculated across n = 3 independent experiments, for carrier or BI2536 treated cells, as indicated. Data are normalized to lane 1 (C), and represented as mean ± SEM. A nonparametric 2-tailed Student t test was applied; ns, nonsignificant. (E) HeLa cells were starved for 1 h for amino acids and growth factors, and stimulated with amino acids and insulin for 35 min. Afterwards, for starvation, amino acids were withdrawn for 30 min. Immunoprecipitation (IP) was performed with PLK1 and control (mock) antibodies. Samples were analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments. (F) Quantification of data shown in (E). Ratio of MTOR:PLK1 was calculated for n = 3 independent experiments. Data are normalized to 1 for amino acids and insulin condition, and represented as mean ± SEM. A nonparametric 2-tailed Student t test was applied; ***, P ≤ 0.001. (G) Immunofluorescence analysis of HeLa cells that were starved for 1 h for amino acids and growth factors, stimulated with amino acids and insulin for 35 min, followed by 30 min of amino acid starvation, as indicated. Staining was performed with PLK1 and MTOR antibodies. White regions in merged image (right) of PLK1 (green) and MTOR (magenta) staining indicate colocalization; insert 1 shows a region with lysosomal MTOR pattern; insert 2 shows a cytoplasmic region without lysosomal MTOR pattern. Nuclei were stained with Hoechst 33342. Scale bar: 20 µm. Representative images are shown for n = 3 independent experiments. (H) Analysis of PLK1-MTOR colocalization by the Pearson correlation coefficient for experiment shown in (G). Ten representative cells were quantified for each condition. Data are represented as mean ± SEM and are representative of n = 3 independent experiments. A nonparametric 2-tailed Student t test was applied; *, P ≤ 0.05. (E, F, G, H) aa, amino acids; ins, insulin.

    Techniques Used: Binding Assay, Cell Culture, Immunoprecipitation, Immunofluorescence, Staining

    PLK1 inhibition hyperactivates MTORC1 and increases lysosomal MTORC1 localization in amino acid-starved interphase cells. (A) HeLa cells were starved for 1 h for amino acids and growth factors, and stimulated with amino acids and insulin for 35 min. Cells were then starved for amino acids for 5, 10, 15 and 30 min and treated with BI2536 or carrier, as indicated. Samples were analyzed by immunoblotting. Data shown are representative of n = 4 independent experiments. (B) Quantification of data shown in (A). Ratio of RPS6KB (p70) phospho-(T389):RPS6KB (p70) was calculated for n = 4 (5 min starvation and 15 min starvation); n = 3 (10 min starvation) independent experiments. Data are normalized to 1 for starvation control condition and represented as mean ± SEM. A nonparametric 2-tailed Student  t  test was applied; *,  P  ≤ 0.05. (C) Immunofluorescence analysis of HeLa cells that were starved for 1 h for amino acids and growth factors, stimulated with amino acids and insulin for 35 min, followed by 30 min amino acid starvation, without or with the PLK1 inhibitor BI2536. Staining was performed with MTOR and LAMP2 antibodies. White regions in merged image (right) of MTOR (green) and LAMP2 (magenta) indicate colocalization. Nuclei were stained with Hoechst 33342. Scale bar 20 µm. Representative images are shown for n = 3 independent experiments. (D) Analysis of MTOR-LAMP2 colocalization by the Pearson correlation coefficient for experiment shown in (C). Data are represented as mean ± SEM, and are representative of n = 3 independent experiments. A nonparametric 2-tailed Student t test was applied; *,  P  ≤ 0.05. (E) Immunofluorescence analysis of HeLa cells that were treated as described in (C). Staining was performed with RRAGC and LAMP2 antibodies. White regions in merged image (right) of RRAGC (green) and LAMP2 (magenta) indicate colocalization. Nuclei were stained with Hoechst 33342. Scale bar 20 µm. Representative images are shown for n = 3 independent experiments. (F) Analysis of RRAGC-LAMP2 colocalization by the Pearson correlation coefficient for experiment shown in (E). Data are represented as mean ± SEM, and are representative of n = 3 independent experiments. A nonparametric 2-tailed Student  t  test was applied; ns, nonsignificant. (G) HeLa cells were either cultured in full medium or starved for amino acids and growth factors for 16 h. Immunoprecipitation (IP) was performed with PLK1 and control (mock) antibodies. Samples were analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments. (H) Quantification of IP samples shown in (G). Ratio of MTOR:PLK1 was calculated for n = 3 independent experiments. Data are normalized to 1 for control condition and represented as mean ± SEM. A nonparametric 2-tailed Student  t  test was applied; *,  P  ≤ 0.05. (I) HeLa cells were treated as described in (G). IP was performed with PLK1 and control (mock) antibodies. Samples were analyzed by immunoblotting. Data shown are representative of n = 4 independent experiments. (J) Quantification of IP samples shown in (I). Ratio of RPTOR:PLK1 was calculated for n = 4 independent experiments. Data are normalized to 1 for control condition and represented as mean ± SEM. A nonparametric 2-tailed Student  t  test was applied; *,  P  ≤ 0.05.
    Figure Legend Snippet: PLK1 inhibition hyperactivates MTORC1 and increases lysosomal MTORC1 localization in amino acid-starved interphase cells. (A) HeLa cells were starved for 1 h for amino acids and growth factors, and stimulated with amino acids and insulin for 35 min. Cells were then starved for amino acids for 5, 10, 15 and 30 min and treated with BI2536 or carrier, as indicated. Samples were analyzed by immunoblotting. Data shown are representative of n = 4 independent experiments. (B) Quantification of data shown in (A). Ratio of RPS6KB (p70) phospho-(T389):RPS6KB (p70) was calculated for n = 4 (5 min starvation and 15 min starvation); n = 3 (10 min starvation) independent experiments. Data are normalized to 1 for starvation control condition and represented as mean ± SEM. A nonparametric 2-tailed Student t test was applied; *, P ≤ 0.05. (C) Immunofluorescence analysis of HeLa cells that were starved for 1 h for amino acids and growth factors, stimulated with amino acids and insulin for 35 min, followed by 30 min amino acid starvation, without or with the PLK1 inhibitor BI2536. Staining was performed with MTOR and LAMP2 antibodies. White regions in merged image (right) of MTOR (green) and LAMP2 (magenta) indicate colocalization. Nuclei were stained with Hoechst 33342. Scale bar 20 µm. Representative images are shown for n = 3 independent experiments. (D) Analysis of MTOR-LAMP2 colocalization by the Pearson correlation coefficient for experiment shown in (C). Data are represented as mean ± SEM, and are representative of n = 3 independent experiments. A nonparametric 2-tailed Student t test was applied; *, P ≤ 0.05. (E) Immunofluorescence analysis of HeLa cells that were treated as described in (C). Staining was performed with RRAGC and LAMP2 antibodies. White regions in merged image (right) of RRAGC (green) and LAMP2 (magenta) indicate colocalization. Nuclei were stained with Hoechst 33342. Scale bar 20 µm. Representative images are shown for n = 3 independent experiments. (F) Analysis of RRAGC-LAMP2 colocalization by the Pearson correlation coefficient for experiment shown in (E). Data are represented as mean ± SEM, and are representative of n = 3 independent experiments. A nonparametric 2-tailed Student t test was applied; ns, nonsignificant. (G) HeLa cells were either cultured in full medium or starved for amino acids and growth factors for 16 h. Immunoprecipitation (IP) was performed with PLK1 and control (mock) antibodies. Samples were analyzed by immunoblotting. Data shown are representative of n = 3 independent experiments. (H) Quantification of IP samples shown in (G). Ratio of MTOR:PLK1 was calculated for n = 3 independent experiments. Data are normalized to 1 for control condition and represented as mean ± SEM. A nonparametric 2-tailed Student t test was applied; *, P ≤ 0.05. (I) HeLa cells were treated as described in (G). IP was performed with PLK1 and control (mock) antibodies. Samples were analyzed by immunoblotting. Data shown are representative of n = 4 independent experiments. (J) Quantification of IP samples shown in (I). Ratio of RPTOR:PLK1 was calculated for n = 4 independent experiments. Data are normalized to 1 for control condition and represented as mean ± SEM. A nonparametric 2-tailed Student t test was applied; *, P ≤ 0.05.

    Techniques Used: Inhibition, Immunofluorescence, Staining, Cell Culture, Immunoprecipitation

    23) Product Images from "Wnt signaling blockage inhibits cell proliferation and migration, and induces apoptosis in triple-negative breast cancer cells"

    Article Title: Wnt signaling blockage inhibits cell proliferation and migration, and induces apoptosis in triple-negative breast cancer cells

    Journal: Journal of Translational Medicine

    doi: 10.1186/1479-5876-11-280

    Wnt signaling pathway is activated in TNBC cells. Subcellular localization of β-catenin in TNBC cells treated with or without human recombinant Wnt-3a (200 ng/ml) for 4 hours was examined using confocal microscopy. Immunofluorescence staining of β-catenin (green) showed cytoplasmic localization in MCF-7 cell line, and was both nuclear and cytoplasmic in TNBC cell lines, MDA-MB-231 and BT-549. Treatment with Wnt-3a resulted in increased nuclear localization of β-catenin in MDA-MB-231 and BT-549 cells. Nuclei were counterstained with Hoechst 33342 (blue). Total magnification was 200×, and the images were zoomed in 500%.
    Figure Legend Snippet: Wnt signaling pathway is activated in TNBC cells. Subcellular localization of β-catenin in TNBC cells treated with or without human recombinant Wnt-3a (200 ng/ml) for 4 hours was examined using confocal microscopy. Immunofluorescence staining of β-catenin (green) showed cytoplasmic localization in MCF-7 cell line, and was both nuclear and cytoplasmic in TNBC cell lines, MDA-MB-231 and BT-549. Treatment with Wnt-3a resulted in increased nuclear localization of β-catenin in MDA-MB-231 and BT-549 cells. Nuclei were counterstained with Hoechst 33342 (blue). Total magnification was 200×, and the images were zoomed in 500%.

    Techniques Used: Recombinant, Confocal Microscopy, Immunofluorescence, Staining, Multiple Displacement Amplification

    24) Product Images from "Improved assays for determining the cytosolic access of peptides, proteins, and their mimetics"

    Article Title: Improved assays for determining the cytosolic access of peptides, proteins, and their mimetics

    Journal: Biochemistry

    doi: 10.1021/bi401069g

     (see Experimental Procedures). (c) GIGI in living cells: Live-cell imaging of eGFP expression in three transiently transfected cell lines. Cells were incubated in the presence or absence of 1  μ M SDex for 24 h before imaging by epifluorescence microscopy. Nuclei were stained using Hoechst 33342. Scale bar = 50  μ m. (d) GIGI in living cells: Quantification of eGFP expression by FACS analysis in transiently transfected HeLa, U2OS, and HEK293T cells treated for 24 h with 1  μ M SDex. The mean cellular fluorescence for untreated (control) transfectants was set to 1 and other values are expressed as the fold-increase in fluorescence emission at 533 nm ± standard deviation (Excel). For panels (a) and (d), statistical analysis was performed using a two-tailed Student's t-test with each cell line treated as a separate population; ** p ≤ 0.005, *** p ≤ 0.001.
    Figure Legend Snippet: (see Experimental Procedures). (c) GIGI in living cells: Live-cell imaging of eGFP expression in three transiently transfected cell lines. Cells were incubated in the presence or absence of 1 μ M SDex for 24 h before imaging by epifluorescence microscopy. Nuclei were stained using Hoechst 33342. Scale bar = 50 μ m. (d) GIGI in living cells: Quantification of eGFP expression by FACS analysis in transiently transfected HeLa, U2OS, and HEK293T cells treated for 24 h with 1 μ M SDex. The mean cellular fluorescence for untreated (control) transfectants was set to 1 and other values are expressed as the fold-increase in fluorescence emission at 533 nm ± standard deviation (Excel). For panels (a) and (d), statistical analysis was performed using a two-tailed Student's t-test with each cell line treated as a separate population; ** p ≤ 0.005, *** p ≤ 0.001.

    Techniques Used: Live Cell Imaging, Expressing, Transfection, Incubation, Imaging, Epifluorescence Microscopy, Staining, FACS, Fluorescence, Standard Deviation, Two Tailed Test

     (see Materials and Methods). Statistical analysis was performed using a two-tailed Student's t-test with each cell line treated as a separate population; ** p ≤ 0.005, *** p ≤ 0.001. (a) GIGI in live cells: Live-cell epifluorescent imaging of U2OS(GIGI) cells treated for 24 h with or without 1 μM SDex. Nuclei were stained with Hoechst 33342. Scale bar = 20  μ m. (b) GIGI in lysates: Comparison of eGFP expression in lysates prepared from transiently transfected U2OS and U2OS(GIGI) cells treated with varying concentrations of SDex for 24 h. (c) GIGI in live cells: Quantification of eGFP expression by FACS analysis in transiently transfected U2OS and U2OS(GIGI) cells treated for 24 h with or without 1  μ M SDex. The mean cellular fluorescence for untreated (control) transfectants was set to 1 and other values are expressed as the fold-increase in fluorescence emission at 533 nm ± standard deviation. (d) GIGI in lysates: Relative eGFP expression levels in U2OS(GIGI) cells treated with 1  μ M of the indicated Dex-tagged miniature protein or peptide. e) GIGI in lysates: Concentration-dependent effect of each miniature protein on eGFP expression in U2OS(GIGI) cells. EC 50 . f) GIGI in lysates: Well-to-well variability of GIGI in U2OS(GIGI) cells treated with 1 μM 5.3 Dex   (see Experimental Procedures).
    Figure Legend Snippet: (see Materials and Methods). Statistical analysis was performed using a two-tailed Student's t-test with each cell line treated as a separate population; ** p ≤ 0.005, *** p ≤ 0.001. (a) GIGI in live cells: Live-cell epifluorescent imaging of U2OS(GIGI) cells treated for 24 h with or without 1 μM SDex. Nuclei were stained with Hoechst 33342. Scale bar = 20 μ m. (b) GIGI in lysates: Comparison of eGFP expression in lysates prepared from transiently transfected U2OS and U2OS(GIGI) cells treated with varying concentrations of SDex for 24 h. (c) GIGI in live cells: Quantification of eGFP expression by FACS analysis in transiently transfected U2OS and U2OS(GIGI) cells treated for 24 h with or without 1 μ M SDex. The mean cellular fluorescence for untreated (control) transfectants was set to 1 and other values are expressed as the fold-increase in fluorescence emission at 533 nm ± standard deviation. (d) GIGI in lysates: Relative eGFP expression levels in U2OS(GIGI) cells treated with 1 μ M of the indicated Dex-tagged miniature protein or peptide. e) GIGI in lysates: Concentration-dependent effect of each miniature protein on eGFP expression in U2OS(GIGI) cells. EC 50 . f) GIGI in lysates: Well-to-well variability of GIGI in U2OS(GIGI) cells treated with 1 μM 5.3 Dex (see Experimental Procedures).

    Techniques Used: Two Tailed Test, Imaging, Staining, Expressing, Transfection, FACS, Fluorescence, Standard Deviation, Concentration Assay

    GIGT validation in stably transfected Saos-2(GIGT) cells. (a) Images of Saos-2(GIGT) cells stably transfected with GR★-GFP with or without treatment with 1 μ M SDex for 30 min. Left images show an overlay of GFP signal (green) with Hoechst 33342 (blue) and right images display GFP signal in grayscale. (b) Effect of SDex on the calculated TR in HeLa cells transiently transfected with GR★-GFP and stable Saos-2(GIGT) cells. (c) Analysis of GR★-GFP nuclear translocation in Saos-2(GIGT) cells after a 30 min treatment with 1 μ M SDex or Dex-labeled peptides. TRs, expressed ± standard deviation, were calculated using Acapella® (see Supporting Information). *** p ≤ 0.001; ANOVA. (d) Well-to-well variability of GIGT in Saos-2(GIGT) cells treated for 30 min with 1 μ M 5.3 Dex (see Experimental Procedures).
    Figure Legend Snippet: GIGT validation in stably transfected Saos-2(GIGT) cells. (a) Images of Saos-2(GIGT) cells stably transfected with GR★-GFP with or without treatment with 1 μ M SDex for 30 min. Left images show an overlay of GFP signal (green) with Hoechst 33342 (blue) and right images display GFP signal in grayscale. (b) Effect of SDex on the calculated TR in HeLa cells transiently transfected with GR★-GFP and stable Saos-2(GIGT) cells. (c) Analysis of GR★-GFP nuclear translocation in Saos-2(GIGT) cells after a 30 min treatment with 1 μ M SDex or Dex-labeled peptides. TRs, expressed ± standard deviation, were calculated using Acapella® (see Supporting Information). *** p ≤ 0.001; ANOVA. (d) Well-to-well variability of GIGT in Saos-2(GIGT) cells treated for 30 min with 1 μ M 5.3 Dex (see Experimental Procedures).

    Techniques Used: Stable Transfection, Transfection, Translocation Assay, Labeling, Standard Deviation

    25) Product Images from "Two Escape Mechanisms of Influenza A Virus to a Broadly Neutralizing Stalk-Binding Antibody"

    Article Title: Two Escape Mechanisms of Influenza A Virus to a Broadly Neutralizing Stalk-Binding Antibody

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005702

    Viruses resistant to mAb 39.29. (A) Micro-neutralization assay was performed on MDCK cells in 96-well plates. WT and mutant A/Perth/16/2009 viruses were incubated with serial dilutions of 39.29 ranging from 0.0032 to 250 μg/ml. Cells were incubated with the virus-antibody mixture for 16 hours prior to immuno-staining with anti-IAV NP and Hoechst 33342. The percentages of infected cells for each virus were normalized to the value at the lowest antibody concentration. The assay was done in triplicate with data presented as Mean +/- SEM (standard error of the mean). (B) The structure is generated from PDB ID, 4KVN with PYMOL. The stalk of A/Perth/16/2009 HA is in red, the light chain of 39.29 Fab is in blue, and the heavy chain is in green. Amino acid side chains for Asn32 (in CDR L1) and Asn93 (in CDR L3) of the light chain, and Asp391 and Gln387 of the HA are represented as sticks.
    Figure Legend Snippet: Viruses resistant to mAb 39.29. (A) Micro-neutralization assay was performed on MDCK cells in 96-well plates. WT and mutant A/Perth/16/2009 viruses were incubated with serial dilutions of 39.29 ranging from 0.0032 to 250 μg/ml. Cells were incubated with the virus-antibody mixture for 16 hours prior to immuno-staining with anti-IAV NP and Hoechst 33342. The percentages of infected cells for each virus were normalized to the value at the lowest antibody concentration. The assay was done in triplicate with data presented as Mean +/- SEM (standard error of the mean). (B) The structure is generated from PDB ID, 4KVN with PYMOL. The stalk of A/Perth/16/2009 HA is in red, the light chain of 39.29 Fab is in blue, and the heavy chain is in green. Amino acid side chains for Asn32 (in CDR L1) and Asn93 (in CDR L3) of the light chain, and Asp391 and Gln387 of the HA are represented as sticks.

    Techniques Used: Neutralization, Mutagenesis, Incubation, Immunostaining, Infection, Concentration Assay, Generated

    26) Product Images from "Identification of ANXA2 (annexin A2) as a specific bleomycin target to induce pulmonary fibrosis by impeding TFEB-mediated autophagic flux"

    Article Title: Identification of ANXA2 (annexin A2) as a specific bleomycin target to induce pulmonary fibrosis by impeding TFEB-mediated autophagic flux

    Journal: Autophagy

    doi: 10.1080/15548627.2017.1409405

    Disruption of bleomycin-ANXA2 interaction accelerates autophagic flux in lung epithelial cells. ( A ) Immunoblots for LC3B in parental and ANXA2 E139A cells treated with bleomycin (50 μM) or cotreated with chloroquine (CQ, 20 μM) or bafilomycin A 1 (BafA1, 100 nM) for 24 h. GAPDH, loading control. S.E., short exposure; L.E., long exposure. Quantification of LC3B levels in parental and ANXA2 E139A cells treated with or without bleomycin is shown. ( B ) LC3B-II:GAPDH levels in parental and ANXA2 E139A cells treated with bleomycin in the presence of CQ or BafA1 shown in ( A ). LC3B-II:GAPDH was calculated as: (LC3B-II level treated with bleomycin combined with CQ or BafA1 − LC3B-II level treated with bleomycin alone) ÷ LC3B-II basal levels. ( C ) Immunoblots for SQSTM1 in parental and ANXA2 E139A cells treated with or without bleomycin (50 μM). GAPDH, loading control. Quantification of SQSTM1 levels is shown. ( D ) Representative images of parental or ANXA2 E139A cells transiently expressing GFP-LC3B plasmids followed by bleomycin (Bleo, 50 μΜ) treatment for 24 h. DNA was counterstained with Hoechst 33342 (blue). Scale bars: 10 μm. ( E ) Quantification of LC3B puncta shown in ( D ). ( F ) Representative images of parental or ANXA2 E139A cells transiently expressing mRFP-GFP-LC3B plasmids followed by treatment with bleomycin (50 μM) for 24 h. Scale bars: 10 μm. ( G ) Quantification of LC3B puncta shown in ( F ). ( H ) Representative images of parental or ANXA2 E139A cells incubated with BODIPY-conjugated bovine serum (DQ-BSA, red) followed by bleomycin treatment (50 μΜ) for 24 h. DNA was counterstained with Hoechst 33342 (blue). Scale bars: 10 μm. Data in ( B , D , F and H ) are means ± s.d., Results are representative of 3 independent experiments. * P
    Figure Legend Snippet: Disruption of bleomycin-ANXA2 interaction accelerates autophagic flux in lung epithelial cells. ( A ) Immunoblots for LC3B in parental and ANXA2 E139A cells treated with bleomycin (50 μM) or cotreated with chloroquine (CQ, 20 μM) or bafilomycin A 1 (BafA1, 100 nM) for 24 h. GAPDH, loading control. S.E., short exposure; L.E., long exposure. Quantification of LC3B levels in parental and ANXA2 E139A cells treated with or without bleomycin is shown. ( B ) LC3B-II:GAPDH levels in parental and ANXA2 E139A cells treated with bleomycin in the presence of CQ or BafA1 shown in ( A ). LC3B-II:GAPDH was calculated as: (LC3B-II level treated with bleomycin combined with CQ or BafA1 − LC3B-II level treated with bleomycin alone) ÷ LC3B-II basal levels. ( C ) Immunoblots for SQSTM1 in parental and ANXA2 E139A cells treated with or without bleomycin (50 μM). GAPDH, loading control. Quantification of SQSTM1 levels is shown. ( D ) Representative images of parental or ANXA2 E139A cells transiently expressing GFP-LC3B plasmids followed by bleomycin (Bleo, 50 μΜ) treatment for 24 h. DNA was counterstained with Hoechst 33342 (blue). Scale bars: 10 μm. ( E ) Quantification of LC3B puncta shown in ( D ). ( F ) Representative images of parental or ANXA2 E139A cells transiently expressing mRFP-GFP-LC3B plasmids followed by treatment with bleomycin (50 μM) for 24 h. Scale bars: 10 μm. ( G ) Quantification of LC3B puncta shown in ( F ). ( H ) Representative images of parental or ANXA2 E139A cells incubated with BODIPY-conjugated bovine serum (DQ-BSA, red) followed by bleomycin treatment (50 μΜ) for 24 h. DNA was counterstained with Hoechst 33342 (blue). Scale bars: 10 μm. Data in ( B , D , F and H ) are means ± s.d., Results are representative of 3 independent experiments. * P

    Techniques Used: Western Blot, Expressing, Incubation

    27) Product Images from "Pemetrexed Induces S-Phase Arrest and Apoptosis via a Deregulated Activation of Akt Signaling Pathway"

    Article Title: Pemetrexed Induces S-Phase Arrest and Apoptosis via a Deregulated Activation of Akt Signaling Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0097888

    Akt translocates to the nucleus during pemetrexed-induced cell death. (A) A549 cells were treated with 0, 0.1, 0.3, and 1 µM pemetrexed for 48 h, and (B) A549 cells were treated with 1 µM pemetrexed for 0, 12, 24, and 48 h. After treatment, the subcellular distribution of p-Akt S473 was detected by confocal microscopy after immunostaining with anti-phospho-Akt S473 and Rhodamine-conjugated secondary antibody. Hoechst 33342 was used to counter stain nuclei, and the images were overlaid to determine the Akt localization within the cell. (C) Inhibiton of Akt activation by Ly294002 and wortmannin blocked Akt nuclear accumulation. A549 cells were pretreated with 10 µM Ly294002 or 3 µM wortmannin for 2 h, and then 1 µM pemetrexed was added for another 48 h. The subcellular distribution of p-Akt S473 was detected by immunocytochemistry.
    Figure Legend Snippet: Akt translocates to the nucleus during pemetrexed-induced cell death. (A) A549 cells were treated with 0, 0.1, 0.3, and 1 µM pemetrexed for 48 h, and (B) A549 cells were treated with 1 µM pemetrexed for 0, 12, 24, and 48 h. After treatment, the subcellular distribution of p-Akt S473 was detected by confocal microscopy after immunostaining with anti-phospho-Akt S473 and Rhodamine-conjugated secondary antibody. Hoechst 33342 was used to counter stain nuclei, and the images were overlaid to determine the Akt localization within the cell. (C) Inhibiton of Akt activation by Ly294002 and wortmannin blocked Akt nuclear accumulation. A549 cells were pretreated with 10 µM Ly294002 or 3 µM wortmannin for 2 h, and then 1 µM pemetrexed was added for another 48 h. The subcellular distribution of p-Akt S473 was detected by immunocytochemistry.

    Techniques Used: Confocal Microscopy, Immunostaining, Staining, Activation Assay, Immunocytochemistry

    Activation of Akt and Cdk2 are occurred in pemetrexed-treated H1299 cells. (A) Pemetrexed stimulates Akt pathway activation. H1299 cells were treated with 0, 0.1, 0.3 and 1 µM pemetrexed for 48 h. After treatment, the levels of total Akt, phosphorylated Akt, total GSK3β, and phosphorylated GSK3β were examined by Western blot analysis. β-Actin was used as an internal loading control. The proportion of S-phase population and apoptotic cells were determined as described in the Materials and Methods section. (B) Nuclear accumulation of Akt occurred in pemetrexed-treated H1299 cells. Cells were treated with 0, 0.1, 0.3, and 1 µM pemetrexed for 48 h, the subcellular distribution of p-Akt S473 was detected by confocal microscopy after immunostaining with anti-phospho-Akt S473 . Hoechst 33342 was used to counterstain nuclei. (C) Pemetrexed activated Cdk2/Cyclin A-associated kinase in H1299cells. H1299 cells were treated with 0, 0.3, and 1 µM pemetrexed for 24 h, and then protein lysates were isolated. The Cdk2 kinase activity and the levels of Cdk2, Cyclin A and Cyclin E were determined.
    Figure Legend Snippet: Activation of Akt and Cdk2 are occurred in pemetrexed-treated H1299 cells. (A) Pemetrexed stimulates Akt pathway activation. H1299 cells were treated with 0, 0.1, 0.3 and 1 µM pemetrexed for 48 h. After treatment, the levels of total Akt, phosphorylated Akt, total GSK3β, and phosphorylated GSK3β were examined by Western blot analysis. β-Actin was used as an internal loading control. The proportion of S-phase population and apoptotic cells were determined as described in the Materials and Methods section. (B) Nuclear accumulation of Akt occurred in pemetrexed-treated H1299 cells. Cells were treated with 0, 0.1, 0.3, and 1 µM pemetrexed for 48 h, the subcellular distribution of p-Akt S473 was detected by confocal microscopy after immunostaining with anti-phospho-Akt S473 . Hoechst 33342 was used to counterstain nuclei. (C) Pemetrexed activated Cdk2/Cyclin A-associated kinase in H1299cells. H1299 cells were treated with 0, 0.3, and 1 µM pemetrexed for 24 h, and then protein lysates were isolated. The Cdk2 kinase activity and the levels of Cdk2, Cyclin A and Cyclin E were determined.

    Techniques Used: Activation Assay, Western Blot, Confocal Microscopy, Immunostaining, Isolation, Activity Assay

    28) Product Images from "Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB4) as a Novel Autophagy Regulator by High Content shRNA Screening"

    Article Title: Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB4) as a Novel Autophagy Regulator by High Content shRNA Screening

    Journal: Oncogene

    doi: 10.1038/onc.2015.23

    Validation of Selected Screen Hits a. Representative low throughput validation images from experiments with uninfected parental Beclin1 +/− iBMK cells (3BC2-clone #D3) or Beclin1 +/− iBMK cells infected with two distinct hairpins targeting red fluorescent protein ( RFP ) or a hairpin targeting Atg7, assaying GFP-p62 clearance following metabolic stress (7.5h) and recovery (18h) in full media. Nuclei were counterstained with Hoechst 33342 to facilitate visualization. Scale bar is 10 μm. Non-validating hairpins were spliced out of the western blots presented for aesthetic reasons. All lanes presented for a given gene were contained within a single gel. Complete gels are shown in Figure S1 . b. Validation of a subset of known autophagy regulators identified in our screens. For a candidate gene to pass validation at least two individual hairpins targeting distinct regions of the message must silence expression of the target gene by western blot and correlate well with GFP-p62 phenotype observed microscopically. Beclin1 +/− iBMK cells were infected with hairpins targeting red fluorescent protein ( RFP ) or mTOR, Ulk1, Prkaa1, Becn1, Ikbkb, Hrs, Rab7, or Etnk1, subjected to selection, and assayed for GFP-p62 clearance following metabolic stress (7.5h) and recovery (18h) in full media by immunofluoresence. Nuclei were counterstained with Hoechst 33342 to facilitate visualization. Scale bar is 10 μm. Confirmation of on-target gene silencing was obtained by western blot analysis. TRC hairpin identifiers and antibody details are contained in the Supplemental Methods section. Non-validating hairpins were spliced out of the western blots presented for aesthetic reasons. All lanes presented for a given gene were contained within a single gel. Complete gels are shown in Figure S1 .
    Figure Legend Snippet: Validation of Selected Screen Hits a. Representative low throughput validation images from experiments with uninfected parental Beclin1 +/− iBMK cells (3BC2-clone #D3) or Beclin1 +/− iBMK cells infected with two distinct hairpins targeting red fluorescent protein ( RFP ) or a hairpin targeting Atg7, assaying GFP-p62 clearance following metabolic stress (7.5h) and recovery (18h) in full media. Nuclei were counterstained with Hoechst 33342 to facilitate visualization. Scale bar is 10 μm. Non-validating hairpins were spliced out of the western blots presented for aesthetic reasons. All lanes presented for a given gene were contained within a single gel. Complete gels are shown in Figure S1 . b. Validation of a subset of known autophagy regulators identified in our screens. For a candidate gene to pass validation at least two individual hairpins targeting distinct regions of the message must silence expression of the target gene by western blot and correlate well with GFP-p62 phenotype observed microscopically. Beclin1 +/− iBMK cells were infected with hairpins targeting red fluorescent protein ( RFP ) or mTOR, Ulk1, Prkaa1, Becn1, Ikbkb, Hrs, Rab7, or Etnk1, subjected to selection, and assayed for GFP-p62 clearance following metabolic stress (7.5h) and recovery (18h) in full media by immunofluoresence. Nuclei were counterstained with Hoechst 33342 to facilitate visualization. Scale bar is 10 μm. Confirmation of on-target gene silencing was obtained by western blot analysis. TRC hairpin identifiers and antibody details are contained in the Supplemental Methods section. Non-validating hairpins were spliced out of the western blots presented for aesthetic reasons. All lanes presented for a given gene were contained within a single gel. Complete gels are shown in Figure S1 .

    Techniques Used: Infection, Western Blot, Expressing, Selection

    Identification of Autophagy Substrate Modulators by High Content shRNA Screening a. Experimental timeline detailing sequence of infection, selection, and assay for p62 elimination. b . Beclin1 +/+ or Beclin1 +/− iBMK cells stably expressing EGFP-p62 were infected with shRNA targeting Luc1510, selected with puromycin, and subjected to a time course of 7 hours ischemia and recovery. Representative images of GFP-p62 accumulation during stress and recovery demonstrating impaired elimination of p62 from the Beclin1 +/− iBMK cell line during recovery in full media. Scale bar is 10 μm. c. Western blot analysis of GFP-p62 accumulation and elimination in Beclin1 +/+ or Beclin1 +/− iBMK cells stably expressing EGFP-p62 infected with shRNA targeting Luc1510, selected with puromycin, and subjected to a time course of 7 hours ischemia and recovery as indicated. d. Schematic representation of the p62 modulator screen. Representative images from the screen are provided for each class of regulator. e. A customized Cellomics algorithm was used to score p62 abundance at the single cell level. Images from pilot studies employing p62 or non-targeting (emptyT) hairpins are provided with and without the overlaid analysis. Cells were counterstained with Hoechst 33342 to visualize nuclei (blue, Channel 1). Channel 2 maps the cell borders, and detects GFP-p62 levels within a designated radius from the nucleus (Ring Spot Total Intensity). 9 images per field were collected to ensure representative coverage. A p62 aggregate score equal to Mean Ring Spot total intensity/Mean nuclei was calculated for each well.
    Figure Legend Snippet: Identification of Autophagy Substrate Modulators by High Content shRNA Screening a. Experimental timeline detailing sequence of infection, selection, and assay for p62 elimination. b . Beclin1 +/+ or Beclin1 +/− iBMK cells stably expressing EGFP-p62 were infected with shRNA targeting Luc1510, selected with puromycin, and subjected to a time course of 7 hours ischemia and recovery. Representative images of GFP-p62 accumulation during stress and recovery demonstrating impaired elimination of p62 from the Beclin1 +/− iBMK cell line during recovery in full media. Scale bar is 10 μm. c. Western blot analysis of GFP-p62 accumulation and elimination in Beclin1 +/+ or Beclin1 +/− iBMK cells stably expressing EGFP-p62 infected with shRNA targeting Luc1510, selected with puromycin, and subjected to a time course of 7 hours ischemia and recovery as indicated. d. Schematic representation of the p62 modulator screen. Representative images from the screen are provided for each class of regulator. e. A customized Cellomics algorithm was used to score p62 abundance at the single cell level. Images from pilot studies employing p62 or non-targeting (emptyT) hairpins are provided with and without the overlaid analysis. Cells were counterstained with Hoechst 33342 to visualize nuclei (blue, Channel 1). Channel 2 maps the cell borders, and detects GFP-p62 levels within a designated radius from the nucleus (Ring Spot Total Intensity). 9 images per field were collected to ensure representative coverage. A p62 aggregate score equal to Mean Ring Spot total intensity/Mean nuclei was calculated for each well.

    Techniques Used: shRNA, Sequencing, Infection, Selection, Stable Transfection, Expressing, Western Blot

    29) Product Images from "Measuring DNA content in live cells by fluorescence microscopy"

    Article Title: Measuring DNA content in live cells by fluorescence microscopy

    Journal: Cell Division

    doi: 10.1186/s13008-018-0039-z

    Determining the length of supravital dye saturation. Asynchronous cells were plated onto 8-well chambered slides and allowed 24 h to adhere. After the addition of Hoechst 33342 into the culture medium, fluorescent images were taken at 20-min intervals. a The integrated fluorescent intensity of cells with approximately 2C, 4C, and 8C DNA content are graphed over time, with error bars representing the standard deviation within groups. b Representative examples of cells with varying amounts of DNA content are presented in a time series with 20-min intervals. Located at the bottom left of each image are the integrated fluorescent units calculated at the corresponding time-point
    Figure Legend Snippet: Determining the length of supravital dye saturation. Asynchronous cells were plated onto 8-well chambered slides and allowed 24 h to adhere. After the addition of Hoechst 33342 into the culture medium, fluorescent images were taken at 20-min intervals. a The integrated fluorescent intensity of cells with approximately 2C, 4C, and 8C DNA content are graphed over time, with error bars representing the standard deviation within groups. b Representative examples of cells with varying amounts of DNA content are presented in a time series with 20-min intervals. Located at the bottom left of each image are the integrated fluorescent units calculated at the corresponding time-point

    Techniques Used: Standard Deviation

    Polyploid cells occasionally experience error prone mitoses. H2B-GFP labeled cells were imaged over a 20-h time course with 3-min intervals between acquisitions. At the 18-h mark, Hoechst 33342 was added to the imaging medium. At the completion of the time-lapse experiment, images were collected for Hoechst 33342 fluorescence. Hoechst 33342 images were analyzed using the ProcessDNA pipeline and concatenated to the time-lapse series. a Highlighted is a polyploid cell progressing through mitosis with asymmetrical separation of DNA between daughter cells. Scale bar = 20 μm. b Mitotic errors such as anaphase bridges (AB, top panel) and lagging chromosomes (LC, bottom panel) with subsequent micronuclei production (MN) were observed. Scale bar = 10 μm. c Asymmetrical separation of DNA occasionally resulted from tripolar (top panel) and quadripolar (bottom panel) spindles. Scale bar = 10 μm
    Figure Legend Snippet: Polyploid cells occasionally experience error prone mitoses. H2B-GFP labeled cells were imaged over a 20-h time course with 3-min intervals between acquisitions. At the 18-h mark, Hoechst 33342 was added to the imaging medium. At the completion of the time-lapse experiment, images were collected for Hoechst 33342 fluorescence. Hoechst 33342 images were analyzed using the ProcessDNA pipeline and concatenated to the time-lapse series. a Highlighted is a polyploid cell progressing through mitosis with asymmetrical separation of DNA between daughter cells. Scale bar = 20 μm. b Mitotic errors such as anaphase bridges (AB, top panel) and lagging chromosomes (LC, bottom panel) with subsequent micronuclei production (MN) were observed. Scale bar = 10 μm. c Asymmetrical separation of DNA occasionally resulted from tripolar (top panel) and quadripolar (bottom panel) spindles. Scale bar = 10 μm

    Techniques Used: Labeling, Imaging, Fluorescence

    Cell cycle profiles obtained with varying cellular densities. Asynchronous cells were plated into 8-well coverglass-bottom chambered slides at varying densities and allowed to adhere for 24 h. Hoechst 33342 was added to the imaging medium and allowed to reach binding saturation (~ 2 h). Images for Hoechst 33342 were then collected at distinct locations and histograms generated from images containing approximately 100, 200, 300, 400, 500 and 600 cells within a single field of view. The x-axis represents normalized integrated nuclear fluorescence for each cell imaged and the y-axis histogram counts
    Figure Legend Snippet: Cell cycle profiles obtained with varying cellular densities. Asynchronous cells were plated into 8-well coverglass-bottom chambered slides at varying densities and allowed to adhere for 24 h. Hoechst 33342 was added to the imaging medium and allowed to reach binding saturation (~ 2 h). Images for Hoechst 33342 were then collected at distinct locations and histograms generated from images containing approximately 100, 200, 300, 400, 500 and 600 cells within a single field of view. The x-axis represents normalized integrated nuclear fluorescence for each cell imaged and the y-axis histogram counts

    Techniques Used: Imaging, Binding Assay, Generated, Fluorescence

    Procedural schematic for measuring DNA content in live cells. Cells of interest are plated in coverglass-bottom chambered slides and are later transferred to an inverted microscope for the collection of time-lapse images. The acquisition is then paused ~ 2 h before the completion of the time-lapse experiment and Hoechst 33342 is added to the imaging medium at a concentration of 1 μg/mL, the acquisition is then resumed. At the completion of the time-lapse experiment, images are collected for Hoechst 33342 fluorescence and analyzed with the ProcessDNA algorithm. The time-lapse images are then concatenated with the analyzed images for DNA content (steps 1–6)
    Figure Legend Snippet: Procedural schematic for measuring DNA content in live cells. Cells of interest are plated in coverglass-bottom chambered slides and are later transferred to an inverted microscope for the collection of time-lapse images. The acquisition is then paused ~ 2 h before the completion of the time-lapse experiment and Hoechst 33342 is added to the imaging medium at a concentration of 1 μg/mL, the acquisition is then resumed. At the completion of the time-lapse experiment, images are collected for Hoechst 33342 fluorescence and analyzed with the ProcessDNA algorithm. The time-lapse images are then concatenated with the analyzed images for DNA content (steps 1–6)

    Techniques Used: Inverted Microscopy, Imaging, Concentration Assay, Fluorescence

    30) Product Images from "Loss of microRNA-27b contributes to breast cancer stem cell generation by activating ENPP1"

    Article Title: Loss of microRNA-27b contributes to breast cancer stem cell generation by activating ENPP1

    Journal: Nature Communications

    doi: 10.1038/ncomms8318

    MiR-27b regulates the resistance of breast cancer cells to docetaxel. ( a ) Overview of the method used to establish miR-27b knockdown MCF7-luc (MCF7-luc anti-miR-27b) cells. ( b , c ) Dose–response curves of MCF7-luc anti-NC, MCF7-luc anti-miR-27b and MCF7-luc miR-27b o.e. cells treated with docetaxel. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC 50 value. Data are represented as the mean±s.d. of n =3 replicates. ( d ) Morphologies of the MCF7-luc anti-NC, MCF7-luc miR-27b o.e. and MCF7-luc anti-miR-27b cells. Scale bar, 100 μm. ( e ) Flow cytometric analyses of the SP fraction of MCF7-luc derivatives in the presence and absence of Ko143. ( f ) Quantification of the SP fraction of MCF7-luc derivatives. The SP fraction was determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n =3 replicates. Statistical significance was determined by Student's t -test.
    Figure Legend Snippet: MiR-27b regulates the resistance of breast cancer cells to docetaxel. ( a ) Overview of the method used to establish miR-27b knockdown MCF7-luc (MCF7-luc anti-miR-27b) cells. ( b , c ) Dose–response curves of MCF7-luc anti-NC, MCF7-luc anti-miR-27b and MCF7-luc miR-27b o.e. cells treated with docetaxel. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC 50 value. Data are represented as the mean±s.d. of n =3 replicates. ( d ) Morphologies of the MCF7-luc anti-NC, MCF7-luc miR-27b o.e. and MCF7-luc anti-miR-27b cells. Scale bar, 100 μm. ( e ) Flow cytometric analyses of the SP fraction of MCF7-luc derivatives in the presence and absence of Ko143. ( f ) Quantification of the SP fraction of MCF7-luc derivatives. The SP fraction was determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n =3 replicates. Statistical significance was determined by Student's t -test.

    Techniques Used: Flow Cytometry, Staining

    Functional analysis of ENPP1 in MCF7-luc cells. ( a ) Flow cytometric analysis of the SP fractions of MCF7-luc cells overexpressing ENPP1-MF or GFP as a control, in the presence and absence of Ko143. ( b ) Quantification of the SP fractions shown in a , determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n =3 replicates. ( c ) Flow cytometric analysis showing the cell surface localization of ABCG2 in the indicated 293T co-transfectants. ( d ) Flow cytometric analyses of the cell surface localization of ABCG2 in MCF7-luc anti-miR-27b cells transfected with a control (shNC) or ENPP1-specific (shENPP1) shRNA. ( e ) Dose–response curves of docetaxel-treated MCF7-luc anti-miR-27b-DR cells transfected with shNC or shENPP1. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC 50 value. Data are represented as the mean±s.d. of n =3 replicates. ( f ) Proximity ligation assay using MCF7-luc anti-NC or MCF7-luc anti-miR-27b cells transiently expressing ABCG2-HA. Scale bar, 50 μm. ( g ) In vitro binding assay using C-terminally Flag-tagged GFP or C-terminally Myc- and Flag-tagged ENPP1 purified from 293T cells and C-terminally HA-tagged ABCG2 purified from Sf21 insect cell extracts.
    Figure Legend Snippet: Functional analysis of ENPP1 in MCF7-luc cells. ( a ) Flow cytometric analysis of the SP fractions of MCF7-luc cells overexpressing ENPP1-MF or GFP as a control, in the presence and absence of Ko143. ( b ) Quantification of the SP fractions shown in a , determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n =3 replicates. ( c ) Flow cytometric analysis showing the cell surface localization of ABCG2 in the indicated 293T co-transfectants. ( d ) Flow cytometric analyses of the cell surface localization of ABCG2 in MCF7-luc anti-miR-27b cells transfected with a control (shNC) or ENPP1-specific (shENPP1) shRNA. ( e ) Dose–response curves of docetaxel-treated MCF7-luc anti-miR-27b-DR cells transfected with shNC or shENPP1. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC 50 value. Data are represented as the mean±s.d. of n =3 replicates. ( f ) Proximity ligation assay using MCF7-luc anti-NC or MCF7-luc anti-miR-27b cells transiently expressing ABCG2-HA. Scale bar, 50 μm. ( g ) In vitro binding assay using C-terminally Flag-tagged GFP or C-terminally Myc- and Flag-tagged ENPP1 purified from 293T cells and C-terminally HA-tagged ABCG2 purified from Sf21 insect cell extracts.

    Techniques Used: Functional Assay, Flow Cytometry, Staining, Transfection, shRNA, Proximity Ligation Assay, Expressing, In Vitro, Binding Assay, Purification

    31) Product Images from "ATP7B expression confers multidrug resistance through drug sequestration"

    Article Title: ATP7B expression confers multidrug resistance through drug sequestration

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8059

    Subcellular localization of doxorubicin in mutant ATP7B expressing cells Subcellular localization of doxorubicin at 4 hours after doxorubicin exposure and washing with PBS in a. KB/M6C/S, b. KB/Cu0, and c. KB/Cu6 cells. GFP-late endosomes are green, doxorubicin is red. The nuclei are stained with Hoechst 33342 (blue).
    Figure Legend Snippet: Subcellular localization of doxorubicin in mutant ATP7B expressing cells Subcellular localization of doxorubicin at 4 hours after doxorubicin exposure and washing with PBS in a. KB/M6C/S, b. KB/Cu0, and c. KB/Cu6 cells. GFP-late endosomes are green, doxorubicin is red. The nuclei are stained with Hoechst 33342 (blue).

    Techniques Used: Mutagenesis, Expressing, Staining

    Subcellular localization of doxorubicin in KB/WD cells Subcellular localization of doxorubicin at 4 hours after its exposure and washing with PBS in a. GFP-Golgi (green) transfected KB/WD cells and b. GFP-late endosome (green) transfected KB/WD cells. Doxorubicin is red. The nuclei are stained with Hoechst 33342 (blue).
    Figure Legend Snippet: Subcellular localization of doxorubicin in KB/WD cells Subcellular localization of doxorubicin at 4 hours after its exposure and washing with PBS in a. GFP-Golgi (green) transfected KB/WD cells and b. GFP-late endosome (green) transfected KB/WD cells. Doxorubicin is red. The nuclei are stained with Hoechst 33342 (blue).

    Techniques Used: Transfection, Staining

    Subcellular localization of doxorubicin and EGFP-ATP7B or EGFP-M6C/S Subcellular localization of doxorubicin and ATP7B and M6C/S at 4 hours after doxorubicin exposure and washing with PBS in EGFP-ATP7B (green) a. or EGFP-M6C/S (green) b. transfected KB-3-1 cells. Doxorubicin is red. The nuclei are stained with Hoechst 33342 (blue).
    Figure Legend Snippet: Subcellular localization of doxorubicin and EGFP-ATP7B or EGFP-M6C/S Subcellular localization of doxorubicin and ATP7B and M6C/S at 4 hours after doxorubicin exposure and washing with PBS in EGFP-ATP7B (green) a. or EGFP-M6C/S (green) b. transfected KB-3-1 cells. Doxorubicin is red. The nuclei are stained with Hoechst 33342 (blue).

    Techniques Used: Transfection, Staining

    Subcellular localization of doxorubicin in NH 4 Cl or tamoxifen treated KB/WD cells Subcellular localization of doxorubicin in a. GFP-late endosome (green) transfected KB/WD cells at 4 hours after exposure, b. in the presence of 10 mM NH 4 Cl, or c. in the presence of 10 μM tamoxifen. Doxorubicin is red. The nuclei are stained with Hoechst 33342 (blue).
    Figure Legend Snippet: Subcellular localization of doxorubicin in NH 4 Cl or tamoxifen treated KB/WD cells Subcellular localization of doxorubicin in a. GFP-late endosome (green) transfected KB/WD cells at 4 hours after exposure, b. in the presence of 10 mM NH 4 Cl, or c. in the presence of 10 μM tamoxifen. Doxorubicin is red. The nuclei are stained with Hoechst 33342 (blue).

    Techniques Used: Transfection, Staining

    Doxorubicin localization in KB/EV and KB/WD cells Cells were exposed to doxorubicin for 1 hour and washed with PBS. Localization was observed in KB/EV cells at 0 time a and b. at 4 hours after washing PBS. Localization was observed in KB/WD cells c. at 0 time and d. 4 hours after washing PBS. Doxorubicin is red. The nuclei are stained with Hoechst 33342 (blue).
    Figure Legend Snippet: Doxorubicin localization in KB/EV and KB/WD cells Cells were exposed to doxorubicin for 1 hour and washed with PBS. Localization was observed in KB/EV cells at 0 time a and b. at 4 hours after washing PBS. Localization was observed in KB/WD cells c. at 0 time and d. 4 hours after washing PBS. Doxorubicin is red. The nuclei are stained with Hoechst 33342 (blue).

    Techniques Used: Staining

    32) Product Images from "Overexpression of Chicken IRF7 Increased Viral Replication and Programmed Cell Death to the Avian Influenza Virus Infection Through TGF-Beta/FoxO Signaling Axis in DF-1"

    Article Title: Overexpression of Chicken IRF7 Increased Viral Replication and Programmed Cell Death to the Avian Influenza Virus Infection Through TGF-Beta/FoxO Signaling Axis in DF-1

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2018.00415

    IRF7 overexpression resulted more cell death. Representative images of Hoechst 33342 and Propidium Iodide (PI) double nuclear staining of control and Cuo- IRF7 cell lines at (A) 6 h ( N = 6) and (B) 12 h ( N = 3) post-infection with H6N2. Images were visualized by 20x subjective lens. (C) The percentage of dead cells (PI positive; red) compared with total cells (Hoechst positive; blue) for Control and CuO- IRF7 cells with mock and H6N2 condition at 6 hpi. All data are shown as mean ± SEM from six replicates. ( ∗∗∗ p
    Figure Legend Snippet: IRF7 overexpression resulted more cell death. Representative images of Hoechst 33342 and Propidium Iodide (PI) double nuclear staining of control and Cuo- IRF7 cell lines at (A) 6 h ( N = 6) and (B) 12 h ( N = 3) post-infection with H6N2. Images were visualized by 20x subjective lens. (C) The percentage of dead cells (PI positive; red) compared with total cells (Hoechst positive; blue) for Control and CuO- IRF7 cells with mock and H6N2 condition at 6 hpi. All data are shown as mean ± SEM from six replicates. ( ∗∗∗ p

    Techniques Used: Over Expression, Staining, Infection

    33) Product Images from "Mineral particles stimulate innate immunity through neutrophil extracellular traps containing HMGB1"

    Article Title: Mineral particles stimulate innate immunity through neutrophil extracellular traps containing HMGB1

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-16778-4

    Mineralo-organic particles induce NET release by neutrophils. Mineralo-organic particles were prepared by adding 3 mM of CaCl 2 and NaH 2 PO 4 each in DMEM containing ( A ) 0.1% or ( B ) 3% FBS, prior to incubation and preparation for TEM without staining as described in Methods . Data are representative of three independent experiments. Scale bars: 200 nm. ( C ) Live cell imaging of human neutrophils stained with Hoechst 33342 (blue) and treated at time 0 with mineral particles (labeled with FITC-bovine serum albumin; FITC-BSA; green). NET-associated DNA was stained with Sytox (red). Data are representative of three independent experiments. Scale bars: 10 μm. Neutrophils were incubated for 2 hours in the absence ( D ) or presence of particles in 0.1% FBS ( E ), 0.12 mg/ml bovine serum fetuin-A (BSF) ( F ), or 40 mg/ml BSF ( G ). NET-associated DNA was stained green by Sytox. Data are representative of three independent experiments. Scale bars: 20 μm. ( H ) Percentage of neutrophils forming NETs 2 hours after addition of particles. Data are shown as means ± standard errors of the mean (SEM) and the results of at least three independent experiments. ** p
    Figure Legend Snippet: Mineralo-organic particles induce NET release by neutrophils. Mineralo-organic particles were prepared by adding 3 mM of CaCl 2 and NaH 2 PO 4 each in DMEM containing ( A ) 0.1% or ( B ) 3% FBS, prior to incubation and preparation for TEM without staining as described in Methods . Data are representative of three independent experiments. Scale bars: 200 nm. ( C ) Live cell imaging of human neutrophils stained with Hoechst 33342 (blue) and treated at time 0 with mineral particles (labeled with FITC-bovine serum albumin; FITC-BSA; green). NET-associated DNA was stained with Sytox (red). Data are representative of three independent experiments. Scale bars: 10 μm. Neutrophils were incubated for 2 hours in the absence ( D ) or presence of particles in 0.1% FBS ( E ), 0.12 mg/ml bovine serum fetuin-A (BSF) ( F ), or 40 mg/ml BSF ( G ). NET-associated DNA was stained green by Sytox. Data are representative of three independent experiments. Scale bars: 20 μm. ( H ) Percentage of neutrophils forming NETs 2 hours after addition of particles. Data are shown as means ± standard errors of the mean (SEM) and the results of at least three independent experiments. ** p

    Techniques Used: Incubation, Transmission Electron Microscopy, Staining, Live Cell Imaging, Labeling

    34) Product Images from "Location, Isolation, and Identification of Mesenchymal Stem Cells from Adult Human Sweat Glands"

    Article Title: Location, Isolation, and Identification of Mesenchymal Stem Cells from Adult Human Sweat Glands

    Journal: Stem Cells International

    doi: 10.1155/2018/2090276

    Histomorphology, immunocytochemical analysis, and ultrastructure of a detached solenoid bulb of ahSGs. (a) Phase contrast image of a detached ahSG solenoid bulb via phase contrast microscopy. (b) H E staining of secretory and duct portions of the detached ahSG solenoid bulb. (c, d) Double immunofluorescence of the detached ahSG solenoid bulbs using antibodies against the following: CK15 ((c): red), CEA ((d): red), and α -SMA ((c, d): green) with cell nuclei stained by Hoechst 33342 (blue). White arrows indicated SMA positive cells. (e) The detached ahSG secretory portion by TEM. The ECM on the outside of the secretory portion of the detached ahSGs disappeared. (f) The detached ahSG secretory portion was observed by immunoelectron microscopy with anti- α -SMA as the primary antibody. Nu: nucleus; Myo: myoepithelial cells. White arrows indicated α -SMA positive immunogold labeling. (a) 40x. (b) Bar: 50 μ m. (c, d) 400x. (e) Bar: 5 μ m. (f) Bar: 2 μ m.
    Figure Legend Snippet: Histomorphology, immunocytochemical analysis, and ultrastructure of a detached solenoid bulb of ahSGs. (a) Phase contrast image of a detached ahSG solenoid bulb via phase contrast microscopy. (b) H E staining of secretory and duct portions of the detached ahSG solenoid bulb. (c, d) Double immunofluorescence of the detached ahSG solenoid bulbs using antibodies against the following: CK15 ((c): red), CEA ((d): red), and α -SMA ((c, d): green) with cell nuclei stained by Hoechst 33342 (blue). White arrows indicated SMA positive cells. (e) The detached ahSG secretory portion by TEM. The ECM on the outside of the secretory portion of the detached ahSGs disappeared. (f) The detached ahSG secretory portion was observed by immunoelectron microscopy with anti- α -SMA as the primary antibody. Nu: nucleus; Myo: myoepithelial cells. White arrows indicated α -SMA positive immunogold labeling. (a) 40x. (b) Bar: 50 μ m. (c, d) 400x. (e) Bar: 5 μ m. (f) Bar: 2 μ m.

    Techniques Used: Microscopy, Staining, Immunofluorescence, Transmission Electron Microscopy, Immuno-Electron Microscopy, Labeling

    In vitro tissue culture from detached ahSG solenoid bulbs. (a) Typical morphology of different cells growing out from an ahSG fragment. The boxed area was magnified to visualize the fibroblast-like cells and epithelioid cells wrapped around them. (b) Double immunofluorescence of the primary cells growing out from the ahSG fragment using antibodies against CK15 and α -SMA. The cells were stained with antibodies to K15 (red) and α -SMA (green) with cell nuclei stained by Hoechst 33342 (blue). (c) FCM showing α -SMA expression by the hSG secretory cells. (a) 40x and the box is 100x. (b) 400x.
    Figure Legend Snippet: In vitro tissue culture from detached ahSG solenoid bulbs. (a) Typical morphology of different cells growing out from an ahSG fragment. The boxed area was magnified to visualize the fibroblast-like cells and epithelioid cells wrapped around them. (b) Double immunofluorescence of the primary cells growing out from the ahSG fragment using antibodies against CK15 and α -SMA. The cells were stained with antibodies to K15 (red) and α -SMA (green) with cell nuclei stained by Hoechst 33342 (blue). (c) FCM showing α -SMA expression by the hSG secretory cells. (a) 40x and the box is 100x. (b) 400x.

    Techniques Used: In Vitro, Immunofluorescence, Staining, Expressing

    Histomorphology, immunocytochemical analysis, and ultrastructure of ahSGs in vivo . (a) Diagram showing each portion of ahSGs, which includes the intraepidermal, intradermal, and intraglandular duct and secretory portion. (b) H E staining of the adult human skin (full thickness). The boxed area was magnified to determine that the solenoid bulb consisted of a duct and secretory portion. (c, d) Double immunofluorescence using the antibody combinations CEA or CK15 and α -SMA. The ahSG duct portion was positive for CEA and negative for CK15 (red) and α -SMA (green), whereas the secretory portion expressed CK15 (red) and α -SMA (green) with cell nuclei stained by Hoechst 33342 (blue). (e) TEM of the ahSG secretory portion. Myoepithelial cells were found in the outer portion of the glandular epithelial cells, and the pyramidal epithelial cells and flattened myoepithelial cells were closely related to each other. The outermost layer of the ahSG secretory portion was abundant ECM. Nuclei of myoepithelial cells were rich in heterochromatin that was darker, on the edge of the nucleus and around the nucleolus. Nu: nucleus; My: myoepithelial cell; G: glandular epithelial cell; E: ECM. (f) Immunoelectron microscopy of the ahSG secretary portion. The boxed area is magnified to visualize the myoepithelial cells. In the cytoplasm of the myoepithelial cells, the dispersive black dots are colloidal gold particles connected to α -SMA (white arrows indicate the colloidal gold particles). (b). Bar: 500 μ m and 50 μ m. (c) 400x. (d) 600x. (e) Bar: 5 μ m. (f) Bar: 2 μ m.
    Figure Legend Snippet: Histomorphology, immunocytochemical analysis, and ultrastructure of ahSGs in vivo . (a) Diagram showing each portion of ahSGs, which includes the intraepidermal, intradermal, and intraglandular duct and secretory portion. (b) H E staining of the adult human skin (full thickness). The boxed area was magnified to determine that the solenoid bulb consisted of a duct and secretory portion. (c, d) Double immunofluorescence using the antibody combinations CEA or CK15 and α -SMA. The ahSG duct portion was positive for CEA and negative for CK15 (red) and α -SMA (green), whereas the secretory portion expressed CK15 (red) and α -SMA (green) with cell nuclei stained by Hoechst 33342 (blue). (e) TEM of the ahSG secretory portion. Myoepithelial cells were found in the outer portion of the glandular epithelial cells, and the pyramidal epithelial cells and flattened myoepithelial cells were closely related to each other. The outermost layer of the ahSG secretory portion was abundant ECM. Nuclei of myoepithelial cells were rich in heterochromatin that was darker, on the edge of the nucleus and around the nucleolus. Nu: nucleus; My: myoepithelial cell; G: glandular epithelial cell; E: ECM. (f) Immunoelectron microscopy of the ahSG secretary portion. The boxed area is magnified to visualize the myoepithelial cells. In the cytoplasm of the myoepithelial cells, the dispersive black dots are colloidal gold particles connected to α -SMA (white arrows indicate the colloidal gold particles). (b). Bar: 500 μ m and 50 μ m. (c) 400x. (d) 600x. (e) Bar: 5 μ m. (f) Bar: 2 μ m.

    Techniques Used: In Vivo, Staining, Immunofluorescence, Transmission Electron Microscopy, Immuno-Electron Microscopy

    35) Product Images from "Brain ischemia downregulates the neuroprotective GDNF-Ret signaling by a calpain-dependent mechanism in cultured hippocampal neurons"

    Article Title: Brain ischemia downregulates the neuroprotective GDNF-Ret signaling by a calpain-dependent mechanism in cultured hippocampal neurons

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.578

    Neuroprotection by GNDF under excitotoxic conditions and  in vitro  ischemia. ( a ) Cultured hippocampal neurons (15 DIV) were challenged with 50  μ M glutamate for 20 min and where indicated they were incubated with 10 ng/ml GDNF (GDNF) during excitotoxic stimulation (co-incubation), or pre-incubated with the neurotrophic factor for 30 min (pre-incubation). ( b ) Cultured hippocampal neurons (15 DIV) were exposed to sham/OGD for 90 min and where indicated they were incubated with 10 ng/ml GDNF (GDNF) during the insult (co-incubation), immediately after (post-incubation) or pre-incubated with the neurotrophic factor for 30 min (pre-incubation). When tested, the neurotrophic factor was also present during the incubation period in culture-conditioned medium that followed glutamate stimulation ( a ) or sham/OGD ( b ). Cell death was assessed 8 h after excitotoxic stimulation ( a ) or 12 h after sham/OGD ( b ) by fluorescence microscopy, using the fluorescent dye Hoechst 33342. ( c ) Hippocampal neurons were transfected with GFP or hRet51-GFP and exposed to sham/OGD for 90 min. After the insult, the cells were further incubated in culture-conditioned medium for 12 h. When tested, 10 ng/ml GDNF (GDNF) was added to the cells immediately after the insult. The transfected cells were identified by immunocytochemistry with an anti-GFP antibody, and the viability of GFP or hRet51-GFP-transfected cells was evaluated with Hoechst 33342. The results are the average±S.E.M. of 3–7 different experiments performed in independent preparations. Statistical analysis was performed using one-way ANOVA followed by Bonferroni's multiple comparison test (*** P
    Figure Legend Snippet: Neuroprotection by GNDF under excitotoxic conditions and in vitro ischemia. ( a ) Cultured hippocampal neurons (15 DIV) were challenged with 50  μ M glutamate for 20 min and where indicated they were incubated with 10 ng/ml GDNF (GDNF) during excitotoxic stimulation (co-incubation), or pre-incubated with the neurotrophic factor for 30 min (pre-incubation). ( b ) Cultured hippocampal neurons (15 DIV) were exposed to sham/OGD for 90 min and where indicated they were incubated with 10 ng/ml GDNF (GDNF) during the insult (co-incubation), immediately after (post-incubation) or pre-incubated with the neurotrophic factor for 30 min (pre-incubation). When tested, the neurotrophic factor was also present during the incubation period in culture-conditioned medium that followed glutamate stimulation ( a ) or sham/OGD ( b ). Cell death was assessed 8 h after excitotoxic stimulation ( a ) or 12 h after sham/OGD ( b ) by fluorescence microscopy, using the fluorescent dye Hoechst 33342. ( c ) Hippocampal neurons were transfected with GFP or hRet51-GFP and exposed to sham/OGD for 90 min. After the insult, the cells were further incubated in culture-conditioned medium for 12 h. When tested, 10 ng/ml GDNF (GDNF) was added to the cells immediately after the insult. The transfected cells were identified by immunocytochemistry with an anti-GFP antibody, and the viability of GFP or hRet51-GFP-transfected cells was evaluated with Hoechst 33342. The results are the average±S.E.M. of 3–7 different experiments performed in independent preparations. Statistical analysis was performed using one-way ANOVA followed by Bonferroni's multiple comparison test (*** P

    Techniques Used: In Vitro, Cell Culture, Incubation, Fluorescence, Microscopy, Transfection, Immunocytochemistry

    36) Product Images from "Pterostilbene modulates the suppression of multidrug resistance protein 1 and triggers autophagic and apoptotic mechanisms in cisplatin-resistant human oral cancer CAR cells via AKT signaling"

    Article Title: Pterostilbene modulates the suppression of multidrug resistance protein 1 and triggers autophagic and apoptotic mechanisms in cisplatin-resistant human oral cancer CAR cells via AKT signaling

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2018.4298

    Effects of pterostilbene on the autophagy and DNA condensation of CAR cells. The cells were treated with 0, 25, 50 and 75 μ M pterostilbene for 24 h and then probed using (A) acridine orange to detect acidic vesicular organelles, indicated by a red color (magnification, ×200). (B) Monodansylcadaverin, an autophagolysosome marker, indicated by a green color (magnification, ×200). (C) LysoTracker Red to determine lysosomal function, indicated by a red color (magnification, ×200). (D) Cathepsin B to detect lysosomal activity, indicated by a red color (magnification, ×200). (E) Hoechst 33342 staining to observe cell nuclei, as indicated by a blue color (magnification, ×400). Representative images were taken from three independent experiments.
    Figure Legend Snippet: Effects of pterostilbene on the autophagy and DNA condensation of CAR cells. The cells were treated with 0, 25, 50 and 75 μ M pterostilbene for 24 h and then probed using (A) acridine orange to detect acidic vesicular organelles, indicated by a red color (magnification, ×200). (B) Monodansylcadaverin, an autophagolysosome marker, indicated by a green color (magnification, ×200). (C) LysoTracker Red to determine lysosomal function, indicated by a red color (magnification, ×200). (D) Cathepsin B to detect lysosomal activity, indicated by a red color (magnification, ×200). (E) Hoechst 33342 staining to observe cell nuclei, as indicated by a blue color (magnification, ×400). Representative images were taken from three independent experiments.

    Techniques Used: Marker, Activity Assay, Staining

    37) Product Images from "Cucurbitacin L 2-O-β-Glucoside Demonstrates Apoptogenesis in Colon Adenocarcinoma Cells (HT-29): Involvement of Reactive Oxygen and Nitrogen Species Regulation"

    Article Title: Cucurbitacin L 2-O-β-Glucoside Demonstrates Apoptogenesis in Colon Adenocarcinoma Cells (HT-29): Involvement of Reactive Oxygen and Nitrogen Species Regulation

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2012/490136

    Fluorescent photomicrographs of cells stained Hoechst 33342 being treated with with CLG (IC 50 ) for 24 and 48 h. (a) Control, (b) chromatin condensation in the nucleus (48 h), and (c) quantitative analysis of apoptosis (total nuclear intensity). Statistical significance is expressed as *, P
    Figure Legend Snippet: Fluorescent photomicrographs of cells stained Hoechst 33342 being treated with with CLG (IC 50 ) for 24 and 48 h. (a) Control, (b) chromatin condensation in the nucleus (48 h), and (c) quantitative analysis of apoptosis (total nuclear intensity). Statistical significance is expressed as *, P

    Techniques Used: Staining

    38) Product Images from "Poly(2-aminoethyl methacrylate) with well-defined chain-length for DNA vaccine delivery to dendritic cells"

    Article Title: Poly(2-aminoethyl methacrylate) with well-defined chain-length for DNA vaccine delivery to dendritic cells

    Journal: Biomacromolecules

    doi: 10.1021/bm201360v

    Intracellular dissociation of polyplexes. (A) Representative confocal fluorescence microscopy images of DC 2.4 cells transfected by polyplexes (N:P ratio of 8) at 24 h. Red: Cy5-labeled plasmid. Green: Oregon Green-labeled polymer. Blue: Hoechst 33342 staining cell nuclei. Arrows point to polyplexes that remained intact or dissociated (labeled “low” or “high”, respectively). (B) Quantification of intracelluar polyplex dissociation. Mean ± SE, 30 cells for each sample were counted, * p
    Figure Legend Snippet: Intracellular dissociation of polyplexes. (A) Representative confocal fluorescence microscopy images of DC 2.4 cells transfected by polyplexes (N:P ratio of 8) at 24 h. Red: Cy5-labeled plasmid. Green: Oregon Green-labeled polymer. Blue: Hoechst 33342 staining cell nuclei. Arrows point to polyplexes that remained intact or dissociated (labeled “low” or “high”, respectively). (B) Quantification of intracelluar polyplex dissociation. Mean ± SE, 30 cells for each sample were counted, * p

    Techniques Used: Fluorescence, Microscopy, Transfection, Labeling, Plasmid Preparation, Staining

    Nuclear localization of plasmid and dissociation of polyplexes. (A) Representative confocal fluorescence microscopy images of DC 2.4 cells transfected by polyplexes (N:P ratio of 8) at 24 h. Red: Cy5-labeled plasmid. Green: Oregon Green-labeled polymer. Blue: Hoechst 33342 staining cell nuclei outlined in dotted white lines. Arrows point to regions where plasmid signal was found in the nucleus.
    Figure Legend Snippet: Nuclear localization of plasmid and dissociation of polyplexes. (A) Representative confocal fluorescence microscopy images of DC 2.4 cells transfected by polyplexes (N:P ratio of 8) at 24 h. Red: Cy5-labeled plasmid. Green: Oregon Green-labeled polymer. Blue: Hoechst 33342 staining cell nuclei outlined in dotted white lines. Arrows point to regions where plasmid signal was found in the nucleus.

    Techniques Used: Plasmid Preparation, Fluorescence, Microscopy, Transfection, Labeling, Staining

    Cellular uptake of plasmid. (A) Representative confocal fluorescence microscopy images of DC 2.4 cells transfected by polyplexes (N:P ratio of 8) at various time points. Red: Cy5-labeled plasmid. Blue: Hoechst 33342 staining cell nuclei. Shown are fluorescence overlaid with white-light images. Scale bar: 25 μm. (B) Quantification of cellular uptake. Mean ± SE, 30 cells for each sample were counted, * p
    Figure Legend Snippet: Cellular uptake of plasmid. (A) Representative confocal fluorescence microscopy images of DC 2.4 cells transfected by polyplexes (N:P ratio of 8) at various time points. Red: Cy5-labeled plasmid. Blue: Hoechst 33342 staining cell nuclei. Shown are fluorescence overlaid with white-light images. Scale bar: 25 μm. (B) Quantification of cellular uptake. Mean ± SE, 30 cells for each sample were counted, * p

    Techniques Used: Plasmid Preparation, Fluorescence, Microscopy, Transfection, Labeling, Staining

    Endolysosomal localization of plasmid. (A) Representative confocal fluorescence microscopy images of DC 2.4 cells transfected by polyplexes (N:P ratio of 8) at 24 h. Red: Cy5-labeled plasmid. Green: LysoTracker Green. Blue: Hoechst 33342 staining cell nuclei. Arrows point to regions where plasmid was or was not localized in the endolysosome (labeled “high” or “low, respectively). low degree of localization plasmid that remained intact (low) or dissociated (high). (B) Quantification of plasmid localized in the endolysosome. Mean ± SE, 30 cells for each sample were counted, * p
    Figure Legend Snippet: Endolysosomal localization of plasmid. (A) Representative confocal fluorescence microscopy images of DC 2.4 cells transfected by polyplexes (N:P ratio of 8) at 24 h. Red: Cy5-labeled plasmid. Green: LysoTracker Green. Blue: Hoechst 33342 staining cell nuclei. Arrows point to regions where plasmid was or was not localized in the endolysosome (labeled “high” or “low, respectively). low degree of localization plasmid that remained intact (low) or dissociated (high). (B) Quantification of plasmid localized in the endolysosome. Mean ± SE, 30 cells for each sample were counted, * p

    Techniques Used: Plasmid Preparation, Fluorescence, Microscopy, Transfection, Labeling, Staining

    39) Product Images from "An accessible organotypic microvessel model using iPSC-derived endothelium"

    Article Title: An accessible organotypic microvessel model using iPSC-derived endothelium

    Journal: Advanced healthcare materials

    doi: 10.1002/adhm.201700497

    iEC microvessels support neutrophil extravasation and migration. (A) Fluorescent microscopy of iEC lumen (green – calcein am) shows successful extravasation and migration of labeled purified neutrophils towards an IL-8 source (top); individual iEC also migrate and sprout from the vessel formed in 5mg/mL collagen I, but can be differentiated due to the lack of calcein stain. Image is displayed as a maximum intensity project across a Z-stack in order to visualize neutrophil migration in multiple planes. (B) Maximum intensity project cell extravasation and migration from whole blood (blue – hoechst 33342) in the iEC lumen. Migratory cells, likely to be neutrophils on this time scale, migrate both towards and away from IL-8 source when left as whole blood. (C) Quantification of maximum migration distance shows purified neutrophils migrating significantly farther in response to 11 µM IL-8 (p=0.0005, n=4) than 10 µM fMLP. (D) A less significant, but opposite trend regarding maximum migration distance (p=0.0192, n=4) is observed when using whole blood for neutrophil extravasation, with greater total migration observed using fMLP. (E) Increased variability and decreased chemotactic effect (number of cells migrating up the gradient compared to total number of migrating cells) is observed with whole blood samples, likely contributing conflicting results. Purified neutrophils trend toward increased chemotactic effect with fMLP (p=0.2925, n.s., n=4) but significantly increase positive chemotaxis with IL-8 gradients (p ≤0.0001, n=4).
    Figure Legend Snippet: iEC microvessels support neutrophil extravasation and migration. (A) Fluorescent microscopy of iEC lumen (green – calcein am) shows successful extravasation and migration of labeled purified neutrophils towards an IL-8 source (top); individual iEC also migrate and sprout from the vessel formed in 5mg/mL collagen I, but can be differentiated due to the lack of calcein stain. Image is displayed as a maximum intensity project across a Z-stack in order to visualize neutrophil migration in multiple planes. (B) Maximum intensity project cell extravasation and migration from whole blood (blue – hoechst 33342) in the iEC lumen. Migratory cells, likely to be neutrophils on this time scale, migrate both towards and away from IL-8 source when left as whole blood. (C) Quantification of maximum migration distance shows purified neutrophils migrating significantly farther in response to 11 µM IL-8 (p=0.0005, n=4) than 10 µM fMLP. (D) A less significant, but opposite trend regarding maximum migration distance (p=0.0192, n=4) is observed when using whole blood for neutrophil extravasation, with greater total migration observed using fMLP. (E) Increased variability and decreased chemotactic effect (number of cells migrating up the gradient compared to total number of migrating cells) is observed with whole blood samples, likely contributing conflicting results. Purified neutrophils trend toward increased chemotactic effect with fMLP (p=0.2925, n.s., n=4) but significantly increase positive chemotaxis with IL-8 gradients (p ≤0.0001, n=4).

    Techniques Used: Migration, Microscopy, Labeling, Purification, Staining, Chemotaxis Assay

    Matrix composition alters iEC vessel barrier function. (A) Fluorescent microscopy (green – FITC-conjugated phalloidin, blue – hoechst 33342) for the visualization of the 10 mg/mL fibrin iEC vessel cytoskeleton reveals alignment parallel to the vessel length, morphological elongation, and strong cortical actin expression, supporting tight cell-cell contact and vessel maturity. (B) Fluorescent visualization of iEC vessels formed in 4 mg/mL collagen I matrices lack obvious cortical actin or morphological elongation and alignment, and they display observable holes in the iEC microvessel monolayer (outlined in red). (C) Permeability testing with 70 kDa FITC-dextran for collagen I ECM vessels highlights the leaky phenotype with observable dextran leakage through holes immediately after loading and significant diffusion by 10 min.
    Figure Legend Snippet: Matrix composition alters iEC vessel barrier function. (A) Fluorescent microscopy (green – FITC-conjugated phalloidin, blue – hoechst 33342) for the visualization of the 10 mg/mL fibrin iEC vessel cytoskeleton reveals alignment parallel to the vessel length, morphological elongation, and strong cortical actin expression, supporting tight cell-cell contact and vessel maturity. (B) Fluorescent visualization of iEC vessels formed in 4 mg/mL collagen I matrices lack obvious cortical actin or morphological elongation and alignment, and they display observable holes in the iEC microvessel monolayer (outlined in red). (C) Permeability testing with 70 kDa FITC-dextran for collagen I ECM vessels highlights the leaky phenotype with observable dextran leakage through holes immediately after loading and significant diffusion by 10 min.

    Techniques Used: Microscopy, Expressing, Permeability, Diffusion-based Assay

    iEC organotypic endothelial models formed using LumeNEXT approach display key characteristics of microvessels when visualized using fluorescent Z-stack microscopy (green – p-selectin, blue – hoechst 33342, red – ZO-1). (A) iEC form the inner lining of the luminal space creating patent, confluent cellular structures through which fluids can flow. (B) Cells display intimate cell-cell contact along the edge of the vessel, and (C) these contacts express moderate levels of ZO-1, suggesting tight junction formation. (D) Ubiquitous P-selectin expression along the surface of the endothelial enables leukocyte capture and regulation of cell adhesion on the endothelium surface for inflammation modeling applications.
    Figure Legend Snippet: iEC organotypic endothelial models formed using LumeNEXT approach display key characteristics of microvessels when visualized using fluorescent Z-stack microscopy (green – p-selectin, blue – hoechst 33342, red – ZO-1). (A) iEC form the inner lining of the luminal space creating patent, confluent cellular structures through which fluids can flow. (B) Cells display intimate cell-cell contact along the edge of the vessel, and (C) these contacts express moderate levels of ZO-1, suggesting tight junction formation. (D) Ubiquitous P-selectin expression along the surface of the endothelial enables leukocyte capture and regulation of cell adhesion on the endothelium surface for inflammation modeling applications.

    Techniques Used: Microscopy, Flow Cytometry, Expressing

    40) Product Images from "Self-Organizing Circuit Assembly through Spatiotemporally Coordinated Neuronal Migration within Geometric Constraints"

    Article Title: Self-Organizing Circuit Assembly through Spatiotemporally Coordinated Neuronal Migration within Geometric Constraints

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028156

    Location and relative number of excitatory and inhibitory neurons within a network. A, A clustered network on a triangle with vertical and horizontal sections. Neuronal dendrites are labeled with MAP2 while axons with Smi312, cell nuclei counterstained with Hoechst 33342. B, Higher magnification of the highlighted cluster in a. Somata are assembled into a ball. C, Immunocytochemistry shows that excitatory neurons tend to cluster more than inhibitory neurons do. Excitatory neurons are labeled with CaMKII while inhibitory neurons are labeled with GABA (refer to the text for details). D, Higher magnification of a cluster. Inhibitory neurons labeled by GABA locate in the periphery of the cluster that is primarily composed of excitatory neurons. E, The location of all cell nuclei within a network. F, The location of excitatory and inhibitory neurons on the same network as E, while most of the neuron somata are clustered in the center of the network, a few somata of inhibitory neurons only are scattered in the periphery of the network. G, The percentage of excitatory neurons in the overall population on geometric constraints of different sizes. Note that there is substantial fluctuation of the total number of neurons among geometric constraints. As the area grows, the ratio gradually approaches unrestrained cultures with large number of cells. (n = 5, 10–15 data points for each size of geometric constraint.) H, Scatterogram showing the distribution of the ratio on geometric constraints of various areas. As the area increases, less fluctuation is present in the percentage. The same set of data as panel G is used. I, Histogram showing that inhibitory neurons tend to stay away from the clusters, indicating a heterogeneous nature in neuronal migration and cluster formation. The green shade represents the range where the diameters of neurons generally fall within. (n = 5, 42 excitatory neurons, 21 inhibitory neurons.) Scale bars: A, 200 µm; B, 25 µm; C, 200 µm; D, 50 µm.
    Figure Legend Snippet: Location and relative number of excitatory and inhibitory neurons within a network. A, A clustered network on a triangle with vertical and horizontal sections. Neuronal dendrites are labeled with MAP2 while axons with Smi312, cell nuclei counterstained with Hoechst 33342. B, Higher magnification of the highlighted cluster in a. Somata are assembled into a ball. C, Immunocytochemistry shows that excitatory neurons tend to cluster more than inhibitory neurons do. Excitatory neurons are labeled with CaMKII while inhibitory neurons are labeled with GABA (refer to the text for details). D, Higher magnification of a cluster. Inhibitory neurons labeled by GABA locate in the periphery of the cluster that is primarily composed of excitatory neurons. E, The location of all cell nuclei within a network. F, The location of excitatory and inhibitory neurons on the same network as E, while most of the neuron somata are clustered in the center of the network, a few somata of inhibitory neurons only are scattered in the periphery of the network. G, The percentage of excitatory neurons in the overall population on geometric constraints of different sizes. Note that there is substantial fluctuation of the total number of neurons among geometric constraints. As the area grows, the ratio gradually approaches unrestrained cultures with large number of cells. (n = 5, 10–15 data points for each size of geometric constraint.) H, Scatterogram showing the distribution of the ratio on geometric constraints of various areas. As the area increases, less fluctuation is present in the percentage. The same set of data as panel G is used. I, Histogram showing that inhibitory neurons tend to stay away from the clusters, indicating a heterogeneous nature in neuronal migration and cluster formation. The green shade represents the range where the diameters of neurons generally fall within. (n = 5, 42 excitatory neurons, 21 inhibitory neurons.) Scale bars: A, 200 µm; B, 25 µm; C, 200 µm; D, 50 µm.

    Techniques Used: Labeling, Immunocytochemistry, Migration

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    Article Snippet: .. After the incubation of cells with FITC-labeled E.coli bioparticles, the cells were stained with 1 μg/ml Hoechst 33342 dye (Invitrogen-Molecular Probes, CA). .. After staining for 1h at room temperature, the cells were fixed and observed under Leica 4000B fluorescence microscope for green (FITC) and blue (Hoechst 33342) fluorescence.

    Article Title: Molecular Responses of Human Retinal Cells to Infection with Dengue Virus
    Article Snippet: .. After washing with PBS, cells were counterstained with Hoechst 33342 nucleic acid stain (Thermo Fisher Scientific-Molecular Probes) for 10 minutes. .. Monolayers were mounted in ProLong Gold Antifade Mountant (Thermo Fisher Scientific-Molecular Probes) and imaged by confocal microscopy (Leica TCS SP5 Confocal Microscope: Leica Microsystems, Mannheim, Germany).

    Article Title: Analysis of the Interaction between Globular Head Modules of Human C1q and Its Candidate Receptor gC1qR
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    Incubation:

    Article Title: Ontogeny and Phagocytic Function of Baboon Lung Dendritic Cells
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    Microscopy:

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  • 92
    Thermo Fisher hoechst 33342 solution
    Image processing workflow of the high content screen for O/A induced pUb(Ser65). Images were loaded as maximum projections. The <t>Hoechst</t> 33342 channel was used to find the nuclei and border nuclei were excluded. The cytoplasm was then found on the calculated Hoechst + TOM20-568 image. The pUb(Ser65)-488 spots were identified within the whole cell and their intensity measured. The outputs of the analysis were the number of nuclei selected and the mean integrated pUb(Ser65) intensity, calculated as the area of the cell covered by pUb(Ser65) spots x corrected intensity of the spots. Analysis parameters for each building block of the Columbus workflow are detailed in the boxes.
    Hoechst 33342 Solution, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hoechst 33342 solution/product/Thermo Fisher
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    97
    Thermo Fisher hoechst 33342
    MC1 (orange) across five rounds of staining, stripping, and re-staining using βME-based stripping along with TSA-developed NeuN (pink) and <t>Hoechst</t> 33342 (blue). (A) Stripping controls which were stained in each round up until the final elution for that section where stripping was confirmed by confirming the lack of signal after development with AF546-conjugated secondary. (B) Signal from MC1 (orange) following staining, stripping, and re-staining across five rounds. NeuN (pink) was only developed in round 1 with AF647-TSA. Hoechst 33342 was stained in each round, as it is in the Prolong mounting media. Signal from MC1 appears across each of the rounds in the same locations. However a decrease in signal intensity is noted by Round 5.. Scale bar: 10 μm
    Hoechst 33342, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 6136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hoechst 33342/product/Thermo Fisher
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    hoechst 33342 - by Bioz Stars, 2020-09
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    92
    Thermo Fisher hoechst 33342 trihydrochloride
    Generating human myotonic dystrophy (DM) myogenic cell lines using direct reprogramming of urine-derived cells with the transcription factor MyoD. ( A ). Urine cells were cultured from healthy controls and individuals with myotonic dystrophy type 1 (DM1) and DM2. Once cell cultures were established, cells were transduced with lentivirus expressing an inducible form of MyoD (iMyoD) in order to induce myogenic reprogramming. This lentivirus produces MyoD in the presence of tamoxifen. Tamoxifen was used to induce MyoD expression, which, in turn, stimulated multinucleated myotube formation in culture. Directly reprogrammed multinucleated myotubes were studied after 28 days in culture. ( B ) MyoD (red) protein expression was readily detected in the nucleus of tamoxifen-treated cells. Nuclei were labeled with <t>Hoechst</t> <t>33342</t> (blue). Scale bar: 100 μm. ( C ) With Hoechst costaining, the transduction efficiency of each transduction (example in B ) could be estimated. Transduction efficiency with the iMyoD construct was similar among control, DM1, and DM2 cell lines and, in each case, averaged greater than 70%. Efficiencies are represented as percentages of MyoD positive nuclei relative to total number of nuclei.
    Hoechst 33342 Trihydrochloride, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Image processing workflow of the high content screen for O/A induced pUb(Ser65). Images were loaded as maximum projections. The Hoechst 33342 channel was used to find the nuclei and border nuclei were excluded. The cytoplasm was then found on the calculated Hoechst + TOM20-568 image. The pUb(Ser65)-488 spots were identified within the whole cell and their intensity measured. The outputs of the analysis were the number of nuclei selected and the mean integrated pUb(Ser65) intensity, calculated as the area of the cell covered by pUb(Ser65) spots x corrected intensity of the spots. Analysis parameters for each building block of the Columbus workflow are detailed in the boxes.

    Journal: bioRxiv

    Article Title: Regulation of mitophagy by the NSL complex underlies genetic risk for Parkinson’s disease at Chr16q11.2 and on the MAPT H1 allele

    doi: 10.1101/2020.01.06.896241

    Figure Lengend Snippet: Image processing workflow of the high content screen for O/A induced pUb(Ser65). Images were loaded as maximum projections. The Hoechst 33342 channel was used to find the nuclei and border nuclei were excluded. The cytoplasm was then found on the calculated Hoechst + TOM20-568 image. The pUb(Ser65)-488 spots were identified within the whole cell and their intensity measured. The outputs of the analysis were the number of nuclei selected and the mean integrated pUb(Ser65) intensity, calculated as the area of the cell covered by pUb(Ser65) spots x corrected intensity of the spots. Analysis parameters for each building block of the Columbus workflow are detailed in the boxes.

    Article Snippet: After 3x PBS washes, AlexaFluor 568 anti-mouse and 488 anti-rabbit secondary antibodies and Hoechst 33342 (Thermo Scientific, 62249) were added (in 10% FBS/PBS, 1:2000 dilution for all) and incubated for 1 h at room temperature.

    Techniques: Blocking Assay

    MC1 (orange) across five rounds of staining, stripping, and re-staining using βME-based stripping along with TSA-developed NeuN (pink) and Hoechst 33342 (blue). (A) Stripping controls which were stained in each round up until the final elution for that section where stripping was confirmed by confirming the lack of signal after development with AF546-conjugated secondary. (B) Signal from MC1 (orange) following staining, stripping, and re-staining across five rounds. NeuN (pink) was only developed in round 1 with AF647-TSA. Hoechst 33342 was stained in each round, as it is in the Prolong mounting media. Signal from MC1 appears across each of the rounds in the same locations. However a decrease in signal intensity is noted by Round 5.. Scale bar: 10 μm

    Journal: bioRxiv

    Article Title: A manual multiplex immunofluorescence method for investigating neurodegenerative diseases

    doi: 10.1101/533547

    Figure Lengend Snippet: MC1 (orange) across five rounds of staining, stripping, and re-staining using βME-based stripping along with TSA-developed NeuN (pink) and Hoechst 33342 (blue). (A) Stripping controls which were stained in each round up until the final elution for that section where stripping was confirmed by confirming the lack of signal after development with AF546-conjugated secondary. (B) Signal from MC1 (orange) following staining, stripping, and re-staining across five rounds. NeuN (pink) was only developed in round 1 with AF647-TSA. Hoechst 33342 was stained in each round, as it is in the Prolong mounting media. Signal from MC1 appears across each of the rounds in the same locations. However a decrease in signal intensity is noted by Round 5.. Scale bar: 10 μm

    Article Snippet: Additionally, we show that the use of Hoechst 33342 in each round of staining or TSA reporting of a neuronal target in the first round of staining are good options as landmarks for co-registration of images in different rounds is feasible with BME.

    Techniques: Staining, Stripping Membranes

    Continuous exposure to 1.7 GHz LTE RF-EMF decreased cell proliferation by inducing intracellular ROS in ASCs and Huh7 cells. ( A–D ) ASCs and Huh7 cells pre-treated or not with 100 μM NAC were exposed to 1.7 GHz RF-EMF for 72 h at 2 SAR, while the sham control cells were incubated for 72 h without RF-EMF exposure. After the exposure, ( A,C ) the cells were collected and counted with a cell counter (Nexcelom Bioscience). Huh7 cells ( B ) and ASCs ( D ) were stained with carboxy-H 2 DCFDA. Cells treat with TBHP were used as a positive control for intracellular ROS generation. ( E,F ) ASCs and Huh7 cells were exposed to 1.7 GHz RF-EMF for 72 h at 1 SAR or 2 SAR, and were stained with MitoSOX. ( B,D–F ) Nuclei were stained with Hoechst 33342. Images were taken with an Axioplan2 fluorescence microscope (Zeiss) under a 200× objective. Scale bar, 25 μm. All experiments consisted of three independent replicates.

    Journal: Scientific Reports

    Article Title: Continuous Exposure to 1.7 GHz LTE Electromagnetic Fields Increases Intracellular Reactive Oxygen Species to Decrease Human Cell Proliferation and Induce Senescence

    doi: 10.1038/s41598-020-65732-4

    Figure Lengend Snippet: Continuous exposure to 1.7 GHz LTE RF-EMF decreased cell proliferation by inducing intracellular ROS in ASCs and Huh7 cells. ( A–D ) ASCs and Huh7 cells pre-treated or not with 100 μM NAC were exposed to 1.7 GHz RF-EMF for 72 h at 2 SAR, while the sham control cells were incubated for 72 h without RF-EMF exposure. After the exposure, ( A,C ) the cells were collected and counted with a cell counter (Nexcelom Bioscience). Huh7 cells ( B ) and ASCs ( D ) were stained with carboxy-H 2 DCFDA. Cells treat with TBHP were used as a positive control for intracellular ROS generation. ( E,F ) ASCs and Huh7 cells were exposed to 1.7 GHz RF-EMF for 72 h at 1 SAR or 2 SAR, and were stained with MitoSOX. ( B,D–F ) Nuclei were stained with Hoechst 33342. Images were taken with an Axioplan2 fluorescence microscope (Zeiss) under a 200× objective. Scale bar, 25 μm. All experiments consisted of three independent replicates.

    Article Snippet: 1 μM Hoechst 33342 was added to the carboxy-H2DCFDA staining solution for the last 5 min of incubation.

    Techniques: Incubation, Staining, Positive Control, Fluorescence, Microscopy

    Generating human myotonic dystrophy (DM) myogenic cell lines using direct reprogramming of urine-derived cells with the transcription factor MyoD. ( A ). Urine cells were cultured from healthy controls and individuals with myotonic dystrophy type 1 (DM1) and DM2. Once cell cultures were established, cells were transduced with lentivirus expressing an inducible form of MyoD (iMyoD) in order to induce myogenic reprogramming. This lentivirus produces MyoD in the presence of tamoxifen. Tamoxifen was used to induce MyoD expression, which, in turn, stimulated multinucleated myotube formation in culture. Directly reprogrammed multinucleated myotubes were studied after 28 days in culture. ( B ) MyoD (red) protein expression was readily detected in the nucleus of tamoxifen-treated cells. Nuclei were labeled with Hoechst 33342 (blue). Scale bar: 100 μm. ( C ) With Hoechst costaining, the transduction efficiency of each transduction (example in B ) could be estimated. Transduction efficiency with the iMyoD construct was similar among control, DM1, and DM2 cell lines and, in each case, averaged greater than 70%. Efficiencies are represented as percentages of MyoD positive nuclei relative to total number of nuclei.

    Journal: JCI Insight

    Article Title: Distinct pathological signatures in human cellular models of myotonic dystrophy subtypes

    doi: 10.1172/jci.insight.122686

    Figure Lengend Snippet: Generating human myotonic dystrophy (DM) myogenic cell lines using direct reprogramming of urine-derived cells with the transcription factor MyoD. ( A ). Urine cells were cultured from healthy controls and individuals with myotonic dystrophy type 1 (DM1) and DM2. Once cell cultures were established, cells were transduced with lentivirus expressing an inducible form of MyoD (iMyoD) in order to induce myogenic reprogramming. This lentivirus produces MyoD in the presence of tamoxifen. Tamoxifen was used to induce MyoD expression, which, in turn, stimulated multinucleated myotube formation in culture. Directly reprogrammed multinucleated myotubes were studied after 28 days in culture. ( B ) MyoD (red) protein expression was readily detected in the nucleus of tamoxifen-treated cells. Nuclei were labeled with Hoechst 33342 (blue). Scale bar: 100 μm. ( C ) With Hoechst costaining, the transduction efficiency of each transduction (example in B ) could be estimated. Transduction efficiency with the iMyoD construct was similar among control, DM1, and DM2 cell lines and, in each case, averaged greater than 70%. Efficiencies are represented as percentages of MyoD positive nuclei relative to total number of nuclei.

    Article Snippet: Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific, H3570), used at final concentration of 1:10,000.

    Techniques: Derivative Assay, Cell Culture, Transduction, Expressing, Labeling, Construct