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A Small-molecule MDMX inhibitor activates p53 leading to activation of pro-apoptotic gene expression A, The chemical structures of XI-006 and Nutlin-3a. B, Recombinant cells carrying the MDMX promoter were treated with varying amounts of XI-006 for 6 h and lysed for luciferase activity assays. Data are depicted as average ± SD values of 3 determinations. C, <t>MCF-7</t> cells were treated with XI-006 overnight and lysed for qRT-PCR assays to determine the MDMX mRNA levels. Data are depicted as average ± SD values of 3 determinations. D, MCF-7 cells were treated with <t>DMSO</t> (Ctrl) or XI-006 for 16 h, and lysed for immunoblotting to measure expression levels of MDMX, p53, p21 and MDM2. E, After treatment with 5 µM of XI-006 for 16 h, MCF-7 cells were incubated with 100 µg/ml of cycloheximide. Cells were lysed at the indicated time and subjected to immunoblotting assays. F, Relative p53 levels in (E) was estimated by densitometry analysis. G, MCF-7 cells were treated as in (D). Total RNA was prepared, reverse transcribed, and cDNA subjected to qRT-PCR assays for measuring levels of PUMA, BAX, PIG3, and p21 mRNA. Data are depicted as average ± SD values of 3 determinations. *, p
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1) Product Images from "A Small-molecule Inhibitor of MDMX Activates p53 and Induces Apoptosis"

Article Title: A Small-molecule Inhibitor of MDMX Activates p53 and Induces Apoptosis

Journal: Molecular cancer therapeutics

doi: 10.1158/1535-7163.MCT-10-0581

A Small-molecule MDMX inhibitor activates p53 leading to activation of pro-apoptotic gene expression A, The chemical structures of XI-006 and Nutlin-3a. B, Recombinant cells carrying the MDMX promoter were treated with varying amounts of XI-006 for 6 h and lysed for luciferase activity assays. Data are depicted as average ± SD values of 3 determinations. C, MCF-7 cells were treated with XI-006 overnight and lysed for qRT-PCR assays to determine the MDMX mRNA levels. Data are depicted as average ± SD values of 3 determinations. D, MCF-7 cells were treated with DMSO (Ctrl) or XI-006 for 16 h, and lysed for immunoblotting to measure expression levels of MDMX, p53, p21 and MDM2. E, After treatment with 5 µM of XI-006 for 16 h, MCF-7 cells were incubated with 100 µg/ml of cycloheximide. Cells were lysed at the indicated time and subjected to immunoblotting assays. F, Relative p53 levels in (E) was estimated by densitometry analysis. G, MCF-7 cells were treated as in (D). Total RNA was prepared, reverse transcribed, and cDNA subjected to qRT-PCR assays for measuring levels of PUMA, BAX, PIG3, and p21 mRNA. Data are depicted as average ± SD values of 3 determinations. *, p
Figure Legend Snippet: A Small-molecule MDMX inhibitor activates p53 leading to activation of pro-apoptotic gene expression A, The chemical structures of XI-006 and Nutlin-3a. B, Recombinant cells carrying the MDMX promoter were treated with varying amounts of XI-006 for 6 h and lysed for luciferase activity assays. Data are depicted as average ± SD values of 3 determinations. C, MCF-7 cells were treated with XI-006 overnight and lysed for qRT-PCR assays to determine the MDMX mRNA levels. Data are depicted as average ± SD values of 3 determinations. D, MCF-7 cells were treated with DMSO (Ctrl) or XI-006 for 16 h, and lysed for immunoblotting to measure expression levels of MDMX, p53, p21 and MDM2. E, After treatment with 5 µM of XI-006 for 16 h, MCF-7 cells were incubated with 100 µg/ml of cycloheximide. Cells were lysed at the indicated time and subjected to immunoblotting assays. F, Relative p53 levels in (E) was estimated by densitometry analysis. G, MCF-7 cells were treated as in (D). Total RNA was prepared, reverse transcribed, and cDNA subjected to qRT-PCR assays for measuring levels of PUMA, BAX, PIG3, and p21 mRNA. Data are depicted as average ± SD values of 3 determinations. *, p

Techniques Used: Activation Assay, Expressing, Recombinant, Luciferase, Activity Assay, Quantitative RT-PCR, Incubation

XI-006 induces apoptosis in MCF-7 cells A B, MCF-7 cells treated with 5 µM of Nutlin-3a, XI-006, or its analog compound 3268, or DMSO for 24 or 48 h were stained with PI and subjected to flow cytometry analysis. Numbers inserted in graphs in (A) indicate percentages of cells at different stages of the cell cycle. The ratios of the numbers of G1 and S phase cells are shown in (B). C, MCF-7 cells treated with XI-006 for 48 h were subjected to TUNEL staining assays. At least 300 cells were randomly chosen and the numbers of TUNEL-positive cells were counted. D, MCF-7 cells were treated with XI-006 for 2 days, and subjected to immunoblotting to detect cleaved (CL-PARP) and full-length (FL-PARP) PARP. E, MCF-7 cells were treated with 5 µM of XI-006, compound 3268, or DMSO for different days and subjected to flow cytometry to determine percentages of apoptotic cells (subG0/G1 cells). F, MCF-7 cells were treated with XI-006, compound 3268, or Nutlin-3a for 4 days. Cell viability was measured by MTT assays.
Figure Legend Snippet: XI-006 induces apoptosis in MCF-7 cells A B, MCF-7 cells treated with 5 µM of Nutlin-3a, XI-006, or its analog compound 3268, or DMSO for 24 or 48 h were stained with PI and subjected to flow cytometry analysis. Numbers inserted in graphs in (A) indicate percentages of cells at different stages of the cell cycle. The ratios of the numbers of G1 and S phase cells are shown in (B). C, MCF-7 cells treated with XI-006 for 48 h were subjected to TUNEL staining assays. At least 300 cells were randomly chosen and the numbers of TUNEL-positive cells were counted. D, MCF-7 cells were treated with XI-006 for 2 days, and subjected to immunoblotting to detect cleaved (CL-PARP) and full-length (FL-PARP) PARP. E, MCF-7 cells were treated with 5 µM of XI-006, compound 3268, or DMSO for different days and subjected to flow cytometry to determine percentages of apoptotic cells (subG0/G1 cells). F, MCF-7 cells were treated with XI-006, compound 3268, or Nutlin-3a for 4 days. Cell viability was measured by MTT assays.

Techniques Used: Staining, Flow Cytometry, Cytometry, TUNEL Assay, MTT Assay

2) Product Images from "Ubiquitination of the GTPase Rap1B by the ubiquitin ligase Smurf2 is required for the establishment of neuronal polarity"

Article Title: Ubiquitination of the GTPase Rap1B by the ubiquitin ligase Smurf2 is required for the establishment of neuronal polarity

Journal: The EMBO Journal

doi: 10.1038/sj.emboj.7601580

Inhibition of the proteasome disrupts neuronal differentiation. ( A–D ) Hippocampal neurons were cultured in the presence of solvent (DMSO), 1.5 μM clasto-Lactacystin β-Lactone (Lactacystin), 40 μM ALLN, or 1.5 μM MG-132 (all dissolved in DMSO) for 48 h and analysed at 3 d.i.v. (stage 3) by staining with an anti-MAP2 antibody (A; red), the Tau-1 (blue), and an anti-Rap1 (red) antibody. Lactacystin induced the formation of multiple axons (asterisks) whose growth cones were all positive for Rap1B (A, arrows). Insets show higher magnifications of the marked growth cones. Axons identified by Tau-1 immunoreactivity are marked by asterisks. The scale bar is 12 μm. (B–D) The effect of Lactacystin (L), ALLN (A), or MG-132 (M) was analysed by determining the number of axons (B) or minor neurites (C) per cell and the length of axons (E) (means±s.e.m.; * P
Figure Legend Snippet: Inhibition of the proteasome disrupts neuronal differentiation. ( A–D ) Hippocampal neurons were cultured in the presence of solvent (DMSO), 1.5 μM clasto-Lactacystin β-Lactone (Lactacystin), 40 μM ALLN, or 1.5 μM MG-132 (all dissolved in DMSO) for 48 h and analysed at 3 d.i.v. (stage 3) by staining with an anti-MAP2 antibody (A; red), the Tau-1 (blue), and an anti-Rap1 (red) antibody. Lactacystin induced the formation of multiple axons (asterisks) whose growth cones were all positive for Rap1B (A, arrows). Insets show higher magnifications of the marked growth cones. Axons identified by Tau-1 immunoreactivity are marked by asterisks. The scale bar is 12 μm. (B–D) The effect of Lactacystin (L), ALLN (A), or MG-132 (M) was analysed by determining the number of axons (B) or minor neurites (C) per cell and the length of axons (E) (means±s.e.m.; * P

Techniques Used: Inhibition, Cell Culture, Staining

3) Product Images from "West Nile Virus Infection Causes Endocytosis of a Specific Subset of Tight Junction Membrane Proteins"

Article Title: West Nile Virus Infection Causes Endocytosis of a Specific Subset of Tight Junction Membrane Proteins

Journal: PLoS ONE

doi: 10.1371/journal.pone.0037886

WNV-induced degradation of claudin-1 and JAM-1 requires dynamin and microtubules. A. CACO-2 cells were infected with WNV and 24 hours later were treated with nocodazole (10 µM), Dynasore (10 µM), paclitaxel (1 µM), latrunculin B (10 µM) or DMSO for a further 8 hours. The corresponding cell lysates then subjected to immunoblot analyses. B. Data from three independent experiments were used to determine the normalized levels of claudin-1 and JAM-1 (relative to β-actin).
Figure Legend Snippet: WNV-induced degradation of claudin-1 and JAM-1 requires dynamin and microtubules. A. CACO-2 cells were infected with WNV and 24 hours later were treated with nocodazole (10 µM), Dynasore (10 µM), paclitaxel (1 µM), latrunculin B (10 µM) or DMSO for a further 8 hours. The corresponding cell lysates then subjected to immunoblot analyses. B. Data from three independent experiments were used to determine the normalized levels of claudin-1 and JAM-1 (relative to β-actin).

Techniques Used: Infection

Internalization of JAM-1 is blocked by disrupting microtubules or inhibiting dynamin function. MDCK cells were infected with WNV and 24 hours later were treated with 10 µM nocodazole, 10 µM Dynasore or DMSO for a further 8 hours. Samples were then processed for indirect immunofluorescence using rabbit anti-JAM-1 and mouse anti-WNV NS3/2B. Primary antibodies were detected using donkey anti-mouse Alexa546 and donkey anti-rabbit Alexa488 secondary antibodies. Nuclei were counter stained with DAPI. Images were captured using a Leica TCS SP5 confocal scanning microscope. Size bars are 10 µm.
Figure Legend Snippet: Internalization of JAM-1 is blocked by disrupting microtubules or inhibiting dynamin function. MDCK cells were infected with WNV and 24 hours later were treated with 10 µM nocodazole, 10 µM Dynasore or DMSO for a further 8 hours. Samples were then processed for indirect immunofluorescence using rabbit anti-JAM-1 and mouse anti-WNV NS3/2B. Primary antibodies were detected using donkey anti-mouse Alexa546 and donkey anti-rabbit Alexa488 secondary antibodies. Nuclei were counter stained with DAPI. Images were captured using a Leica TCS SP5 confocal scanning microscope. Size bars are 10 µm.

Techniques Used: Infection, Immunofluorescence, Staining, Microscopy

Internalization of claudin-1 is blocked by disrupting microtubules or inhibiting dynamin function. MDCK cells were infected with WNV and 24 hours later were treated with 10 µM nocodazole, 10 µM Dynasore or DMSO for a further 8 hours. Samples were then processed for indirect immunofluorescence using mouse anti-claudin-1 and rabbit anti-WNV capsid. Primary antibodies were detected using donkey anti-mouse Alexa546 and donkey anti-rabbit Alexa488 secondary antibodies. Nuclei were counter stained with DAPI. Images were captured using a Leica TCS SP5 confocal scanning microscope. Size bars are 10 µm.
Figure Legend Snippet: Internalization of claudin-1 is blocked by disrupting microtubules or inhibiting dynamin function. MDCK cells were infected with WNV and 24 hours later were treated with 10 µM nocodazole, 10 µM Dynasore or DMSO for a further 8 hours. Samples were then processed for indirect immunofluorescence using mouse anti-claudin-1 and rabbit anti-WNV capsid. Primary antibodies were detected using donkey anti-mouse Alexa546 and donkey anti-rabbit Alexa488 secondary antibodies. Nuclei were counter stained with DAPI. Images were captured using a Leica TCS SP5 confocal scanning microscope. Size bars are 10 µm.

Techniques Used: Infection, Immunofluorescence, Staining, Microscopy

4) Product Images from "Evolutionarily Repurposed Networks Reveal the Well-Known Antifungal Drug Thiabendazole to Be a Novel Vascular Disrupting AgentEvolution-Based Drug Discovery: Antifungal Also Disrupts Blood Vessel Formation"

Article Title: Evolutionarily Repurposed Networks Reveal the Well-Known Antifungal Drug Thiabendazole to Be a Novel Vascular Disrupting AgentEvolution-Based Drug Discovery: Antifungal Also Disrupts Blood Vessel Formation

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1001379

TBZ inhibits angiogenesis in vivo in Xenopus embryos. Formation of Xenopus embryo veins is disrupted, marked by expression of vascular reporter genes (A, B) erg and (C, D) aplnr , contrasting treatment with 1% DMSO control only (A, C) with 1% DMSO, 250 µM TBZ, treated at stage 31 and imaged at stages 35–36 (B, D). PCV, posterior cardinal vein; ISV, intersomitic vein; VV, vitellin veins. Similarly, TBZ disrupts vasculature imaged within a living Xenopus embryo and visualized by vascular specific GFP in kdr:GFP frogs from [19] , contrasting the vasculature of stage 46 animals treated from stage 41 with the 1% DMSO control (E) or 1% DMSO, 250 µM TBZ (F). Scale bar, 200 µm.
Figure Legend Snippet: TBZ inhibits angiogenesis in vivo in Xenopus embryos. Formation of Xenopus embryo veins is disrupted, marked by expression of vascular reporter genes (A, B) erg and (C, D) aplnr , contrasting treatment with 1% DMSO control only (A, C) with 1% DMSO, 250 µM TBZ, treated at stage 31 and imaged at stages 35–36 (B, D). PCV, posterior cardinal vein; ISV, intersomitic vein; VV, vitellin veins. Similarly, TBZ disrupts vasculature imaged within a living Xenopus embryo and visualized by vascular specific GFP in kdr:GFP frogs from [19] , contrasting the vasculature of stage 46 animals treated from stage 41 with the 1% DMSO control (E) or 1% DMSO, 250 µM TBZ (F). Scale bar, 200 µm.

Techniques Used: In Vivo, Expressing

TBZ significantly disrupts tube formation in cultured human umbilical vein endothelial cells (HUVECs), an in vitro capillary model. Here, we show effects of 1% DMSO-treated control (A) versus 1% DMSO, 100 µM TBZ (B) and 1% DMSO, 250 µM TBZ (C). Scale bar, 100 µm. (D) Tube disruption is dose-dependent and comparable to that from silencing known pro-angiogenic gene HOXA9 .
Figure Legend Snippet: TBZ significantly disrupts tube formation in cultured human umbilical vein endothelial cells (HUVECs), an in vitro capillary model. Here, we show effects of 1% DMSO-treated control (A) versus 1% DMSO, 100 µM TBZ (B) and 1% DMSO, 250 µM TBZ (C). Scale bar, 100 µm. (D) Tube disruption is dose-dependent and comparable to that from silencing known pro-angiogenic gene HOXA9 .

Techniques Used: Cell Culture, In Vitro

TBZ impedes migration of HUVECs in a wound scratch assay, but treatment with the Rho Kinase inhibitor Y27632 reverses TBZ's effects. (A) The effects of 1% DMSO-treated control versus 1% DMSO, 250 µM TBZ, and 1% DMSO, 250 µM TBZ, 10 µM Y27632. Scale bar, 200 µm. (B) quantifies the dose-dependent suppression of TBZ inhibition by Y27632. Error bars represent the mean ± 1 s.d. across 3 wells (1 of 3 trials). TBZ results in disorganization of actin stress fibers, as shown in (C) for 1% DMSO-treated control versus 1% DMSO, 250 µM TBZ-treated cells. Scale bar, 20 µm.
Figure Legend Snippet: TBZ impedes migration of HUVECs in a wound scratch assay, but treatment with the Rho Kinase inhibitor Y27632 reverses TBZ's effects. (A) The effects of 1% DMSO-treated control versus 1% DMSO, 250 µM TBZ, and 1% DMSO, 250 µM TBZ, 10 µM Y27632. Scale bar, 200 µm. (B) quantifies the dose-dependent suppression of TBZ inhibition by Y27632. Error bars represent the mean ± 1 s.d. across 3 wells (1 of 3 trials). TBZ results in disorganization of actin stress fibers, as shown in (C) for 1% DMSO-treated control versus 1% DMSO, 250 µM TBZ-treated cells. Scale bar, 20 µm.

Techniques Used: Migration, Wound Healing Assay, Inhibition

5) Product Images from "The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain"

Article Title: The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain

Journal: Biochemical Journal

doi: 10.1042/BJ20130133

26S proteasomes and 19S proteasome caps are chaperones for denatured RTA ( A ) 26S proteasomes maintain the solubility of denatured RTA. Top panels: GdnHCl-denatured RTA (40 nM) was diluted in the absence (−) or presence (+) of 26S proteasomes (25 nM, equivalent to 40 nM 19S RP) and, after centrifugation, aggregated (P) and soluble (S) fractions were examined by anti-RTA immunoblot after SDS/PAGE. Bottom panels: samples were treated in the same way as above, but were incubated at 37°C for 2 h before SDS/PAGE. ( B ) The solubilizing activity of the 26S proteasome is independent of its proteolytic activity. GdnHCl-denatured RTA (40 nM) was diluted in the absence (−) or presence (+) of 40 nM 26S proteasomes pretreated with the vehicle DMSO (top panel) or the proteasome inhibitor cLβ-l (bottom panel), and aggregated (P) and soluble (S) fractions were examined by anti-RTA immunoblot after SDS/PAGE. ( C ) Efficacy of cLβ-l was confirmed in vitro by its ability to block degradation of casein (arrowhead) in the presence of mammalian 26S proteasomes. Casein (40 nM) was incubated in the presence (+) or absence (−) of 26S proteasomes that had been pretreated with vehicle DMSO or cLβ-l. ( D ) 20S proteasome cores do not maintain the solubility of denatured RTA. GdnHCl-denatured RTA (40 nM) was diluted in the absence (−) or presence of 40 nM 20S proteasomes (+ 20S) or 40 nM BSA (+ BSA) and, after centrifugation, aggregated (P) and soluble (S) fractions were examined by anti-RTA immunoblot after SDS/PAGE. ( E ) 19S proteasome RP maintain the solubility of denatured RTA. Heat (45°C)-denatured and GdnHCl-denatured RTA (40 nM) were diluted in the absence (−) or presence (+) of 40 nM 19S RP and, after centrifugation, aggregated (P) and soluble (S) fractions were examined by anti-RTA immunoblot after SDS/PAGE. ( F ) Catalytic activity can be recovered from proteasome-solubilized RTA. Native RTA (RTA), GdnHCl-denatured RTA (denRTA) and a mixture of denRTA and 19S RP were centrifuged to remove aggregates and the soluble fractions were incubated with 20 μg of yeast ribosomes for 2 h at 30°C. After cleavage of any depurinated 28S rRNA with acetic-aniline, rRNAs were extracted, electrophoresed in denaturing conditions (1.2% agarose/50% formamide), and the gel was stained with ethidium bromide for visualization. Aniline fragment, grey arrowhead; 5.8S rRNA, white arrowhead.
Figure Legend Snippet: 26S proteasomes and 19S proteasome caps are chaperones for denatured RTA ( A ) 26S proteasomes maintain the solubility of denatured RTA. Top panels: GdnHCl-denatured RTA (40 nM) was diluted in the absence (−) or presence (+) of 26S proteasomes (25 nM, equivalent to 40 nM 19S RP) and, after centrifugation, aggregated (P) and soluble (S) fractions were examined by anti-RTA immunoblot after SDS/PAGE. Bottom panels: samples were treated in the same way as above, but were incubated at 37°C for 2 h before SDS/PAGE. ( B ) The solubilizing activity of the 26S proteasome is independent of its proteolytic activity. GdnHCl-denatured RTA (40 nM) was diluted in the absence (−) or presence (+) of 40 nM 26S proteasomes pretreated with the vehicle DMSO (top panel) or the proteasome inhibitor cLβ-l (bottom panel), and aggregated (P) and soluble (S) fractions were examined by anti-RTA immunoblot after SDS/PAGE. ( C ) Efficacy of cLβ-l was confirmed in vitro by its ability to block degradation of casein (arrowhead) in the presence of mammalian 26S proteasomes. Casein (40 nM) was incubated in the presence (+) or absence (−) of 26S proteasomes that had been pretreated with vehicle DMSO or cLβ-l. ( D ) 20S proteasome cores do not maintain the solubility of denatured RTA. GdnHCl-denatured RTA (40 nM) was diluted in the absence (−) or presence of 40 nM 20S proteasomes (+ 20S) or 40 nM BSA (+ BSA) and, after centrifugation, aggregated (P) and soluble (S) fractions were examined by anti-RTA immunoblot after SDS/PAGE. ( E ) 19S proteasome RP maintain the solubility of denatured RTA. Heat (45°C)-denatured and GdnHCl-denatured RTA (40 nM) were diluted in the absence (−) or presence (+) of 40 nM 19S RP and, after centrifugation, aggregated (P) and soluble (S) fractions were examined by anti-RTA immunoblot after SDS/PAGE. ( F ) Catalytic activity can be recovered from proteasome-solubilized RTA. Native RTA (RTA), GdnHCl-denatured RTA (denRTA) and a mixture of denRTA and 19S RP were centrifuged to remove aggregates and the soluble fractions were incubated with 20 μg of yeast ribosomes for 2 h at 30°C. After cleavage of any depurinated 28S rRNA with acetic-aniline, rRNAs were extracted, electrophoresed in denaturing conditions (1.2% agarose/50% formamide), and the gel was stained with ethidium bromide for visualization. Aniline fragment, grey arrowhead; 5.8S rRNA, white arrowhead.

Techniques Used: Solubility, Centrifugation, SDS Page, Incubation, Activity Assay, In Vitro, Blocking Assay, Staining

Inhibition of proteasome proteolytic activities sensitizes HeLa cells to ricin only after long incubations ( A ) HeLa cells were treated for 1, 2, 4, 6 or 8 h with graded doses of ricin in growth medium containing 1 μM Pi1 (●) or vehicle (DMSO, ○), and their subsequent ability to synthesize proteins was determined by measuring incorporation of [ 35 S]methionine into acid-precipitable material. Typical single assays are shown. ( B ) Top panel: cells were treated as described in ( A ), sensitivities to toxin (IC 50 , toxin concentration required to reduce protein synthesis to 50% that of non-toxin treated controls) were determined, and fold protection (IC 50 Pi1-treated cells/IC 50 DMSO-treated cells) is displayed. Middle and bottom panels: cells were treated as described above, substituting the proteasome inhibitor ALLN (middle panel) or a mixture of the cathepsin inhibitors leupeptin and pepstatin (L+P, bottom panel) for Pi1, and substituting water for the vehicle for L+P treatment. Values are means±S.D. for three independent experiments. Broken line, no protective effect over that of treatment with vehicle only; n.d., not determined. ( C ) Cells were treated with a saturating dose of ricin for 4 h in the presence (+) or absence (−) of Pi1 (top panel), L+P (middle panel) or the secretion inhibitor BFA (bottom panel). Detergent-soluble extracts were separated by SDS/PAGE, and RTA (black arrowhead) and a proteolytically clipped RTA (white arrowhead) were revealed by immunoblotting. ( D ) HeLa cells were treated for 2, 4 or 6 h as described in ( A ), substituting the proteasome inhibitor cLβ-l for Pi1. Values are means±S.D. for three independent experiments. Broken line, no protective effect over that of treatment with DMSO only. ( E ) Cells were treated with a single low dose of ricin (1 ng/ml , top panel) for increasing times in the presence of cLβ-l (●) or vehicle (DMSO, ○) and in parallel with cLβ-l (●) or vehicle (DMSO, ○) in the absence of ricin (bottom panel) and their subsequent ability to synthesize proteins was determined by measuring incorporation of [ 35 S]methionine into acid-precipitable material. Typical single assays are shown. Inset: toxin trafficking times, determined as in the top panel, in the presence of DMSO (D, ▼) or cLβ-l (c, ▽). Values are means±S.D. for three independent experiments.
Figure Legend Snippet: Inhibition of proteasome proteolytic activities sensitizes HeLa cells to ricin only after long incubations ( A ) HeLa cells were treated for 1, 2, 4, 6 or 8 h with graded doses of ricin in growth medium containing 1 μM Pi1 (●) or vehicle (DMSO, ○), and their subsequent ability to synthesize proteins was determined by measuring incorporation of [ 35 S]methionine into acid-precipitable material. Typical single assays are shown. ( B ) Top panel: cells were treated as described in ( A ), sensitivities to toxin (IC 50 , toxin concentration required to reduce protein synthesis to 50% that of non-toxin treated controls) were determined, and fold protection (IC 50 Pi1-treated cells/IC 50 DMSO-treated cells) is displayed. Middle and bottom panels: cells were treated as described above, substituting the proteasome inhibitor ALLN (middle panel) or a mixture of the cathepsin inhibitors leupeptin and pepstatin (L+P, bottom panel) for Pi1, and substituting water for the vehicle for L+P treatment. Values are means±S.D. for three independent experiments. Broken line, no protective effect over that of treatment with vehicle only; n.d., not determined. ( C ) Cells were treated with a saturating dose of ricin for 4 h in the presence (+) or absence (−) of Pi1 (top panel), L+P (middle panel) or the secretion inhibitor BFA (bottom panel). Detergent-soluble extracts were separated by SDS/PAGE, and RTA (black arrowhead) and a proteolytically clipped RTA (white arrowhead) were revealed by immunoblotting. ( D ) HeLa cells were treated for 2, 4 or 6 h as described in ( A ), substituting the proteasome inhibitor cLβ-l for Pi1. Values are means±S.D. for three independent experiments. Broken line, no protective effect over that of treatment with DMSO only. ( E ) Cells were treated with a single low dose of ricin (1 ng/ml , top panel) for increasing times in the presence of cLβ-l (●) or vehicle (DMSO, ○) and in parallel with cLβ-l (●) or vehicle (DMSO, ○) in the absence of ricin (bottom panel) and their subsequent ability to synthesize proteins was determined by measuring incorporation of [ 35 S]methionine into acid-precipitable material. Typical single assays are shown. Inset: toxin trafficking times, determined as in the top panel, in the presence of DMSO (D, ▼) or cLβ-l (c, ▽). Values are means±S.D. for three independent experiments.

Techniques Used: Inhibition, Concentration Assay, SDS Page

6) Product Images from "Dynamic Chromatin Modification Sustains Epithelial-Mesenchymal Transition following Inducible Expression of Snail-1"

Article Title: Dynamic Chromatin Modification Sustains Epithelial-Mesenchymal Transition following Inducible Expression of Snail-1

Journal: Cell reports

doi: 10.1016/j.celrep.2013.11.034

Suppression of Mesenchymal Markers and Migration by Small-Molecule Inhibitors in Triple-Negative Cancer Lines (A) Reversibility of mesenchymal markers in established cancer cell lines (triple-negative breast cancer) following treatment with small-molecule inhibitors for 24 hr. Expression of mesenchymal (FN1, SERPINE1, and VIM) genes were assayed (Fluidigm qRT-PCR) at 24 hr. *p%0.05. Student’s t test for mean of mesenchymal gene markers, for each drug-treated cell line compared with control DMSO-treated cell line (data in Figures S6B and S6C ). (B) Real-time cell migration (Roche xCElligence system) of HCC1937, MDA-MB-231, and Hs578T following treatment (24 hr) with and without TSA. Migration of BT549 and MDA-MB-468 is not shown because there was minimal effect. See also Figure S6 .
Figure Legend Snippet: Suppression of Mesenchymal Markers and Migration by Small-Molecule Inhibitors in Triple-Negative Cancer Lines (A) Reversibility of mesenchymal markers in established cancer cell lines (triple-negative breast cancer) following treatment with small-molecule inhibitors for 24 hr. Expression of mesenchymal (FN1, SERPINE1, and VIM) genes were assayed (Fluidigm qRT-PCR) at 24 hr. *p%0.05. Student’s t test for mean of mesenchymal gene markers, for each drug-treated cell line compared with control DMSO-treated cell line (data in Figures S6B and S6C ). (B) Real-time cell migration (Roche xCElligence system) of HCC1937, MDA-MB-231, and Hs578T following treatment (24 hr) with and without TSA. Migration of BT549 and MDA-MB-468 is not shown because there was minimal effect. See also Figure S6 .

Techniques Used: Migration, Expressing, Quantitative RT-PCR, Multiple Displacement Amplification

7) Product Images from "Dynamic Chromatin Modification Sustains Epithelial-Mesenchymal Transition following Inducible Expression of Snail-1"

Article Title: Dynamic Chromatin Modification Sustains Epithelial-Mesenchymal Transition following Inducible Expression of Snail-1

Journal: Cell reports

doi: 10.1016/j.celrep.2013.11.034

Suppression of Mesenchymal Markers and Migration by Small-Molecule Inhibitors in Triple-Negative Cancer Lines (A) Reversibility of mesenchymal markers in established cancer cell lines (triple-negative breast cancer) following treatment with small-molecule inhibitors for 24 hr. Expression of mesenchymal (FN1, SERPINE1, and VIM) genes were assayed (Fluidigm qRT-PCR) at 24 hr. *p%0.05. Student’s t test for mean of mesenchymal gene markers, for each drug-treated cell line compared with control DMSO-treated cell line (data in Figures S6B and S6C ). (B) Real-time cell migration (Roche xCElligence system) of HCC1937, MDA-MB-231, and Hs578T following treatment (24 hr) with and without TSA. Migration of BT549 and MDA-MB-468 is not shown because there was minimal effect. See also Figure S6 .
Figure Legend Snippet: Suppression of Mesenchymal Markers and Migration by Small-Molecule Inhibitors in Triple-Negative Cancer Lines (A) Reversibility of mesenchymal markers in established cancer cell lines (triple-negative breast cancer) following treatment with small-molecule inhibitors for 24 hr. Expression of mesenchymal (FN1, SERPINE1, and VIM) genes were assayed (Fluidigm qRT-PCR) at 24 hr. *p%0.05. Student’s t test for mean of mesenchymal gene markers, for each drug-treated cell line compared with control DMSO-treated cell line (data in Figures S6B and S6C ). (B) Real-time cell migration (Roche xCElligence system) of HCC1937, MDA-MB-231, and Hs578T following treatment (24 hr) with and without TSA. Migration of BT549 and MDA-MB-468 is not shown because there was minimal effect. See also Figure S6 .

Techniques Used: Migration, Expressing, Quantitative RT-PCR, Multiple Displacement Amplification

8) Product Images from "Activation of Cdc42 by trans interactions of the cell adhesion molecules nectins through c-Src and Cdc42-GEF FRG"

Article Title: Activation of Cdc42 by trans interactions of the cell adhesion molecules nectins through c-Src and Cdc42-GEF FRG

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200401093

Inhibition by PP2 and Csk of the nectin-induced formation of filopodia and lamellipodia in nectin-1-L cells. (A) Nectin-induced formation of filopodia and lamellipodia. Nectin-1-L or wild-type L cells were cultured on the Nef-3–, IgG-, or PLL-coated coverslips for 30 min and stained for F-actin with rhodamine-phalloidin. Bars in the quantitative analysis represent the number of cells attached on the coverslips per millimeter squared and the percentage of cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments. (B) Inhibitory effect of PP2. Nectin-1-L cells were cultured on the Nef-3– or IgG-coated coverslips in the presence of PP2, PP3, or DMSO for 30 min and stained with rhodamine-phalloidin. (C) Inhibitory effect of Csk. Nectin-1-L cells infected with Av1CATcsk or Av1CATlacZ were cultured on the Nef-3–coated coverslips for 30 min and stained with rhodamine-phalloidin and the anti-Csk or the anti–β-galactosidase mAbs, respectively. β-Gal, β-galactosidase. Bars in the quantitative analysis of B and C represent the percentage of cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of three independent experiments. Bars, 10 μm.
Figure Legend Snippet: Inhibition by PP2 and Csk of the nectin-induced formation of filopodia and lamellipodia in nectin-1-L cells. (A) Nectin-induced formation of filopodia and lamellipodia. Nectin-1-L or wild-type L cells were cultured on the Nef-3–, IgG-, or PLL-coated coverslips for 30 min and stained for F-actin with rhodamine-phalloidin. Bars in the quantitative analysis represent the number of cells attached on the coverslips per millimeter squared and the percentage of cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments. (B) Inhibitory effect of PP2. Nectin-1-L cells were cultured on the Nef-3– or IgG-coated coverslips in the presence of PP2, PP3, or DMSO for 30 min and stained with rhodamine-phalloidin. (C) Inhibitory effect of Csk. Nectin-1-L cells infected with Av1CATcsk or Av1CATlacZ were cultured on the Nef-3–coated coverslips for 30 min and stained with rhodamine-phalloidin and the anti-Csk or the anti–β-galactosidase mAbs, respectively. β-Gal, β-galactosidase. Bars in the quantitative analysis of B and C represent the percentage of cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of three independent experiments. Bars, 10 μm.

Techniques Used: Inhibition, Cell Culture, Staining, Infection

Inhibition by PP2 and Csk of the nectin-induced formation of filopodia and lamellipodia in nectin-1-MDCK cells. (A) Nectin-induced formation of filopodia and lamellipodia. Nectin-1-MDCK or wild-type MDCK cells were cultured on the Nef-3–, IgG-, Cef-, or PLL-coated coverslips for 2 h and stained for F-actin with rhodamine-phalloidin. Bars in the quantitative analysis represent number of the cells attached on the coverslips per millimeter squared and percentage of the cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments. (B) Inhibitory effect of PP2. Nectin-1-MDCK cells were cultured on the Nef-3– or IgG-coated coverslips in the presence of PP2, PP3, or DMSO for 2 h and stained with rhodamine-phalloidin. (C) Inhibitory effect of Csk. Nectin-1-MDCK cells infected with Av1CATcsk or Av1CATlacZ were cultured on the Nef-3–coated coverslips for 2 h and stained with rhodamine-phalloidin and the anti-Csk or the anti–β-galactosidase mAb, respectively. β-Gal, β-galactosidase. Bars in the quantitative analysis of B and C represent the percentage of cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments. Bars, 10 μm.
Figure Legend Snippet: Inhibition by PP2 and Csk of the nectin-induced formation of filopodia and lamellipodia in nectin-1-MDCK cells. (A) Nectin-induced formation of filopodia and lamellipodia. Nectin-1-MDCK or wild-type MDCK cells were cultured on the Nef-3–, IgG-, Cef-, or PLL-coated coverslips for 2 h and stained for F-actin with rhodamine-phalloidin. Bars in the quantitative analysis represent number of the cells attached on the coverslips per millimeter squared and percentage of the cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments. (B) Inhibitory effect of PP2. Nectin-1-MDCK cells were cultured on the Nef-3– or IgG-coated coverslips in the presence of PP2, PP3, or DMSO for 2 h and stained with rhodamine-phalloidin. (C) Inhibitory effect of Csk. Nectin-1-MDCK cells infected with Av1CATcsk or Av1CATlacZ were cultured on the Nef-3–coated coverslips for 2 h and stained with rhodamine-phalloidin and the anti-Csk or the anti–β-galactosidase mAb, respectively. β-Gal, β-galactosidase. Bars in the quantitative analysis of B and C represent the percentage of cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments. Bars, 10 μm.

Techniques Used: Inhibition, Cell Culture, Staining, Infection

9) Product Images from "Inhibition of Melanogenesis by the Pyridinyl Imidazole Class of Compounds: Possible Involvement of the Wnt/?-Catenin Signaling Pathway"

Article Title: Inhibition of Melanogenesis by the Pyridinyl Imidazole Class of Compounds: Possible Involvement of the Wnt/?-Catenin Signaling Pathway

Journal: PLoS ONE

doi: 10.1371/journal.pone.0033021

Effect of pyridinyl imidazoles compounds on melanin synthesis in B16-F0 melanoma cells. (A) Following incubation with α-MSH (0.1 µM) and increasing concentrations (1, 2.5, 5, 10, 20 µM) of pyridinyl imidazoles for 72 h, the extracellular and intracellular levels of melanin were determined separately by measuring the absorbance at 405 nm. Standard curves of synthetic melanin were used to extrapolate the absolute values of melanin content. The total amount of melanin was calculated for each experimental point by adding the extracellular and intracellular melanin values after normalization for protein content. Total melanin produced at the end-point by control (DMSO-treated cells) and hormone-stimulated cells (α-MSH plus DMSO-treated cells) is reported for comparison. (B) B16-F0 cells were also treated with pyridinyl imidazoles compounds for 96 h in absence of α-MSH. Results are expressed as percentage of untreated control samples. The data show the mean±SD of three experiments performed in duplicate. *P≤0.05; #P≤0.01 versus control.
Figure Legend Snippet: Effect of pyridinyl imidazoles compounds on melanin synthesis in B16-F0 melanoma cells. (A) Following incubation with α-MSH (0.1 µM) and increasing concentrations (1, 2.5, 5, 10, 20 µM) of pyridinyl imidazoles for 72 h, the extracellular and intracellular levels of melanin were determined separately by measuring the absorbance at 405 nm. Standard curves of synthetic melanin were used to extrapolate the absolute values of melanin content. The total amount of melanin was calculated for each experimental point by adding the extracellular and intracellular melanin values after normalization for protein content. Total melanin produced at the end-point by control (DMSO-treated cells) and hormone-stimulated cells (α-MSH plus DMSO-treated cells) is reported for comparison. (B) B16-F0 cells were also treated with pyridinyl imidazoles compounds for 96 h in absence of α-MSH. Results are expressed as percentage of untreated control samples. The data show the mean±SD of three experiments performed in duplicate. *P≤0.05; #P≤0.01 versus control.

Techniques Used: Incubation, Produced

Regulation of Wnt/β-catenin signaling by pyridinyl imidazoles. (A) Inhibition of the β-catenin/Tcf/Lef1-responsive luciferase reporter gene by PI compounds. The pTK-Renilla was inserted as an internal control. Twenty-four hours after transfection, cells were treated with PI compounds (SB202474, SB202190, SB203580, SB220025, PD169316 20 µM: MAPK Inh III 10 µM) for 6 h. Firefly luciferase activity, normalized to the corresponding renilla luciferase activity was expressed as fold decrease compared with control cells. Values represent mean ± SD of three representative experiments performed in duplicate. (B) Semi-qunatitative real-time PCR was used to measure Wnt/β-catenin-target genes Axin2, Lef1 and Wisp1 mRNAs expression in B16-F0 cells after 6 h of treatments with PI compounds. The graphs show fold differences in transcript abundance in comparison with untreated cells. Results shown were normalized by the β-actin mRNA levels. The data show the mean±SD of three experiments performed in triplicate. *P≤0.05; #P≤0.01 versus control. (C) Expression of β-catenin in B16-F0 cells after 6 h of treatment with PI compounds (SB202474, SB202190, SB203580, SB220025, PD169316 20 µM: MAPK Inh III 10 µM). Total cellular proteins (30 µg/lane) were subject to 10% SDS-PAGE. Variation of loading was determined by blotting with anti-β-tubulin antibody. Western blot assays are representative of at least three experiments. (D) Immunofluorescence analysis of β-catenin. B16-F0 cells were grown on glass coverslips and then treated with SB202474, PD169316 (20 µM) or DMSO respectively. Six hours later, cells were fixed and analyzed by immunofluorescence labelling with a mouse monoclonal anti-β-catenin followed by Alexa-Fluor-546-conjugated goat anti-mouse IgG antibody. Nuclei were labelled with bisbenzidine (DAPI). Original magnification 20×.
Figure Legend Snippet: Regulation of Wnt/β-catenin signaling by pyridinyl imidazoles. (A) Inhibition of the β-catenin/Tcf/Lef1-responsive luciferase reporter gene by PI compounds. The pTK-Renilla was inserted as an internal control. Twenty-four hours after transfection, cells were treated with PI compounds (SB202474, SB202190, SB203580, SB220025, PD169316 20 µM: MAPK Inh III 10 µM) for 6 h. Firefly luciferase activity, normalized to the corresponding renilla luciferase activity was expressed as fold decrease compared with control cells. Values represent mean ± SD of three representative experiments performed in duplicate. (B) Semi-qunatitative real-time PCR was used to measure Wnt/β-catenin-target genes Axin2, Lef1 and Wisp1 mRNAs expression in B16-F0 cells after 6 h of treatments with PI compounds. The graphs show fold differences in transcript abundance in comparison with untreated cells. Results shown were normalized by the β-actin mRNA levels. The data show the mean±SD of three experiments performed in triplicate. *P≤0.05; #P≤0.01 versus control. (C) Expression of β-catenin in B16-F0 cells after 6 h of treatment with PI compounds (SB202474, SB202190, SB203580, SB220025, PD169316 20 µM: MAPK Inh III 10 µM). Total cellular proteins (30 µg/lane) were subject to 10% SDS-PAGE. Variation of loading was determined by blotting with anti-β-tubulin antibody. Western blot assays are representative of at least three experiments. (D) Immunofluorescence analysis of β-catenin. B16-F0 cells were grown on glass coverslips and then treated with SB202474, PD169316 (20 µM) or DMSO respectively. Six hours later, cells were fixed and analyzed by immunofluorescence labelling with a mouse monoclonal anti-β-catenin followed by Alexa-Fluor-546-conjugated goat anti-mouse IgG antibody. Nuclei were labelled with bisbenzidine (DAPI). Original magnification 20×.

Techniques Used: Inhibition, Luciferase, Transfection, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, SDS Page, Western Blot, Immunofluorescence

10) Product Images from "Polarization of the endomembrane system is an early event in fucoid zygote development"

Article Title: Polarization of the endomembrane system is an early event in fucoid zygote development

Journal: BMC Plant Biology

doi: 10.1186/1471-2229-6-5

Perturbation of microtubule dynamics or membrane trafficking alters endomembrane distribution. (a) Experimental design. Zygotes on coverslips in Petri dishes were treated chronically with drug beginning at 6 h AF. At 30 min intervals thereafter a separate coverslip was labeled with FM4-64 for 30 min. Arrows at the end of the FM4-64 labeling period indicate time at which zygotes were scored for endomembrane asymmetry. (b-d) Time course of changes in endomembrane asymmetry following treatment with 5 μ M paclitaxel (b), 5 μ M oryzalin (c), or 5 μ g/ml BFA (d). Y axis indicates percent of zygotes with polar endomembrane distribution. Each experiment was repeated three times and over 100 zygotes were scored for each time point. Bars are standard errors. Images are zygotes treated with 0.05% DMSO (e), paclitaxel (f), oryzalin (g) or BFA (h). Bar = 50 μ m
Figure Legend Snippet: Perturbation of microtubule dynamics or membrane trafficking alters endomembrane distribution. (a) Experimental design. Zygotes on coverslips in Petri dishes were treated chronically with drug beginning at 6 h AF. At 30 min intervals thereafter a separate coverslip was labeled with FM4-64 for 30 min. Arrows at the end of the FM4-64 labeling period indicate time at which zygotes were scored for endomembrane asymmetry. (b-d) Time course of changes in endomembrane asymmetry following treatment with 5 μ M paclitaxel (b), 5 μ M oryzalin (c), or 5 μ g/ml BFA (d). Y axis indicates percent of zygotes with polar endomembrane distribution. Each experiment was repeated three times and over 100 zygotes were scored for each time point. Bars are standard errors. Images are zygotes treated with 0.05% DMSO (e), paclitaxel (f), oryzalin (g) or BFA (h). Bar = 50 μ m

Techniques Used: Labeling

Perturbation of microtubule dynamics or membrane trafficking alters endomembrane distribution. (a) Experimental design. Zygotes on coverslips in Petri dishes were treated chronically with drug beginning at 6 h AF. At 30 min intervals thereafter a separate coverslip was labeled with FM4-64 for 30 min. Arrows at the end of the FM4-64 labeling period indicate time at which zygotes were scored for endomembrane asymmetry. (b-d) Time course of changes in endomembrane asymmetry following treatment with 5 μ M paclitaxel (b), 5 μ M oryzalin (c), or 5 μ g/ml BFA (d). Y axis indicates percent of zygotes with polar endomembrane distribution. Each experiment was repeated three times and over 100 zygotes were scored for each time point. Bars are standard errors. Images are zygotes treated with 0.05% DMSO (e), paclitaxel (f), oryzalin (g) or BFA (h). Bar = 50 μ m
Figure Legend Snippet: Perturbation of microtubule dynamics or membrane trafficking alters endomembrane distribution. (a) Experimental design. Zygotes on coverslips in Petri dishes were treated chronically with drug beginning at 6 h AF. At 30 min intervals thereafter a separate coverslip was labeled with FM4-64 for 30 min. Arrows at the end of the FM4-64 labeling period indicate time at which zygotes were scored for endomembrane asymmetry. (b-d) Time course of changes in endomembrane asymmetry following treatment with 5 μ M paclitaxel (b), 5 μ M oryzalin (c), or 5 μ g/ml BFA (d). Y axis indicates percent of zygotes with polar endomembrane distribution. Each experiment was repeated three times and over 100 zygotes were scored for each time point. Bars are standard errors. Images are zygotes treated with 0.05% DMSO (e), paclitaxel (f), oryzalin (g) or BFA (h). Bar = 50 μ m

Techniques Used: Labeling

11) Product Images from "Dopaminergic neurons regenerate following chemogenetic ablation in the olfactory bulb of adult Zebrafish (Danio rerio)"

Article Title: Dopaminergic neurons regenerate following chemogenetic ablation in the olfactory bulb of adult Zebrafish (Danio rerio)

Journal: Scientific Reports

doi: 10.1038/s41598-020-69734-0

Relative swimming activity of Mtz and DMSO treated adult zebrafish . All zebrafish were transferred to individual tanks to acclimate for 30 min prior to behavioural analyses. ( a – c ) Effects of Mtz and DMSO on total distance travelled, average velocity, and freezing bout duration. ( d / d ′– f / f ′) Overhead path images of adult zebrafish assessed at 3 time points; pre-treatment, 1 dpt, and 7 dpt. Inactive movements (0–4 cm/s), moderate movement (4–8 cm/s) and fast movement ( > 8 cm/s). The graphs were made with GraphPad Prism 7.0 and the path images were generated with the ZebraLab software and ZebraCube tracking system (ViewPoint Life Science, Lyon, France). Sample sizes of n = 10; bars represent the Mean ± the SEM; statistical power calculated using a two-way ANOVA followed by Tukey’s multiple comparison test.
Figure Legend Snippet: Relative swimming activity of Mtz and DMSO treated adult zebrafish . All zebrafish were transferred to individual tanks to acclimate for 30 min prior to behavioural analyses. ( a – c ) Effects of Mtz and DMSO on total distance travelled, average velocity, and freezing bout duration. ( d / d ′– f / f ′) Overhead path images of adult zebrafish assessed at 3 time points; pre-treatment, 1 dpt, and 7 dpt. Inactive movements (0–4 cm/s), moderate movement (4–8 cm/s) and fast movement ( > 8 cm/s). The graphs were made with GraphPad Prism 7.0 and the path images were generated with the ZebraLab software and ZebraCube tracking system (ViewPoint Life Science, Lyon, France). Sample sizes of n = 10; bars represent the Mean ± the SEM; statistical power calculated using a two-way ANOVA followed by Tukey’s multiple comparison test.

Techniques Used: Activity Assay, Generated, Software

12) Product Images from "Actomyosin forces and the energetics of red blood cell invasion by the malaria parasite Plasmodium falciparum"

Article Title: Actomyosin forces and the energetics of red blood cell invasion by the malaria parasite Plasmodium falciparum

Journal: bioRxiv

doi: 10.1101/2020.06.25.171900

In the absence of PfMyoA or PfELC, merozoites cannot strongly deform or internalize A Comparison of event types from PfMyoA-cKO, PfMyoA-comp and PfELC-cKO lines after DMSO and RAP treatment. PfMyoA-cKO parasites show neither deformation nor internalisation after RAP treatment (p
Figure Legend Snippet: In the absence of PfMyoA or PfELC, merozoites cannot strongly deform or internalize A Comparison of event types from PfMyoA-cKO, PfMyoA-comp and PfELC-cKO lines after DMSO and RAP treatment. PfMyoA-cKO parasites show neither deformation nor internalisation after RAP treatment (p

Techniques Used:

PfMyoA drives invasion pore closure, while PfMyoA and PfMyoB both help initiation of internalization A RAP treated PfMyoA-K764E parasites showed a significantly shifted distribution of event types, with more Type B and Type C events. This is consistent with these parasites having insufficient motor function to overcome a third barrier, at completion of internalisation. Significance assessed by chi-square test. PfMyoB-cKO parasites showed a significant increase in Type C failures, consistent with an impairment at initiation of internalisation. Significance assessed by Fisher’ s exact test, comparing successful invasion to pooled invasion failures. B PfMyoA-K764E parasites show a slight weakening of deformation, though not significant. There is no difference between the deformation scores in PfMyoB-cKO parasites after RAP treatment. Significance assessed by chi-square test. C For PfMyoA-K764E parasites undergoing Type A events (black bars and data points) RAP treatment induces a longer pause pre-internalisation. Only in Type B events (cyan bars) after RAP treatment is internalisation significantly slower and the pause post-internalisation shorter. Bars show median and interquartile range, or median only for Type B events. Significance assessed between Type A DMSO and RAP treatments by Mann-Whitney test, shown when p
Figure Legend Snippet: PfMyoA drives invasion pore closure, while PfMyoA and PfMyoB both help initiation of internalization A RAP treated PfMyoA-K764E parasites showed a significantly shifted distribution of event types, with more Type B and Type C events. This is consistent with these parasites having insufficient motor function to overcome a third barrier, at completion of internalisation. Significance assessed by chi-square test. PfMyoB-cKO parasites showed a significant increase in Type C failures, consistent with an impairment at initiation of internalisation. Significance assessed by Fisher’ s exact test, comparing successful invasion to pooled invasion failures. B PfMyoA-K764E parasites show a slight weakening of deformation, though not significant. There is no difference between the deformation scores in PfMyoB-cKO parasites after RAP treatment. Significance assessed by chi-square test. C For PfMyoA-K764E parasites undergoing Type A events (black bars and data points) RAP treatment induces a longer pause pre-internalisation. Only in Type B events (cyan bars) after RAP treatment is internalisation significantly slower and the pause post-internalisation shorter. Bars show median and interquartile range, or median only for Type B events. Significance assessed between Type A DMSO and RAP treatments by Mann-Whitney test, shown when p

Techniques Used: MANN-WHITNEY

Disruption of PfMyoB produces a mild parasite growth defect A Schematic showing generation of a PfMyoB-cKO targeting construct. A region of Pfmyob encoding the C-terminal 204 residues was synthesised with re-optimised codons (rcz) and a 3xHA tag, and is placed between two loxPint modules, with sfgfp out-of-frame downstream. Guide RNA sites (scissors) and homology regions were chosen to start as close as possible to the start and end of the modified region. B A structural model of PfMyoB indicating the region excised in PfMyoB-cKO (in black). C Genotyping PCR confirms that transfectants contain only the integrated locus (IN, purple half arrow), while the WT locus (blue half arrow) is completely lost. D Growth of PfMyoB-cKO parasites over 96 h is no different to the parental, DiCre-expressing, B11 line. Line shows mean parasitaemia, N=3, each experiment in triplicate. E Western blot analysis of WT, PfMyoB-cKO or Cas9-3xHA-expressing controls (where Cas9 was the only 3xHA-tagged protein or PfMyoA-3xHA was also expressed). In all lanes with PfMyoB-cKO or Cas9-3xHA-expressing controls a band around the expected size of Cas9-3xHA (168 kDa) and a presumed Cas9-3xHA breakdown product (∼95 kDa) is observed. In PfMyoB-cKO+DMSO, but not +RAP, a slightly larger band is detected around the expected size for PfMyoB-3xHA (97 kDa), confirming that PfMyoB-3xHA is properly expressed and lost after RAP treatment. The PfMyoB-3xHA band runs at a similar size to PfMyoA-3xHA control (96 kDa). F Genotyping PCR shows the loss of much of the integrated, unexcised locus (IN, purple half arrow) after RAP treatment and detection of the excised locus (EX, green half arrow). G Measuring the parasitaemia of PfMyoB-cKO parasites in each of the three cycles following RAP treatment shows a small, steady growth defect, of 93% on average. Lines show mean parasitaemia, normalised to DMSO for each line/cycle. N=3, each experiment in triplicate.
Figure Legend Snippet: Disruption of PfMyoB produces a mild parasite growth defect A Schematic showing generation of a PfMyoB-cKO targeting construct. A region of Pfmyob encoding the C-terminal 204 residues was synthesised with re-optimised codons (rcz) and a 3xHA tag, and is placed between two loxPint modules, with sfgfp out-of-frame downstream. Guide RNA sites (scissors) and homology regions were chosen to start as close as possible to the start and end of the modified region. B A structural model of PfMyoB indicating the region excised in PfMyoB-cKO (in black). C Genotyping PCR confirms that transfectants contain only the integrated locus (IN, purple half arrow), while the WT locus (blue half arrow) is completely lost. D Growth of PfMyoB-cKO parasites over 96 h is no different to the parental, DiCre-expressing, B11 line. Line shows mean parasitaemia, N=3, each experiment in triplicate. E Western blot analysis of WT, PfMyoB-cKO or Cas9-3xHA-expressing controls (where Cas9 was the only 3xHA-tagged protein or PfMyoA-3xHA was also expressed). In all lanes with PfMyoB-cKO or Cas9-3xHA-expressing controls a band around the expected size of Cas9-3xHA (168 kDa) and a presumed Cas9-3xHA breakdown product (∼95 kDa) is observed. In PfMyoB-cKO+DMSO, but not +RAP, a slightly larger band is detected around the expected size for PfMyoB-3xHA (97 kDa), confirming that PfMyoB-3xHA is properly expressed and lost after RAP treatment. The PfMyoB-3xHA band runs at a similar size to PfMyoA-3xHA control (96 kDa). F Genotyping PCR shows the loss of much of the integrated, unexcised locus (IN, purple half arrow) after RAP treatment and detection of the excised locus (EX, green half arrow). G Measuring the parasitaemia of PfMyoB-cKO parasites in each of the three cycles following RAP treatment shows a small, steady growth defect, of 93% on average. Lines show mean parasitaemia, normalised to DMSO for each line/cycle. N=3, each experiment in triplicate.

Techniques Used: Construct, Modification, Polymerase Chain Reaction, Expressing, Western Blot

Conditional complementation and mutagenesis of PfMyoA A RAP treatment of PfMyoA-comp and PfMyoA-K764E show that the complementing line has no growth defect, while the RAP-treated K764E line grows at around 55% of DMSO-treated control per cycle under static conditions. B Genotyping PCR of the endogenous Pfmyoa locus in PfMyoA-comp and PfMyoA-K764E after RAP treatment shows that the replacement of the unexcised, integrated allele (IN) with the excised allele (EX) is almost complete. C Western blot of WT, PfMyoA-cKO, PfMyoA-comp and PfMyoA-K764E schizonts confirms that PfMyoA-FLAG, expressed from the endogenous Pfmyoa locus, is almost completely lost in favour of truncated PfMyoA-GFP. PfMyoA-3xHA, expressed at the ectopic locus, is unaffected by RAP treatment. D Parasites were treated with DMSO or RAP and cultured under static or suspension conditions. Parasitaemia was then measured in the following cycle by flow cytometry and normalised to DMSO control for each line and condition, showing that suspension conditions partially alleviate the growth defect caused by K764E mutation, from 67% of DMSO-treated to 77%. Bars show mean parasitaemia, N=4 (or 2 for PfELC-cKO, tested separately), each experiment in triplicate. Significance assessed by paired t test, two tailed.
Figure Legend Snippet: Conditional complementation and mutagenesis of PfMyoA A RAP treatment of PfMyoA-comp and PfMyoA-K764E show that the complementing line has no growth defect, while the RAP-treated K764E line grows at around 55% of DMSO-treated control per cycle under static conditions. B Genotyping PCR of the endogenous Pfmyoa locus in PfMyoA-comp and PfMyoA-K764E after RAP treatment shows that the replacement of the unexcised, integrated allele (IN) with the excised allele (EX) is almost complete. C Western blot of WT, PfMyoA-cKO, PfMyoA-comp and PfMyoA-K764E schizonts confirms that PfMyoA-FLAG, expressed from the endogenous Pfmyoa locus, is almost completely lost in favour of truncated PfMyoA-GFP. PfMyoA-3xHA, expressed at the ectopic locus, is unaffected by RAP treatment. D Parasites were treated with DMSO or RAP and cultured under static or suspension conditions. Parasitaemia was then measured in the following cycle by flow cytometry and normalised to DMSO control for each line and condition, showing that suspension conditions partially alleviate the growth defect caused by K764E mutation, from 67% of DMSO-treated to 77%. Bars show mean parasitaemia, N=4 (or 2 for PfELC-cKO, tested separately), each experiment in triplicate. Significance assessed by paired t test, two tailed.

Techniques Used: Mutagenesis, Polymerase Chain Reaction, Western Blot, Cell Culture, Flow Cytometry, Two Tailed Test

13) Product Images from "Intramitochondrial proteostasis is directly coupled to α-synuclein and amyloid β1-42 pathologies"

Article Title: Intramitochondrial proteostasis is directly coupled to α-synuclein and amyloid β1-42 pathologies

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA119.011650

Mitochondrial proteostasis influences amyloid-β 1-42 pathology. A, Aβ42-mCherry overexpressing HEK cells were treated with DMSO (control), 1 μ m FCCP, or 10 μ m BAPTA-AM for 24 h. The aggregation of Aβ42 was increased upon treatment with FCCP and BAPTA-AM. Data are presented as mean ± S.D. *, p = 0.0298 and ****, p
Figure Legend Snippet: Mitochondrial proteostasis influences amyloid-β 1-42 pathology. A, Aβ42-mCherry overexpressing HEK cells were treated with DMSO (control), 1 μ m FCCP, or 10 μ m BAPTA-AM for 24 h. The aggregation of Aβ42 was increased upon treatment with FCCP and BAPTA-AM. Data are presented as mean ± S.D. *, p = 0.0298 and ****, p

Techniques Used:

Downstream effectors of mitochondrial dysfunction do not influence α-synuclein pathology. A, YFP–α-synuclein SH-SY5Y cells were treated with DMSO (control), 500 μ m MPP + , 1 μ m ionomycin, or 3 μ m menadione for 3 days (1 h before, during α-synuclein fibrillar seed incubation, and during the 3-day period until evaluation). Scale bars : 20 μ m . α-Synuclein seeding was not significantly increased (one-way ANOVA with Dunnett's post hoc correction). Data are presented as mean ± S.D., n = 11, 8, 8, and 7 with n = regions analyzed, three biological repeats. B, fluorescence lifetime images and graphs for ATP levels (Ateam1.03 donor fluorescence lifetime) in SH-SY5Y cells treated with DMSO (control), 500 μ m MPP + , 10 μ m FCCP, and 10 μ m BAPTA-AM for 1 h. MPP + and FCCP significantly decreased ATP levels, BAPTA-AM had no significant effect. ****, p
Figure Legend Snippet: Downstream effectors of mitochondrial dysfunction do not influence α-synuclein pathology. A, YFP–α-synuclein SH-SY5Y cells were treated with DMSO (control), 500 μ m MPP + , 1 μ m ionomycin, or 3 μ m menadione for 3 days (1 h before, during α-synuclein fibrillar seed incubation, and during the 3-day period until evaluation). Scale bars : 20 μ m . α-Synuclein seeding was not significantly increased (one-way ANOVA with Dunnett's post hoc correction). Data are presented as mean ± S.D., n = 11, 8, 8, and 7 with n = regions analyzed, three biological repeats. B, fluorescence lifetime images and graphs for ATP levels (Ateam1.03 donor fluorescence lifetime) in SH-SY5Y cells treated with DMSO (control), 500 μ m MPP + , 10 μ m FCCP, and 10 μ m BAPTA-AM for 1 h. MPP + and FCCP significantly decreased ATP levels, BAPTA-AM had no significant effect. ****, p

Techniques Used: Incubation, Fluorescence

Downstream effectors of mitochondrial dysfunction do not influence α-synuclein pathology. A, YFP–α-synuclein SH-SY5Y cells were treated with DMSO (control), 500 μ m MPP + , 1 μ m ionomycin, or 3 μ m menadione for 3 days (1 h before, during α-synuclein fibrillar seed incubation, and during the 3-day period until evaluation). Scale bars : 20 μ m . α-Synuclein seeding was not significantly increased (one-way ANOVA with Dunnett's post hoc correction). Data are presented as mean ± S.D., n = 11, 8, 8, and 7 with n = regions analyzed, three biological repeats. B, fluorescence lifetime images and graphs for ATP levels (Ateam1.03 donor fluorescence lifetime) in SH-SY5Y cells treated with DMSO (control), 500 μ m MPP + , 10 μ m FCCP, and 10 μ m BAPTA-AM for 1 h. MPP + and FCCP significantly decreased ATP levels, BAPTA-AM had no significant effect. ****, p
Figure Legend Snippet: Downstream effectors of mitochondrial dysfunction do not influence α-synuclein pathology. A, YFP–α-synuclein SH-SY5Y cells were treated with DMSO (control), 500 μ m MPP + , 1 μ m ionomycin, or 3 μ m menadione for 3 days (1 h before, during α-synuclein fibrillar seed incubation, and during the 3-day period until evaluation). Scale bars : 20 μ m . α-Synuclein seeding was not significantly increased (one-way ANOVA with Dunnett's post hoc correction). Data are presented as mean ± S.D., n = 11, 8, 8, and 7 with n = regions analyzed, three biological repeats. B, fluorescence lifetime images and graphs for ATP levels (Ateam1.03 donor fluorescence lifetime) in SH-SY5Y cells treated with DMSO (control), 500 μ m MPP + , 10 μ m FCCP, and 10 μ m BAPTA-AM for 1 h. MPP + and FCCP significantly decreased ATP levels, BAPTA-AM had no significant effect. ****, p

Techniques Used: Incubation, Fluorescence

BAPTA-AM treatment increases α-synuclein pathology. A, YFP–α-synuclein SH-SY5Y cells were treated with DMSO (control), 10 μ m BAPTA-AM for 1 h (before fibrillar seed incubation) and for 5 h (before plus during the incubation with α-synuclein fibrillar seeds). Scale bars : 20 μ m . α-Synuclein seeding was increased upon 1 h pre-treatment and 5 h treatment with BAPTA-AM. Data are presented as mean ± S.D. *, p = 0.0127 and ****, p
Figure Legend Snippet: BAPTA-AM treatment increases α-synuclein pathology. A, YFP–α-synuclein SH-SY5Y cells were treated with DMSO (control), 10 μ m BAPTA-AM for 1 h (before fibrillar seed incubation) and for 5 h (before plus during the incubation with α-synuclein fibrillar seeds). Scale bars : 20 μ m . α-Synuclein seeding was increased upon 1 h pre-treatment and 5 h treatment with BAPTA-AM. Data are presented as mean ± S.D. *, p = 0.0127 and ****, p

Techniques Used: Incubation

Mitochondrial proteostasis influences amyloid-β 1-42 pathology. A, Aβ42-mCherry overexpressing HEK cells were treated with DMSO (control), 1 μ m FCCP, or 10 μ m BAPTA-AM for 24 h. The aggregation of Aβ42 was increased upon treatment with FCCP and BAPTA-AM. Data are presented as mean ± S.D. *, p = 0.0298 and ****, p
Figure Legend Snippet: Mitochondrial proteostasis influences amyloid-β 1-42 pathology. A, Aβ42-mCherry overexpressing HEK cells were treated with DMSO (control), 1 μ m FCCP, or 10 μ m BAPTA-AM for 24 h. The aggregation of Aβ42 was increased upon treatment with FCCP and BAPTA-AM. Data are presented as mean ± S.D. *, p = 0.0298 and ****, p

Techniques Used:

Downstream effectors of mitochondrial dysfunction do not influence α-synuclein pathology. A, YFP–α-synuclein SH-SY5Y cells were treated with DMSO (control), 500 μ m MPP + , 1 μ m ionomycin, or 3 μ m menadione for 3 days (1 h before, during α-synuclein fibrillar seed incubation, and during the 3-day period until evaluation). Scale bars : 20 μ m . α-Synuclein seeding was not significantly increased (one-way ANOVA with Dunnett's post hoc correction). Data are presented as mean ± S.D., n = 11, 8, 8, and 7 with n = regions analyzed, three biological repeats. B, fluorescence lifetime images and graphs for ATP levels (Ateam1.03 donor fluorescence lifetime) in SH-SY5Y cells treated with DMSO (control), 500 μ m MPP + , 10 μ m FCCP, and 10 μ m BAPTA-AM for 1 h. MPP + and FCCP significantly decreased ATP levels, BAPTA-AM had no significant effect. ****, p
Figure Legend Snippet: Downstream effectors of mitochondrial dysfunction do not influence α-synuclein pathology. A, YFP–α-synuclein SH-SY5Y cells were treated with DMSO (control), 500 μ m MPP + , 1 μ m ionomycin, or 3 μ m menadione for 3 days (1 h before, during α-synuclein fibrillar seed incubation, and during the 3-day period until evaluation). Scale bars : 20 μ m . α-Synuclein seeding was not significantly increased (one-way ANOVA with Dunnett's post hoc correction). Data are presented as mean ± S.D., n = 11, 8, 8, and 7 with n = regions analyzed, three biological repeats. B, fluorescence lifetime images and graphs for ATP levels (Ateam1.03 donor fluorescence lifetime) in SH-SY5Y cells treated with DMSO (control), 500 μ m MPP + , 10 μ m FCCP, and 10 μ m BAPTA-AM for 1 h. MPP + and FCCP significantly decreased ATP levels, BAPTA-AM had no significant effect. ****, p

Techniques Used: Incubation, Fluorescence

14) Product Images from "Epigenetic Vulnerability of Insulator CTCF Motifs at Parkinson’s Disease-Associated Genes in Response to Neurotoxicant Rotenone"

Article Title: Epigenetic Vulnerability of Insulator CTCF Motifs at Parkinson’s Disease-Associated Genes in Response to Neurotoxicant Rotenone

Journal: Frontiers in Genetics

doi: 10.3389/fgene.2020.00627

CTCF site H3K27 enhancer activation in response to rotenone. (A) The local abundance of H3K27ac within CTCF binding sites at Parkinson’s disease-associated genes was measured with ChIP-qPCR and expressed as the percent of total DNA input used for immunoprecipitation. (B) The negative control for ChIP analysis was Rabbit IgG. Significance was tested with paired Student’s t -test using the percent input of vehicle (DMSO) vs rotenone, and post hoc analysis for multiple hypotheses was done using the false discovery method. *FDR
Figure Legend Snippet: CTCF site H3K27 enhancer activation in response to rotenone. (A) The local abundance of H3K27ac within CTCF binding sites at Parkinson’s disease-associated genes was measured with ChIP-qPCR and expressed as the percent of total DNA input used for immunoprecipitation. (B) The negative control for ChIP analysis was Rabbit IgG. Significance was tested with paired Student’s t -test using the percent input of vehicle (DMSO) vs rotenone, and post hoc analysis for multiple hypotheses was done using the false discovery method. *FDR

Techniques Used: Activation Assay, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Negative Control

Global epigenetic patterns in rotenone treated HEK293 cells. (A) DNA methylation was visualized by dot blot method using the anti-5mC antibody. Three biological replicates are shown in this image. The standard ladder using 100% 5mC standard is shown in Supplementary Figure 1 . (B) Global histone H3K27ac levels were measured from total extracted histones using Western blot. Total histone H3 was used as the loading control. Three biological replicates are shown in this image. This western blot was quantified using ImageJ software and is shown as the fold change in the amount of H3K27ac relative to the vehicle (DMSO) control. ∗ p
Figure Legend Snippet: Global epigenetic patterns in rotenone treated HEK293 cells. (A) DNA methylation was visualized by dot blot method using the anti-5mC antibody. Three biological replicates are shown in this image. The standard ladder using 100% 5mC standard is shown in Supplementary Figure 1 . (B) Global histone H3K27ac levels were measured from total extracted histones using Western blot. Total histone H3 was used as the loading control. Three biological replicates are shown in this image. This western blot was quantified using ImageJ software and is shown as the fold change in the amount of H3K27ac relative to the vehicle (DMSO) control. ∗ p

Techniques Used: DNA Methylation Assay, Dot Blot, Western Blot, Software

CTCF binding at PD-associated genes in response to rotenone. (A) The local abundance of CTCF binding at Parkinson’s disease-associated genes was measured with ChIP-qPCR and expressed as the percent of total DNA input used for immunoprecipitation. (B) The negative control for ChIP analysis was Rabbit IgG. Significance was tested with paired student’s t -test using the percent input of vehicle (DMSO) vs rotenone and post hoc analysis for multiple hypotheses was done using the false discovery method. *FDR
Figure Legend Snippet: CTCF binding at PD-associated genes in response to rotenone. (A) The local abundance of CTCF binding at Parkinson’s disease-associated genes was measured with ChIP-qPCR and expressed as the percent of total DNA input used for immunoprecipitation. (B) The negative control for ChIP analysis was Rabbit IgG. Significance was tested with paired student’s t -test using the percent input of vehicle (DMSO) vs rotenone and post hoc analysis for multiple hypotheses was done using the false discovery method. *FDR

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Negative Control

15) Product Images from "A Simple and Highly Effective Method for Slow-Freezing Human Pluripotent Stem Cells Using Dimethyl Sulfoxide, Hydroxyethyl Starch and Ethylene Glycol"

Article Title: A Simple and Highly Effective Method for Slow-Freezing Human Pluripotent Stem Cells Using Dimethyl Sulfoxide, Hydroxyethyl Starch and Ethylene Glycol

Journal: PLoS ONE

doi: 10.1371/journal.pone.0088696

Selection of a cell dissociation reagent suitable for slow-freezing. (A) hiPSC 201B7 colonies were dissociated with Pronase/EDTA, trypsin/EDTA, Dispase II, collagenase IV, or CTK, followed by cryopreservation with Formula A medium (6% HES, 5% DMSO, 4% BSA, and 50% D-MEM/F12 in saline). Recovery frequencies (rate, %) were determined by scoring the number of post-thaw ALP+ colonies at day 5 in 6-well dish for comparison against non-frozen cells at day 5 in 6-well dishes that had been passaged with the same dissociation buffer. The results of 3 independent experiments are shown with standard deviation bars [SD]). *; P
Figure Legend Snippet: Selection of a cell dissociation reagent suitable for slow-freezing. (A) hiPSC 201B7 colonies were dissociated with Pronase/EDTA, trypsin/EDTA, Dispase II, collagenase IV, or CTK, followed by cryopreservation with Formula A medium (6% HES, 5% DMSO, 4% BSA, and 50% D-MEM/F12 in saline). Recovery frequencies (rate, %) were determined by scoring the number of post-thaw ALP+ colonies at day 5 in 6-well dish for comparison against non-frozen cells at day 5 in 6-well dishes that had been passaged with the same dissociation buffer. The results of 3 independent experiments are shown with standard deviation bars [SD]). *; P

Techniques Used: Selection, ALP Assay, Standard Deviation

Selection of cryopreservation medium for slow-freezing. (A) Recovery frequencies (rate, %) of iPSC (201B7) colonies treated with Pronase/EDTA dissociation followed by cryopreservation with 5 different media (Formulas A–E). Recovery frequencies (rate, %) were determined by the percentage of ALP+ colonies 5 days after thawing compared with those at day 5 after passaging with Pronase/EDTA without cryopreservation. Recovery frequencies (rate, %) are shown as bars with S.D. Formula A: [6% HES, 5% DMSO, 4% BSA, and 50% D-MEM/F12 in saline]; B: [6% HES, 5% DMSO, and 50% D-MEM/F12 in saline]; C: [6% HES, 5% DMSO, and 4% BSA in saline]; D: [6% HES and 5% DMSO in saline]; E: [6% HES, 5% DMSO, and 5% ethylene glycol (EG) in saline]. Results of 3 independent experiments are shown. Differences between E and the others are significant. *; P
Figure Legend Snippet: Selection of cryopreservation medium for slow-freezing. (A) Recovery frequencies (rate, %) of iPSC (201B7) colonies treated with Pronase/EDTA dissociation followed by cryopreservation with 5 different media (Formulas A–E). Recovery frequencies (rate, %) were determined by the percentage of ALP+ colonies 5 days after thawing compared with those at day 5 after passaging with Pronase/EDTA without cryopreservation. Recovery frequencies (rate, %) are shown as bars with S.D. Formula A: [6% HES, 5% DMSO, 4% BSA, and 50% D-MEM/F12 in saline]; B: [6% HES, 5% DMSO, and 50% D-MEM/F12 in saline]; C: [6% HES, 5% DMSO, and 4% BSA in saline]; D: [6% HES and 5% DMSO in saline]; E: [6% HES, 5% DMSO, and 5% ethylene glycol (EG) in saline]. Results of 3 independent experiments are shown. Differences between E and the others are significant. *; P

Techniques Used: Selection, ALP Assay, Passaging

16) Product Images from "Stability of the Aryl hydrocarbon Receptor and its Regulated Genes in the Low activity Variant of Hepa-1 cell line"

Article Title: Stability of the Aryl hydrocarbon Receptor and its Regulated Genes in the Low activity Variant of Hepa-1 cell line

Journal: Toxicology letters

doi: 10.1016/j.toxlet.2015.01.016

The time course of TCDD-induced nuclear accumulation of AhR in Hepa- WT and LA1 and LA2 variants. Cells were treated with 10 nM TCDD or DMSO (vehicle) in fresh growth media, and were collected at the indicated times after treatment by scraping in cold PBS, pelleted and lyzed. Nuclei were separated from supernatant (SN), 100 μg of nuclear lysates at each treatment were electrophoresed (in regular size gels) and immunoblotted with anti-AhR antibody. Approximately 100 μg of SN of each respective cell line at time zero after TCDD exposure, were included as a reference control for total cellular AhR protein. The nuclear AhR immuno-detectable band (95 kDa) was quantified by densitometric scanning of the blots and density of the nuclear AhR bands was corrected relative to the respective value of SN, and values from duplicate blots of two experiments were averaged and the mean values and standard deviation (n=4) were plotted (A). Time course of TCDD- depletion of cytosolic AhR in Hepa-1 WT, LA1 and LA2 variants. Fifty μg of the non-nuclear fraction (SN) of cellular lysates of similar treatments as in Fig 3-A were electrophoresed and immunoblotted for cytosolic AhR. The AhR immuno-detectable band (95 kDa) was quantified by densitometric scanning and the AhR estimated relative levels were plotted against time of TCDD exposure (B).
Figure Legend Snippet: The time course of TCDD-induced nuclear accumulation of AhR in Hepa- WT and LA1 and LA2 variants. Cells were treated with 10 nM TCDD or DMSO (vehicle) in fresh growth media, and were collected at the indicated times after treatment by scraping in cold PBS, pelleted and lyzed. Nuclei were separated from supernatant (SN), 100 μg of nuclear lysates at each treatment were electrophoresed (in regular size gels) and immunoblotted with anti-AhR antibody. Approximately 100 μg of SN of each respective cell line at time zero after TCDD exposure, were included as a reference control for total cellular AhR protein. The nuclear AhR immuno-detectable band (95 kDa) was quantified by densitometric scanning of the blots and density of the nuclear AhR bands was corrected relative to the respective value of SN, and values from duplicate blots of two experiments were averaged and the mean values and standard deviation (n=4) were plotted (A). Time course of TCDD- depletion of cytosolic AhR in Hepa-1 WT, LA1 and LA2 variants. Fifty μg of the non-nuclear fraction (SN) of cellular lysates of similar treatments as in Fig 3-A were electrophoresed and immunoblotted for cytosolic AhR. The AhR immuno-detectable band (95 kDa) was quantified by densitometric scanning and the AhR estimated relative levels were plotted against time of TCDD exposure (B).

Techniques Used: Standard Deviation

A. Comparison of AhR protein levels in LA1 cells at early (p4) and late (p21) passage relative to WT hepa-1 cells (at p15). Protein samples (15 μg) from each treatment were analyzed by Western blotting (mini-gels) as described in Materials Methods, using anti-AhR antibodies. The same membrane was stripped and re-probed with anti-actin antibodies, for protein loading. B. Expression of CYP1A1 mRNA in LA1 cells at early (p4) and late passage (p21), in comparison to Hepa-1 WT. Total RNA was isolated by Trizol method from cells treated with 10 nM TCDD or vehicle (DMSO) for 20 h. Approximately 30 μg total RNA was subjected to Northern blot analysis as described in Materials Methods. Membranes were first hybridized with a mouse CYP1A1 cDNA probe, stripped and re-probed with a human actin cDNA probe. Relative CYP1A1 RNA levels (calculated as the corrected intensity of CYP1A1 band divided by the intensity of actin band) from two separate experiments were averaged and plotted. C. Expression of CYP1A1 mRNA in Hepa -1 WT at early (p8) and late passage (p24) following TCDD treatment for 20h (upper panel). CYP1A1 mRNA expression was determined by RT-PCR as reported previously ( Dale and Eltom 2006 ). In lower panel, proteins isolated from trizol extract of the same treatment points used for CYP1A1 mRNA determination, were analyzed by Western blotting and probed by AhR antibody. Protein loading was verified by Ponceau S staining of the membrane.
Figure Legend Snippet: A. Comparison of AhR protein levels in LA1 cells at early (p4) and late (p21) passage relative to WT hepa-1 cells (at p15). Protein samples (15 μg) from each treatment were analyzed by Western blotting (mini-gels) as described in Materials Methods, using anti-AhR antibodies. The same membrane was stripped and re-probed with anti-actin antibodies, for protein loading. B. Expression of CYP1A1 mRNA in LA1 cells at early (p4) and late passage (p21), in comparison to Hepa-1 WT. Total RNA was isolated by Trizol method from cells treated with 10 nM TCDD or vehicle (DMSO) for 20 h. Approximately 30 μg total RNA was subjected to Northern blot analysis as described in Materials Methods. Membranes were first hybridized with a mouse CYP1A1 cDNA probe, stripped and re-probed with a human actin cDNA probe. Relative CYP1A1 RNA levels (calculated as the corrected intensity of CYP1A1 band divided by the intensity of actin band) from two separate experiments were averaged and plotted. C. Expression of CYP1A1 mRNA in Hepa -1 WT at early (p8) and late passage (p24) following TCDD treatment for 20h (upper panel). CYP1A1 mRNA expression was determined by RT-PCR as reported previously ( Dale and Eltom 2006 ). In lower panel, proteins isolated from trizol extract of the same treatment points used for CYP1A1 mRNA determination, were analyzed by Western blotting and probed by AhR antibody. Protein loading was verified by Ponceau S staining of the membrane.

Techniques Used: Western Blot, Expressing, Isolation, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Staining

Total cellular AhR levels in Hepa-1 wild-type and LA1 variant cells following actinomycin D treatment of TCDD-treated or untreated cells. Parallel plates to experiments outlined in Fig 4 were treated simultaneously and cells were lysed in Trizol at the indicated times after actinomycin D treatment and used for protein isolation. Approximately 10 μg (WT) or 50 μg (LA1) protein aliquots of each treatment were immunoblotted for AhR and actin. Cells were either pre-treated with 10 nM TCDD (A) or vehicle (DMSO) (B) for 20 h, then treated with actinomycin D. The AhR immuno-detectable bands (95 kDa) were quantified by densitometric scanning of multiple blots and values were corrected for actin loading and normalized to the respective value at the time of actinomycin D addition (0 h) of each cell line and plotted (lower panel).
Figure Legend Snippet: Total cellular AhR levels in Hepa-1 wild-type and LA1 variant cells following actinomycin D treatment of TCDD-treated or untreated cells. Parallel plates to experiments outlined in Fig 4 were treated simultaneously and cells were lysed in Trizol at the indicated times after actinomycin D treatment and used for protein isolation. Approximately 10 μg (WT) or 50 μg (LA1) protein aliquots of each treatment were immunoblotted for AhR and actin. Cells were either pre-treated with 10 nM TCDD (A) or vehicle (DMSO) (B) for 20 h, then treated with actinomycin D. The AhR immuno-detectable bands (95 kDa) were quantified by densitometric scanning of multiple blots and values were corrected for actin loading and normalized to the respective value at the time of actinomycin D addition (0 h) of each cell line and plotted (lower panel).

Techniques Used: Variant Assay, Isolation

17) Product Images from "Early ERK1/2 activation promotes DRP1-dependent mitochondrial fission necessary for cell reprogramming"

Article Title: Early ERK1/2 activation promotes DRP1-dependent mitochondrial fission necessary for cell reprogramming

Journal: Nature Communications

doi: 10.1038/ncomms11124

Reprogramming-induced mitochondrial fission depends on Erk1/2 phosphorylation of Drp1. ( a ) Lysates of mock- or OSKM-transduced MEFs for the indicated days were analysed by immunoblotting using the indicated antibodies. Graphs on the right show the quantification of the indicated ratios ( n =3). ( b ) MEFs were OSKM-transduced and 3 days post-infection cells were treated with DMSO (black bars), as vehicle control, or the MEK1/2 inhibitor PD0325901 (1 μM) (iMek, green bars) for 16 h. Then, cell lysates were prepared and analysed by immunoblotting using the indicated antibodies (left). Graphs on the right show the quantification of the indicated ratios ( n =3). ( c ) (left) Representative confocal images of OSKM-expressing MEFs for 3 days, treated as in b and stained with anti-Tom20 antibody (red) to assess the different mitochondrial morphologies. Insets show a black and white magnification of the pictures. DAPI (blue) was used as a nuclear counterstaining. Scale bars, 24 μm. Graph on the right shows the quantification of the indicated mitochondrial morphologies observed in the cells treated as indicated ( n =3). ( d ) (left) Representative confocal images of MEFs expressing the reprogramming factors, together with Drp1 wild type (Drp1 WT ) or the phosphomimetic S579D mutation (Drp1 S579D ), during 4 days. Cells were then treated, fixed and stained as in c . Insets show a black and white magnification of the pictures. DAPI (blue) was used as a nuclear counterstaining. Scale bars, 24 μm. Graph on the right shows the quantification of the indicated mitochondrial morphologies observed in the cells treated as indicated ( n =3). Data are represented as mean±s.e.m. (* P
Figure Legend Snippet: Reprogramming-induced mitochondrial fission depends on Erk1/2 phosphorylation of Drp1. ( a ) Lysates of mock- or OSKM-transduced MEFs for the indicated days were analysed by immunoblotting using the indicated antibodies. Graphs on the right show the quantification of the indicated ratios ( n =3). ( b ) MEFs were OSKM-transduced and 3 days post-infection cells were treated with DMSO (black bars), as vehicle control, or the MEK1/2 inhibitor PD0325901 (1 μM) (iMek, green bars) for 16 h. Then, cell lysates were prepared and analysed by immunoblotting using the indicated antibodies (left). Graphs on the right show the quantification of the indicated ratios ( n =3). ( c ) (left) Representative confocal images of OSKM-expressing MEFs for 3 days, treated as in b and stained with anti-Tom20 antibody (red) to assess the different mitochondrial morphologies. Insets show a black and white magnification of the pictures. DAPI (blue) was used as a nuclear counterstaining. Scale bars, 24 μm. Graph on the right shows the quantification of the indicated mitochondrial morphologies observed in the cells treated as indicated ( n =3). ( d ) (left) Representative confocal images of MEFs expressing the reprogramming factors, together with Drp1 wild type (Drp1 WT ) or the phosphomimetic S579D mutation (Drp1 S579D ), during 4 days. Cells were then treated, fixed and stained as in c . Insets show a black and white magnification of the pictures. DAPI (blue) was used as a nuclear counterstaining. Scale bars, 24 μm. Graph on the right shows the quantification of the indicated mitochondrial morphologies observed in the cells treated as indicated ( n =3). Data are represented as mean±s.e.m. (* P

Techniques Used: Infection, Expressing, Staining, Mutagenesis

Effect of Dusp6 in cell reprogramming. ( a ) Dusp6 gene expression in untreated (control) or OSKM-infected MEFs was assessed by qPCR ( n =3). ( b ) MEFs were transduced with OSKM together with empty vector- or Dusp6-encoding retroviruses. Four days after, cell lysates were prepared and analysed by immunoblotting using the indicated antibodies. Graphs on the right show the quantification of the data ( n =3). ( c ) (left) IF images of MEFs transduced as in b and stained with anti-Tom20 antibody (red) 4 days post infection to assess the different mitochondrial morphologies. Insets show a black and white magnification of the pictures. DAPI (blue) was used as a nuclear counterstaining. Scale bars, 24 μm. Graph on the right shows the quantification of the observed mitochondrial morphologies in the cells ( n =3). ( d ) (left) IF images of MEFs co-expressing the reprogramming factors and Dusp6 together with Drp1 wild type (Drp1 WT ) or the phosphomimetic S579D mutation (Drp1 S579D ) during 4 days. Cells were then fixed and stained as in c . Insets show a black and white magnification of the pictures. DAPI (blue) was used as a nuclear counterstaining. Scale bars, 24 μm. Graph on the right, quantification of the indicated mitochondrial morphologies observed in the cells ( n =3). ( e ) Graphs showing the number of AP-positive colonies obtained in MEFs after 25 days of retroviral delivery of the OSKM factors either together with empty vector- (control) or Dusp6-encoding retroviruses (Dusp6) (left) or in the presence of DMSO (as vehicle control) or the MEK1/2 inhibitor PD0325901 (1 μM) (iMek) (right) ( n =3). Panels in the right, bright-field images from the plates of the indicated cultures after AP-staining. Insets show a magnification of a selected area from the AP-stained plates. Data are represented as mean±s.e.m. (* P
Figure Legend Snippet: Effect of Dusp6 in cell reprogramming. ( a ) Dusp6 gene expression in untreated (control) or OSKM-infected MEFs was assessed by qPCR ( n =3). ( b ) MEFs were transduced with OSKM together with empty vector- or Dusp6-encoding retroviruses. Four days after, cell lysates were prepared and analysed by immunoblotting using the indicated antibodies. Graphs on the right show the quantification of the data ( n =3). ( c ) (left) IF images of MEFs transduced as in b and stained with anti-Tom20 antibody (red) 4 days post infection to assess the different mitochondrial morphologies. Insets show a black and white magnification of the pictures. DAPI (blue) was used as a nuclear counterstaining. Scale bars, 24 μm. Graph on the right shows the quantification of the observed mitochondrial morphologies in the cells ( n =3). ( d ) (left) IF images of MEFs co-expressing the reprogramming factors and Dusp6 together with Drp1 wild type (Drp1 WT ) or the phosphomimetic S579D mutation (Drp1 S579D ) during 4 days. Cells were then fixed and stained as in c . Insets show a black and white magnification of the pictures. DAPI (blue) was used as a nuclear counterstaining. Scale bars, 24 μm. Graph on the right, quantification of the indicated mitochondrial morphologies observed in the cells ( n =3). ( e ) Graphs showing the number of AP-positive colonies obtained in MEFs after 25 days of retroviral delivery of the OSKM factors either together with empty vector- (control) or Dusp6-encoding retroviruses (Dusp6) (left) or in the presence of DMSO (as vehicle control) or the MEK1/2 inhibitor PD0325901 (1 μM) (iMek) (right) ( n =3). Panels in the right, bright-field images from the plates of the indicated cultures after AP-staining. Insets show a magnification of a selected area from the AP-stained plates. Data are represented as mean±s.e.m. (* P

Techniques Used: Expressing, Infection, Real-time Polymerase Chain Reaction, Transduction, Plasmid Preparation, Staining, Mutagenesis

18) Product Images from "Trichloromethane fraction of Incarvillea compacta induces lytic cytotoxicity and apoptosis in Epstein-Barr virus-positive gastric cancer AGS cells"

Article Title: Trichloromethane fraction of Incarvillea compacta induces lytic cytotoxicity and apoptosis in Epstein-Barr virus-positive gastric cancer AGS cells

Journal: BMC Complementary and Alternative Medicine

doi: 10.1186/s12906-016-1331-6

Induction of apoptosis after R2 treatment in AGS-EBV cells. Micrographs show apoptotic cells after treatment with R2 at different concentrations and staining by Hoechst 33342 for 24 h ( a ). R2 induced apoptosis in AGS-EBV cells was also detected by the Annexin V-FITC/7-AAD staining test ( b and c ). Cells were treated with R2 (0, 5, 10, 20 μg/mL) for 24 h. DMSO treatment was used as a vehicle control. The apoptotic rates were determined by Annexin V-FITC/7-AAD staining. * P
Figure Legend Snippet: Induction of apoptosis after R2 treatment in AGS-EBV cells. Micrographs show apoptotic cells after treatment with R2 at different concentrations and staining by Hoechst 33342 for 24 h ( a ). R2 induced apoptosis in AGS-EBV cells was also detected by the Annexin V-FITC/7-AAD staining test ( b and c ). Cells were treated with R2 (0, 5, 10, 20 μg/mL) for 24 h. DMSO treatment was used as a vehicle control. The apoptotic rates were determined by Annexin V-FITC/7-AAD staining. * P

Techniques Used: Staining

R2 induced mitochondrial membrane potential depolarization ( a and b ) in AGS-EBV cells. AGS-EBV cells were cultured in R2 (0, 5, 10, 20 μg/mL) for 24 h. DMSO treatment was used as vehicle control. Cells were labeled with DIOC6 and then analyzed by flow cytometry to detect the levels of mitochondrial membrane potential. Relative expression levels were showed in b . Results obtained from a representative experiment are shown ( n = 3). ** p
Figure Legend Snippet: R2 induced mitochondrial membrane potential depolarization ( a and b ) in AGS-EBV cells. AGS-EBV cells were cultured in R2 (0, 5, 10, 20 μg/mL) for 24 h. DMSO treatment was used as vehicle control. Cells were labeled with DIOC6 and then analyzed by flow cytometry to detect the levels of mitochondrial membrane potential. Relative expression levels were showed in b . Results obtained from a representative experiment are shown ( n = 3). ** p

Techniques Used: Cell Culture, Labeling, Flow Cytometry, Cytometry, Expressing

19) Product Images from "Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus"

Article Title: Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus

Journal: mBio

doi: 10.1128/mBio.01998-16

Stability of quasi-enveloped eHAV in human bile acids. (A) Quasi-enveloped eHAV virions were incubated with chenodeoxycholic acid (CDCA; 24 mM), taurocholic acid (TCA; 93 mM or 930 mM), or DMSO (10%) at 37°C for 2 h. The virus was then centrifuged to equilibrium in isopycnic iodixanol gradients. The buoyant density of virions was determined by measuring the quantity of HAV RNA in fractions 6 to 19 by RT-qPCR. The distribution of virus in the DMSO-treated sample matched that of virus incubated in PBS alone (not shown). The shaded zone indicates the expected density of nonenveloped HAV virions. (B) HAV capsid antigen ELISA of gradient-purified eHAV following treatment with bile acids. Virus samples (approximately 2 × 10 7 GE) were subjected to diafiltration to remove bile acids and then tested for the presence of detectable (exposed) capsid antigen by ELISA (see Materials and Methods). (C) Infectivity of eHAV following treatment with bile acids. Samples were subjected to diafiltration to remove bile acids and then diluted and inoculated onto FRhK-4 cells for IR-FIFA. The content of the 10 −2 dilution of the CDCA-treated sample was 3.8 × 10 4 GE/ml, that of the 10 −2 dilution of the TCA-treated sample was 3.1 × 10 4 GE/ml, that of the 10 −2 dilution of the DMSO-treated sample was 2.3 × 10 4 GE/ml, and that of the 10 −2 dilution of the PBS-treated sample was 3.6 × 10 4 GE/ml.
Figure Legend Snippet: Stability of quasi-enveloped eHAV in human bile acids. (A) Quasi-enveloped eHAV virions were incubated with chenodeoxycholic acid (CDCA; 24 mM), taurocholic acid (TCA; 93 mM or 930 mM), or DMSO (10%) at 37°C for 2 h. The virus was then centrifuged to equilibrium in isopycnic iodixanol gradients. The buoyant density of virions was determined by measuring the quantity of HAV RNA in fractions 6 to 19 by RT-qPCR. The distribution of virus in the DMSO-treated sample matched that of virus incubated in PBS alone (not shown). The shaded zone indicates the expected density of nonenveloped HAV virions. (B) HAV capsid antigen ELISA of gradient-purified eHAV following treatment with bile acids. Virus samples (approximately 2 × 10 7 GE) were subjected to diafiltration to remove bile acids and then tested for the presence of detectable (exposed) capsid antigen by ELISA (see Materials and Methods). (C) Infectivity of eHAV following treatment with bile acids. Samples were subjected to diafiltration to remove bile acids and then diluted and inoculated onto FRhK-4 cells for IR-FIFA. The content of the 10 −2 dilution of the CDCA-treated sample was 3.8 × 10 4 GE/ml, that of the 10 −2 dilution of the TCA-treated sample was 3.1 × 10 4 GE/ml, that of the 10 −2 dilution of the DMSO-treated sample was 2.3 × 10 4 GE/ml, and that of the 10 −2 dilution of the PBS-treated sample was 3.6 × 10 4 GE/ml.

Techniques Used: Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Purification, Diafiltration Assay, Infection

20) Product Images from "Proteomic and Systems Biology Analysis of Monocytes Exposed to Securinine, a GABAA Receptor Antagonist and Immune Adjuvant"

Article Title: Proteomic and Systems Biology Analysis of Monocytes Exposed to Securinine, a GABAA Receptor Antagonist and Immune Adjuvant

Journal: PLoS ONE

doi: 10.1371/journal.pone.0041278

Securinine induces rapid phosphorylation of p38 MAPK in MonoMac-1 cells. MonoMac-1 cells were treated with DMSO, 50 uM securinine or 20 ug/mL anisomycin for the indicated times. Lysates were prepared and subjected to Western blot with anti-phospho-p38 MAPK (top panel) or anti-p38 MAPK (bottom panel). Blots were developed with ECL (GE Healthcare) and exposed to film for autoradiography. Anisomycin was used as a positive control.
Figure Legend Snippet: Securinine induces rapid phosphorylation of p38 MAPK in MonoMac-1 cells. MonoMac-1 cells were treated with DMSO, 50 uM securinine or 20 ug/mL anisomycin for the indicated times. Lysates were prepared and subjected to Western blot with anti-phospho-p38 MAPK (top panel) or anti-p38 MAPK (bottom panel). Blots were developed with ECL (GE Healthcare) and exposed to film for autoradiography. Anisomycin was used as a positive control.

Techniques Used: Western Blot, Autoradiography, Positive Control

21) Product Images from "Plasticity between MyoC- and MyoA-Glideosomes: An Example of Functional Compensation in Toxoplasma gondii Invasion"

Article Title: Plasticity between MyoC- and MyoA-Glideosomes: An Example of Functional Compensation in Toxoplasma gondii Invasion

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004504

MyoC-glideosome is not involved in cell division but in invasion. A. Intracellular growth assay performed on GAP45-iKO and GAP45-iKO/GAP80-KO strains by determining the number of parasites per vacuole after 48 hours ± ATc. Data are represented as mean ± SD. B. Gliding assay performed on poly-L-lysine coated coverslips with GAP45-iKO and GAP45-iKO/GAP80-KO strains after 42 hours ± ATc. C. Ionophore-induced egress assay of GAP45-iKO and GAP45-iKO/GAP80-KO strains was performed by treating the parasites with DMSO or Ca2+-ionophore A23187 for 5 min after 56 hours ± ATc before The results are expressed as a percentage of ruptured vacuoles and represented as mean ± SD. D. Invasiveness of GAP45-iKO and GAP45-iKO/GAP80-KO strains was determined using a two-color immunofluorescence assay performed after 42 hours ± ATc. Intracellular: invaded parasites, extracellular: attached parasites. Data are represented as mean ± SD. The significance of the data was evaluated using a parametric paired t-test and the two-tailed p-value is written on the graph. E. Co-IP performed on metabolically labeled wild type and MyoA-KO parasites using anti-MLC1 antibodies. F. In MyoA-KO, MycMyoC-iKO relocalized to the periphery of the parasites up to the apical basal ring in addition to its basal localization. Two exposures are presented for MycMyoC localization. Scale bars: 2 µm. G. Western-blot of total extract of MycMyoC-iKO and MycMyoC-iKO/MyoA-iKO analyzed using anti-MyoA, anti-MLC1 and anti-Myc antibodies. The loading control was done at the same time with anti-PRF and fluorescent secondary antibodies on the same membrane as MLC1 for the upper panel and as Myc for the lower panel. H. Ionophore-induced egress assay of MyoC-iKO and MyoC-iKO/MyoA-KO strains performed by treating the parasites with DMSO or Ca2+-ionophore A23187 for 5 min after 54 hours ± ATc before The results are expressed as a percentage of ruptured vacuoles and represented as mean ± SD. I. Red/green invasion assay performed after 42 hours ± ATc. Intracellular: invaded parasites, extracellular: attached parasites. Data are represented as mean ± SD. The significance of the data was evaluated using a parametric paired t-test and the two-tailed p-value is written on the graph.
Figure Legend Snippet: MyoC-glideosome is not involved in cell division but in invasion. A. Intracellular growth assay performed on GAP45-iKO and GAP45-iKO/GAP80-KO strains by determining the number of parasites per vacuole after 48 hours ± ATc. Data are represented as mean ± SD. B. Gliding assay performed on poly-L-lysine coated coverslips with GAP45-iKO and GAP45-iKO/GAP80-KO strains after 42 hours ± ATc. C. Ionophore-induced egress assay of GAP45-iKO and GAP45-iKO/GAP80-KO strains was performed by treating the parasites with DMSO or Ca2+-ionophore A23187 for 5 min after 56 hours ± ATc before The results are expressed as a percentage of ruptured vacuoles and represented as mean ± SD. D. Invasiveness of GAP45-iKO and GAP45-iKO/GAP80-KO strains was determined using a two-color immunofluorescence assay performed after 42 hours ± ATc. Intracellular: invaded parasites, extracellular: attached parasites. Data are represented as mean ± SD. The significance of the data was evaluated using a parametric paired t-test and the two-tailed p-value is written on the graph. E. Co-IP performed on metabolically labeled wild type and MyoA-KO parasites using anti-MLC1 antibodies. F. In MyoA-KO, MycMyoC-iKO relocalized to the periphery of the parasites up to the apical basal ring in addition to its basal localization. Two exposures are presented for MycMyoC localization. Scale bars: 2 µm. G. Western-blot of total extract of MycMyoC-iKO and MycMyoC-iKO/MyoA-iKO analyzed using anti-MyoA, anti-MLC1 and anti-Myc antibodies. The loading control was done at the same time with anti-PRF and fluorescent secondary antibodies on the same membrane as MLC1 for the upper panel and as Myc for the lower panel. H. Ionophore-induced egress assay of MyoC-iKO and MyoC-iKO/MyoA-KO strains performed by treating the parasites with DMSO or Ca2+-ionophore A23187 for 5 min after 54 hours ± ATc before The results are expressed as a percentage of ruptured vacuoles and represented as mean ± SD. I. Red/green invasion assay performed after 42 hours ± ATc. Intracellular: invaded parasites, extracellular: attached parasites. Data are represented as mean ± SD. The significance of the data was evaluated using a parametric paired t-test and the two-tailed p-value is written on the graph.

Techniques Used: Growth Assay, Gliding Assay, Immunofluorescence, Two Tailed Test, Co-Immunoprecipitation Assay, Metabolic Labelling, Labeling, Western Blot, Invasion Assay

22) Product Images from "The Epidermal Growth Factor Receptor (EGFR) Promotes Uptake of Influenza A Viruses (IAV) into Host Cells"

Article Title: The Epidermal Growth Factor Receptor (EGFR) Promotes Uptake of Influenza A Viruses (IAV) into Host Cells

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1001099

EGFR kinase activity is induced upon viral attachment and is required for efficient IAV internalization. (A) A549 cells were incubated with PD153035 (2.8 µM) or DMSO for 1 h at 37°C and were subsequently treated for 10 min with EGF (30 ng ml −1 ). PI3K dependent Akt phosphorlyation (pAktS473) and phosphorylated EGFR (pEGFRY1068) were detected by WB. (B) A549 cells were mock infected [−] or infected with FPV (MOI = 100) [+] for the indicated times at 37°C. In (C and E) cells were treated with sialidase (± sialidase; 0.01 units ml −1 , 3 h, 37°C), and inoculated with PR8 (MOI = 100) for 1 h at 4°C. Subsequently cells were incubated at 37°C for the indicated times. (C, lower panel) The amount of pEGFR(Y1068) was quantified. Relative pEGFR(Y1068) densities are expressed as mean ±SD of three independent experiments. (D, G) A549 cells were infected with PR8 (MOI = 4) for 30 min upon pre-treatment with (D) an EGFR inhibitor (10 µM), or (G) PD153035 (2.8 µM) or DMSO for 1 h at 37°C. (E) EGFR was immuno-precipitated. Co-immunoprecipitated p85 and the input-control was analysed in WB. (F) Cells were stimulated with EGF (30 ng ml −1 ) 10 min prior to infection and infected with PR8 (MOI = 4) for 30 min at 37°C; an acidic wash (PBS; pH 1.3, 4°C) was performed. WBs were probed with the indicated antibodies. (H) Biotinylated Sambucus nigra agglutinin (SNA) or Maackia amurensis agglutinin II (MAAII) (1 µg ml −1 ) were incubated for 60 min at 4°C with A549 cells. Subsequently cells were either incubated at 37°C for 30 min and lysed for detection of pEGFR(Y1068) levels by WB or incubated with Cy3-conjugated streptavidin to detect lectins for fluorescence microscopy (scale bar 10 µm). See also Figure S3 .
Figure Legend Snippet: EGFR kinase activity is induced upon viral attachment and is required for efficient IAV internalization. (A) A549 cells were incubated with PD153035 (2.8 µM) or DMSO for 1 h at 37°C and were subsequently treated for 10 min with EGF (30 ng ml −1 ). PI3K dependent Akt phosphorlyation (pAktS473) and phosphorylated EGFR (pEGFRY1068) were detected by WB. (B) A549 cells were mock infected [−] or infected with FPV (MOI = 100) [+] for the indicated times at 37°C. In (C and E) cells were treated with sialidase (± sialidase; 0.01 units ml −1 , 3 h, 37°C), and inoculated with PR8 (MOI = 100) for 1 h at 4°C. Subsequently cells were incubated at 37°C for the indicated times. (C, lower panel) The amount of pEGFR(Y1068) was quantified. Relative pEGFR(Y1068) densities are expressed as mean ±SD of three independent experiments. (D, G) A549 cells were infected with PR8 (MOI = 4) for 30 min upon pre-treatment with (D) an EGFR inhibitor (10 µM), or (G) PD153035 (2.8 µM) or DMSO for 1 h at 37°C. (E) EGFR was immuno-precipitated. Co-immunoprecipitated p85 and the input-control was analysed in WB. (F) Cells were stimulated with EGF (30 ng ml −1 ) 10 min prior to infection and infected with PR8 (MOI = 4) for 30 min at 37°C; an acidic wash (PBS; pH 1.3, 4°C) was performed. WBs were probed with the indicated antibodies. (H) Biotinylated Sambucus nigra agglutinin (SNA) or Maackia amurensis agglutinin II (MAAII) (1 µg ml −1 ) were incubated for 60 min at 4°C with A549 cells. Subsequently cells were either incubated at 37°C for 30 min and lysed for detection of pEGFR(Y1068) levels by WB or incubated with Cy3-conjugated streptavidin to detect lectins for fluorescence microscopy (scale bar 10 µm). See also Figure S3 .

Techniques Used: Activity Assay, Incubation, Western Blot, Infection, Immunoprecipitation, Fluorescence, Microscopy

23) Product Images from "Bacillus anthracis’ PA63 Delivers the Tumor Metastasis Suppressor Protein NDPK-A/NME1 into Breast Cancer Cells"

Article Title: Bacillus anthracis’ PA63 Delivers the Tumor Metastasis Suppressor Protein NDPK-A/NME1 into Breast Cancer Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms21093295

PA 63 mediates delivery of His 6 -NDPK-A and His 6 -DTA into HeLa cells. ( A ) PA 63 delivers His 6 -NDPK-A into HeLa cells in a PA 63 -dependent manner. After incubation of the cells with His 6 -NDPK-A (10 µg/mL) and PA 63 (1 µg/mL) or His 6 -NDPK-A only for 6 h at 37 °C, the cells were analyzed by immunoblotting. The uptake of His 6 -NDPK-A was detected with an anti-His-antibody. Hsp90 was detected as loading control and densitometric analysis performed using the ImageJ software. Signal intensity of His 6 -NDPK-A was quantified against Hsp90. ( B ) Cell viability is not reduced by treatment of cells with His 6 -NDPK-A plus PA 63 . After incubation of the cells with His 6 -NDPK-A plus PA 63 or His 6 -NDPK-A alone for 48 h at 37 °C, the cell viability was determined by the MTS assay. Data were normalized to untreated cells (control). DMSO served as positive control. Data are given as mean ± SD, n = 3. ( C ) Uptake of His 6 -DTA is mediated by PA 63 and is sensitive to BafA1. Cells were incubated with BafA1 (30 nM) for 30 min at 37 °C followed by 24 h incubation with His 6 -DTA (5 µg/mL) with or without PA 63 (1 µg/mL). Representative pictures are shown. Thereafter, cell lysates were analyzed by immunoblotting for the presence of His 6 -DTA with an anti-His-antibody. Detection of Hsp90 was used as loading control. ( D ) PA 63 mediates delivery of His 6 -DTA into the cytosol of HeLa cells. Substrate modification was determined via strep-POD after sequential ADP-ribosylation of EF-2 as described. To confirm comparable protein loading, GAPDH was again used as loading control. ( E ) Cell viability is decreased after treatment of cells with His 6 -DTA plus PA 63 . Cells were incubated with His 6 -DTA alone or in combination with PA 63 for 24 h at 37 °C. Cell viability was determined by the MTS assay. Staurosporine (1 µM) served as positive control. Data are given as mean ± SD, n = 3. Significance was tested using One-Way ANOVA followed by Dunnett’s multiple comparison test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. untreated controls.
Figure Legend Snippet: PA 63 mediates delivery of His 6 -NDPK-A and His 6 -DTA into HeLa cells. ( A ) PA 63 delivers His 6 -NDPK-A into HeLa cells in a PA 63 -dependent manner. After incubation of the cells with His 6 -NDPK-A (10 µg/mL) and PA 63 (1 µg/mL) or His 6 -NDPK-A only for 6 h at 37 °C, the cells were analyzed by immunoblotting. The uptake of His 6 -NDPK-A was detected with an anti-His-antibody. Hsp90 was detected as loading control and densitometric analysis performed using the ImageJ software. Signal intensity of His 6 -NDPK-A was quantified against Hsp90. ( B ) Cell viability is not reduced by treatment of cells with His 6 -NDPK-A plus PA 63 . After incubation of the cells with His 6 -NDPK-A plus PA 63 or His 6 -NDPK-A alone for 48 h at 37 °C, the cell viability was determined by the MTS assay. Data were normalized to untreated cells (control). DMSO served as positive control. Data are given as mean ± SD, n = 3. ( C ) Uptake of His 6 -DTA is mediated by PA 63 and is sensitive to BafA1. Cells were incubated with BafA1 (30 nM) for 30 min at 37 °C followed by 24 h incubation with His 6 -DTA (5 µg/mL) with or without PA 63 (1 µg/mL). Representative pictures are shown. Thereafter, cell lysates were analyzed by immunoblotting for the presence of His 6 -DTA with an anti-His-antibody. Detection of Hsp90 was used as loading control. ( D ) PA 63 mediates delivery of His 6 -DTA into the cytosol of HeLa cells. Substrate modification was determined via strep-POD after sequential ADP-ribosylation of EF-2 as described. To confirm comparable protein loading, GAPDH was again used as loading control. ( E ) Cell viability is decreased after treatment of cells with His 6 -DTA plus PA 63 . Cells were incubated with His 6 -DTA alone or in combination with PA 63 for 24 h at 37 °C. Cell viability was determined by the MTS assay. Staurosporine (1 µM) served as positive control. Data are given as mean ± SD, n = 3. Significance was tested using One-Way ANOVA followed by Dunnett’s multiple comparison test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. untreated controls.

Techniques Used: Incubation, Software, MTS Assay, Positive Control, Modification

PA 63 mediates delivery of His 6 -NDPK-A and His 6 -DTA into MDA-MB-231 cells. ( A ) PA 63 delivers His 6 -NDPK-A into MDA-MB-231 cells in a concentration-dependent manner. After incubation of the cells with His 6 -NDPK-A and PA 63 (10 + 1, 18 + 6 or 30 + 3 µg/mL) or His 6 -NDPK-A only (10, 18 or 30 µg/mL) for 48 h at 37 °C Western blot analysis was performed. The uptake of His 6 -NDPK-A (18 kDa) was detected with the specific anti-nm23-H1 antibody. The lane on the far left contains purified recombinant His 6 -taged NDPK-A only which was detected at 18 kDa. Endogenous NDPK-A is detected in the cell lysates at 17 kDa. Hsp90 was detected as loading control and densitometric analysis was performed using the ImageJ software. The signal intensity of His 6 -NDPK-A was quantified against Hsp90. ( B ) Cell viability is not affected by the treatment of cells with His 6 -NDPK-A and PA 63 . After incubation of the cells with His 6 -NDPK-A and PA 63 or His 6 -NDPK-A-only for 48 h at 37 °C, cell viability was determined by the MTS assay. Data were normalized to control (untreated cells). The known reduction by 20% (by vol.) DMSO was used as control. Data are given as mean ± SD, n = 3. ( C ) Uptake of His 6 -DTA is mediated by PA 63 and can be blocked by BafA1. Cells were incubated with BafA1 (30 nM) for 30 min at 37 °C followed by 12 h incubation with His 6 -DTA (5 µg/mL) with or without PA 63 (1 µg/mL). Representative pictures depict no morphological changes after incubation and before sample preparation. His 6 -DTA was detected by an anti- His 6 -antibody. Detection of Hsp90 served as loading control. ( D ) PA 63 mediates cytosolic localization His 6 -DTA in MDA-MB-231 cells. After incubation with BafA1 (30 nM) for 30 min, His 6 -DTA (5 µg/mL) with or without PA 63 (1 µg/mL) was added for 12 h at 37 °C. After cell lysis, In Vitro ADP-ribosylation was performed using biotinylated NAD + (10 mM) as substrate. Modified proteins were precipitated by strep-POD and biotinylated EF-2 was detected by immunoblotting. GAPDH expression was used as loading control. ( E ) Cell viability is decreased after treatment of cells with His 6 -DTA and PA 63 . Cell viability was determined via the MTS assay after 24 h of incubation at 37 °C with His 6 -DTA with or without PA 63 . Staurosporine (1 µM) was used as positive control. Data are normalized to untreated controls and given as mean ± SD, n = 3. Significance was tested using One-Way ANOVA followed by Dunnett’s multiple comparison test. ** p ≤ 0.01 vs. untreated controls.
Figure Legend Snippet: PA 63 mediates delivery of His 6 -NDPK-A and His 6 -DTA into MDA-MB-231 cells. ( A ) PA 63 delivers His 6 -NDPK-A into MDA-MB-231 cells in a concentration-dependent manner. After incubation of the cells with His 6 -NDPK-A and PA 63 (10 + 1, 18 + 6 or 30 + 3 µg/mL) or His 6 -NDPK-A only (10, 18 or 30 µg/mL) for 48 h at 37 °C Western blot analysis was performed. The uptake of His 6 -NDPK-A (18 kDa) was detected with the specific anti-nm23-H1 antibody. The lane on the far left contains purified recombinant His 6 -taged NDPK-A only which was detected at 18 kDa. Endogenous NDPK-A is detected in the cell lysates at 17 kDa. Hsp90 was detected as loading control and densitometric analysis was performed using the ImageJ software. The signal intensity of His 6 -NDPK-A was quantified against Hsp90. ( B ) Cell viability is not affected by the treatment of cells with His 6 -NDPK-A and PA 63 . After incubation of the cells with His 6 -NDPK-A and PA 63 or His 6 -NDPK-A-only for 48 h at 37 °C, cell viability was determined by the MTS assay. Data were normalized to control (untreated cells). The known reduction by 20% (by vol.) DMSO was used as control. Data are given as mean ± SD, n = 3. ( C ) Uptake of His 6 -DTA is mediated by PA 63 and can be blocked by BafA1. Cells were incubated with BafA1 (30 nM) for 30 min at 37 °C followed by 12 h incubation with His 6 -DTA (5 µg/mL) with or without PA 63 (1 µg/mL). Representative pictures depict no morphological changes after incubation and before sample preparation. His 6 -DTA was detected by an anti- His 6 -antibody. Detection of Hsp90 served as loading control. ( D ) PA 63 mediates cytosolic localization His 6 -DTA in MDA-MB-231 cells. After incubation with BafA1 (30 nM) for 30 min, His 6 -DTA (5 µg/mL) with or without PA 63 (1 µg/mL) was added for 12 h at 37 °C. After cell lysis, In Vitro ADP-ribosylation was performed using biotinylated NAD + (10 mM) as substrate. Modified proteins were precipitated by strep-POD and biotinylated EF-2 was detected by immunoblotting. GAPDH expression was used as loading control. ( E ) Cell viability is decreased after treatment of cells with His 6 -DTA and PA 63 . Cell viability was determined via the MTS assay after 24 h of incubation at 37 °C with His 6 -DTA with or without PA 63 . Staurosporine (1 µM) was used as positive control. Data are normalized to untreated controls and given as mean ± SD, n = 3. Significance was tested using One-Way ANOVA followed by Dunnett’s multiple comparison test. ** p ≤ 0.01 vs. untreated controls.

Techniques Used: Multiple Displacement Amplification, Concentration Assay, Incubation, Western Blot, Purification, Recombinant, Software, MTS Assay, Sample Prep, Lysis, In Vitro, Modification, Expressing, Positive Control

24) Product Images from "Hakin-1, a New Specific Small-Molecule Inhibitor for the E3 Ubiquitin-Ligase Hakai, Inhibits Carcinoma Growth and Progression"

Article Title: Hakin-1, a New Specific Small-Molecule Inhibitor for the E3 Ubiquitin-Ligase Hakai, Inhibits Carcinoma Growth and Progression

Journal: Cancers

doi: 10.3390/cancers12051340

In silico and in vivo screening of E3 ubiquitin-ligase Hakai inhibitors ( a ) Chemical structure of Hakin-1 and Hakin-5. ( b ) Predicted binding poses for Hakin-1 (upper panel, in yellow) and Hakin-5 (bottom panel, in orange) molecules docked within Hakai dimers (represented in blue and green), as determined by the CRDOCK docking program. ( c ) Hakai-dependent ubiquitination assay in HEK293 cells transfected with Flag-Hakai, v-Src and HA-ubiquitin in the presence of either DMSO or compound Hakin-1. ( d ) Ubiquitination assay in HEK293T cells transfected with v-Src and HA-ubiquitin in presence of DMSO or compound Hakin-1. ( e ) Hakai-dependent ubiquitination assay in HEK293T cells transfected with Flag-Hakai, v-Src and HA-ubiquitin in the presence of either DMSO or compound Hakin-5. ( f ) Effects of Hakin-1 on Hakai-dependent ubiquitination of the E-cadherin complex. Flag-Hakai, myc-E-cadherin, v-Src and HA–ubiquitin were transiently transfected into HEK293 cells. Immunoprecipitation was performed with the anti-E-cadherin antibody before Western blotting using the indicated antibodies.
Figure Legend Snippet: In silico and in vivo screening of E3 ubiquitin-ligase Hakai inhibitors ( a ) Chemical structure of Hakin-1 and Hakin-5. ( b ) Predicted binding poses for Hakin-1 (upper panel, in yellow) and Hakin-5 (bottom panel, in orange) molecules docked within Hakai dimers (represented in blue and green), as determined by the CRDOCK docking program. ( c ) Hakai-dependent ubiquitination assay in HEK293 cells transfected with Flag-Hakai, v-Src and HA-ubiquitin in the presence of either DMSO or compound Hakin-1. ( d ) Ubiquitination assay in HEK293T cells transfected with v-Src and HA-ubiquitin in presence of DMSO or compound Hakin-1. ( e ) Hakai-dependent ubiquitination assay in HEK293T cells transfected with Flag-Hakai, v-Src and HA-ubiquitin in the presence of either DMSO or compound Hakin-5. ( f ) Effects of Hakin-1 on Hakai-dependent ubiquitination of the E-cadherin complex. Flag-Hakai, myc-E-cadherin, v-Src and HA–ubiquitin were transiently transfected into HEK293 cells. Immunoprecipitation was performed with the anti-E-cadherin antibody before Western blotting using the indicated antibodies.

Techniques Used: In Silico, In Vivo, Binding Assay, Ubiquitin Assay, Transfection, Immunoprecipitation, Western Blot

Hakin-1 reduces cell invasion and cell migration of epithelial tumour cells. ( a ) Invasion assay in LoVo cell line was performed as described in Materials and Methods. Cells were treated in the presence of DMSO or Hakin-1 for 48 h before being seeded into an invasion chamber. Representative images were taken using the 20× objective (right panel) and quantification of the photographed invasive cells are shown (left panel). ( b ) Invasion assay was performed as previously indicated by using MDCK and Hakai-MDCK cells. ( c ) Migration assay in HT-29 cells was analysed after treatment with DMSO or Hakin-1 during 48 h. Cells were seeded in a migration chamber as described in Materials and Methods. Representative images taken with 20× objective (right panel) and quantification of migrating cells (left panel) are shown. Results are represented as mean ± SD of triplicates of three independent experiments (*** p
Figure Legend Snippet: Hakin-1 reduces cell invasion and cell migration of epithelial tumour cells. ( a ) Invasion assay in LoVo cell line was performed as described in Materials and Methods. Cells were treated in the presence of DMSO or Hakin-1 for 48 h before being seeded into an invasion chamber. Representative images were taken using the 20× objective (right panel) and quantification of the photographed invasive cells are shown (left panel). ( b ) Invasion assay was performed as previously indicated by using MDCK and Hakai-MDCK cells. ( c ) Migration assay in HT-29 cells was analysed after treatment with DMSO or Hakin-1 during 48 h. Cells were seeded in a migration chamber as described in Materials and Methods. Representative images taken with 20× objective (right panel) and quantification of migrating cells (left panel) are shown. Results are represented as mean ± SD of triplicates of three independent experiments (*** p

Techniques Used: Migration, Invasion Assay

25) Product Images from "Intra-mitochondrial proteostasis is directly coupled to alpha-synuclein and Amyloid β 1-42 pathology"

Article Title: Intra-mitochondrial proteostasis is directly coupled to alpha-synuclein and Amyloid β 1-42 pathology

Journal: bioRxiv

doi: 10.1101/561134

Mitochondrial proteostasis influences Amyloid β 1-42 pathology. (A) Aβ42-mCherry overexpressing HEK cells were treated with DMSO (control), 1 µM FCCP or 10 µM BAPTA-AM for 24 h. The aggregation of Aβ42 was increased upon treatment with FCCP and BAPTA-AM. Data are presented as mean ± SEM. *p = 0.0298 and ****p
Figure Legend Snippet: Mitochondrial proteostasis influences Amyloid β 1-42 pathology. (A) Aβ42-mCherry overexpressing HEK cells were treated with DMSO (control), 1 µM FCCP or 10 µM BAPTA-AM for 24 h. The aggregation of Aβ42 was increased upon treatment with FCCP and BAPTA-AM. Data are presented as mean ± SEM. *p = 0.0298 and ****p

Techniques Used:

Downstream effectors of mitochondrial dysfunction do not influence alpha-synuclein pathology. (A) YFP-alpha-synuclein SH-SY5Y cells were treated with DMSO (control), 500 µM MPP + , 1 µM ionomycin, or 3 µM menadione for 3 days (1h before, during alpha-synuclein fibrillar seed incubation, and during the 3 day period until evaluation). Scale bars: 20 µm. Alpha-synuclein seeding was not significantly increased (one-way ANOVA with Dunnett’s post-hoc correction). Data are presented as mean ± SEM, N = 11, 8, 8, 7 with n = regions analyzed, three biological repeats. (B) Fluorescence lifetime images and graphs for ATP levels (Ateam1.03 fluorescence lifetime) in SH-SY5Y cells treated with DMSO (control), 500 µM MPP + , 10 µM FCCP and 10 µM BAPTA-AM for 1h. The effect of 500 µM MPP + and FCCP was significant, BAPTA-AM had no significant effect. ****p
Figure Legend Snippet: Downstream effectors of mitochondrial dysfunction do not influence alpha-synuclein pathology. (A) YFP-alpha-synuclein SH-SY5Y cells were treated with DMSO (control), 500 µM MPP + , 1 µM ionomycin, or 3 µM menadione for 3 days (1h before, during alpha-synuclein fibrillar seed incubation, and during the 3 day period until evaluation). Scale bars: 20 µm. Alpha-synuclein seeding was not significantly increased (one-way ANOVA with Dunnett’s post-hoc correction). Data are presented as mean ± SEM, N = 11, 8, 8, 7 with n = regions analyzed, three biological repeats. (B) Fluorescence lifetime images and graphs for ATP levels (Ateam1.03 fluorescence lifetime) in SH-SY5Y cells treated with DMSO (control), 500 µM MPP + , 10 µM FCCP and 10 µM BAPTA-AM for 1h. The effect of 500 µM MPP + and FCCP was significant, BAPTA-AM had no significant effect. ****p

Techniques Used: Incubation, Fluorescence

Downstream effectors of mitochondrial dysfunction do not influence alpha-synuclein pathology. (A) YFP-alpha-synuclein SH-SY5Y cells were treated with DMSO (control), 500 µM MPP + , 1 µM ionomycin, or 3 µM menadione for 3 days (1h before, during alpha-synuclein fibrillar seed incubation, and during the 3 day period until evaluation). Scale bars: 20 µm. Alpha-synuclein seeding was not significantly increased (one-way ANOVA with Dunnett’s post-hoc correction). Data are presented as mean ± SEM, N = 11, 8, 8, 7 with n = regions analyzed, three biological repeats. (B) Fluorescence lifetime images and graphs for ATP levels (Ateam1.03 fluorescence lifetime) in SH-SY5Y cells treated with DMSO (control), 500 µM MPP + , 10 µM FCCP and 10 µM BAPTA-AM for 1h. The effect of 500 µM MPP + and FCCP was significant, BAPTA-AM had no significant effect. ****p
Figure Legend Snippet: Downstream effectors of mitochondrial dysfunction do not influence alpha-synuclein pathology. (A) YFP-alpha-synuclein SH-SY5Y cells were treated with DMSO (control), 500 µM MPP + , 1 µM ionomycin, or 3 µM menadione for 3 days (1h before, during alpha-synuclein fibrillar seed incubation, and during the 3 day period until evaluation). Scale bars: 20 µm. Alpha-synuclein seeding was not significantly increased (one-way ANOVA with Dunnett’s post-hoc correction). Data are presented as mean ± SEM, N = 11, 8, 8, 7 with n = regions analyzed, three biological repeats. (B) Fluorescence lifetime images and graphs for ATP levels (Ateam1.03 fluorescence lifetime) in SH-SY5Y cells treated with DMSO (control), 500 µM MPP + , 10 µM FCCP and 10 µM BAPTA-AM for 1h. The effect of 500 µM MPP + and FCCP was significant, BAPTA-AM had no significant effect. ****p

Techniques Used: Incubation, Fluorescence

Mitochondrial proteostasis influences Amyloid β 1-42 pathology. (A) Aβ42-mCherry overexpressing HEK cells were treated with DMSO (control), 1 µM FCCP or 10 µM BAPTA-AM for 24 h. The aggregation of Aβ42 was increased upon treatment with FCCP and BAPTA-AM. Data are presented as mean ± SEM. *p = 0.0298 and ****p
Figure Legend Snippet: Mitochondrial proteostasis influences Amyloid β 1-42 pathology. (A) Aβ42-mCherry overexpressing HEK cells were treated with DMSO (control), 1 µM FCCP or 10 µM BAPTA-AM for 24 h. The aggregation of Aβ42 was increased upon treatment with FCCP and BAPTA-AM. Data are presented as mean ± SEM. *p = 0.0298 and ****p

Techniques Used:

BAPTA-AM increases alpha-synuclein pathology. (A) YFP-alpha-synuclein SH-SY5Y cells were treated with DMSO (control), 10 µM BAPTA-AM for 1h (before fibrillar seed incubation) and for 5h (before plus during the incubation with alpha-synuclein fibrillar seeds). Scale bars: 20 µm. Alpha-synuclein seeding was increased upon 1h pre-treatment and 5h treatment with BAPTA-AM. Data are presented as mean ± SEM. *p = 0.0127 and ****p
Figure Legend Snippet: BAPTA-AM increases alpha-synuclein pathology. (A) YFP-alpha-synuclein SH-SY5Y cells were treated with DMSO (control), 10 µM BAPTA-AM for 1h (before fibrillar seed incubation) and for 5h (before plus during the incubation with alpha-synuclein fibrillar seeds). Scale bars: 20 µm. Alpha-synuclein seeding was increased upon 1h pre-treatment and 5h treatment with BAPTA-AM. Data are presented as mean ± SEM. *p = 0.0127 and ****p

Techniques Used: Incubation

Downstream effectors of mitochondrial dysfunction do not influence alpha-synuclein pathology. (A) YFP-alpha-synuclein SH-SY5Y cells were treated with DMSO (control), 500 µM MPP + , 1 µM ionomycin, or 3 µM menadione for 3 days (1h before, during alpha-synuclein fibrillar seed incubation, and during the 3 day period until evaluation). Scale bars: 20 µm. Alpha-synuclein seeding was not significantly increased (one-way ANOVA with Dunnett’s post-hoc correction). Data are presented as mean ± SEM, N = 11, 8, 8, 7 with n = regions analyzed, three biological repeats. (B) Fluorescence lifetime images and graphs for ATP levels (Ateam1.03 fluorescence lifetime) in SH-SY5Y cells treated with DMSO (control), 500 µM MPP + , 10 µM FCCP and 10 µM BAPTA-AM for 1h. The effect of 500 µM MPP + and FCCP was significant, BAPTA-AM had no significant effect. ****p
Figure Legend Snippet: Downstream effectors of mitochondrial dysfunction do not influence alpha-synuclein pathology. (A) YFP-alpha-synuclein SH-SY5Y cells were treated with DMSO (control), 500 µM MPP + , 1 µM ionomycin, or 3 µM menadione for 3 days (1h before, during alpha-synuclein fibrillar seed incubation, and during the 3 day period until evaluation). Scale bars: 20 µm. Alpha-synuclein seeding was not significantly increased (one-way ANOVA with Dunnett’s post-hoc correction). Data are presented as mean ± SEM, N = 11, 8, 8, 7 with n = regions analyzed, three biological repeats. (B) Fluorescence lifetime images and graphs for ATP levels (Ateam1.03 fluorescence lifetime) in SH-SY5Y cells treated with DMSO (control), 500 µM MPP + , 10 µM FCCP and 10 µM BAPTA-AM for 1h. The effect of 500 µM MPP + and FCCP was significant, BAPTA-AM had no significant effect. ****p

Techniques Used: Incubation, Fluorescence

26) Product Images from "Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules"

Article Title: Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

Journal: bioRxiv

doi: 10.1101/2020.03.13.980946

dTAG V -1 is an exclusively selective, in vivo -compatible degrader of FKBP12 F36V -tagged proteins. ( a ) Schematic depiction of the dTAG system using VHL-recruiting dTAG molecules. VHL-recruiting dTAG molecules promote ternary complex formation between the FKBP12 F36V -tagged target protein and E3 ubiquitin ligase complex, inducing target protein ubiquitination and degradation. ( b ) Chemical structures of dTAG V -1 and dTAG V -1-NEG. ( c ) DMSO-normalized ratio of Nluc/Fluc signal of 293FT FKBP12 WT -Nluc or FKBP12 F36V -Nluc cells treated with dTAG V -1 or dTAG V -1-NEG molecules for 24 h. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. ( d ) Protein abundance after treatment of PATU-8902 LACZ-FKBP12 F36V clone with 500 nM dTAG V -1 for 4 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value. Significance designations derived from a permutation-based FDR estimation (q
Figure Legend Snippet: dTAG V -1 is an exclusively selective, in vivo -compatible degrader of FKBP12 F36V -tagged proteins. ( a ) Schematic depiction of the dTAG system using VHL-recruiting dTAG molecules. VHL-recruiting dTAG molecules promote ternary complex formation between the FKBP12 F36V -tagged target protein and E3 ubiquitin ligase complex, inducing target protein ubiquitination and degradation. ( b ) Chemical structures of dTAG V -1 and dTAG V -1-NEG. ( c ) DMSO-normalized ratio of Nluc/Fluc signal of 293FT FKBP12 WT -Nluc or FKBP12 F36V -Nluc cells treated with dTAG V -1 or dTAG V -1-NEG molecules for 24 h. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. ( d ) Protein abundance after treatment of PATU-8902 LACZ-FKBP12 F36V clone with 500 nM dTAG V -1 for 4 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value. Significance designations derived from a permutation-based FDR estimation (q

Techniques Used: In Vivo, Derivative Assay

EWS/FLI degradation reverses abnormal proteomic signaling and proliferation. ( a-b ) Immunoblot analysis of EWS502 FKBP12 F36V -GFP or FKBP12 F36V -EWS/FLI; EWS/FLI -/- cells treated with DMSO (a-b), dTAG-13 (a), or dTAG V -1 (b) for 24 h. Data in a - b are representative of n = 3 independent experiments. ( c ) Protein abundance after treatment of EWS502 FKBP12 F36V -EWS/FLI; EWS/FLI -/- cells with dTAG V -1 for 6 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value. Significance designations derived from a permutation-based FDR estimation (q
Figure Legend Snippet: EWS/FLI degradation reverses abnormal proteomic signaling and proliferation. ( a-b ) Immunoblot analysis of EWS502 FKBP12 F36V -GFP or FKBP12 F36V -EWS/FLI; EWS/FLI -/- cells treated with DMSO (a-b), dTAG-13 (a), or dTAG V -1 (b) for 24 h. Data in a - b are representative of n = 3 independent experiments. ( c ) Protein abundance after treatment of EWS502 FKBP12 F36V -EWS/FLI; EWS/FLI -/- cells with dTAG V -1 for 6 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value. Significance designations derived from a permutation-based FDR estimation (q

Techniques Used: Derivative Assay

27) Product Images from "Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules"

Article Title: Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

Journal: bioRxiv

doi: 10.1101/2020.03.13.980946

dTAG V -1 is an exclusively selective, in vivo -compatible degrader of FKBP12 F36V -tagged proteins. ( a ) Schematic depiction of the dTAG system using VHL-recruiting dTAG molecules. VHL-recruiting dTAG molecules promote ternary complex formation between the FKBP12 F36V -tagged target protein and E3 ubiquitin ligase complex, inducing target protein ubiquitination and degradation. ( b ) Chemical structures of dTAG V -1 and dTAG V -1-NEG. ( c ) DMSO-normalized ratio of Nluc/Fluc signal of 293FT FKBP12 WT -Nluc or FKBP12 F36V -Nluc cells treated with dTAG V -1 or dTAG V -1-NEG molecules for 24 h. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. ( d ) Protein abundance after treatment of PATU-8902 LACZ-FKBP12 F36V clone with 500 nM dTAG V -1 for 4 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value. Significance designations derived from a permutation-based FDR estimation (q
Figure Legend Snippet: dTAG V -1 is an exclusively selective, in vivo -compatible degrader of FKBP12 F36V -tagged proteins. ( a ) Schematic depiction of the dTAG system using VHL-recruiting dTAG molecules. VHL-recruiting dTAG molecules promote ternary complex formation between the FKBP12 F36V -tagged target protein and E3 ubiquitin ligase complex, inducing target protein ubiquitination and degradation. ( b ) Chemical structures of dTAG V -1 and dTAG V -1-NEG. ( c ) DMSO-normalized ratio of Nluc/Fluc signal of 293FT FKBP12 WT -Nluc or FKBP12 F36V -Nluc cells treated with dTAG V -1 or dTAG V -1-NEG molecules for 24 h. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. ( d ) Protein abundance after treatment of PATU-8902 LACZ-FKBP12 F36V clone with 500 nM dTAG V -1 for 4 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value. Significance designations derived from a permutation-based FDR estimation (q

Techniques Used: In Vivo, Derivative Assay

EWS/FLI degradation reverses abnormal proteomic signaling and proliferation. ( a-b ) Immunoblot analysis of EWS502 FKBP12 F36V -GFP or FKBP12 F36V -EWS/FLI; EWS/FLI -/- cells treated with DMSO (a-b), dTAG-13 (a), or dTAG V -1 (b) for 24 h. Data in a - b are representative of n = 3 independent experiments. ( c ) Protein abundance after treatment of EWS502 FKBP12 F36V -EWS/FLI; EWS/FLI -/- cells with dTAG V -1 for 6 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value. Significance designations derived from a permutation-based FDR estimation (q
Figure Legend Snippet: EWS/FLI degradation reverses abnormal proteomic signaling and proliferation. ( a-b ) Immunoblot analysis of EWS502 FKBP12 F36V -GFP or FKBP12 F36V -EWS/FLI; EWS/FLI -/- cells treated with DMSO (a-b), dTAG-13 (a), or dTAG V -1 (b) for 24 h. Data in a - b are representative of n = 3 independent experiments. ( c ) Protein abundance after treatment of EWS502 FKBP12 F36V -EWS/FLI; EWS/FLI -/- cells with dTAG V -1 for 6 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value. Significance designations derived from a permutation-based FDR estimation (q

Techniques Used: Derivative Assay

28) Product Images from "Cryopreservation of Human Mesenchymal Stem Cells in an Allogeneic Bioscaffold based on Platelet Rich Plasma and Synovial Fluid"

Article Title: Cryopreservation of Human Mesenchymal Stem Cells in an Allogeneic Bioscaffold based on Platelet Rich Plasma and Synovial Fluid

Journal: Scientific Reports

doi: 10.1038/s41598-017-16134-6

In vitro characterization of cryopreserved embedded MSCs within PRP-SF bioscaffolds 7 days after thawing. Quantification by flow cytometry of ( A ) live cell percentage after calcein/ethidium staining and ( B ) early apoptotic cell percentage after Annexin/PI staining. ( C ) Quantification of viable cell number in proliferation by CCK-8. ( D ) Quantification of membrane integrity by LDH release. ( E ). Micrographs of calcein/ethidium stained samples. Note: Control: non-cryopreserved bioscaffolds; without CPA and additives:wo CPA; DMSO: DMSO 10%; Sucrose: Sucrose 0,2 M; DMSO + Sucrose: DMSO 10% + Sucrose 0,2 M; Values represent mean ± SD. *p
Figure Legend Snippet: In vitro characterization of cryopreserved embedded MSCs within PRP-SF bioscaffolds 7 days after thawing. Quantification by flow cytometry of ( A ) live cell percentage after calcein/ethidium staining and ( B ) early apoptotic cell percentage after Annexin/PI staining. ( C ) Quantification of viable cell number in proliferation by CCK-8. ( D ) Quantification of membrane integrity by LDH release. ( E ). Micrographs of calcein/ethidium stained samples. Note: Control: non-cryopreserved bioscaffolds; without CPA and additives:wo CPA; DMSO: DMSO 10%; Sucrose: Sucrose 0,2 M; DMSO + Sucrose: DMSO 10% + Sucrose 0,2 M; Values represent mean ± SD. *p

Techniques Used: In Vitro, Flow Cytometry, Cytometry, Staining, CCK-8 Assay

Cryoprotective comparison of PRP and PRP-SF bioscaffolds. ( A ) Macroscopic images of 1 day cultured PRP (1) and PRP-SF (2) bioscaffolds. ( B ) Viable cell number in proliferation of embedded MSCs within PRP and PRP-SF bioscaffolds 1 and 7 days after thawing. ( C ) Micrographs of calcein/ethidium stained MSCs within PRP and PRP-SF bioscaffolds 7 days after thawing. Note: PRP control: non-cryopreserved PRP bioscaffold; PRP wo CPA: PRP bioscaffold cryopreserved without CPA and additives; PRP DMSO: PRP bioscaffold cryopreserved DMSO 10%: PRP-SF control: non-cryopreserved PRP-SF bioscaffold; PRP-SF wo CPA: PRP-SF bioscaffold cryopreserved without CPA and additives; PRP-SF DMSO: PRP-SF bioscaffold cryopreserved DMSO 10%. Scale bar: 100 µm.
Figure Legend Snippet: Cryoprotective comparison of PRP and PRP-SF bioscaffolds. ( A ) Macroscopic images of 1 day cultured PRP (1) and PRP-SF (2) bioscaffolds. ( B ) Viable cell number in proliferation of embedded MSCs within PRP and PRP-SF bioscaffolds 1 and 7 days after thawing. ( C ) Micrographs of calcein/ethidium stained MSCs within PRP and PRP-SF bioscaffolds 7 days after thawing. Note: PRP control: non-cryopreserved PRP bioscaffold; PRP wo CPA: PRP bioscaffold cryopreserved without CPA and additives; PRP DMSO: PRP bioscaffold cryopreserved DMSO 10%: PRP-SF control: non-cryopreserved PRP-SF bioscaffold; PRP-SF wo CPA: PRP-SF bioscaffold cryopreserved without CPA and additives; PRP-SF DMSO: PRP-SF bioscaffold cryopreserved DMSO 10%. Scale bar: 100 µm.

Techniques Used: Cell Culture, Staining

Effects and interactions of CPAs and human serum in the viable cell number in proliferation of cryopreserved embedded MSCs within PRP-SF. Viable cell number in proliferation of embedded MSCs within PRP-SF bioscaffolds 7 ( A ) and 14 ( B ) days after thawing. ( C ) Micrographs of calcein/ethidium stained MSCs within PRP-SF bioscaffolds 7 and 14 days after thawing Note: Control: non-cryopreserved bioscaffolds; wo CPA: without CPA and additives; DMSO: DMSO 10%; HS: Human Serum 10%; DMSO + HS: DMSO 10% + Human serum 10%; Sucrose: Sucrose 0,2 M; DMSO + Sucrose: DMSO 10% + Sucrose 0,2 M; HS + Sucrose: Human Serum 10% + Sucrose 0,2 M; DMSO + HS + Sucrose: DMSO 10% + Human Serum 10% + Sucrose 0,2 M. Scale bar: 100 µm.
Figure Legend Snippet: Effects and interactions of CPAs and human serum in the viable cell number in proliferation of cryopreserved embedded MSCs within PRP-SF. Viable cell number in proliferation of embedded MSCs within PRP-SF bioscaffolds 7 ( A ) and 14 ( B ) days after thawing. ( C ) Micrographs of calcein/ethidium stained MSCs within PRP-SF bioscaffolds 7 and 14 days after thawing Note: Control: non-cryopreserved bioscaffolds; wo CPA: without CPA and additives; DMSO: DMSO 10%; HS: Human Serum 10%; DMSO + HS: DMSO 10% + Human serum 10%; Sucrose: Sucrose 0,2 M; DMSO + Sucrose: DMSO 10% + Sucrose 0,2 M; HS + Sucrose: Human Serum 10% + Sucrose 0,2 M; DMSO + HS + Sucrose: DMSO 10% + Human Serum 10% + Sucrose 0,2 M. Scale bar: 100 µm.

Techniques Used: Staining

Differentiation potential of cryopreserved MSCs within PRP-SF bioscaffolds. Microscopic images of 3 weeks MSCs released from thawed PRP-SF bioscaffolds and differentiated into osteocytes, adypocites or chondrocytes. Note: Control: non cryopreserved; DMSO: DMSO 10%; Sucrose: Sucrose 0,2 M; DMSO + Sucrose: DMSO 10% + Sucrose 0,2 M. Osteo: osteogenic differentiation; Contr Osteo: non osteogenic differentiation (control); Adipo: adipogenic differentiation; Contr Osteo: non adipogenic differentiation (control); Chondro: chondrogenic differentiation; Contr Chondro: non chonrogenic differentiation (control). Scale bar: 100 µm.
Figure Legend Snippet: Differentiation potential of cryopreserved MSCs within PRP-SF bioscaffolds. Microscopic images of 3 weeks MSCs released from thawed PRP-SF bioscaffolds and differentiated into osteocytes, adypocites or chondrocytes. Note: Control: non cryopreserved; DMSO: DMSO 10%; Sucrose: Sucrose 0,2 M; DMSO + Sucrose: DMSO 10% + Sucrose 0,2 M. Osteo: osteogenic differentiation; Contr Osteo: non osteogenic differentiation (control); Adipo: adipogenic differentiation; Contr Osteo: non adipogenic differentiation (control); Chondro: chondrogenic differentiation; Contr Chondro: non chonrogenic differentiation (control). Scale bar: 100 µm.

Techniques Used:

29) Product Images from "FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-π Interactions"

Article Title: FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-π Interactions

Journal: Cell

doi: 10.1016/j.cell.2018.03.056

25 μM AdOx Treatment Significantly Reduces Asymmetric Dimethylation of FUS Purified from Sf9 Cells, Related to Figure 1 (A) Left: Representative western blot of MBP-FUS-EmGFP protein purified from Sf9 cells after 4 days of 25μM AdOx, or DMSO control. Right: Quantification of western blots. n = 4 per experimental group, one-tailed, Mann-Whitney U test, ∗ p ≤ 0.05, error bars = SEM. (B) Top: FUS dimethylation sites differ in their apparent susceptibility to AdOx treatment. Depicted are the relative proportion of peptide-to-spectrum matches (CID MS2 spectra of unlabeled peptides identified with the PEAKS algorithm) comprising a given FUS arginine residue observed in an unmodified, mono- or dimethylated state. For most FUS arginine residues, methylation decreased in AdOx treated relative to mock treated cells, except for R394 and R481 which were consistently observed to be dimethylated. Middle: Orbitrap CID MS2 spectrum of a chymotryptic FUS peptide dimethylated at R407 identified with confidence exceeding 99%. The other arginine residue present (R422) was unmodified, as evidenced by a continuous y-ion series indicating no post-translational modification in this part of the peptide. The spectrum is representative of 77 peptide-to-spectrum matches for this region of FUS, which consistently identified (mono or di) methylated R407 accompanied by unmodified R422. Bottom left: Evidence for AdOx-dependent inhibition of arginine methylation. The graph depicts the relative ratios of unmethylated, monomethylated and dimethylated versions of the FUS peptides 209-GQQDRGGRGRGGSGGGGGGGGGGY-232 and 398-GGGGSGGGGRGGFPSGGGGGGGQQRAGDW-426 in untreated and AdOx-treated FUS preparations. Circles represent individual quantifications. The number of quantifications (based on separate peptide-to-spectrum matches) underlying each cumulative quantification are listed above the graph. Horizontal marks depict median Log2 ratios for a given peptide and modification. Bottom right: AdOx treatment did not affect overall abundance of peptides in untreated versus AdOx-treated samples. Abundance ratios of nine chymotryptic peptides from the MBP-FUS-EmGFP. All peptides depicted either lack arginine residues or contain arginine residues but were not observed to be methylated or dimethylated.
Figure Legend Snippet: 25 μM AdOx Treatment Significantly Reduces Asymmetric Dimethylation of FUS Purified from Sf9 Cells, Related to Figure 1 (A) Left: Representative western blot of MBP-FUS-EmGFP protein purified from Sf9 cells after 4 days of 25μM AdOx, or DMSO control. Right: Quantification of western blots. n = 4 per experimental group, one-tailed, Mann-Whitney U test, ∗ p ≤ 0.05, error bars = SEM. (B) Top: FUS dimethylation sites differ in their apparent susceptibility to AdOx treatment. Depicted are the relative proportion of peptide-to-spectrum matches (CID MS2 spectra of unlabeled peptides identified with the PEAKS algorithm) comprising a given FUS arginine residue observed in an unmodified, mono- or dimethylated state. For most FUS arginine residues, methylation decreased in AdOx treated relative to mock treated cells, except for R394 and R481 which were consistently observed to be dimethylated. Middle: Orbitrap CID MS2 spectrum of a chymotryptic FUS peptide dimethylated at R407 identified with confidence exceeding 99%. The other arginine residue present (R422) was unmodified, as evidenced by a continuous y-ion series indicating no post-translational modification in this part of the peptide. The spectrum is representative of 77 peptide-to-spectrum matches for this region of FUS, which consistently identified (mono or di) methylated R407 accompanied by unmodified R422. Bottom left: Evidence for AdOx-dependent inhibition of arginine methylation. The graph depicts the relative ratios of unmethylated, monomethylated and dimethylated versions of the FUS peptides 209-GQQDRGGRGRGGSGGGGGGGGGGY-232 and 398-GGGGSGGGGRGGFPSGGGGGGGQQRAGDW-426 in untreated and AdOx-treated FUS preparations. Circles represent individual quantifications. The number of quantifications (based on separate peptide-to-spectrum matches) underlying each cumulative quantification are listed above the graph. Horizontal marks depict median Log2 ratios for a given peptide and modification. Bottom right: AdOx treatment did not affect overall abundance of peptides in untreated versus AdOx-treated samples. Abundance ratios of nine chymotryptic peptides from the MBP-FUS-EmGFP. All peptides depicted either lack arginine residues or contain arginine residues but were not observed to be methylated or dimethylated.

Techniques Used: Purification, Western Blot, One-tailed Test, MANN-WHITNEY, Methylation, Modification, Inhibition

30) Product Images from "Medial HOXA genes demarcate haematopoietic stem cell fate during human development"

Article Title: Medial HOXA genes demarcate haematopoietic stem cell fate during human development

Journal: Nature cell biology

doi: 10.1038/ncb3354

Retinoic acid signalling pulse in EB haemato-vascular cells induces transcriptional programs associated with definitive haemogenic endothelium and HSC fate (A) FPKM quantification from RNA-seq for RAR-α targets RAR-β and RARA-γ and (B) HOXA genes in sorted AM580-treated and DMSO control hESC-HSPCs (mean from 2 independent experiments). (C) RNA-seq genome browser screen shot for HOXA cluster and RUNX1 in hESC- and FL-HSPCs after 6 days of AM580 treatment. (D) GO categories of biological processes significantly upregulated in hESC-HSPCs by AM580 treatment at day 6. (E) FPKM quantification values from representative genes from vasculature development and transcription GO categories from genes significantly upregulated in hESC-HSPCs by 6 day AM580 treatment (2-fold or greater change, p-value
Figure Legend Snippet: Retinoic acid signalling pulse in EB haemato-vascular cells induces transcriptional programs associated with definitive haemogenic endothelium and HSC fate (A) FPKM quantification from RNA-seq for RAR-α targets RAR-β and RARA-γ and (B) HOXA genes in sorted AM580-treated and DMSO control hESC-HSPCs (mean from 2 independent experiments). (C) RNA-seq genome browser screen shot for HOXA cluster and RUNX1 in hESC- and FL-HSPCs after 6 days of AM580 treatment. (D) GO categories of biological processes significantly upregulated in hESC-HSPCs by AM580 treatment at day 6. (E) FPKM quantification values from representative genes from vasculature development and transcription GO categories from genes significantly upregulated in hESC-HSPCs by 6 day AM580 treatment (2-fold or greater change, p-value

Techniques Used: RNA Sequencing Assay

31) Product Images from "Regulated intramembrane proteolysis of the AXL receptor kinase generates an intracellular domain that localizes in the nucleus of cancer cells"

Article Title: Regulated intramembrane proteolysis of the AXL receptor kinase generates an intracellular domain that localizes in the nucleus of cancer cells

Journal: The FASEB Journal

doi: 10.1096/fj.201600702R

Endogenous AXL in cancer cells is sequentially processed by α- and γ-secretases. A – C ) NSCLC cell lines (H1299 and A549; A ), pancreatic cancer cell lines (AsPC-1, Panc-1, and Panc-28; B ), and glioma cell lines (LN-18 and LN-229; C ) were treated overnight with DMSO, 100 ng/ml PMA, 20 μM TAPI-1, 5 μM C3, or 200 nM Epo in the presence or absence of 10 μM DAPT. Cell lysates were collected for detection of AXL-FL, -CTF, or -ICD using the C-20 Ab targeting the C terminus of AXL. D , E ) α-Secretases ADAM10 and TACE are in charge of the production of AXL-CTF in Panc-28 ( D ) and HEK293T ( E ) cells. Panc-28 and HEK293T cells were transfected with the indicated siRNA in the absence and presence of Flag-tagged AXL-FL, respectively, before the 16-h incubation with DAPT (for details, see Materials and Methods). Cells were then lysed to analyze AXL-FL and AXL-CTF by Western blotting using an anti-AXL Ab (B-2) and an anti-Flag Ab for Panc-28 and HEK293T cells, respectively. AXL-CTF levels in DAPT-treated groups were quantified and expressed as the percentage of control siRNA (siCTR). Data are presented as means ± sem ( n = 3). Knockdown efficiencies of siRNAs for ADAM10, TACE, and BACE were validated by quantitative RT-PCR and/or Western blots in these samples, and data are shown in Supplemental Fig. 1 . GAPDH and β-actin were used as equal loading controls. Asterisk indicates nonspecific band. All blots were performed at least twice, and a representative experiment is shown. * P
Figure Legend Snippet: Endogenous AXL in cancer cells is sequentially processed by α- and γ-secretases. A – C ) NSCLC cell lines (H1299 and A549; A ), pancreatic cancer cell lines (AsPC-1, Panc-1, and Panc-28; B ), and glioma cell lines (LN-18 and LN-229; C ) were treated overnight with DMSO, 100 ng/ml PMA, 20 μM TAPI-1, 5 μM C3, or 200 nM Epo in the presence or absence of 10 μM DAPT. Cell lysates were collected for detection of AXL-FL, -CTF, or -ICD using the C-20 Ab targeting the C terminus of AXL. D , E ) α-Secretases ADAM10 and TACE are in charge of the production of AXL-CTF in Panc-28 ( D ) and HEK293T ( E ) cells. Panc-28 and HEK293T cells were transfected with the indicated siRNA in the absence and presence of Flag-tagged AXL-FL, respectively, before the 16-h incubation with DAPT (for details, see Materials and Methods). Cells were then lysed to analyze AXL-FL and AXL-CTF by Western blotting using an anti-AXL Ab (B-2) and an anti-Flag Ab for Panc-28 and HEK293T cells, respectively. AXL-CTF levels in DAPT-treated groups were quantified and expressed as the percentage of control siRNA (siCTR). Data are presented as means ± sem ( n = 3). Knockdown efficiencies of siRNAs for ADAM10, TACE, and BACE were validated by quantitative RT-PCR and/or Western blots in these samples, and data are shown in Supplemental Fig. 1 . GAPDH and β-actin were used as equal loading controls. Asterisk indicates nonspecific band. All blots were performed at least twice, and a representative experiment is shown. * P

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

Pharmacological and genetic tools uncover the sequential cleavage of AXL mediated by α- and γ-secretases. A , C ) HEK293T cells either transiently ( A ) or stably ( C ) expressing AXL-FL were treated overnight with DMSO (final concentration, 0.3% w/v), 100 ng/ml PMA, 20 μM α-secretase inhibitor TAPI-1, 5 μM BACE1 inhibitor C3, or 200 nM Epo in the presence or absence of 10 μM DAPT. Cells were then lysed, and whole-cell extracts were separated by PAGE on a 10% Tris-glycine SDS gel for detection of AXL-FL and AXL-CTF with an anti-Flag Ab (Abmart). β-Actin was used as an equal loading control. B ) Densitometric estimation of the relative levels of AXL-CTF in panel A . AXL-CTF levels were quantified by using ImageJ software and expressed as fold-change of DMSO controls. Data are expressed as means ± sem of triplicate measurements; the statistical analyses between the treated and DMSO groups, or between other lined groups are shown with asterisks ( n = 3). *** P
Figure Legend Snippet: Pharmacological and genetic tools uncover the sequential cleavage of AXL mediated by α- and γ-secretases. A , C ) HEK293T cells either transiently ( A ) or stably ( C ) expressing AXL-FL were treated overnight with DMSO (final concentration, 0.3% w/v), 100 ng/ml PMA, 20 μM α-secretase inhibitor TAPI-1, 5 μM BACE1 inhibitor C3, or 200 nM Epo in the presence or absence of 10 μM DAPT. Cells were then lysed, and whole-cell extracts were separated by PAGE on a 10% Tris-glycine SDS gel for detection of AXL-FL and AXL-CTF with an anti-Flag Ab (Abmart). β-Actin was used as an equal loading control. B ) Densitometric estimation of the relative levels of AXL-CTF in panel A . AXL-CTF levels were quantified by using ImageJ software and expressed as fold-change of DMSO controls. Data are expressed as means ± sem of triplicate measurements; the statistical analyses between the treated and DMSO groups, or between other lined groups are shown with asterisks ( n = 3). *** P

Techniques Used: Stable Transfection, Expressing, Concentration Assay, Polyacrylamide Gel Electrophoresis, SDS-Gel, Software

32) Product Images from "Dimethyl Fumarate ameliorates pulmonary arterial hypertension and lung fibrosis by targeting multiple pathways"

Article Title: Dimethyl Fumarate ameliorates pulmonary arterial hypertension and lung fibrosis by targeting multiple pathways

Journal: Scientific Reports

doi: 10.1038/srep41605

DMF inhibits pro-inflammatory gene expression in endothelial cells by suppressing NFκB, STAT3 and cJUN signaling. (A,B,C,E) HPAECs were incubated for up to 24 h in (H) hypoxia (2.5% O 2 ) or (N) normoxia (21% O 2 ) with 10 μM DMF (D) or DMSO. Relative gene expression was measured with qPCR: (A) HO-1 , (B) Nox4, (C) pro-inflammatory genes: OSM, IL-6 and ICAM1. n = 4 independent experiments. (D) 10 μM DMF treatment reduced the phosphorylation of STAT3 and cJUN in HPAECs exposed to hypoxia for 4 h. Representative immunoblots and densitometry quantification are shown. n = 3 independent experiments. (E) NFκB activity luciferase reporter assay was performed in HPAECs in presence of 10 ng/ml LPS (L) and/or 30 μM DMF ( D ), control cells treated with DMSO ( C ). n = 3 independent experiments. Data shown as mean ± SD. *P
Figure Legend Snippet: DMF inhibits pro-inflammatory gene expression in endothelial cells by suppressing NFκB, STAT3 and cJUN signaling. (A,B,C,E) HPAECs were incubated for up to 24 h in (H) hypoxia (2.5% O 2 ) or (N) normoxia (21% O 2 ) with 10 μM DMF (D) or DMSO. Relative gene expression was measured with qPCR: (A) HO-1 , (B) Nox4, (C) pro-inflammatory genes: OSM, IL-6 and ICAM1. n = 4 independent experiments. (D) 10 μM DMF treatment reduced the phosphorylation of STAT3 and cJUN in HPAECs exposed to hypoxia for 4 h. Representative immunoblots and densitometry quantification are shown. n = 3 independent experiments. (E) NFκB activity luciferase reporter assay was performed in HPAECs in presence of 10 ng/ml LPS (L) and/or 30 μM DMF ( D ), control cells treated with DMSO ( C ). n = 3 independent experiments. Data shown as mean ± SD. *P

Techniques Used: Expressing, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Luciferase, Reporter Assay

DMF inhibits vascular muscularization in hypoxic, hypoxic/SU5416 mice and suppresses hypoxia-induced gene expression in human PASMCs. (A,B) Intra-acinar vessels ranging from 20 to 70 μm in size were counted and categorized into non muscularized, partially muscularized, or fully muscularized. n = 4 mice per group. (C) Representative pictures of immunostaining of mouse lung sections with anti-vWF and anti-αSMA antibodies. (D) Smooth muscle cells were incubated in hypoxia ( H ) (1% O 2 ) or normoxia (N) for 24 h with 50 μM DMF or DMSO. Relative gene expression was measured with qPCR. n = 1 donor for healthy control (HC) and n = 2 donors for pulmonary hypertension (PH) cell lines. Two independent experiments were performed, each in triplicate. *P versus normoxia(N); † P versus hypoxia(H). Data shown as mean ± SD. *P
Figure Legend Snippet: DMF inhibits vascular muscularization in hypoxic, hypoxic/SU5416 mice and suppresses hypoxia-induced gene expression in human PASMCs. (A,B) Intra-acinar vessels ranging from 20 to 70 μm in size were counted and categorized into non muscularized, partially muscularized, or fully muscularized. n = 4 mice per group. (C) Representative pictures of immunostaining of mouse lung sections with anti-vWF and anti-αSMA antibodies. (D) Smooth muscle cells were incubated in hypoxia ( H ) (1% O 2 ) or normoxia (N) for 24 h with 50 μM DMF or DMSO. Relative gene expression was measured with qPCR. n = 1 donor for healthy control (HC) and n = 2 donors for pulmonary hypertension (PH) cell lines. Two independent experiments were performed, each in triplicate. *P versus normoxia(N); † P versus hypoxia(H). Data shown as mean ± SD. *P

Techniques Used: Mouse Assay, Expressing, Immunostaining, Incubation, Real-time Polymerase Chain Reaction

TGFβ –induced lung fibroblast activation is inhibited by DMF. ( A,B ) Human primary lung fibroblasts from healthy controls (HC) and scleroderma patients (SSc) were treated for ( A ) 24 h with 2.5 ng/ml TGFβ(T) and 90 μM DMF(D), control cells ( C ) were treated with vehicle (DMSO), or ( B ) pre-treated for 1 h with DMF( D ) and then exposed to TGFβ(T) for 30 min. (A) Relative gene expression was measured with qPCR. CTGF and COLI proteins were measured by densitometry of immunoblots from the whole cell lysates. (B) Representative immunoblots and densitometry measurements of pSMAD2 and pSMAD1/5 levels. n = 3 cell lines per group. Data shown as mean ± SD. *P versus control ( C ); † P versus TGFβ(T). *P
Figure Legend Snippet: TGFβ –induced lung fibroblast activation is inhibited by DMF. ( A,B ) Human primary lung fibroblasts from healthy controls (HC) and scleroderma patients (SSc) were treated for ( A ) 24 h with 2.5 ng/ml TGFβ(T) and 90 μM DMF(D), control cells ( C ) were treated with vehicle (DMSO), or ( B ) pre-treated for 1 h with DMF( D ) and then exposed to TGFβ(T) for 30 min. (A) Relative gene expression was measured with qPCR. CTGF and COLI proteins were measured by densitometry of immunoblots from the whole cell lysates. (B) Representative immunoblots and densitometry measurements of pSMAD2 and pSMAD1/5 levels. n = 3 cell lines per group. Data shown as mean ± SD. *P versus control ( C ); † P versus TGFβ(T). *P

Techniques Used: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot

33) Product Images from "FiloQuant reveals increased filopodia density during breast cancer progression"

Article Title: FiloQuant reveals increased filopodia density during breast cancer progression

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201704045

Filopodia can be detected and quantified in sprouting endothelia in the developing zebrafish embryo using intravital imaging and FiloQuant. (A) Simplified cartoon of a zebrafish embryo. Insets represent magnified regions of interest (ROIs). In particular, endothelial tip cells in the sprouting intersegmental arteries are highlighted to indicate the region being imaged in 29 h postfertilization (hpf) transgenic embryos expressing GFP in the endothelium (Tg(fli1:EGFP)y1; roy −/− ; mitfa −/− ). (B and C) Transgenic zebrafish embryos (Tg(fli1:EGFP)y1; roy −/− ; mitfa −/− ) were treated with 1% DMSO or150 ng/ml latrunculin B from 25 hpf to 29 hpf. The embryos were then anesthetized and mounted in low-melting point agarose on glass-bottom dishes. Z stack images of the sprouting segmental arteries were obtained live using an SDC microscope (long working distance 63× water objective, CMOS camera). Representative maximal z projection and the z projection used for FiloQuant analyses are shown (B). In addition, ROIs (yellow square) used for FiloQuant analyses, magnified area (red squares), and filopodia detected using FiloQuant (magenta) are displayed. Bars: (main) 20 µm; (inset) 5 µm. Quantification of filopodia density and filopodia length using the semiautomated version of FiloQuant are displayed as Tukey box plots; C; DMSO, 15 embryos imaged; latrunculin B, 17 embryos imaged; filopodia density: DMSO, 60 ROIs analyzed; latrunculin B, 71 ROIs analyzed; filopodia length: DMSO, 2,232 filopodia measured; latrunculin B, 138 filopodia measured; ***, P
Figure Legend Snippet: Filopodia can be detected and quantified in sprouting endothelia in the developing zebrafish embryo using intravital imaging and FiloQuant. (A) Simplified cartoon of a zebrafish embryo. Insets represent magnified regions of interest (ROIs). In particular, endothelial tip cells in the sprouting intersegmental arteries are highlighted to indicate the region being imaged in 29 h postfertilization (hpf) transgenic embryos expressing GFP in the endothelium (Tg(fli1:EGFP)y1; roy −/− ; mitfa −/− ). (B and C) Transgenic zebrafish embryos (Tg(fli1:EGFP)y1; roy −/− ; mitfa −/− ) were treated with 1% DMSO or150 ng/ml latrunculin B from 25 hpf to 29 hpf. The embryos were then anesthetized and mounted in low-melting point agarose on glass-bottom dishes. Z stack images of the sprouting segmental arteries were obtained live using an SDC microscope (long working distance 63× water objective, CMOS camera). Representative maximal z projection and the z projection used for FiloQuant analyses are shown (B). In addition, ROIs (yellow square) used for FiloQuant analyses, magnified area (red squares), and filopodia detected using FiloQuant (magenta) are displayed. Bars: (main) 20 µm; (inset) 5 µm. Quantification of filopodia density and filopodia length using the semiautomated version of FiloQuant are displayed as Tukey box plots; C; DMSO, 15 embryos imaged; latrunculin B, 17 embryos imaged; filopodia density: DMSO, 60 ROIs analyzed; latrunculin B, 71 ROIs analyzed; filopodia length: DMSO, 2,232 filopodia measured; latrunculin B, 138 filopodia measured; ***, P

Techniques Used: Imaging, Transgenic Assay, Expressing, Microscopy

34) Product Images from "Combined BET bromodomain and CDK2 inhibition in MYC-driven medulloblastoma"

Article Title: Combined BET bromodomain and CDK2 inhibition in MYC-driven medulloblastoma

Journal: Oncogene

doi: 10.1038/s41388-018-0135-1

JQ1 and Milciclib treatment synergistically target MYC-driven medulloblastoma cells. a Survival of GTML2 cells after treatment with the indicated concentration of JQ1 and Milciclib. Combination index (CI) was calculated using the CompuSyn software for drug combinations and for general dose effect analysis, ComboSyn, Inc. Paramus, NJ, 2007. [ www.combosyn.com ] . *Indicate CI: 0.5–0.8 moderate synergy; **CI: 0.1–0.5 strong synergy. b Dose response (survival) of JQ1, Milciclib, or Palbociclib treatment together with single-dose irradiation in GTML2, response compared to non-irradiated DMSO control, analyzed 5 days postirradiation and/or posttreatment. c Long-term treatment of GTML2 cells with JQ1, Milciclib, or Palbociclib alone or in combination. d Long-term treatment of MB002 cells with JQ1 (500 nM), Milciclib (500 nM), or Palbociclib (2 μM) alone or in combination. c , d Cells treated one time (OT) or every other day for 10 days and monitored for tumor cell recovery until 16 days posttreatment start
Figure Legend Snippet: JQ1 and Milciclib treatment synergistically target MYC-driven medulloblastoma cells. a Survival of GTML2 cells after treatment with the indicated concentration of JQ1 and Milciclib. Combination index (CI) was calculated using the CompuSyn software for drug combinations and for general dose effect analysis, ComboSyn, Inc. Paramus, NJ, 2007. [ www.combosyn.com ] . *Indicate CI: 0.5–0.8 moderate synergy; **CI: 0.1–0.5 strong synergy. b Dose response (survival) of JQ1, Milciclib, or Palbociclib treatment together with single-dose irradiation in GTML2, response compared to non-irradiated DMSO control, analyzed 5 days postirradiation and/or posttreatment. c Long-term treatment of GTML2 cells with JQ1, Milciclib, or Palbociclib alone or in combination. d Long-term treatment of MB002 cells with JQ1 (500 nM), Milciclib (500 nM), or Palbociclib (2 μM) alone or in combination. c , d Cells treated one time (OT) or every other day for 10 days and monitored for tumor cell recovery until 16 days posttreatment start

Techniques Used: Concentration Assay, Software, Irradiation

Combined BET and CDK2 inhibition targets the MYC transcriptional output in MYC-amplified human medulloblastoma. a – d GSEA result for the oncogenic signature-related gene set (MYC_UP.V1_UP) most strongly downregulated in cerebellar cells ( a ) and MB002-JQ1+Milciclib-6h ( d ) as compared to MB002-DMSO-6h; the GSEAs for the same gene set are shown comparing MB002-DMSO-6h with MB002-JQ1-6h ( b ) and MB002-Milciclib-6h ( c ). Enrichments were considered significant if FDR
Figure Legend Snippet: Combined BET and CDK2 inhibition targets the MYC transcriptional output in MYC-amplified human medulloblastoma. a – d GSEA result for the oncogenic signature-related gene set (MYC_UP.V1_UP) most strongly downregulated in cerebellar cells ( a ) and MB002-JQ1+Milciclib-6h ( d ) as compared to MB002-DMSO-6h; the GSEAs for the same gene set are shown comparing MB002-DMSO-6h with MB002-JQ1-6h ( b ) and MB002-Milciclib-6h ( c ). Enrichments were considered significant if FDR

Techniques Used: Inhibition, Amplification

Comparing the transcriptional output from MYCN suppression with BET and CDK inhibition in order to identify essential gene targets. Characterizing the transcriptional changes induced after 6 h treatments. a – d GSEA results for the top MYCN target gene-related gene set (NMYC_01) downregulated in GTML-DOX-6h as compared to GTML–DMSO-6h ( a ); the GSEAs for the same gene set are shown comparing GTML-DMSO-6h with GTML-JQ1-6h ( b ), GTML-Milciclib-6h ( c ), and GTML-JQ1+Milciclib-6h ( d ). Enrichments were considered significant if FDR
Figure Legend Snippet: Comparing the transcriptional output from MYCN suppression with BET and CDK inhibition in order to identify essential gene targets. Characterizing the transcriptional changes induced after 6 h treatments. a – d GSEA results for the top MYCN target gene-related gene set (NMYC_01) downregulated in GTML-DOX-6h as compared to GTML–DMSO-6h ( a ); the GSEAs for the same gene set are shown comparing GTML-DMSO-6h with GTML-JQ1-6h ( b ), GTML-Milciclib-6h ( c ), and GTML-JQ1+Milciclib-6h ( d ). Enrichments were considered significant if FDR

Techniques Used: Inhibition

35) Product Images from "Ferroptosis: A Novel Anti-tumor Action for Cisplatin"

Article Title: Ferroptosis: A Novel Anti-tumor Action for Cisplatin

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

doi: 10.4143/crt.2016.572

Cisplatin led to intracellular glutathione (GSH) depletion and glutathione peroxidases (GPXs) inactivation in A549 and HCT116 cells. (A) GSH level analysis of A549 and HCT116 cells. (B) GPXs activity analysis of A549 and HCT116 cells. Cells were treated with cisplatin (5 μg/mL) or erastin (10 μmol/L, as a positive control) for 48 hours, respectively. DMSO, dimethyl sulfoxide. Standard error represents three independent experiments (n=3). * p
Figure Legend Snippet: Cisplatin led to intracellular glutathione (GSH) depletion and glutathione peroxidases (GPXs) inactivation in A549 and HCT116 cells. (A) GSH level analysis of A549 and HCT116 cells. (B) GPXs activity analysis of A549 and HCT116 cells. Cells were treated with cisplatin (5 μg/mL) or erastin (10 μmol/L, as a positive control) for 48 hours, respectively. DMSO, dimethyl sulfoxide. Standard error represents three independent experiments (n=3). * p

Techniques Used: Activity Assay, Positive Control

Cisplatin led to intracellular glutathione (GSH) depletion and glutathione peroxidases (GPXs) inactivation in A549 and HCT116 cells. (A) GSH level analysis of A549 and HCT116 cells. (B) GPXs activity analysis of A549 and HCT116 cells. Cells were treated with cisplatin (5 μg/mL) or erastin (10 μmol/L, as a positive control) for 48 hours, respectively. DMSO, dimethyl sulfoxide. Standard error represents three independent experiments (n=3). * p
Figure Legend Snippet: Cisplatin led to intracellular glutathione (GSH) depletion and glutathione peroxidases (GPXs) inactivation in A549 and HCT116 cells. (A) GSH level analysis of A549 and HCT116 cells. (B) GPXs activity analysis of A549 and HCT116 cells. Cells were treated with cisplatin (5 μg/mL) or erastin (10 μmol/L, as a positive control) for 48 hours, respectively. DMSO, dimethyl sulfoxide. Standard error represents three independent experiments (n=3). * p

Techniques Used: Activity Assay, Positive Control

36) Product Images from "Dexmedetomidine attenuates persistent postsurgical pain by upregulating K+–Cl− cotransporter-2 in the spinal dorsal horn in rats"

Article Title: Dexmedetomidine attenuates persistent postsurgical pain by upregulating K+–Cl− cotransporter-2 in the spinal dorsal horn in rats

Journal: Journal of Pain Research

doi: 10.2147/JPR.S158737

KCC2 inhibitor VU0240551 intrathecally administered at 15 min presurgery and at PODs 1–3 reduced the effect of DEX on SMIR rats. Notes: ( A ) The influence of VU0240551 on the analgesic effect of DEX in SMIR-induced PPSP rats. ( B and C ) KCC2 expression in the SMIR + DEX + VU0240551 group was significantly inhibited compared with the SMIR + DEX + DMSO group. ( D and E ) KCC2 expression in the ipsilateral spinal dorsal horn (a) and KCC2 expression in the superficial dorsal horn layers (b). Magnification: a =50× and b =400×. Scale bar: 20 mm. * P
Figure Legend Snippet: KCC2 inhibitor VU0240551 intrathecally administered at 15 min presurgery and at PODs 1–3 reduced the effect of DEX on SMIR rats. Notes: ( A ) The influence of VU0240551 on the analgesic effect of DEX in SMIR-induced PPSP rats. ( B and C ) KCC2 expression in the SMIR + DEX + VU0240551 group was significantly inhibited compared with the SMIR + DEX + DMSO group. ( D and E ) KCC2 expression in the ipsilateral spinal dorsal horn (a) and KCC2 expression in the superficial dorsal horn layers (b). Magnification: a =50× and b =400×. Scale bar: 20 mm. * P

Techniques Used: Expressing

37) Product Images from "Salmonella Typhimurium Invalidated for the Three Currently Known Invasion Factors Keeps Its Ability to Invade Several Cell Models"

Article Title: Salmonella Typhimurium Invalidated for the Three Currently Known Invasion Factors Keeps Its Ability to Invade Several Cell Models

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2018.00273

Both clathrin- and non-clathrin-dependent pathways are used by the S . Typhimurium 14028 strain and its triple mutant during interaction with AML-12 cells. Invasion abilities of the S . Typhimurium 14028 and STM-3Δ mutant were compared using gentamicin protection assays performed on AML-12 cells in the presence of different drugs. AML-12 cells were pre-treated with inhibitors at the indicated concentrations or with the appropriate solvent used for the dilution of the drugs for 30 min with amiloride (culture medium), filipin (ethanol) and genistein (DMSO), for 1 h with CPZ (culture medium) and MDC (DMSO). Bacteria (MOI = 10) were deposited on cells for 1.5 h followed by the addition of gentamicin (100 μg/mL) for 1.5 h. The dilution-effect of each drug was performed and viability of the bacteria was checked in the presence of all dilutions of the drugs used. The numbers of internalized bacteria were determined and expressed relative to values obtained for cells treated with control-diluted reagent arbitrarily set at 100%. Data are the mean ± SEM in duplicate and repeated at least twice for each strain. Data were analyzed using asymptotic two-sample Fisher-Pitman permutation tests. Significance was *** p
Figure Legend Snippet: Both clathrin- and non-clathrin-dependent pathways are used by the S . Typhimurium 14028 strain and its triple mutant during interaction with AML-12 cells. Invasion abilities of the S . Typhimurium 14028 and STM-3Δ mutant were compared using gentamicin protection assays performed on AML-12 cells in the presence of different drugs. AML-12 cells were pre-treated with inhibitors at the indicated concentrations or with the appropriate solvent used for the dilution of the drugs for 30 min with amiloride (culture medium), filipin (ethanol) and genistein (DMSO), for 1 h with CPZ (culture medium) and MDC (DMSO). Bacteria (MOI = 10) were deposited on cells for 1.5 h followed by the addition of gentamicin (100 μg/mL) for 1.5 h. The dilution-effect of each drug was performed and viability of the bacteria was checked in the presence of all dilutions of the drugs used. The numbers of internalized bacteria were determined and expressed relative to values obtained for cells treated with control-diluted reagent arbitrarily set at 100%. Data are the mean ± SEM in duplicate and repeated at least twice for each strain. Data were analyzed using asymptotic two-sample Fisher-Pitman permutation tests. Significance was *** p

Techniques Used: Mutagenesis

38) Product Images from "Intrathecal cGMP elicits pressor responses and maintains mean blood pressure during haemorrhage in anaesthetized rats"

Article Title: Intrathecal cGMP elicits pressor responses and maintains mean blood pressure during haemorrhage in anaesthetized rats

Journal:

doi: 10.1113/jphysiol.2006.125690

The effect of intrathecal ODQ (100 m m , 10 μl) and vehicle (DMSO, 10 μl intrathecal) injected prior to haemorrhage (1.5% body weight) on MAP (mmHg ± s.e.m. ) measured throughout the removal of blood
Figure Legend Snippet: The effect of intrathecal ODQ (100 m m , 10 μl) and vehicle (DMSO, 10 μl intrathecal) injected prior to haemorrhage (1.5% body weight) on MAP (mmHg ± s.e.m. ) measured throughout the removal of blood

Techniques Used: Injection

39) Product Images from "The cyl Genes Reveal the Biosynthetic and Evolutionary Origins of the Group B Streptococcus Hemolytic Lipid, Granadaene"

Article Title: The cyl Genes Reveal the Biosynthetic and Evolutionary Origins of the Group B Streptococcus Hemolytic Lipid, Granadaene

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2019.03123

Pigment extracted and purified from L. lactis p cylX-K is identical to Granadaene extracted from WT GBS. (A) High resolution mass spectrometry was performed on pigment extracted from L. lactis p cylX-K and demonstrated an M + H ion at m/z 677.3795, which is associated with an ion formula of C 39 H 53 N 2 O 8 (expected mass of 677.3796). (B) 1 H NMR analyses on L. lactis p cylX-K pigment revealed a signal corresponding to a polyene structure (7.20–5.5 ppm), as well as signals corresponding to an ornithine (4.32, 2.77, 1.80, 1.59, and 1.64 ppm) and a rhamnose (4.63, 3.52, 3.43, 3.39, 3.16, and 1.09 ppm). (C) HPLC-purified pigment from L. lactis p cylX-K or WT GBS in DMSO + trifluoroacetic acid + starch (DTS) was added to human erythrocytes in twofold serial dilutions starting from 2.5 to 0.0024 μM for 1 h. As controls, equivalent amounts of extracts from L. lactis pEmpty and from non-pigmented/hemolytic GBSΔ cylE were tested. Mean and standard error from three independent experiments performed in triplicate are shown. The EC 50 for L. lactis p cylX-K pigment (0.128 μM, 95% CI: 0.108, 0.153) is no different than that of WT GBS pigment/Granadaene (0.127 μM, 95% CI: 0.100, 0.162). (D) Primary human neutrophils were incubated with 0.5 μM purified pigment from L. lactis p cylX-K for 4 h at 37°C. As a control, neutrophils were treated with an equivalent amount of L. lactis pEmpty extract. Neutrophil death was measured by LDH release into cell supernatants relative to Triton X-100 and PBS-only controls. Mean and standard error from three independent experiments performed in triplicate are shown. An unpaired t -test was performed, and p = 0.0013. ∗∗ indicates that p
Figure Legend Snippet: Pigment extracted and purified from L. lactis p cylX-K is identical to Granadaene extracted from WT GBS. (A) High resolution mass spectrometry was performed on pigment extracted from L. lactis p cylX-K and demonstrated an M + H ion at m/z 677.3795, which is associated with an ion formula of C 39 H 53 N 2 O 8 (expected mass of 677.3796). (B) 1 H NMR analyses on L. lactis p cylX-K pigment revealed a signal corresponding to a polyene structure (7.20–5.5 ppm), as well as signals corresponding to an ornithine (4.32, 2.77, 1.80, 1.59, and 1.64 ppm) and a rhamnose (4.63, 3.52, 3.43, 3.39, 3.16, and 1.09 ppm). (C) HPLC-purified pigment from L. lactis p cylX-K or WT GBS in DMSO + trifluoroacetic acid + starch (DTS) was added to human erythrocytes in twofold serial dilutions starting from 2.5 to 0.0024 μM for 1 h. As controls, equivalent amounts of extracts from L. lactis pEmpty and from non-pigmented/hemolytic GBSΔ cylE were tested. Mean and standard error from three independent experiments performed in triplicate are shown. The EC 50 for L. lactis p cylX-K pigment (0.128 μM, 95% CI: 0.108, 0.153) is no different than that of WT GBS pigment/Granadaene (0.127 μM, 95% CI: 0.100, 0.162). (D) Primary human neutrophils were incubated with 0.5 μM purified pigment from L. lactis p cylX-K for 4 h at 37°C. As a control, neutrophils were treated with an equivalent amount of L. lactis pEmpty extract. Neutrophil death was measured by LDH release into cell supernatants relative to Triton X-100 and PBS-only controls. Mean and standard error from three independent experiments performed in triplicate are shown. An unpaired t -test was performed, and p = 0.0013. ∗∗ indicates that p

Techniques Used: Purification, Mass Spectrometry, Nuclear Magnetic Resonance, High Performance Liquid Chromatography, Incubation

40) Product Images from "Nuclear Xenobiotic Receptor Pregnane X Receptor Locks Corepressor Silencing Mediator for Retinoid and Thyroid Hormone Receptors (SMRT) onto theCYP24A1 Promoter to Attenuate Vitamin D3 Activation S⃞"

Article Title: Nuclear Xenobiotic Receptor Pregnane X Receptor Locks Corepressor Silencing Mediator for Retinoid and Thyroid Hormone Receptors (SMRT) onto theCYP24A1 Promoter to Attenuate Vitamin D3 Activation S⃞

Journal: Molecular Pharmacology

doi: 10.1124/mol.108.051904

CAR repression of vitamin D 3 activation via SMRT. A, Huh7 cells were transfected with or without pcDNA3.1/mVDR and pCR3/hCAR. After 24 h, cells were treated with DMSO, VD 3 (10 nM), CITCO (250 nM), and/or PK11195 (10 μM) for an additional 24 h before being harvested for qRT-PCR. Relative CYP24A1 mRNA levels were expressed by taking the level of the DMSO-treated cells transfected with empty plasmid as 1. Bars, mean ± S.D. B, pGL3/CYP24A1-3 kb was transfected with or without pcDNA3.1/mVDR and pCR3/hCAR into Huh7 cells. After 24 h, cells were treated with DMSO, VD 3 (10 nM), CITCO (25, 75, and 250 nM), and PK11195 (1, 3, and 10 μM) for an additional 24 h before being harvested for luciferase assays. Relative luciferase activities were expressed relative to the activity of the DMSO-treated cells transfected with empty plasmid as 1. Bars, mean ± S.D. C, Huh7 cells were transfected with pcDNA3.1/mVDR and pcDNA3.1/mVDR plus pCR3/hCAR. After 24 h, cells were treated with DMSO, VD 3 (10 nM), CITCO (250 nM), and/or PK11195 (10 μM) for an additional 2 h before being harvested for ChIP assays. Cross-linked chromatin-protein complexes were immunoprecipitated with anti-VDR (αVDR), anti-CAR (αCAR), anti-SMRT (αSMRT), or normal mouse IgG (αIgG). By scanning three independent assays, the relative intensities of the bands were calculated based the inputs: in the VDR binding, 3.4 ± 0.8, 3.3 ± 1.0, 3.4 ± 1.3, and 3.4 ± 1.2% for VDR plus VD 3 , VDR plus CAR and VD 3 , VDR plus CAR, VD 3 , and CITCO, and VDR plus CAR, VD 3 , and PK11195, respectively; in the CAR binding, 0.1 ± 0.3, 1.0 ± 0.3, 2.9 ± 0.5, and 3.7 ± 1.2% for VDR plus VD 3 , VDR plus CAR and VD 3 , VDR plus CAR, VD 3 , and CITCO, and VDR plus CAR, VD 3 , and PK11195, respectively; in the SMRT binding, 0.0 ± 0.0, 0.8 ± 0.1, 2.0 ± 0.5, and 2.1 ± 0.3% for VDR plus VD 3 , VDR plus CAR and VD 3 , VDR plus CAR, VD 3 , and CITCO, and VDR plus CAR, VD 3 , and PK11195, respectively.
Figure Legend Snippet: CAR repression of vitamin D 3 activation via SMRT. A, Huh7 cells were transfected with or without pcDNA3.1/mVDR and pCR3/hCAR. After 24 h, cells were treated with DMSO, VD 3 (10 nM), CITCO (250 nM), and/or PK11195 (10 μM) for an additional 24 h before being harvested for qRT-PCR. Relative CYP24A1 mRNA levels were expressed by taking the level of the DMSO-treated cells transfected with empty plasmid as 1. Bars, mean ± S.D. B, pGL3/CYP24A1-3 kb was transfected with or without pcDNA3.1/mVDR and pCR3/hCAR into Huh7 cells. After 24 h, cells were treated with DMSO, VD 3 (10 nM), CITCO (25, 75, and 250 nM), and PK11195 (1, 3, and 10 μM) for an additional 24 h before being harvested for luciferase assays. Relative luciferase activities were expressed relative to the activity of the DMSO-treated cells transfected with empty plasmid as 1. Bars, mean ± S.D. C, Huh7 cells were transfected with pcDNA3.1/mVDR and pcDNA3.1/mVDR plus pCR3/hCAR. After 24 h, cells were treated with DMSO, VD 3 (10 nM), CITCO (250 nM), and/or PK11195 (10 μM) for an additional 2 h before being harvested for ChIP assays. Cross-linked chromatin-protein complexes were immunoprecipitated with anti-VDR (αVDR), anti-CAR (αCAR), anti-SMRT (αSMRT), or normal mouse IgG (αIgG). By scanning three independent assays, the relative intensities of the bands were calculated based the inputs: in the VDR binding, 3.4 ± 0.8, 3.3 ± 1.0, 3.4 ± 1.3, and 3.4 ± 1.2% for VDR plus VD 3 , VDR plus CAR and VD 3 , VDR plus CAR, VD 3 , and CITCO, and VDR plus CAR, VD 3 , and PK11195, respectively; in the CAR binding, 0.1 ± 0.3, 1.0 ± 0.3, 2.9 ± 0.5, and 3.7 ± 1.2% for VDR plus VD 3 , VDR plus CAR and VD 3 , VDR plus CAR, VD 3 , and CITCO, and VDR plus CAR, VD 3 , and PK11195, respectively; in the SMRT binding, 0.0 ± 0.0, 0.8 ± 0.1, 2.0 ± 0.5, and 2.1 ± 0.3% for VDR plus VD 3 , VDR plus CAR and VD 3 , VDR plus CAR, VD 3 , and CITCO, and VDR plus CAR, VD 3 , and PK11195, respectively.

Techniques Used: Activation Assay, Transfection, Quantitative RT-PCR, Plasmid Preparation, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Binding Assay

Related Articles

Transfection:

Article Title: West Nile Virus Infection Causes Endocytosis of a Specific Subset of Tight Junction Membrane Proteins
Article Snippet: .. Reagents and antibodies The following reagents were purchased from the respective suppliers: protein A-sepharose, protein G-sepharose from GE Healthcare Bio-Sciences AB (Piscataway, NJ); general lab chemicals, DMSO, Nocodazole, Dynasore, Paclitaxel and Latrunculin B from Sigma-Aldrich (St. Louis, MO); Complete™ EDTA-free protease inhibitor cocktail, FuGENE 6 transfection regent from Roche Diagnostics (Laval, Quebec); ProLong® Gold Antifade reagent with 4′-6-diamidino-2-phenylindole (DAPI), media and fetal bovine serum (FBS) for cell culture from Invitrogen; Pierce BCA Protein Assay Kit from Thermo Scientific (Rockford, IL); High Capacity RNA-to-cDNA Master Mix and Fast SYBR Green Master Mix from Applied Biosystems (Streetsville, ON); qScript One-Step SYBR Green qRT-PCR Kit from Quanta Biosciences (Gaithersville, MD); HEK-293T, CACO-2 and MDCK cells from the American Type Culture Collection (Manassas, VA). .. Human umbilical vein endothelial cells (HUVECs) isolated from individual umbilical cords as described were obtained from Dr. Denise Hemmings (Obstetrics & Gynaecology, University of Alberta).

Homogenization:

Article Title: Evolutionarily Repurposed Networks Reveal the Well-Known Antifungal Drug Thiabendazole to Be a Novel Vascular Disrupting AgentEvolution-Based Drug Discovery: Antifungal Also Disrupts Blood Vessel Formation
Article Snippet: .. Mass Spectrometry HUVECs were treated with 1% DMSO or 1% DMSO, 250 µM TBZ for 24 h, and lysed by Dounce homogenization in low salt buffer (10 mM Tris-HCl, pH 8.8, 10 mM KCl, 1.5 mM MgCl2 ) with 0.5 mM DTT and protease inhibitor mixture (Calbiochem). ..

Protease Inhibitor:

Article Title: Evolutionarily Repurposed Networks Reveal the Well-Known Antifungal Drug Thiabendazole to Be a Novel Vascular Disrupting AgentEvolution-Based Drug Discovery: Antifungal Also Disrupts Blood Vessel Formation
Article Snippet: .. Mass Spectrometry HUVECs were treated with 1% DMSO or 1% DMSO, 250 µM TBZ for 24 h, and lysed by Dounce homogenization in low salt buffer (10 mM Tris-HCl, pH 8.8, 10 mM KCl, 1.5 mM MgCl2 ) with 0.5 mM DTT and protease inhibitor mixture (Calbiochem). ..

Article Title: West Nile Virus Infection Causes Endocytosis of a Specific Subset of Tight Junction Membrane Proteins
Article Snippet: .. Reagents and antibodies The following reagents were purchased from the respective suppliers: protein A-sepharose, protein G-sepharose from GE Healthcare Bio-Sciences AB (Piscataway, NJ); general lab chemicals, DMSO, Nocodazole, Dynasore, Paclitaxel and Latrunculin B from Sigma-Aldrich (St. Louis, MO); Complete™ EDTA-free protease inhibitor cocktail, FuGENE 6 transfection regent from Roche Diagnostics (Laval, Quebec); ProLong® Gold Antifade reagent with 4′-6-diamidino-2-phenylindole (DAPI), media and fetal bovine serum (FBS) for cell culture from Invitrogen; Pierce BCA Protein Assay Kit from Thermo Scientific (Rockford, IL); High Capacity RNA-to-cDNA Master Mix and Fast SYBR Green Master Mix from Applied Biosystems (Streetsville, ON); qScript One-Step SYBR Green qRT-PCR Kit from Quanta Biosciences (Gaithersville, MD); HEK-293T, CACO-2 and MDCK cells from the American Type Culture Collection (Manassas, VA). .. Human umbilical vein endothelial cells (HUVECs) isolated from individual umbilical cords as described were obtained from Dr. Denise Hemmings (Obstetrics & Gynaecology, University of Alberta).

Cell Culture:

Article Title: West Nile Virus Infection Causes Endocytosis of a Specific Subset of Tight Junction Membrane Proteins
Article Snippet: .. Reagents and antibodies The following reagents were purchased from the respective suppliers: protein A-sepharose, protein G-sepharose from GE Healthcare Bio-Sciences AB (Piscataway, NJ); general lab chemicals, DMSO, Nocodazole, Dynasore, Paclitaxel and Latrunculin B from Sigma-Aldrich (St. Louis, MO); Complete™ EDTA-free protease inhibitor cocktail, FuGENE 6 transfection regent from Roche Diagnostics (Laval, Quebec); ProLong® Gold Antifade reagent with 4′-6-diamidino-2-phenylindole (DAPI), media and fetal bovine serum (FBS) for cell culture from Invitrogen; Pierce BCA Protein Assay Kit from Thermo Scientific (Rockford, IL); High Capacity RNA-to-cDNA Master Mix and Fast SYBR Green Master Mix from Applied Biosystems (Streetsville, ON); qScript One-Step SYBR Green qRT-PCR Kit from Quanta Biosciences (Gaithersville, MD); HEK-293T, CACO-2 and MDCK cells from the American Type Culture Collection (Manassas, VA). .. Human umbilical vein endothelial cells (HUVECs) isolated from individual umbilical cords as described were obtained from Dr. Denise Hemmings (Obstetrics & Gynaecology, University of Alberta).

SYBR Green Assay:

Article Title: West Nile Virus Infection Causes Endocytosis of a Specific Subset of Tight Junction Membrane Proteins
Article Snippet: .. Reagents and antibodies The following reagents were purchased from the respective suppliers: protein A-sepharose, protein G-sepharose from GE Healthcare Bio-Sciences AB (Piscataway, NJ); general lab chemicals, DMSO, Nocodazole, Dynasore, Paclitaxel and Latrunculin B from Sigma-Aldrich (St. Louis, MO); Complete™ EDTA-free protease inhibitor cocktail, FuGENE 6 transfection regent from Roche Diagnostics (Laval, Quebec); ProLong® Gold Antifade reagent with 4′-6-diamidino-2-phenylindole (DAPI), media and fetal bovine serum (FBS) for cell culture from Invitrogen; Pierce BCA Protein Assay Kit from Thermo Scientific (Rockford, IL); High Capacity RNA-to-cDNA Master Mix and Fast SYBR Green Master Mix from Applied Biosystems (Streetsville, ON); qScript One-Step SYBR Green qRT-PCR Kit from Quanta Biosciences (Gaithersville, MD); HEK-293T, CACO-2 and MDCK cells from the American Type Culture Collection (Manassas, VA). .. Human umbilical vein endothelial cells (HUVECs) isolated from individual umbilical cords as described were obtained from Dr. Denise Hemmings (Obstetrics & Gynaecology, University of Alberta).

Concentration Assay:

Article Title: The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain
Article Snippet: .. Agents, their final concentrations and vehicles were Pi1 [proteasome inhibitor 1 (Calbiochem); used at a final concentration of 50 μM with DMSO as a vehicle], the proteasome inhibitor ALLN [N -acetyl-L -leucyl-L -leucylnorleucinal (Calbiochem); used at a final concentration of 20 μM with DMSO as a vehicle], the proteasome inhibitor cLβ-l (clasto lactacystin β-lactone; used at a final concentration of 20 μM with DMSO as a vehicle), a mixture of the cathepsin inhibitors pepstatin and leupeptin (used at final concentrations of 100 μM and 1 μM respectively with water as a vehicle) and the secretion inhibitor BFA (brefeldin A; used at a final concentration of 10 μg/ml with ethanol as a vehicle). ..

Article Title: Ubiquitination of the GTPase Rap1B by the ubiquitin ligase Smurf2 is required for the establishment of neuronal polarity
Article Snippet: .. DMSO, ALLN, clasto-Lactacystin β-Lactone, and MG-132 (Calbiochem) were directly added to neuronal cultures 16 h after plating to a final concentration of 30, 40, 1.5, and 1.5 μM, respectively ( ). .. To analyse the distribution of Smurf1 and Smurf2, the normalised immunofluorescence intensity in the growth cones was calculated as the ratio of Smurf1 or Smurf2 fluorescence intensity and cell volume as measured by the CMFDA signal.

Inhibition:

Article Title: Activation of Cdc42 by trans interactions of the cell adhesion molecules nectins through c-Src and Cdc42-GEF FRG
Article Snippet: .. For inhibition of the activity of SFKs, the cells were treated with 0.2% DMSO, 20 μM PP2 (Calbiochem-Novabiochem) containing 0.2% DMSO, or 20 μM PP3 (Calbiochem-Novabiochem) containing 0.2% DMSO in DME for 1 h before the trypsin-EDTA treatment and during the culture on the Nef-3– or IgG-coated coverslips. ..

Activity Assay:

Article Title: Activation of Cdc42 by trans interactions of the cell adhesion molecules nectins through c-Src and Cdc42-GEF FRG
Article Snippet: .. For inhibition of the activity of SFKs, the cells were treated with 0.2% DMSO, 20 μM PP2 (Calbiochem-Novabiochem) containing 0.2% DMSO, or 20 μM PP3 (Calbiochem-Novabiochem) containing 0.2% DMSO in DME for 1 h before the trypsin-EDTA treatment and during the culture on the Nef-3– or IgG-coated coverslips. ..

Quantitative RT-PCR:

Article Title: West Nile Virus Infection Causes Endocytosis of a Specific Subset of Tight Junction Membrane Proteins
Article Snippet: .. Reagents and antibodies The following reagents were purchased from the respective suppliers: protein A-sepharose, protein G-sepharose from GE Healthcare Bio-Sciences AB (Piscataway, NJ); general lab chemicals, DMSO, Nocodazole, Dynasore, Paclitaxel and Latrunculin B from Sigma-Aldrich (St. Louis, MO); Complete™ EDTA-free protease inhibitor cocktail, FuGENE 6 transfection regent from Roche Diagnostics (Laval, Quebec); ProLong® Gold Antifade reagent with 4′-6-diamidino-2-phenylindole (DAPI), media and fetal bovine serum (FBS) for cell culture from Invitrogen; Pierce BCA Protein Assay Kit from Thermo Scientific (Rockford, IL); High Capacity RNA-to-cDNA Master Mix and Fast SYBR Green Master Mix from Applied Biosystems (Streetsville, ON); qScript One-Step SYBR Green qRT-PCR Kit from Quanta Biosciences (Gaithersville, MD); HEK-293T, CACO-2 and MDCK cells from the American Type Culture Collection (Manassas, VA). .. Human umbilical vein endothelial cells (HUVECs) isolated from individual umbilical cords as described were obtained from Dr. Denise Hemmings (Obstetrics & Gynaecology, University of Alberta).

Mass Spectrometry:

Article Title: Evolutionarily Repurposed Networks Reveal the Well-Known Antifungal Drug Thiabendazole to Be a Novel Vascular Disrupting AgentEvolution-Based Drug Discovery: Antifungal Also Disrupts Blood Vessel Formation
Article Snippet: .. Mass Spectrometry HUVECs were treated with 1% DMSO or 1% DMSO, 250 µM TBZ for 24 h, and lysed by Dounce homogenization in low salt buffer (10 mM Tris-HCl, pH 8.8, 10 mM KCl, 1.5 mM MgCl2 ) with 0.5 mM DTT and protease inhibitor mixture (Calbiochem). ..

Article Title: Dynamic Chromatin Modification Sustains Epithelial-Mesenchymal Transition following Inducible Expression of Snail-1
Article Snippet: .. Chemical inhibitor studies for Snail-1 or TGF-β induction experiments were performed by treatment of cells with 50 µMTCP (Sigma-Aldrich) in DMSO, 1 µM TSA (Sigma-Aldrich) in DMSO, 5 µM 5-azacytidine (Sigma-Aldrich) in acetic acid/H2 O (1:1 v/v), 10 mM nicotinamide (Sigma-Aldrich) in H2 O, 1 µM MS-275 (Selleck Chemicals) in DMSO, 1 µM LBH589 (Selleck Chemicals) in DMSO, 1 µM PXD101 (Selleck Chemicals) in DMSO, 1 µM SAHA (Selleck Chemicals) in DMSO, 1 µM Tubastatin A (Selleck Chemicals) in DMSO, and 12.5 µM of Pargyline HCl (Sigma-Aldrich) in H2 O for 24 hr, prior to addition of 4-OHT for another 48 hr. ..

BIA-KA:

Article Title: West Nile Virus Infection Causes Endocytosis of a Specific Subset of Tight Junction Membrane Proteins
Article Snippet: .. Reagents and antibodies The following reagents were purchased from the respective suppliers: protein A-sepharose, protein G-sepharose from GE Healthcare Bio-Sciences AB (Piscataway, NJ); general lab chemicals, DMSO, Nocodazole, Dynasore, Paclitaxel and Latrunculin B from Sigma-Aldrich (St. Louis, MO); Complete™ EDTA-free protease inhibitor cocktail, FuGENE 6 transfection regent from Roche Diagnostics (Laval, Quebec); ProLong® Gold Antifade reagent with 4′-6-diamidino-2-phenylindole (DAPI), media and fetal bovine serum (FBS) for cell culture from Invitrogen; Pierce BCA Protein Assay Kit from Thermo Scientific (Rockford, IL); High Capacity RNA-to-cDNA Master Mix and Fast SYBR Green Master Mix from Applied Biosystems (Streetsville, ON); qScript One-Step SYBR Green qRT-PCR Kit from Quanta Biosciences (Gaithersville, MD); HEK-293T, CACO-2 and MDCK cells from the American Type Culture Collection (Manassas, VA). .. Human umbilical vein endothelial cells (HUVECs) isolated from individual umbilical cords as described were obtained from Dr. Denise Hemmings (Obstetrics & Gynaecology, University of Alberta).

Staining:

Article Title: A Small-molecule Inhibitor of MDMX Activates p53 and Induces Apoptosis
Article Snippet: .. MCF-7 cells treated with DMSO, Nutlin-3a or the MDMX inhibitor were permeabilized with cold 70% ethanol overnight, and stained with a solution containing 50 µg/ml propidium iodide (PI) (Sigma-Aldrich) and 20 µg/ml RNase A at 37°C for 20 min. .. The cells were then subjected to flow cytometry analysis as described previously ( ).

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    Inhibition of the proteasome disrupts neuronal differentiation. ( A–D ) Hippocampal neurons were cultured in the presence of solvent <t>(DMSO),</t> 1.5 μM clasto-Lactacystin β-Lactone (Lactacystin), 40 μM <t>ALLN,</t> or 1.5 μM MG-132 (all dissolved in DMSO) for 48 h and analysed at 3 d.i.v. (stage 3) by staining with an anti-MAP2 antibody (A; red), the Tau-1 (blue), and an anti-Rap1 (red) antibody. Lactacystin induced the formation of multiple axons (asterisks) whose growth cones were all positive for Rap1B (A, arrows). Insets show higher magnifications of the marked growth cones. Axons identified by Tau-1 immunoreactivity are marked by asterisks. The scale bar is 12 μm. (B–D) The effect of Lactacystin (L), ALLN (A), or MG-132 (M) was analysed by determining the number of axons (B) or minor neurites (C) per cell and the length of axons (E) (means±s.e.m.; * P
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    Inhibition of the proteasome disrupts neuronal differentiation. ( A–D ) Hippocampal neurons were cultured in the presence of solvent (DMSO), 1.5 μM clasto-Lactacystin β-Lactone (Lactacystin), 40 μM ALLN, or 1.5 μM MG-132 (all dissolved in DMSO) for 48 h and analysed at 3 d.i.v. (stage 3) by staining with an anti-MAP2 antibody (A; red), the Tau-1 (blue), and an anti-Rap1 (red) antibody. Lactacystin induced the formation of multiple axons (asterisks) whose growth cones were all positive for Rap1B (A, arrows). Insets show higher magnifications of the marked growth cones. Axons identified by Tau-1 immunoreactivity are marked by asterisks. The scale bar is 12 μm. (B–D) The effect of Lactacystin (L), ALLN (A), or MG-132 (M) was analysed by determining the number of axons (B) or minor neurites (C) per cell and the length of axons (E) (means±s.e.m.; * P

    Journal: The EMBO Journal

    Article Title: Ubiquitination of the GTPase Rap1B by the ubiquitin ligase Smurf2 is required for the establishment of neuronal polarity

    doi: 10.1038/sj.emboj.7601580

    Figure Lengend Snippet: Inhibition of the proteasome disrupts neuronal differentiation. ( A–D ) Hippocampal neurons were cultured in the presence of solvent (DMSO), 1.5 μM clasto-Lactacystin β-Lactone (Lactacystin), 40 μM ALLN, or 1.5 μM MG-132 (all dissolved in DMSO) for 48 h and analysed at 3 d.i.v. (stage 3) by staining with an anti-MAP2 antibody (A; red), the Tau-1 (blue), and an anti-Rap1 (red) antibody. Lactacystin induced the formation of multiple axons (asterisks) whose growth cones were all positive for Rap1B (A, arrows). Insets show higher magnifications of the marked growth cones. Axons identified by Tau-1 immunoreactivity are marked by asterisks. The scale bar is 12 μm. (B–D) The effect of Lactacystin (L), ALLN (A), or MG-132 (M) was analysed by determining the number of axons (B) or minor neurites (C) per cell and the length of axons (E) (means±s.e.m.; * P

    Article Snippet: DMSO, ALLN, clasto-Lactacystin β-Lactone, and MG-132 (Calbiochem) were directly added to neuronal cultures 16 h after plating to a final concentration of 30, 40, 1.5, and 1.5 μM, respectively ( ).

    Techniques: Inhibition, Cell Culture, Staining

    TBZ inhibits angiogenesis in vivo in Xenopus embryos. Formation of Xenopus embryo veins is disrupted, marked by expression of vascular reporter genes (A, B) erg and (C, D) aplnr , contrasting treatment with 1% DMSO control only (A, C) with 1% DMSO, 250 µM TBZ, treated at stage 31 and imaged at stages 35–36 (B, D). PCV, posterior cardinal vein; ISV, intersomitic vein; VV, vitellin veins. Similarly, TBZ disrupts vasculature imaged within a living Xenopus embryo and visualized by vascular specific GFP in kdr:GFP frogs from [19] , contrasting the vasculature of stage 46 animals treated from stage 41 with the 1% DMSO control (E) or 1% DMSO, 250 µM TBZ (F). Scale bar, 200 µm.

    Journal: PLoS Biology

    Article Title: Evolutionarily Repurposed Networks Reveal the Well-Known Antifungal Drug Thiabendazole to Be a Novel Vascular Disrupting AgentEvolution-Based Drug Discovery: Antifungal Also Disrupts Blood Vessel Formation

    doi: 10.1371/journal.pbio.1001379

    Figure Lengend Snippet: TBZ inhibits angiogenesis in vivo in Xenopus embryos. Formation of Xenopus embryo veins is disrupted, marked by expression of vascular reporter genes (A, B) erg and (C, D) aplnr , contrasting treatment with 1% DMSO control only (A, C) with 1% DMSO, 250 µM TBZ, treated at stage 31 and imaged at stages 35–36 (B, D). PCV, posterior cardinal vein; ISV, intersomitic vein; VV, vitellin veins. Similarly, TBZ disrupts vasculature imaged within a living Xenopus embryo and visualized by vascular specific GFP in kdr:GFP frogs from [19] , contrasting the vasculature of stage 46 animals treated from stage 41 with the 1% DMSO control (E) or 1% DMSO, 250 µM TBZ (F). Scale bar, 200 µm.

    Article Snippet: Mass Spectrometry HUVECs were treated with 1% DMSO or 1% DMSO, 250 µM TBZ for 24 h, and lysed by Dounce homogenization in low salt buffer (10 mM Tris-HCl, pH 8.8, 10 mM KCl, 1.5 mM MgCl2 ) with 0.5 mM DTT and protease inhibitor mixture (Calbiochem).

    Techniques: In Vivo, Expressing

    TBZ significantly disrupts tube formation in cultured human umbilical vein endothelial cells (HUVECs), an in vitro capillary model. Here, we show effects of 1% DMSO-treated control (A) versus 1% DMSO, 100 µM TBZ (B) and 1% DMSO, 250 µM TBZ (C). Scale bar, 100 µm. (D) Tube disruption is dose-dependent and comparable to that from silencing known pro-angiogenic gene HOXA9 .

    Journal: PLoS Biology

    Article Title: Evolutionarily Repurposed Networks Reveal the Well-Known Antifungal Drug Thiabendazole to Be a Novel Vascular Disrupting AgentEvolution-Based Drug Discovery: Antifungal Also Disrupts Blood Vessel Formation

    doi: 10.1371/journal.pbio.1001379

    Figure Lengend Snippet: TBZ significantly disrupts tube formation in cultured human umbilical vein endothelial cells (HUVECs), an in vitro capillary model. Here, we show effects of 1% DMSO-treated control (A) versus 1% DMSO, 100 µM TBZ (B) and 1% DMSO, 250 µM TBZ (C). Scale bar, 100 µm. (D) Tube disruption is dose-dependent and comparable to that from silencing known pro-angiogenic gene HOXA9 .

    Article Snippet: Mass Spectrometry HUVECs were treated with 1% DMSO or 1% DMSO, 250 µM TBZ for 24 h, and lysed by Dounce homogenization in low salt buffer (10 mM Tris-HCl, pH 8.8, 10 mM KCl, 1.5 mM MgCl2 ) with 0.5 mM DTT and protease inhibitor mixture (Calbiochem).

    Techniques: Cell Culture, In Vitro

    TBZ impedes migration of HUVECs in a wound scratch assay, but treatment with the Rho Kinase inhibitor Y27632 reverses TBZ's effects. (A) The effects of 1% DMSO-treated control versus 1% DMSO, 250 µM TBZ, and 1% DMSO, 250 µM TBZ, 10 µM Y27632. Scale bar, 200 µm. (B) quantifies the dose-dependent suppression of TBZ inhibition by Y27632. Error bars represent the mean ± 1 s.d. across 3 wells (1 of 3 trials). TBZ results in disorganization of actin stress fibers, as shown in (C) for 1% DMSO-treated control versus 1% DMSO, 250 µM TBZ-treated cells. Scale bar, 20 µm.

    Journal: PLoS Biology

    Article Title: Evolutionarily Repurposed Networks Reveal the Well-Known Antifungal Drug Thiabendazole to Be a Novel Vascular Disrupting AgentEvolution-Based Drug Discovery: Antifungal Also Disrupts Blood Vessel Formation

    doi: 10.1371/journal.pbio.1001379

    Figure Lengend Snippet: TBZ impedes migration of HUVECs in a wound scratch assay, but treatment with the Rho Kinase inhibitor Y27632 reverses TBZ's effects. (A) The effects of 1% DMSO-treated control versus 1% DMSO, 250 µM TBZ, and 1% DMSO, 250 µM TBZ, 10 µM Y27632. Scale bar, 200 µm. (B) quantifies the dose-dependent suppression of TBZ inhibition by Y27632. Error bars represent the mean ± 1 s.d. across 3 wells (1 of 3 trials). TBZ results in disorganization of actin stress fibers, as shown in (C) for 1% DMSO-treated control versus 1% DMSO, 250 µM TBZ-treated cells. Scale bar, 20 µm.

    Article Snippet: Mass Spectrometry HUVECs were treated with 1% DMSO or 1% DMSO, 250 µM TBZ for 24 h, and lysed by Dounce homogenization in low salt buffer (10 mM Tris-HCl, pH 8.8, 10 mM KCl, 1.5 mM MgCl2 ) with 0.5 mM DTT and protease inhibitor mixture (Calbiochem).

    Techniques: Migration, Wound Healing Assay, Inhibition

    Mef2d is necessary for the activating effect of Fgf on Myod expression at the neurula stage. (A) Embryos were injected unilaterally either with Fgf8b synthetic mRNA alone (5 pg), or with moMyod1 or with moMef2d1, fixed at the gastrula stage and submitted to in situ hybridization for Xbra , Myod or Mef2d . Co-injection of Mef2dF mRNA with moMef2d1 was able to rescue the phenotype of moMef2d1 embryos (+Mef2dF). (B) Unilateral injection of Fgf8b mRNA induced the lateral expansion of Mef2d expression domain at stage 16. (C) Embryos were injected unilaterally with 20 ng of XIMOF8 and fixed at stage 16. Co-injection with Mef2dF mRNA was able to rescue the phenotype of XIMOF8 embryos (+Mef2dF). (D) Embryos were injected at stage 11.5/12 with DMSO or 5 mM SU5402, an inhibitor of Fgf signaling, and fixed at stage 16. A first unilateral injection of Mef2dF mRNA at the two-cell stage was able to rescue the phenotype of treated embryos (+Mef2dF). Injected side (*) at the bottom except in (A), on the left. Bracket indicates the position of lateral myogenic cells. Probes are in a framed box and indicated for each panel. For complete statistical data, see supporting information, figure S2 .

    Journal: PLoS ONE

    Article Title: Mef2d Acts Upstream of Muscle Identity Genes and Couples Lateral Myogenesis to Dermomyotome Formation in Xenopus laevis

    doi: 10.1371/journal.pone.0052359

    Figure Lengend Snippet: Mef2d is necessary for the activating effect of Fgf on Myod expression at the neurula stage. (A) Embryos were injected unilaterally either with Fgf8b synthetic mRNA alone (5 pg), or with moMyod1 or with moMef2d1, fixed at the gastrula stage and submitted to in situ hybridization for Xbra , Myod or Mef2d . Co-injection of Mef2dF mRNA with moMef2d1 was able to rescue the phenotype of moMef2d1 embryos (+Mef2dF). (B) Unilateral injection of Fgf8b mRNA induced the lateral expansion of Mef2d expression domain at stage 16. (C) Embryos were injected unilaterally with 20 ng of XIMOF8 and fixed at stage 16. Co-injection with Mef2dF mRNA was able to rescue the phenotype of XIMOF8 embryos (+Mef2dF). (D) Embryos were injected at stage 11.5/12 with DMSO or 5 mM SU5402, an inhibitor of Fgf signaling, and fixed at stage 16. A first unilateral injection of Mef2dF mRNA at the two-cell stage was able to rescue the phenotype of treated embryos (+Mef2dF). Injected side (*) at the bottom except in (A), on the left. Bracket indicates the position of lateral myogenic cells. Probes are in a framed box and indicated for each panel. For complete statistical data, see supporting information, figure S2 .

    Article Snippet: 20 nanoliters of DMSO solution or a 5 mM DMSO stock of the Fgf receptor kinase inhibitor SU5402 (Calbiochem) were injected into the archenteron.

    Techniques: Expressing, Injection, In Situ Hybridization

    26S proteasomes and 19S proteasome caps are chaperones for denatured RTA ( A ) 26S proteasomes maintain the solubility of denatured RTA. Top panels: GdnHCl-denatured RTA (40 nM) was diluted in the absence (−) or presence (+) of 26S proteasomes (25 nM, equivalent to 40 nM 19S RP) and, after centrifugation, aggregated (P) and soluble (S) fractions were examined by anti-RTA immunoblot after SDS/PAGE. Bottom panels: samples were treated in the same way as above, but were incubated at 37°C for 2 h before SDS/PAGE. ( B ) The solubilizing activity of the 26S proteasome is independent of its proteolytic activity. GdnHCl-denatured RTA (40 nM) was diluted in the absence (−) or presence (+) of 40 nM 26S proteasomes pretreated with the vehicle DMSO (top panel) or the proteasome inhibitor cLβ-l (bottom panel), and aggregated (P) and soluble (S) fractions were examined by anti-RTA immunoblot after SDS/PAGE. ( C ) Efficacy of cLβ-l was confirmed in vitro by its ability to block degradation of casein (arrowhead) in the presence of mammalian 26S proteasomes. Casein (40 nM) was incubated in the presence (+) or absence (−) of 26S proteasomes that had been pretreated with vehicle DMSO or cLβ-l. ( D ) 20S proteasome cores do not maintain the solubility of denatured RTA. GdnHCl-denatured RTA (40 nM) was diluted in the absence (−) or presence of 40 nM 20S proteasomes (+ 20S) or 40 nM BSA (+ BSA) and, after centrifugation, aggregated (P) and soluble (S) fractions were examined by anti-RTA immunoblot after SDS/PAGE. ( E ) 19S proteasome RP maintain the solubility of denatured RTA. Heat (45°C)-denatured and GdnHCl-denatured RTA (40 nM) were diluted in the absence (−) or presence (+) of 40 nM 19S RP and, after centrifugation, aggregated (P) and soluble (S) fractions were examined by anti-RTA immunoblot after SDS/PAGE. ( F ) Catalytic activity can be recovered from proteasome-solubilized RTA. Native RTA (RTA), GdnHCl-denatured RTA (denRTA) and a mixture of denRTA and 19S RP were centrifuged to remove aggregates and the soluble fractions were incubated with 20 μg of yeast ribosomes for 2 h at 30°C. After cleavage of any depurinated 28S rRNA with acetic-aniline, rRNAs were extracted, electrophoresed in denaturing conditions (1.2% agarose/50% formamide), and the gel was stained with ethidium bromide for visualization. Aniline fragment, grey arrowhead; 5.8S rRNA, white arrowhead.

    Journal: Biochemical Journal

    Article Title: The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain

    doi: 10.1042/BJ20130133

    Figure Lengend Snippet: 26S proteasomes and 19S proteasome caps are chaperones for denatured RTA ( A ) 26S proteasomes maintain the solubility of denatured RTA. Top panels: GdnHCl-denatured RTA (40 nM) was diluted in the absence (−) or presence (+) of 26S proteasomes (25 nM, equivalent to 40 nM 19S RP) and, after centrifugation, aggregated (P) and soluble (S) fractions were examined by anti-RTA immunoblot after SDS/PAGE. Bottom panels: samples were treated in the same way as above, but were incubated at 37°C for 2 h before SDS/PAGE. ( B ) The solubilizing activity of the 26S proteasome is independent of its proteolytic activity. GdnHCl-denatured RTA (40 nM) was diluted in the absence (−) or presence (+) of 40 nM 26S proteasomes pretreated with the vehicle DMSO (top panel) or the proteasome inhibitor cLβ-l (bottom panel), and aggregated (P) and soluble (S) fractions were examined by anti-RTA immunoblot after SDS/PAGE. ( C ) Efficacy of cLβ-l was confirmed in vitro by its ability to block degradation of casein (arrowhead) in the presence of mammalian 26S proteasomes. Casein (40 nM) was incubated in the presence (+) or absence (−) of 26S proteasomes that had been pretreated with vehicle DMSO or cLβ-l. ( D ) 20S proteasome cores do not maintain the solubility of denatured RTA. GdnHCl-denatured RTA (40 nM) was diluted in the absence (−) or presence of 40 nM 20S proteasomes (+ 20S) or 40 nM BSA (+ BSA) and, after centrifugation, aggregated (P) and soluble (S) fractions were examined by anti-RTA immunoblot after SDS/PAGE. ( E ) 19S proteasome RP maintain the solubility of denatured RTA. Heat (45°C)-denatured and GdnHCl-denatured RTA (40 nM) were diluted in the absence (−) or presence (+) of 40 nM 19S RP and, after centrifugation, aggregated (P) and soluble (S) fractions were examined by anti-RTA immunoblot after SDS/PAGE. ( F ) Catalytic activity can be recovered from proteasome-solubilized RTA. Native RTA (RTA), GdnHCl-denatured RTA (denRTA) and a mixture of denRTA and 19S RP were centrifuged to remove aggregates and the soluble fractions were incubated with 20 μg of yeast ribosomes for 2 h at 30°C. After cleavage of any depurinated 28S rRNA with acetic-aniline, rRNAs were extracted, electrophoresed in denaturing conditions (1.2% agarose/50% formamide), and the gel was stained with ethidium bromide for visualization. Aniline fragment, grey arrowhead; 5.8S rRNA, white arrowhead.

    Article Snippet: Agents, their final concentrations and vehicles were Pi1 [proteasome inhibitor 1 (Calbiochem); used at a final concentration of 50 μM with DMSO as a vehicle], the proteasome inhibitor ALLN [N -acetyl-L -leucyl-L -leucylnorleucinal (Calbiochem); used at a final concentration of 20 μM with DMSO as a vehicle], the proteasome inhibitor cLβ-l (clasto lactacystin β-lactone; used at a final concentration of 20 μM with DMSO as a vehicle), a mixture of the cathepsin inhibitors pepstatin and leupeptin (used at final concentrations of 100 μM and 1 μM respectively with water as a vehicle) and the secretion inhibitor BFA (brefeldin A; used at a final concentration of 10 μg/ml with ethanol as a vehicle).

    Techniques: Solubility, Centrifugation, SDS Page, Incubation, Activity Assay, In Vitro, Blocking Assay, Staining

    Inhibition of proteasome proteolytic activities sensitizes HeLa cells to ricin only after long incubations ( A ) HeLa cells were treated for 1, 2, 4, 6 or 8 h with graded doses of ricin in growth medium containing 1 μM Pi1 (●) or vehicle (DMSO, ○), and their subsequent ability to synthesize proteins was determined by measuring incorporation of [ 35 S]methionine into acid-precipitable material. Typical single assays are shown. ( B ) Top panel: cells were treated as described in ( A ), sensitivities to toxin (IC 50 , toxin concentration required to reduce protein synthesis to 50% that of non-toxin treated controls) were determined, and fold protection (IC 50 Pi1-treated cells/IC 50 DMSO-treated cells) is displayed. Middle and bottom panels: cells were treated as described above, substituting the proteasome inhibitor ALLN (middle panel) or a mixture of the cathepsin inhibitors leupeptin and pepstatin (L+P, bottom panel) for Pi1, and substituting water for the vehicle for L+P treatment. Values are means±S.D. for three independent experiments. Broken line, no protective effect over that of treatment with vehicle only; n.d., not determined. ( C ) Cells were treated with a saturating dose of ricin for 4 h in the presence (+) or absence (−) of Pi1 (top panel), L+P (middle panel) or the secretion inhibitor BFA (bottom panel). Detergent-soluble extracts were separated by SDS/PAGE, and RTA (black arrowhead) and a proteolytically clipped RTA (white arrowhead) were revealed by immunoblotting. ( D ) HeLa cells were treated for 2, 4 or 6 h as described in ( A ), substituting the proteasome inhibitor cLβ-l for Pi1. Values are means±S.D. for three independent experiments. Broken line, no protective effect over that of treatment with DMSO only. ( E ) Cells were treated with a single low dose of ricin (1 ng/ml , top panel) for increasing times in the presence of cLβ-l (●) or vehicle (DMSO, ○) and in parallel with cLβ-l (●) or vehicle (DMSO, ○) in the absence of ricin (bottom panel) and their subsequent ability to synthesize proteins was determined by measuring incorporation of [ 35 S]methionine into acid-precipitable material. Typical single assays are shown. Inset: toxin trafficking times, determined as in the top panel, in the presence of DMSO (D, ▼) or cLβ-l (c, ▽). Values are means±S.D. for three independent experiments.

    Journal: Biochemical Journal

    Article Title: The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain

    doi: 10.1042/BJ20130133

    Figure Lengend Snippet: Inhibition of proteasome proteolytic activities sensitizes HeLa cells to ricin only after long incubations ( A ) HeLa cells were treated for 1, 2, 4, 6 or 8 h with graded doses of ricin in growth medium containing 1 μM Pi1 (●) or vehicle (DMSO, ○), and their subsequent ability to synthesize proteins was determined by measuring incorporation of [ 35 S]methionine into acid-precipitable material. Typical single assays are shown. ( B ) Top panel: cells were treated as described in ( A ), sensitivities to toxin (IC 50 , toxin concentration required to reduce protein synthesis to 50% that of non-toxin treated controls) were determined, and fold protection (IC 50 Pi1-treated cells/IC 50 DMSO-treated cells) is displayed. Middle and bottom panels: cells were treated as described above, substituting the proteasome inhibitor ALLN (middle panel) or a mixture of the cathepsin inhibitors leupeptin and pepstatin (L+P, bottom panel) for Pi1, and substituting water for the vehicle for L+P treatment. Values are means±S.D. for three independent experiments. Broken line, no protective effect over that of treatment with vehicle only; n.d., not determined. ( C ) Cells were treated with a saturating dose of ricin for 4 h in the presence (+) or absence (−) of Pi1 (top panel), L+P (middle panel) or the secretion inhibitor BFA (bottom panel). Detergent-soluble extracts were separated by SDS/PAGE, and RTA (black arrowhead) and a proteolytically clipped RTA (white arrowhead) were revealed by immunoblotting. ( D ) HeLa cells were treated for 2, 4 or 6 h as described in ( A ), substituting the proteasome inhibitor cLβ-l for Pi1. Values are means±S.D. for three independent experiments. Broken line, no protective effect over that of treatment with DMSO only. ( E ) Cells were treated with a single low dose of ricin (1 ng/ml , top panel) for increasing times in the presence of cLβ-l (●) or vehicle (DMSO, ○) and in parallel with cLβ-l (●) or vehicle (DMSO, ○) in the absence of ricin (bottom panel) and their subsequent ability to synthesize proteins was determined by measuring incorporation of [ 35 S]methionine into acid-precipitable material. Typical single assays are shown. Inset: toxin trafficking times, determined as in the top panel, in the presence of DMSO (D, ▼) or cLβ-l (c, ▽). Values are means±S.D. for three independent experiments.

    Article Snippet: Agents, their final concentrations and vehicles were Pi1 [proteasome inhibitor 1 (Calbiochem); used at a final concentration of 50 μM with DMSO as a vehicle], the proteasome inhibitor ALLN [N -acetyl-L -leucyl-L -leucylnorleucinal (Calbiochem); used at a final concentration of 20 μM with DMSO as a vehicle], the proteasome inhibitor cLβ-l (clasto lactacystin β-lactone; used at a final concentration of 20 μM with DMSO as a vehicle), a mixture of the cathepsin inhibitors pepstatin and leupeptin (used at final concentrations of 100 μM and 1 μM respectively with water as a vehicle) and the secretion inhibitor BFA (brefeldin A; used at a final concentration of 10 μg/ml with ethanol as a vehicle).

    Techniques: Inhibition, Concentration Assay, SDS Page