Structured Review

Millipore eipa
Cellular factors required for EV infection. ( A – D ) HeLa cells were left untreated or treated with DMSO (highest used concentration), staurosporine (Stau), genistein (Geni), wortmannin (Wort), calphostin C (CalC), rottlerin (Rott), 3-indolepropionic acid (IPA-3), Iressa (Ires), chlorpromazine (Chlo), ML-7 (all 30 min), cytochalasin D (Cyto), jasplakinolide (Jasp), or ethylisopropyl amiloride <t>(EIPA)</t> (all 15 min). MVs or EVs of IHD-J GFP ( A ), WR GFP ( C ), or WR ΔA34R GFP ( D ) were preincubated in the presence of drugs and in the case of EVs with 7D11 (MOI 2). Cells were infected and green fluorescent cells quantified 4 h p.i. by flow cytometry. Infection levels were normalized to untreated samples. ( B ) EV (IHD-J F13–GFP) and MV (IHD-J EGFP–A5) internalization in the presence of various drugs was quantified as described in Figure 3 . The geometric mean of green fluorescence intensity of 0°C samples was substracted from 37°C samples and internalization normalized to untreated samples. All experiments were performed three times independently, mean values±s.e.m. are shown.
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Images

1) Product Images from "Vaccinia extracellular virions enter cells by macropinocytosis and acid-activated membrane rupture"

Article Title: Vaccinia extracellular virions enter cells by macropinocytosis and acid-activated membrane rupture

Journal: The EMBO Journal

doi: 10.1038/emboj.2011.245

Cellular factors required for EV infection. ( A – D ) HeLa cells were left untreated or treated with DMSO (highest used concentration), staurosporine (Stau), genistein (Geni), wortmannin (Wort), calphostin C (CalC), rottlerin (Rott), 3-indolepropionic acid (IPA-3), Iressa (Ires), chlorpromazine (Chlo), ML-7 (all 30 min), cytochalasin D (Cyto), jasplakinolide (Jasp), or ethylisopropyl amiloride (EIPA) (all 15 min). MVs or EVs of IHD-J GFP ( A ), WR GFP ( C ), or WR ΔA34R GFP ( D ) were preincubated in the presence of drugs and in the case of EVs with 7D11 (MOI 2). Cells were infected and green fluorescent cells quantified 4 h p.i. by flow cytometry. Infection levels were normalized to untreated samples. ( B ) EV (IHD-J F13–GFP) and MV (IHD-J EGFP–A5) internalization in the presence of various drugs was quantified as described in Figure 3 . The geometric mean of green fluorescence intensity of 0°C samples was substracted from 37°C samples and internalization normalized to untreated samples. All experiments were performed three times independently, mean values±s.e.m. are shown.
Figure Legend Snippet: Cellular factors required for EV infection. ( A – D ) HeLa cells were left untreated or treated with DMSO (highest used concentration), staurosporine (Stau), genistein (Geni), wortmannin (Wort), calphostin C (CalC), rottlerin (Rott), 3-indolepropionic acid (IPA-3), Iressa (Ires), chlorpromazine (Chlo), ML-7 (all 30 min), cytochalasin D (Cyto), jasplakinolide (Jasp), or ethylisopropyl amiloride (EIPA) (all 15 min). MVs or EVs of IHD-J GFP ( A ), WR GFP ( C ), or WR ΔA34R GFP ( D ) were preincubated in the presence of drugs and in the case of EVs with 7D11 (MOI 2). Cells were infected and green fluorescent cells quantified 4 h p.i. by flow cytometry. Infection levels were normalized to untreated samples. ( B ) EV (IHD-J F13–GFP) and MV (IHD-J EGFP–A5) internalization in the presence of various drugs was quantified as described in Figure 3 . The geometric mean of green fluorescence intensity of 0°C samples was substracted from 37°C samples and internalization normalized to untreated samples. All experiments were performed three times independently, mean values±s.e.m. are shown.

Techniques Used: Infection, Concentration Assay, Indirect Immunoperoxidase Assay, Flow Cytometry, Cytometry, Fluorescence

EV internalization and acid-mediated EV membrane disruption. ( A ) IHD-J—internalization. EV (IHD-J F13–GFP) and MV (IHD-J EGFP–A5) internalization in the presence of BafA, MonA, and EIPA was quantified as described in Figure 4B . ( B ) In vitro EV membrane disruption. IHD-J, WR, and WR ΔA34R EVs were incubated at pH 5.0 or pH 7.4 and 37°C for 5 min. EVs were titrated with or without 7D11 at pH 7.4 as described in Figure 1A . The percentage of intact EVs was calculated by normalizing plaque numbers after 7D11 neutralization to plaque numbers in untreated samples; mean values±s.e.m. of three independent experiments are presented. ( C ) EV infection—acid bypass of BafA block. HeLa cells were left untreated or treated with BafA for 1 h. EVs of IHD-J GFP, WR GFP, or WR ΔA34R GFP were preincubated in the presence of drugs and 7D11 for 1 h at 37°C (MOI 2). Virus particles were bound to cells on ice. Cells were shifted to 37°C in pH 7.4 or pH 4.5 medium for 5 min and then incubated in full medium with drugs for 4 h. Infected cells were quantified by flow cytometry. The percentage of infected cells was normalized to the untreated samples. All experiments were performed three times independently, mean values±s.e.m. are shown, asterisks mark significant differences ( P
Figure Legend Snippet: EV internalization and acid-mediated EV membrane disruption. ( A ) IHD-J—internalization. EV (IHD-J F13–GFP) and MV (IHD-J EGFP–A5) internalization in the presence of BafA, MonA, and EIPA was quantified as described in Figure 4B . ( B ) In vitro EV membrane disruption. IHD-J, WR, and WR ΔA34R EVs were incubated at pH 5.0 or pH 7.4 and 37°C for 5 min. EVs were titrated with or without 7D11 at pH 7.4 as described in Figure 1A . The percentage of intact EVs was calculated by normalizing plaque numbers after 7D11 neutralization to plaque numbers in untreated samples; mean values±s.e.m. of three independent experiments are presented. ( C ) EV infection—acid bypass of BafA block. HeLa cells were left untreated or treated with BafA for 1 h. EVs of IHD-J GFP, WR GFP, or WR ΔA34R GFP were preincubated in the presence of drugs and 7D11 for 1 h at 37°C (MOI 2). Virus particles were bound to cells on ice. Cells were shifted to 37°C in pH 7.4 or pH 4.5 medium for 5 min and then incubated in full medium with drugs for 4 h. Infected cells were quantified by flow cytometry. The percentage of infected cells was normalized to the untreated samples. All experiments were performed three times independently, mean values±s.e.m. are shown, asterisks mark significant differences ( P

Techniques Used: In Vitro, Incubation, Neutralization, Infection, Blocking Assay, Flow Cytometry, Cytometry

2) Product Images from "Salt secretion is linked to acid-base regulation of ionocytes in seawater-acclimated medaka: new insights into the salt-secreting mechanism"

Article Title: Salt secretion is linked to acid-base regulation of ionocytes in seawater-acclimated medaka: new insights into the salt-secreting mechanism

Journal: Scientific Reports

doi: 10.1038/srep31433

Effects of DIDS (400 μM) ( A ), AZ (200 μM) ( B ), EIPA (400 μM) ( C ), and bumetanide (Bumex; 400 μM) and/or DIDS (400 μM) ( D ) on Cl − flux at ionocytes of larval skin. Data are presented as the means ± SEM. * p
Figure Legend Snippet: Effects of DIDS (400 μM) ( A ), AZ (200 μM) ( B ), EIPA (400 μM) ( C ), and bumetanide (Bumex; 400 μM) and/or DIDS (400 μM) ( D ) on Cl − flux at ionocytes of larval skin. Data are presented as the means ± SEM. * p

Techniques Used:

3) Product Images from "Benzimidazolones enhance the function of epithelial Na+ transport"

Article Title: Benzimidazolones enhance the function of epithelial Na+ transport

Journal: British Journal of Pharmacology

doi: 10.1111/bph.12027

Benzimidazolones enhance benzamil-sensitive I SC . Effects of DC-EBIO (0.3 mM) on benzamil- and EIPA-sensitive I SC of rat FDLE cell monolayers. The figure shows the current reduction caused by 10 μM benzamil and 100 μM EIPA. Mean treatment-associated changes in current ± SEM of 12 (control/benzamil), 9 (DC-EBIO/benzamil), 11 (control/EIPA) and 14 (DC-EBIO/EIPA) monolayers (* P
Figure Legend Snippet: Benzimidazolones enhance benzamil-sensitive I SC . Effects of DC-EBIO (0.3 mM) on benzamil- and EIPA-sensitive I SC of rat FDLE cell monolayers. The figure shows the current reduction caused by 10 μM benzamil and 100 μM EIPA. Mean treatment-associated changes in current ± SEM of 12 (control/benzamil), 9 (DC-EBIO/benzamil), 11 (control/EIPA) and 14 (DC-EBIO/EIPA) monolayers (* P

Techniques Used:

4) Product Images from "Nox2-Mediated PI3K and Cofilin Activation Confers Alternate Redox Control of Macrophage Pinocytosis"

Article Title: Nox2-Mediated PI3K and Cofilin Activation Confers Alternate Redox Control of Macrophage Pinocytosis

Journal: Antioxidants & Redox Signaling

doi: 10.1089/ars.2016.6639

Physiological stimulation of macropinocytosis by M-CSF involves Nox2 activation. (A) WT macrophages were pretreated for 1 h with EIPA (10 μ M ), DPI (10 μ M ), or EUK 134 (10 μ M ), and challenged with M-CSF (100 ng/ml, 4 h) in the presence of FITC-dextran. FITC fluorescence was analyzed by FACS. (B) WT and Nox2 y/− macrophages were treated with vehicle and M-CSF, and FITC-dextran incorporation was analyzed by FACS. Three independent experiments were executed. Data represent the mean ± SEM. * p ≤ 0.05 versus vehicle, p ≤ 0.05 versus WT M-CSF or Ctrl MCSF. (C) WT macrophages were either treated with M-CSF alone or pretreated with inhibitors mentioned in (A) . Lysates were subjected to Western blot analysis for phospho- or total cofilin, Akt, and PTEN proteins. Representative Western blot images are shown ( n = 3). (D) Nox2 y/− macrophages were incubated with vehicle, or M-CSF. Cell lysates were processed for Western blot analysis for phospho- and total cofilin and PTEN expression ( n
Figure Legend Snippet: Physiological stimulation of macropinocytosis by M-CSF involves Nox2 activation. (A) WT macrophages were pretreated for 1 h with EIPA (10 μ M ), DPI (10 μ M ), or EUK 134 (10 μ M ), and challenged with M-CSF (100 ng/ml, 4 h) in the presence of FITC-dextran. FITC fluorescence was analyzed by FACS. (B) WT and Nox2 y/− macrophages were treated with vehicle and M-CSF, and FITC-dextran incorporation was analyzed by FACS. Three independent experiments were executed. Data represent the mean ± SEM. * p ≤ 0.05 versus vehicle, p ≤ 0.05 versus WT M-CSF or Ctrl MCSF. (C) WT macrophages were either treated with M-CSF alone or pretreated with inhibitors mentioned in (A) . Lysates were subjected to Western blot analysis for phospho- or total cofilin, Akt, and PTEN proteins. Representative Western blot images are shown ( n = 3). (D) Nox2 y/− macrophages were incubated with vehicle, or M-CSF. Cell lysates were processed for Western blot analysis for phospho- and total cofilin and PTEN expression ( n

Techniques Used: Activation Assay, Fluorescence, FACS, Western Blot, Incubation, Expressing

5) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Utilizes an Actin Polymerization-Dependent Macropinocytic Pathway To Enter Human Dermal Microvascular Endothelial and Human Umbilical Vein Endothelial Cells ▿"

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Utilizes an Actin Polymerization-Dependent Macropinocytic Pathway To Enter Human Dermal Microvascular Endothelial and Human Umbilical Vein Endothelial Cells ▿

Journal: Journal of Virology

doi: 10.1128/JVI.02498-08

Effect of endocytic inhibitors on KSHV gene expression, binding, and entry in HMVEC-d and HFF cells. (A and B) Cells were left untreated or pretreated with endocytic inhibitors for 1 h at 37°C, washed, and infected with KSHV at an MOI of 10. Total RNA was isolated at 2 h and 24 h p.i., and 50 ng of DNase-treated RNA/μl was subjected to real-time RT-PCR with ORF73 and ORF50 gene-specific primers and TaqMan probes. Known concentrations of DNase-treated, in vitro-transcribed ORF50 and ORF73 transcripts were used in real-time RT-PCR to construct a standard graph from which the relative copy numbers of viral transcripts were calculated and normalized to the amount of GAPDH. Histograms depict KSHV ORF73 and ORF50 gene RNA copy numbers in untreated cells (KSHV) or cells in the presence of indicated nontoxic concentrations of chlorpromazine (Chlor), EIPA, rottlerin (Rot), cytochalasin D (CytoD), filipin, cholera toxin B (CTB), MβCD, or nystatin (nys) in HMVEC-d (A) and HFF (B) cells. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three independent experiments. (C, D, and E) Effect of endocytic inhibitors on KSHV binding (C) and internalization (D) in HMVEC-d cells and on internalization (E) in HFF cells. (C) HMVEC-d cells grown in 24-well plates were either left untreated or pretreated with various nontoxic concentrations of agents for 1 h at 37°C and incubated with a fixed concentration of [ 3 ). Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments.
Figure Legend Snippet: Effect of endocytic inhibitors on KSHV gene expression, binding, and entry in HMVEC-d and HFF cells. (A and B) Cells were left untreated or pretreated with endocytic inhibitors for 1 h at 37°C, washed, and infected with KSHV at an MOI of 10. Total RNA was isolated at 2 h and 24 h p.i., and 50 ng of DNase-treated RNA/μl was subjected to real-time RT-PCR with ORF73 and ORF50 gene-specific primers and TaqMan probes. Known concentrations of DNase-treated, in vitro-transcribed ORF50 and ORF73 transcripts were used in real-time RT-PCR to construct a standard graph from which the relative copy numbers of viral transcripts were calculated and normalized to the amount of GAPDH. Histograms depict KSHV ORF73 and ORF50 gene RNA copy numbers in untreated cells (KSHV) or cells in the presence of indicated nontoxic concentrations of chlorpromazine (Chlor), EIPA, rottlerin (Rot), cytochalasin D (CytoD), filipin, cholera toxin B (CTB), MβCD, or nystatin (nys) in HMVEC-d (A) and HFF (B) cells. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three independent experiments. (C, D, and E) Effect of endocytic inhibitors on KSHV binding (C) and internalization (D) in HMVEC-d cells and on internalization (E) in HFF cells. (C) HMVEC-d cells grown in 24-well plates were either left untreated or pretreated with various nontoxic concentrations of agents for 1 h at 37°C and incubated with a fixed concentration of [ 3 ). Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments.

Techniques Used: Expressing, Binding Assay, Infection, Isolation, Quantitative RT-PCR, In Vitro, Construct, CtB Assay, Standard Deviation, Incubation, Concentration Assay

6) Product Images from "NHERF2 is necessary for basal activity, second messenger inhibition, and LPA stimulation of NHE3 in mouse distal ileum"

Article Title: NHERF2 is necessary for basal activity, second messenger inhibition, and LPA stimulation of NHE3 in mouse distal ileum

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00311.2010

Ileal brush border (BB) NHE3 activity is NHERF2 dependent. A : the initial rate of NHE3 activity was decreased in NHERF2 −/− ileal epithelial cells compared with WT ileum. The tissue was loaded with 20 μM SNARF-4F, and EIPA-sensitive
Figure Legend Snippet: Ileal brush border (BB) NHE3 activity is NHERF2 dependent. A : the initial rate of NHE3 activity was decreased in NHERF2 −/− ileal epithelial cells compared with WT ileum. The tissue was loaded with 20 μM SNARF-4F, and EIPA-sensitive

Techniques Used: Activity Assay

7) Product Images from "Exploring the role of stromal osmoregulation in cancer and disease using executable modelling"

Article Title: Exploring the role of stromal osmoregulation in cancer and disease using executable modelling

Journal: Nature Communications

doi: 10.1038/s41467-018-05414-y

Application of the qualitative network to murine embryonic fibroblasts. Expansion of the QN to p53 −/− , Kras G12D/+ (HET) and p53 −/− , Kras G12D/G12D (HOM) MEFs. a Transport proteins deregulated within HOM MEFs when compared to HET MEFs. Proteins are upregulated (red arrows), downregulated (blue arrows), or unchanged (grey). b Phenotype change for MEFS when HOM MEFs are compared to HET MEFs. The model output represents the physiological behaviour predicted by the model when proteins from ( a , where it is known. The model predicts that attachment will be significantly different between the two cell types, but viability and cell size will remain unchanged. c Effects of application of channel inhibitors to HOM MEFs in vitro showing application of 10 µM DIDs for 72 h, resulting in decreased cellular attachment (left), and application of 10 nM EIPA ± 10 µM DIDs (right). Data are technical replicates. Dotted lines represent calculated Bliss independence values. d Effects of application of 10 µM DIDs ± 10 nM EIPA on viability in HOM MEFs. e Effects of application of 10 µM DIDs ± 10 nM AHCL on attachment in HOM MEFs. f Effects of application of 10 µM DIDs ± 10 nM AHCL on viability in HOM MEFs.* P
Figure Legend Snippet: Application of the qualitative network to murine embryonic fibroblasts. Expansion of the QN to p53 −/− , Kras G12D/+ (HET) and p53 −/− , Kras G12D/G12D (HOM) MEFs. a Transport proteins deregulated within HOM MEFs when compared to HET MEFs. Proteins are upregulated (red arrows), downregulated (blue arrows), or unchanged (grey). b Phenotype change for MEFS when HOM MEFs are compared to HET MEFs. The model output represents the physiological behaviour predicted by the model when proteins from ( a , where it is known. The model predicts that attachment will be significantly different between the two cell types, but viability and cell size will remain unchanged. c Effects of application of channel inhibitors to HOM MEFs in vitro showing application of 10 µM DIDs for 72 h, resulting in decreased cellular attachment (left), and application of 10 nM EIPA ± 10 µM DIDs (right). Data are technical replicates. Dotted lines represent calculated Bliss independence values. d Effects of application of 10 µM DIDs ± 10 nM EIPA on viability in HOM MEFs. e Effects of application of 10 µM DIDs ± 10 nM AHCL on attachment in HOM MEFs. f Effects of application of 10 µM DIDs ± 10 nM AHCL on viability in HOM MEFs.* P

Techniques Used: In Vitro, Cell Attachment Assay

8) Product Images from "Computational Analysis of Amiloride Analogue Inhibitors of Coxsackievirus B3 RNA Polymerase"

Article Title: Computational Analysis of Amiloride Analogue Inhibitors of Coxsackievirus B3 RNA Polymerase

Journal: Journal of proteomics & bioinformatics

doi: 10.4172/jpb.S9-004

Inhibitor docking to 3D pol All active compounds interact in the same general orientation in the nucleotide binding site. The residues involved in significant interactions with inhibitors are shown (grey sticks) with polar interactions indicated as yellow dashed lines. The schematic 2D representations shown in the right column were created by LigPlot + ] with hydrophobic interactions shown as red semi-circles with lines radiating out in the direction of the interaction. (a) Amiloride (magenta carbons); (b) DMA (orange carbons); (c) EIPA (cyan carbons); (d) HMA (green carbons); (e) Benzamil (yellow carbons); (f) DCB (peach carbons); and (g) HMB (purple carbons).
Figure Legend Snippet: Inhibitor docking to 3D pol All active compounds interact in the same general orientation in the nucleotide binding site. The residues involved in significant interactions with inhibitors are shown (grey sticks) with polar interactions indicated as yellow dashed lines. The schematic 2D representations shown in the right column were created by LigPlot + ] with hydrophobic interactions shown as red semi-circles with lines radiating out in the direction of the interaction. (a) Amiloride (magenta carbons); (b) DMA (orange carbons); (c) EIPA (cyan carbons); (d) HMA (green carbons); (e) Benzamil (yellow carbons); (f) DCB (peach carbons); and (g) HMB (purple carbons).

Techniques Used: Binding Assay

9) Product Images from "Visualization of Macropinocytosis in Prostate Fibroblasts"

Article Title: Visualization of Macropinocytosis in Prostate Fibroblasts

Journal: Bio-protocol

doi: 10.21769/BioProtoc.3235

Reduction of macropinocytosis by EIPA. Macropinosomes visualization in Ras V12 prostatic mouse fibroblasts was carried out with 30 min pretreatment with 5-(N-ethyl-N-isopropyl) amiloride (EIPA). Scale bar = 24 μm.
Figure Legend Snippet: Reduction of macropinocytosis by EIPA. Macropinosomes visualization in Ras V12 prostatic mouse fibroblasts was carried out with 30 min pretreatment with 5-(N-ethyl-N-isopropyl) amiloride (EIPA). Scale bar = 24 μm.

Techniques Used:

10) Product Images from "Exosomes Exploit the Virus Entry Machinery and Pathway To Transmit Alpha Interferon-Induced Antiviral Activity"

Article Title: Exosomes Exploit the Virus Entry Machinery and Pathway To Transmit Alpha Interferon-Induced Antiviral Activity

Journal: Journal of Virology

doi: 10.1128/JVI.01578-18

Exosome internalization involves macropinocytosis. (A) Preincubation with exosomes increased dextran uptake in HepG2 cells. RhoB-dextran (RhoB-DEX) uptake by HepG2 cells pretreated with exosomes [EXO(+)] was analyzed by flow cytometry, and the MFI is normalized to that of untreated cells [EXO(−)]. (B and C) Confocal images (B) and flow cytometry analysis (C) of exosome and dextran internalization by HepG2 cells treated with EIPA. Scale bars: 10 µm. For flow cytometry analysis, MFIs (right) are normalized to that of DMSO-treated cells. (D to F) Confocal images (D) and flow cytometry analysis (E and F) of exosome and dextran internalization by HepG2 cells treated with IPA-3 or rottlerin. Scale bars: 10 µm. For flow cytometry analysis, MFIs (right) are normalized to that of DMSO-treated cells. (G and H) Exosome uptake is independent of Rac1 or Cdc42. Flow cytometry analysis of exosome internalization by HepG2 cells transfected with EGFP-Rac1 dominant negative mutant (G) or EGFP-Cdc42 dominant negative mutant (H), followed by incubation with PKH26-labeled exosomes. Transfected cells (EGFP + ) are gated, and the uptake of exosomes among transfected cells (EGFP + PKH26 + ) was analyzed as described above. (I and J) Internalized exosome colocalized with dextran 30 min (I) and 1 h (J) after internalization. The colocalization of Rho-dextran (red) with PKH67-labeled exosomes (green) was analyzed as described above. Scale bars: 5 µm. (K) HepG2.2.15 cells were treated with DMSO or EIPA. The level of HBsAg in the culture medium (supernatant) was detected by ELISA. HBV DNA in the culture medium and intracellular core particle DNA were quantified by qPCR. (L) Blockade of IFN-α-induced anti-HBV activity transmission by EIPA treatment. HepG2.2.15 cells were pretreated with DMSO or EIPA, and the drugs were present continuously during following incubation with IFN-EXO or Ctrl-EXO. HBV DNA levels in the medium were quantified by qPCR. The error bars indicate the SD. *, P
Figure Legend Snippet: Exosome internalization involves macropinocytosis. (A) Preincubation with exosomes increased dextran uptake in HepG2 cells. RhoB-dextran (RhoB-DEX) uptake by HepG2 cells pretreated with exosomes [EXO(+)] was analyzed by flow cytometry, and the MFI is normalized to that of untreated cells [EXO(−)]. (B and C) Confocal images (B) and flow cytometry analysis (C) of exosome and dextran internalization by HepG2 cells treated with EIPA. Scale bars: 10 µm. For flow cytometry analysis, MFIs (right) are normalized to that of DMSO-treated cells. (D to F) Confocal images (D) and flow cytometry analysis (E and F) of exosome and dextran internalization by HepG2 cells treated with IPA-3 or rottlerin. Scale bars: 10 µm. For flow cytometry analysis, MFIs (right) are normalized to that of DMSO-treated cells. (G and H) Exosome uptake is independent of Rac1 or Cdc42. Flow cytometry analysis of exosome internalization by HepG2 cells transfected with EGFP-Rac1 dominant negative mutant (G) or EGFP-Cdc42 dominant negative mutant (H), followed by incubation with PKH26-labeled exosomes. Transfected cells (EGFP + ) are gated, and the uptake of exosomes among transfected cells (EGFP + PKH26 + ) was analyzed as described above. (I and J) Internalized exosome colocalized with dextran 30 min (I) and 1 h (J) after internalization. The colocalization of Rho-dextran (red) with PKH67-labeled exosomes (green) was analyzed as described above. Scale bars: 5 µm. (K) HepG2.2.15 cells were treated with DMSO or EIPA. The level of HBsAg in the culture medium (supernatant) was detected by ELISA. HBV DNA in the culture medium and intracellular core particle DNA were quantified by qPCR. (L) Blockade of IFN-α-induced anti-HBV activity transmission by EIPA treatment. HepG2.2.15 cells were pretreated with DMSO or EIPA, and the drugs were present continuously during following incubation with IFN-EXO or Ctrl-EXO. HBV DNA levels in the medium were quantified by qPCR. The error bars indicate the SD. *, P

Techniques Used: Flow Cytometry, Cytometry, Indirect Immunoperoxidase Assay, Transfection, Dominant Negative Mutation, Incubation, Labeling, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Activity Assay, Transmission Assay

11) Product Images from "Non-invasive monitoring of single-cell mechanics by acoustic scattering"

Article Title: Non-invasive monitoring of single-cell mechanics by acoustic scattering

Journal: Nature methods

doi: 10.1038/s41592-019-0326-x

Mitotic swelling and actin remodeling are responsible for SNACS dynamics in mitosis. a, L1210 cell volume (blue circles) and SNACS (red) in mitosis. b, Buoyant mass (black) and SNACS (red) of a L1210 cell arrested in metaphase by treating with S-trityl-cysteine (STLC, 5 μM). c, Left, mean SNACS change of control (red) and ethylisopropylamiloride (EIPA, 10 μM, blue) treated L1210 cells in early mitosis. Right, quantification of SNACS change with STLC (5 μM, n = 7 cells from 7 independent experiments, P = 0.58) and EIPA (10 μM, n = 5 cells from 5 independent experiments. P = 3.0 × 10 -5 ) treatments. Data depicts mean ± s.d. of the maximal SNACS change in prophase and metaphase. Statistical comparisons (two-sided Welch’s t-test) were made to the control (n = 24 cells from 13 independent experiments). **** P
Figure Legend Snippet: Mitotic swelling and actin remodeling are responsible for SNACS dynamics in mitosis. a, L1210 cell volume (blue circles) and SNACS (red) in mitosis. b, Buoyant mass (black) and SNACS (red) of a L1210 cell arrested in metaphase by treating with S-trityl-cysteine (STLC, 5 μM). c, Left, mean SNACS change of control (red) and ethylisopropylamiloride (EIPA, 10 μM, blue) treated L1210 cells in early mitosis. Right, quantification of SNACS change with STLC (5 μM, n = 7 cells from 7 independent experiments, P = 0.58) and EIPA (10 μM, n = 5 cells from 5 independent experiments. P = 3.0 × 10 -5 ) treatments. Data depicts mean ± s.d. of the maximal SNACS change in prophase and metaphase. Statistical comparisons (two-sided Welch’s t-test) were made to the control (n = 24 cells from 13 independent experiments). **** P

Techniques Used:

12) Product Images from "Mosquito Cellular Factors and Functions in Mediating the Infectious entry of Chikungunya Virus"

Article Title: Mosquito Cellular Factors and Functions in Mediating the Infectious entry of Chikungunya Virus

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0002050

Effects of clathrin-mediated endocytic inhibitors on the entry of CHIKV into C6/36 cells. C6/36 cells were pre-treated with different inhibitors for 3 hours before CHIKV infection. Supernatants were harvested 24 hours p.i for viral plaque assays. The log virus titre is plotted against the concentrations of drug used. Dose-dependent inhibition of CHIKV entry into (a) monodansylcadaverine-, (b) chlorpromazine- and (c) dynasore-treated cells is observed. In contrast, minimal inhibition of CHIKV infectious entry into (d) filipin- and (e) nystatin-treated cells is noted. Cholesterol-dependent endocytosis of CHIKV into C6/36 cells is further analysed. Dose-dependent inhibition of CHIKV infection is observed with (f) methyl-β cyclodextrin treatment of C6/36 cells. Furthermore, minimal inhibition on the infectious entry of CHIKV into (g) EIPA-treated C6/36 cells is observed. Cell viability upon drug treatments is represented by the line graphs. The asterisk indicates * p values
Figure Legend Snippet: Effects of clathrin-mediated endocytic inhibitors on the entry of CHIKV into C6/36 cells. C6/36 cells were pre-treated with different inhibitors for 3 hours before CHIKV infection. Supernatants were harvested 24 hours p.i for viral plaque assays. The log virus titre is plotted against the concentrations of drug used. Dose-dependent inhibition of CHIKV entry into (a) monodansylcadaverine-, (b) chlorpromazine- and (c) dynasore-treated cells is observed. In contrast, minimal inhibition of CHIKV infectious entry into (d) filipin- and (e) nystatin-treated cells is noted. Cholesterol-dependent endocytosis of CHIKV into C6/36 cells is further analysed. Dose-dependent inhibition of CHIKV infection is observed with (f) methyl-β cyclodextrin treatment of C6/36 cells. Furthermore, minimal inhibition on the infectious entry of CHIKV into (g) EIPA-treated C6/36 cells is observed. Cell viability upon drug treatments is represented by the line graphs. The asterisk indicates * p values

Techniques Used: Infection, Inhibition

13) Product Images from "Transcription Factor Eb Is Required for Macropinocytosis-Mediated Growth Recovery of Nutrient-Deprived Kras-Mutant Cells"

Article Title: Transcription Factor Eb Is Required for Macropinocytosis-Mediated Growth Recovery of Nutrient-Deprived Kras-Mutant Cells

Journal: Nutrients

doi: 10.3390/nu10111638

Transcription factor EB (TFEB) plays a role in macropinocytosis-mediated recovery of mTORC1 activity and cell growth in KRAS-mutant cells. ( A ) Representative Western blot showing mTORC1 activation in KRAS-mutant cells. Cells were transfected with scrambled siRNA or si TFEB for 24 h, and then maintained in leucine-free medium for 24 h with or without 3% BSA. ( B ) Quantitative densitometric data of phospho/total S6K abundance shown in ( A ). The intensity of each band was measured using ImageJ software. ( C ) Cells were transfected with scrambled siRNA or si TFEB for 24 h, and then maintained in leucine-free medium with or without 3% BSA or 10 μM EIPA for 72 h. Cell proliferation was measured using a CCK-8 assay. Data were normalized with control siRNA-transfected cells in leucine-replete media and expressed as means ±SEM of three independent experiments. n.s., not significant. * p
Figure Legend Snippet: Transcription factor EB (TFEB) plays a role in macropinocytosis-mediated recovery of mTORC1 activity and cell growth in KRAS-mutant cells. ( A ) Representative Western blot showing mTORC1 activation in KRAS-mutant cells. Cells were transfected with scrambled siRNA or si TFEB for 24 h, and then maintained in leucine-free medium for 24 h with or without 3% BSA. ( B ) Quantitative densitometric data of phospho/total S6K abundance shown in ( A ). The intensity of each band was measured using ImageJ software. ( C ) Cells were transfected with scrambled siRNA or si TFEB for 24 h, and then maintained in leucine-free medium with or without 3% BSA or 10 μM EIPA for 72 h. Cell proliferation was measured using a CCK-8 assay. Data were normalized with control siRNA-transfected cells in leucine-replete media and expressed as means ±SEM of three independent experiments. n.s., not significant. * p

Techniques Used: Activity Assay, Mutagenesis, Western Blot, Activation Assay, Transfection, Software, CCK-8 Assay

14) Product Images from "Salt secretion is linked to acid-base regulation of ionocytes in seawater-acclimated medaka: new insights into the salt-secreting mechanism"

Article Title: Salt secretion is linked to acid-base regulation of ionocytes in seawater-acclimated medaka: new insights into the salt-secreting mechanism

Journal: Scientific Reports

doi: 10.1038/srep31433

Effects of DIDS (400 μM) ( A ), AZ (200 μM) ( B ), EIPA (400 μM) ( C ), and bumetanide (Bumex; 400 μM) and/or DIDS (400 μM) ( D ) on Cl − flux at ionocytes of larval skin. Data are presented as the means ± SEM. * p
Figure Legend Snippet: Effects of DIDS (400 μM) ( A ), AZ (200 μM) ( B ), EIPA (400 μM) ( C ), and bumetanide (Bumex; 400 μM) and/or DIDS (400 μM) ( D ) on Cl − flux at ionocytes of larval skin. Data are presented as the means ± SEM. * p

Techniques Used:

15) Product Images from "Rab5 and Rab11 Are Required for Clathrin-Dependent Endocytosis of Japanese Encephalitis Virus in BHK-21 Cells"

Article Title: Rab5 and Rab11 Are Required for Clathrin-Dependent Endocytosis of Japanese Encephalitis Virus in BHK-21 Cells

Journal: Journal of Virology

doi: 10.1128/JVI.01113-17

Role of macropinocytosis on JEV binding, entry, and infection. Cells were pretreated with subtoxic doses of EIPA or wortmannin, as indicated, for 1 h at 37°C. Drugs were present in the medium during the adsorption period. BHK-21 cells were infected with JEV (MOI of 5) (A and D) or DF-1 cells were infected with NDV (MOI = 5) (C and F) at 4°C for 1 h and then at 37°C for 0 h (binding) or 1 h (entry); the infected cells were lysed to determine viral RNA copy number by RT-qPCR. (B and E) cells were infected with JEV (MOI of 0.05) at 37°C; at 24 hpi, viral RNA copy number was determined by RT-qPCR. The horizontal lines show results of subtoxic doses of EIPA and wortmannin on BHK-21 cells as determined by cell viability assay as described in Materials and Methods. Results are presented as the means ± SD of three independent experiments. **, P
Figure Legend Snippet: Role of macropinocytosis on JEV binding, entry, and infection. Cells were pretreated with subtoxic doses of EIPA or wortmannin, as indicated, for 1 h at 37°C. Drugs were present in the medium during the adsorption period. BHK-21 cells were infected with JEV (MOI of 5) (A and D) or DF-1 cells were infected with NDV (MOI = 5) (C and F) at 4°C for 1 h and then at 37°C for 0 h (binding) or 1 h (entry); the infected cells were lysed to determine viral RNA copy number by RT-qPCR. (B and E) cells were infected with JEV (MOI of 0.05) at 37°C; at 24 hpi, viral RNA copy number was determined by RT-qPCR. The horizontal lines show results of subtoxic doses of EIPA and wortmannin on BHK-21 cells as determined by cell viability assay as described in Materials and Methods. Results are presented as the means ± SD of three independent experiments. **, P

Techniques Used: Binding Assay, Infection, Adsorption, Quantitative RT-PCR, Viability Assay

16) Product Images from "Ebolavirus Is Internalized into Host Cells via Macropinocytosis in a Viral Glycoprotein-Dependent Manner"

Article Title: Ebolavirus Is Internalized into Host Cells via Macropinocytosis in a Viral Glycoprotein-Dependent Manner

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1001121

Macropinocytosis-associated events occur during Ebola virion internalization. (A) The effect of the internalization of DiI-labeled Ebola VLPs on dextran uptake. Vero cells were incubated with 0.5 mg/ml Alexa Fluor 647-Dex Mw 10K in the absence or presence of Ebola VLPs for 60 min at 37°C. The uptake of Alexa Fluor 647-Dex Mw 10K was analyzed by using flow cytometry. The effect of EIPA pretreatment was assessed in parallel. Each experiment was performed in triplicate and the results are presented as the mean ± SD. (B) Co-localization of internalized DiI-labeled Ebola VLPs and Dex Mw 10K. DiI-Ebola VLPs were adsorbed to Vero cells for 30 min on ice. The cells were cultured in the presence of 0.5 mg/ml Alexa Fluor 647-Dex Mw 10K for 10 min at 37°C. Co-localization of DiI-virions (red) and Alexa Fluor-Dex Mw 10K (green) was analyzed by using confocal laser scanning microscope. Co-localized virions are shown by arrows. Outlines of individual cells were drawn. Scale bar, 10 µm. (C) Effect of a dominant-negative form of Rac1 on the internalization of DiI-labeled Ebola virions. The eGFP-fused, wild-type Rac1 (wtRac1, upper panels) or the dominant-negative form of Rac1 (dnRac1, lower panels) was expressed in Vero cells. DiI-labeled Ebola VLPs were adsorbed to the cells for 30 min on ice. After incubation for 2 h at 37°C, surface-bound virions were removed by trypsin and the internalization of DiI-virions was analyzed by using confocal laser scanning microscope. Expression of dnRac1 interfered with the internalization of Alexa Fluor 647-Dex Mw 10K (blue; lower middle panel), attesting to its functionality. Scale bars, 10 µm. (D) Quantitative analysis of the internalization of DiI-labeled Ebola virions in wtRac1 or dnRac1-expressed Vero cells. The internalized DiI-virions were measured in 10 individual wtRac1 or dnRac1-expressed cells. Each experiment was performed in triplicate and the results are presented as the mean ± SD. (E) Effect of PKC inhibitors on the internalization of DiI-labeled Ebola virions. Vero cells were treated with DMSO or staurosporine (Stauro) for 30 min at 37°C. Labeled Ebola VLPs were adsorbed to the cells for 30 min on ice and incubated for 2 h at 37°C in the absence or presence of inhibitor. Surface-bound virions were removed by trypsin and the internalization of DiI-virions was analyzed by using confocal laser scanning microscope. The internalized DiI-virions were analyzed in 10 individual DMSO- or staurosporine-treated cells (red bars). The efficiency of Alexa Fluor-Dex Mw 10K uptake in inhibitor-treated cells was measured by using flow cytometry (blue bars). Each experiment was performed in triplicate and relative uptake efficiencies are presented as the mean ± SD (red bars). Staurosporine treatment interfered with the internalization of Alexa Fluor 633-Tf (blue bars), attesting to its functionality. (F) The down-regulation of Cdc42 and Pak1 by siRNA. The efficiencies of Cdc42 and Pak1 knock-down were assessed by use of RT-PCR. Total cellular RNA was isolated from siRNA-transfected Vero cells 48 h post-transfection by using the TRI reagent (Sigma-Aldrich) according to the manufacturer's instructions. cDNA synthesis was performed with Molony murine leukemia virus RTase using a random hexamer (Invitrogen) according to the manufacturer's protocol. PCR was carried out for 25–30 cycles consisting of a DNA denaturing step for 30 s at 94°C, annealing for 30 s at 55°C, and extension for 1 min at 72°C by use of Taq DNA polymerase (Promega). Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as an endogenous control. The oligonucleotides used for amplification of individual genes are shown in Table S1 . (G) Effect of down-regulation of Cdc42 and Pak1 on the internalization of DiI-labeled Ebola virions. Vero cells were transfected with control (Cont) non-targeting siRNA or siRNA to down-regulate Cdc42 and Pak1 expression. Labeled Ebola VLPs were adsorbed to the siRNA-transfected cells for 30 min on ice, 48 h post-transfection. After incubation for 2 h at 37°C, surface-bound virions were removed by trypsin for 5 min at 37°C and the internalization of Ebola VLPs was analyzed by using confocal laser scanning microscope, and the number of DiI-virions in 10 individual siRNA-transfected cells was measured. The efficiency of Alexa Fluor-Dex Mw 10K uptake in siRNA-transfected cells was measured by use of flow cytometry (blue bars). Each experiment was performed in triplicate and the relative uptake efficiencies are presented as the mean ± SD. (H) The internalization of Ebola virions is associated with plasma membrane ruffling. DiI-EbolaΔVP30 virions were adsorbed to eGFP-actin-expressing Vero cells for 30 min on ice. The cells were then incubated at 37°C and time-lapse images were acquired at 15-second intervals over a period of 10 min by using confocal laser scanning microscope. Still frames at the indicated times (sec) after the temperature shift to 37°C are shown. Scale bar, 10 µm.
Figure Legend Snippet: Macropinocytosis-associated events occur during Ebola virion internalization. (A) The effect of the internalization of DiI-labeled Ebola VLPs on dextran uptake. Vero cells were incubated with 0.5 mg/ml Alexa Fluor 647-Dex Mw 10K in the absence or presence of Ebola VLPs for 60 min at 37°C. The uptake of Alexa Fluor 647-Dex Mw 10K was analyzed by using flow cytometry. The effect of EIPA pretreatment was assessed in parallel. Each experiment was performed in triplicate and the results are presented as the mean ± SD. (B) Co-localization of internalized DiI-labeled Ebola VLPs and Dex Mw 10K. DiI-Ebola VLPs were adsorbed to Vero cells for 30 min on ice. The cells were cultured in the presence of 0.5 mg/ml Alexa Fluor 647-Dex Mw 10K for 10 min at 37°C. Co-localization of DiI-virions (red) and Alexa Fluor-Dex Mw 10K (green) was analyzed by using confocal laser scanning microscope. Co-localized virions are shown by arrows. Outlines of individual cells were drawn. Scale bar, 10 µm. (C) Effect of a dominant-negative form of Rac1 on the internalization of DiI-labeled Ebola virions. The eGFP-fused, wild-type Rac1 (wtRac1, upper panels) or the dominant-negative form of Rac1 (dnRac1, lower panels) was expressed in Vero cells. DiI-labeled Ebola VLPs were adsorbed to the cells for 30 min on ice. After incubation for 2 h at 37°C, surface-bound virions were removed by trypsin and the internalization of DiI-virions was analyzed by using confocal laser scanning microscope. Expression of dnRac1 interfered with the internalization of Alexa Fluor 647-Dex Mw 10K (blue; lower middle panel), attesting to its functionality. Scale bars, 10 µm. (D) Quantitative analysis of the internalization of DiI-labeled Ebola virions in wtRac1 or dnRac1-expressed Vero cells. The internalized DiI-virions were measured in 10 individual wtRac1 or dnRac1-expressed cells. Each experiment was performed in triplicate and the results are presented as the mean ± SD. (E) Effect of PKC inhibitors on the internalization of DiI-labeled Ebola virions. Vero cells were treated with DMSO or staurosporine (Stauro) for 30 min at 37°C. Labeled Ebola VLPs were adsorbed to the cells for 30 min on ice and incubated for 2 h at 37°C in the absence or presence of inhibitor. Surface-bound virions were removed by trypsin and the internalization of DiI-virions was analyzed by using confocal laser scanning microscope. The internalized DiI-virions were analyzed in 10 individual DMSO- or staurosporine-treated cells (red bars). The efficiency of Alexa Fluor-Dex Mw 10K uptake in inhibitor-treated cells was measured by using flow cytometry (blue bars). Each experiment was performed in triplicate and relative uptake efficiencies are presented as the mean ± SD (red bars). Staurosporine treatment interfered with the internalization of Alexa Fluor 633-Tf (blue bars), attesting to its functionality. (F) The down-regulation of Cdc42 and Pak1 by siRNA. The efficiencies of Cdc42 and Pak1 knock-down were assessed by use of RT-PCR. Total cellular RNA was isolated from siRNA-transfected Vero cells 48 h post-transfection by using the TRI reagent (Sigma-Aldrich) according to the manufacturer's instructions. cDNA synthesis was performed with Molony murine leukemia virus RTase using a random hexamer (Invitrogen) according to the manufacturer's protocol. PCR was carried out for 25–30 cycles consisting of a DNA denaturing step for 30 s at 94°C, annealing for 30 s at 55°C, and extension for 1 min at 72°C by use of Taq DNA polymerase (Promega). Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as an endogenous control. The oligonucleotides used for amplification of individual genes are shown in Table S1 . (G) Effect of down-regulation of Cdc42 and Pak1 on the internalization of DiI-labeled Ebola virions. Vero cells were transfected with control (Cont) non-targeting siRNA or siRNA to down-regulate Cdc42 and Pak1 expression. Labeled Ebola VLPs were adsorbed to the siRNA-transfected cells for 30 min on ice, 48 h post-transfection. After incubation for 2 h at 37°C, surface-bound virions were removed by trypsin for 5 min at 37°C and the internalization of Ebola VLPs was analyzed by using confocal laser scanning microscope, and the number of DiI-virions in 10 individual siRNA-transfected cells was measured. The efficiency of Alexa Fluor-Dex Mw 10K uptake in siRNA-transfected cells was measured by use of flow cytometry (blue bars). Each experiment was performed in triplicate and the relative uptake efficiencies are presented as the mean ± SD. (H) The internalization of Ebola virions is associated with plasma membrane ruffling. DiI-EbolaΔVP30 virions were adsorbed to eGFP-actin-expressing Vero cells for 30 min on ice. The cells were then incubated at 37°C and time-lapse images were acquired at 15-second intervals over a period of 10 min by using confocal laser scanning microscope. Still frames at the indicated times (sec) after the temperature shift to 37°C are shown. Scale bar, 10 µm.

Techniques Used: Labeling, Incubation, Flow Cytometry, Cytometry, Cell Culture, Laser-Scanning Microscopy, Dominant Negative Mutation, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Transfection, Random Hexamer Labeling, Polymerase Chain Reaction, Amplification, Size-exclusion Chromatography

Macropinocytotic internalization of Ebola virions is GP-dependent. (A) Co-localization of SNX5 with VSV pseudotyped with EBOV GP. Labeled VSV particles pseudotyped with EBOV GP (DiI-VSVΔ*G-GP) or VSV G (DiI-VSVΔ*G-G) were adsorbed to eGFP-SNX5-expressing Vero cells for 30 min on ice. The cells were then incubated at 37°C and time-lapse images were acquired at 20-second intervals over a period of 30 min by using confocal laser scanning microscope. Still frames of DiI-VSVΔ*G-GP (left panel) and DiI-VSVΔ*G-G (right panel) at 10 min after the temperature shift are shown. DiI-pseudovirions that co-localize with eGFP-SNX5 are indicated by arrows. Scale bars, 10 µm. (B) Graphic representation of the co-localization of EBOV GP-pseudotyped VSV virions with Rab7-positive vesicles. Co-localization of DiI-VSVΔ*G-GP (green bars) with Rab7-positive vesicles was analyzed at the indicated time points as indicated in the Materials and Methods . Experiments were performed in triplicate and the results are presented as the mean ± standard deviation. Results obtained for DiI-EbolaΔVP30 (blue bars) and DiI-VSVΔ*G-G (red bars) are shown for comparison. (C) Effect of macropinocytosis inhibitors on the co-localization of DiI-labeled VSV pseudovirions with eGFP-Rab7-positive vesicles. Vero cells expressing eGFP-Rab7 were pretreated with CytoD, Wort, LY294002 or EIPA for 30 min at 37°C; control cells were treated with DMSO. DiI-labeled VSVΔ*G-GP (green bars) or VSVΔ*G-G (red bars) were adsorbed to cells for 30 min on ice. The cells were then incubated at 37°C in the presence of inhibitors for 2 h. Co-localization of DiI-pseudovirions with eGFP-Rab7-positive vesicles was analyzed as described in the Materials and Methods . Experiments were carried out in triplicate and the results are presented as the mean ± standard deviation. (D) Effect of macropinocytosis inhibitors on the infectivity of VSV pseudovirions. Vero cells were treated with individual inhibitors for 30 min at 37°C and infected with VSVΔG*-GP (green bars) or VSVΔG*-G (red bars) in the presence of the inhibitor. 1 h post-infection, surface-bound virions were removed by trypsin and the cells were cultured for 24 h in the absence of inhibitors. The infection efficiency of each pseudovirus was determined by measuring the number of GFP-positive cells using with conventional fluorescent microscope. Each experiment was performed in triplicate and the relative infection efficiencies are presented as the mean ± SD.
Figure Legend Snippet: Macropinocytotic internalization of Ebola virions is GP-dependent. (A) Co-localization of SNX5 with VSV pseudotyped with EBOV GP. Labeled VSV particles pseudotyped with EBOV GP (DiI-VSVΔ*G-GP) or VSV G (DiI-VSVΔ*G-G) were adsorbed to eGFP-SNX5-expressing Vero cells for 30 min on ice. The cells were then incubated at 37°C and time-lapse images were acquired at 20-second intervals over a period of 30 min by using confocal laser scanning microscope. Still frames of DiI-VSVΔ*G-GP (left panel) and DiI-VSVΔ*G-G (right panel) at 10 min after the temperature shift are shown. DiI-pseudovirions that co-localize with eGFP-SNX5 are indicated by arrows. Scale bars, 10 µm. (B) Graphic representation of the co-localization of EBOV GP-pseudotyped VSV virions with Rab7-positive vesicles. Co-localization of DiI-VSVΔ*G-GP (green bars) with Rab7-positive vesicles was analyzed at the indicated time points as indicated in the Materials and Methods . Experiments were performed in triplicate and the results are presented as the mean ± standard deviation. Results obtained for DiI-EbolaΔVP30 (blue bars) and DiI-VSVΔ*G-G (red bars) are shown for comparison. (C) Effect of macropinocytosis inhibitors on the co-localization of DiI-labeled VSV pseudovirions with eGFP-Rab7-positive vesicles. Vero cells expressing eGFP-Rab7 were pretreated with CytoD, Wort, LY294002 or EIPA for 30 min at 37°C; control cells were treated with DMSO. DiI-labeled VSVΔ*G-GP (green bars) or VSVΔ*G-G (red bars) were adsorbed to cells for 30 min on ice. The cells were then incubated at 37°C in the presence of inhibitors for 2 h. Co-localization of DiI-pseudovirions with eGFP-Rab7-positive vesicles was analyzed as described in the Materials and Methods . Experiments were carried out in triplicate and the results are presented as the mean ± standard deviation. (D) Effect of macropinocytosis inhibitors on the infectivity of VSV pseudovirions. Vero cells were treated with individual inhibitors for 30 min at 37°C and infected with VSVΔG*-GP (green bars) or VSVΔG*-G (red bars) in the presence of the inhibitor. 1 h post-infection, surface-bound virions were removed by trypsin and the cells were cultured for 24 h in the absence of inhibitors. The infection efficiency of each pseudovirus was determined by measuring the number of GFP-positive cells using with conventional fluorescent microscope. Each experiment was performed in triplicate and the relative infection efficiencies are presented as the mean ± SD.

Techniques Used: Labeling, Expressing, Incubation, Laser-Scanning Microscopy, Standard Deviation, Infection, Cell Culture, Microscopy

Effect of macropinocytosis inhibitors on the co-localization of DiI-labeled viral particles with Rab7-positive vesicles. Vero cells expressing eGFP-Rab7 were pretreated with cytochalasin D (CytoD), wortmannin (Wort), LY294002, or EIPA for 30 min at 37°C as described in the Materials and Methods . DiI-EbolaΔVP30 virions, DiI-Ebola VLPs and DiI-influenza virus were adsorbed to the cells for 30 min on ice. The cells were then incubated at 37°C in the presence of inhibitors for 2 h. As a control, DMSO-treated cells were incubated with labeled EBOV particles. Representative images of the co-localization of DiI-EbolaΔVP30 virions with eGFP-Rab7 acquired 2 h after the temperature shift are shown (A). DiI-labeled EbolaΔVP30 virions that co-localize with eGFP-Rab7-positive vesicles are indicated by arrows. Scale bars, 10 µm. (B) shows a graphic representation of the data. The number of DiI-labeled EbolaΔVP30 virions (blue bars), Ebola VLPs (yellow bars) and influenza virions (red bars) co-localized with eGFP-Rab7-positive vesicles was measured in 10 individual cells and the percentage of co-localization in the total DiI-virions is shown for each time point. Each experiment was carried out in triplicate and the results are presented as the mean ± SD.
Figure Legend Snippet: Effect of macropinocytosis inhibitors on the co-localization of DiI-labeled viral particles with Rab7-positive vesicles. Vero cells expressing eGFP-Rab7 were pretreated with cytochalasin D (CytoD), wortmannin (Wort), LY294002, or EIPA for 30 min at 37°C as described in the Materials and Methods . DiI-EbolaΔVP30 virions, DiI-Ebola VLPs and DiI-influenza virus were adsorbed to the cells for 30 min on ice. The cells were then incubated at 37°C in the presence of inhibitors for 2 h. As a control, DMSO-treated cells were incubated with labeled EBOV particles. Representative images of the co-localization of DiI-EbolaΔVP30 virions with eGFP-Rab7 acquired 2 h after the temperature shift are shown (A). DiI-labeled EbolaΔVP30 virions that co-localize with eGFP-Rab7-positive vesicles are indicated by arrows. Scale bars, 10 µm. (B) shows a graphic representation of the data. The number of DiI-labeled EbolaΔVP30 virions (blue bars), Ebola VLPs (yellow bars) and influenza virions (red bars) co-localized with eGFP-Rab7-positive vesicles was measured in 10 individual cells and the percentage of co-localization in the total DiI-virions is shown for each time point. Each experiment was carried out in triplicate and the results are presented as the mean ± SD.

Techniques Used: Labeling, Expressing, Incubation

17) Product Images from "Macropinocytic entry of isolated mitochondria in epidermal growth factor-activated human osteosarcoma cells"

Article Title: Macropinocytic entry of isolated mitochondria in epidermal growth factor-activated human osteosarcoma cells

Journal: Scientific Reports

doi: 10.1038/s41598-017-13227-0

Internalisation of mitochondria occurs by endocytosis in HOS cells, with a macropinocytic activator EGF upregulating the process. ( A – F ) Internalisation of EGFP-labelled mitochondria in HOS cells, as quantified by FACS, in the presence and absence of the endocytic inhibitors: chlorpromazine (CPZ) (100 μm) ( A ), MβCD (5 mM) ( B ), dynasore (120 μm) ( C ), EIPA (50 μm) ( D ), wortmannin (300 nM) ( E ) and latrunculin A (0.5 µM) ( F ). ( G , H ) EGF upregulates mitochondrial internalisation. Internalisation of EGFP-labelled mitochondria by HOS cells, as quantified by FACS, in the presence and absence of 50 nM and 100 nM EGF ( G ), 50 nM FGF and 100 nM FGF ( H ). The Y axis shows the percentage of green fluorescent cells normalised to the numbers of green cells in a control sample (cells incubated with only EGFP-labelled mitochondria for 90 min). The number of green fluorescent cells in this control sample was taken to represent 100%. The green fluorescence in all samples for inhibitor assays of mitochondrial internalisation were normalised to levels in their respective control samples. Data shown as mean values +/− s.e.m. n = 3 ( A – D and F – H ) n = 6 ( E ). *p
Figure Legend Snippet: Internalisation of mitochondria occurs by endocytosis in HOS cells, with a macropinocytic activator EGF upregulating the process. ( A – F ) Internalisation of EGFP-labelled mitochondria in HOS cells, as quantified by FACS, in the presence and absence of the endocytic inhibitors: chlorpromazine (CPZ) (100 μm) ( A ), MβCD (5 mM) ( B ), dynasore (120 μm) ( C ), EIPA (50 μm) ( D ), wortmannin (300 nM) ( E ) and latrunculin A (0.5 µM) ( F ). ( G , H ) EGF upregulates mitochondrial internalisation. Internalisation of EGFP-labelled mitochondria by HOS cells, as quantified by FACS, in the presence and absence of 50 nM and 100 nM EGF ( G ), 50 nM FGF and 100 nM FGF ( H ). The Y axis shows the percentage of green fluorescent cells normalised to the numbers of green cells in a control sample (cells incubated with only EGFP-labelled mitochondria for 90 min). The number of green fluorescent cells in this control sample was taken to represent 100%. The green fluorescence in all samples for inhibitor assays of mitochondrial internalisation were normalised to levels in their respective control samples. Data shown as mean values +/− s.e.m. n = 3 ( A – D and F – H ) n = 6 ( E ). *p

Techniques Used: FACS, Incubation, Fluorescence

18) Product Images from "Exploring the role of stromal osmoregulation in cancer and disease using executable modelling"

Article Title: Exploring the role of stromal osmoregulation in cancer and disease using executable modelling

Journal: Nature Communications

doi: 10.1038/s41467-018-05414-y

Application of the qualitative network to murine embryonic fibroblasts. Expansion of the QN to p53 −/− , Kras G12D/+ (HET) and p53 −/− , Kras G12D/G12D (HOM) MEFs. a Transport proteins deregulated within HOM MEFs when compared to HET MEFs. Proteins are upregulated (red arrows), downregulated (blue arrows), or unchanged (grey). b Phenotype change for MEFS when HOM MEFs are compared to HET MEFs. The model output represents the physiological behaviour predicted by the model when proteins from ( a ) are deregulated in the model in the same manner as in the gene array. Experimental phenotype represents behaviour reported previously 42 , where it is known. The model predicts that attachment will be significantly different between the two cell types, but viability and cell size will remain unchanged. c Effects of application of channel inhibitors to HOM MEFs in vitro showing application of 10 µM DIDs for 72 h, resulting in decreased cellular attachment (left), and application of 10 nM EIPA ± 10 µM DIDs (right). Data are technical replicates. Dotted lines represent calculated Bliss independence values. d Effects of application of 10 µM DIDs ± 10 nM EIPA on viability in HOM MEFs. e Effects of application of 10 µM DIDs ± 10 nM AHCL on attachment in HOM MEFs. f Effects of application of 10 µM DIDs ± 10 nM AHCL on viability in HOM MEFs.* P
Figure Legend Snippet: Application of the qualitative network to murine embryonic fibroblasts. Expansion of the QN to p53 −/− , Kras G12D/+ (HET) and p53 −/− , Kras G12D/G12D (HOM) MEFs. a Transport proteins deregulated within HOM MEFs when compared to HET MEFs. Proteins are upregulated (red arrows), downregulated (blue arrows), or unchanged (grey). b Phenotype change for MEFS when HOM MEFs are compared to HET MEFs. The model output represents the physiological behaviour predicted by the model when proteins from ( a ) are deregulated in the model in the same manner as in the gene array. Experimental phenotype represents behaviour reported previously 42 , where it is known. The model predicts that attachment will be significantly different between the two cell types, but viability and cell size will remain unchanged. c Effects of application of channel inhibitors to HOM MEFs in vitro showing application of 10 µM DIDs for 72 h, resulting in decreased cellular attachment (left), and application of 10 nM EIPA ± 10 µM DIDs (right). Data are technical replicates. Dotted lines represent calculated Bliss independence values. d Effects of application of 10 µM DIDs ± 10 nM EIPA on viability in HOM MEFs. e Effects of application of 10 µM DIDs ± 10 nM AHCL on attachment in HOM MEFs. f Effects of application of 10 µM DIDs ± 10 nM AHCL on viability in HOM MEFs.* P

Techniques Used: In Vitro, Cell Attachment Assay

19) Product Images from "Vaccinia virus strains use distinct forms of macropinocytosis for host-cell entry"

Article Title: Vaccinia virus strains use distinct forms of macropinocytosis for host-cell entry

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

doi: 10.1073/pnas.1004618107

IHD-J MVs enter cells by PS-mediated macropinocytosis. ( A–C ) Cells were pretreated with staurosporine (Stau), genistein (Geni), wortmannin (Wort), rottlerin (Rott), Gö6979, IPA-3 and PIR 3.5, or EIPA and infected with IHD-J- or WR-EGFP-MVs.
Figure Legend Snippet: IHD-J MVs enter cells by PS-mediated macropinocytosis. ( A–C ) Cells were pretreated with staurosporine (Stau), genistein (Geni), wortmannin (Wort), rottlerin (Rott), Gö6979, IPA-3 and PIR 3.5, or EIPA and infected with IHD-J- or WR-EGFP-MVs.

Techniques Used: Indirect Immunoperoxidase Assay, Infection

20) Product Images from "The J-Domain of Heat Shock Protein 40 Can Enhance the Transduction Efficiency of Arginine-Rich Cell-Penetrating Peptides"

Article Title: The J-Domain of Heat Shock Protein 40 Can Enhance the Transduction Efficiency of Arginine-Rich Cell-Penetrating Peptides

Journal: BioMed Research International

doi: 10.1155/2015/698067

Macropinocytosis is involved in the cell-penetration pathway of PTD-J-DsRed. Huh-7 cells were pretreated with filipin (5 μ g/mL), EIPA (100 μ M), or cytochalasin D (10 μ M) for 1 hour before the treatment of the PTD-J-DsRed recombinant protein (40 μ g/mL) for 2 hours. The fluorescence values of 100 μ L cleared lysate samples were measured (three experiments were performed and the P values of filipin, cytochalasin D, and EIPA data relative to the control are 0.0257, 0.000812, and 0.000165, resp.).
Figure Legend Snippet: Macropinocytosis is involved in the cell-penetration pathway of PTD-J-DsRed. Huh-7 cells were pretreated with filipin (5 μ g/mL), EIPA (100 μ M), or cytochalasin D (10 μ M) for 1 hour before the treatment of the PTD-J-DsRed recombinant protein (40 μ g/mL) for 2 hours. The fluorescence values of 100 μ L cleared lysate samples were measured (three experiments were performed and the P values of filipin, cytochalasin D, and EIPA data relative to the control are 0.0257, 0.000812, and 0.000165, resp.).

Techniques Used: Recombinant, Fluorescence

21) Product Images from "Rapid elevation of sodium transport through insulin is mediated by AKT in alveolar cells"

Article Title: Rapid elevation of sodium transport through insulin is mediated by AKT in alveolar cells

Journal: Physiological Reports

doi: 10.1002/phy2.269

Insulin enhances benzamil‐sensitive I SC . Effects of insulin (200 nmol/L) on benzamil‐ and EIPA‐sensitive I SC of FDLE cell monolayers. (A) 10 μ mol/L benzamil ( n = 24 and 25, ** P
Figure Legend Snippet: Insulin enhances benzamil‐sensitive I SC . Effects of insulin (200 nmol/L) on benzamil‐ and EIPA‐sensitive I SC of FDLE cell monolayers. (A) 10 μ mol/L benzamil ( n = 24 and 25, ** P

Techniques Used:

22) Product Images from "Dual Targeting of BRAF and mTOR Signaling in Melanoma Cells with Pyridinyl Imidazole Compounds"

Article Title: Dual Targeting of BRAF and mTOR Signaling in Melanoma Cells with Pyridinyl Imidazole Compounds

Journal: Cancers

doi: 10.3390/cancers12061516

Vacuole-like vesicles induced by pyridinyl imidazole compounds in melanoma cells have an endolysosomal origin. A375 cells were treated with SB202190 (SB202; 15 μM) or the equivalent amount of vehicle (DMSO) in control. ( A ) Cells were treated with SB202190 for 20 h and stained with acridine orange (5 µg/mL) for 15 min. Scale bar: 50 µm. The graph shows the relative change in the yellow stained area in response to SB202190. Similar results were obtained in two independent experiments. ( B ) Cells were treated with SB202190 for 20 h and stained with LysoTracker Green (50 nM) for 15 min. Scale bar: 50 µm. The graph shows the relative change of the LysoTracker Green signal in response to SB202190. Similar results were obtained in two independent experiments. ( C ) The confocal microscopy detection of endogenous early endosomal marker RAB5 after 24 h treatment with SB202190. Scale bar: 20 µm. The graph shows the number of RAB5-positive vacuoles per cell with a diameter larger than 1 µm. Similar results were obtained in three independent experiments. ( D ) Deconvolved wide-field fluorescence microscopy imaging of EGFP-tagged late endosomal/lysosomal marker RAB7A after 24 h SB202190 treatment. Scale bar: 10 µm. The diameter of RAB7-positive structures was determined, and the number of vacuoles larger than 1 μm per cell was plotted in the graph. The experiment was performed three times with similar results. ( E ) Effect on vacuolization was visualized by bright-field images in cells treated for 20 h with SB202190 alone and in combination with sucrose (0.5 M) or EIPA (50 μM). Scale bar: 50 µm. The graph shows the number of vacuoles per cell, quantified using ImageJ/Fiji (find maxima—bright spots above a certain threshold). Dying rounded cells were excluded from the analysis. The experiment was repeated three times with a similar response to the addition of sucrose and EIPA.
Figure Legend Snippet: Vacuole-like vesicles induced by pyridinyl imidazole compounds in melanoma cells have an endolysosomal origin. A375 cells were treated with SB202190 (SB202; 15 μM) or the equivalent amount of vehicle (DMSO) in control. ( A ) Cells were treated with SB202190 for 20 h and stained with acridine orange (5 µg/mL) for 15 min. Scale bar: 50 µm. The graph shows the relative change in the yellow stained area in response to SB202190. Similar results were obtained in two independent experiments. ( B ) Cells were treated with SB202190 for 20 h and stained with LysoTracker Green (50 nM) for 15 min. Scale bar: 50 µm. The graph shows the relative change of the LysoTracker Green signal in response to SB202190. Similar results were obtained in two independent experiments. ( C ) The confocal microscopy detection of endogenous early endosomal marker RAB5 after 24 h treatment with SB202190. Scale bar: 20 µm. The graph shows the number of RAB5-positive vacuoles per cell with a diameter larger than 1 µm. Similar results were obtained in three independent experiments. ( D ) Deconvolved wide-field fluorescence microscopy imaging of EGFP-tagged late endosomal/lysosomal marker RAB7A after 24 h SB202190 treatment. Scale bar: 10 µm. The diameter of RAB7-positive structures was determined, and the number of vacuoles larger than 1 μm per cell was plotted in the graph. The experiment was performed three times with similar results. ( E ) Effect on vacuolization was visualized by bright-field images in cells treated for 20 h with SB202190 alone and in combination with sucrose (0.5 M) or EIPA (50 μM). Scale bar: 50 µm. The graph shows the number of vacuoles per cell, quantified using ImageJ/Fiji (find maxima—bright spots above a certain threshold). Dying rounded cells were excluded from the analysis. The experiment was repeated three times with a similar response to the addition of sucrose and EIPA.

Techniques Used: Staining, Confocal Microscopy, Marker, Fluorescence, Microscopy, Imaging

23) Product Images from "Hyperosmotic Stress Induces Nuclear Factor-?B Activation and Interleukin-8 Production in Human Intestinal Epithelial Cells"

Article Title: Hyperosmotic Stress Induces Nuclear Factor-?B Activation and Interleukin-8 Production in Human Intestinal Epithelial Cells

Journal: The American Journal of Pathology

doi:

Treatment of HT-29 cells with the selective NHE inhibitors amiloride ( A ), MIA ( B ), or EIPA ( C ) suppresses mannitol-induced IL-8 production. Treatment of HT-29 cells with the nonamiloride NHE inhibitors cimetidine ( D ), clonidine ( E ), or harmaline ( F ), reproduces the suppressive effect of selective NHE inhibitors on the production of IL-8 by mannitol-induced HT-29 cells. When harmaline was used, supernatants for IL-8 measurement were taken 4 hours after the hyperosmotic challenge. In the case of amiloride, MIA, EIPA, cimetidine, and clonidine, IL-8 levels were measured from supernatants obtained 18 hours after the hyperosmotic challenge. Hyperosmotic medium prepared by the addition of 100 mmol/L of mannitol to isosmolar growth medium was used for hyperosmotic stimulation. Data are mean ± SEM of n = 6 to 12 wells from two different experiments. *, P
Figure Legend Snippet: Treatment of HT-29 cells with the selective NHE inhibitors amiloride ( A ), MIA ( B ), or EIPA ( C ) suppresses mannitol-induced IL-8 production. Treatment of HT-29 cells with the nonamiloride NHE inhibitors cimetidine ( D ), clonidine ( E ), or harmaline ( F ), reproduces the suppressive effect of selective NHE inhibitors on the production of IL-8 by mannitol-induced HT-29 cells. When harmaline was used, supernatants for IL-8 measurement were taken 4 hours after the hyperosmotic challenge. In the case of amiloride, MIA, EIPA, cimetidine, and clonidine, IL-8 levels were measured from supernatants obtained 18 hours after the hyperosmotic challenge. Hyperosmotic medium prepared by the addition of 100 mmol/L of mannitol to isosmolar growth medium was used for hyperosmotic stimulation. Data are mean ± SEM of n = 6 to 12 wells from two different experiments. *, P

Techniques Used:

24) Product Images from "Mechanistic studies of intracellular delivery of proteins by cell-penetrating peptides in cyanobacteria"

Article Title: Mechanistic studies of intracellular delivery of proteins by cell-penetrating peptides in cyanobacteria

Journal: BMC Microbiology

doi: 10.1186/1471-2180-13-57

The mechanism of the CPP - mediated GFP delivery in 6803 and 7942 strains of cyanobacteria. Cells were treated with NEM and R9/GFP mixtures in the absence or presence of CytD, EIPA, or wortmannin (Wort), as indicated. Results were observed in the GFP channel using a confocal microscope, and fluorescent intensities were analyzed by the UN-SCAN-IT software. Data are presented as mean ± SD from three independent experiments. Significant differences of P
Figure Legend Snippet: The mechanism of the CPP - mediated GFP delivery in 6803 and 7942 strains of cyanobacteria. Cells were treated with NEM and R9/GFP mixtures in the absence or presence of CytD, EIPA, or wortmannin (Wort), as indicated. Results were observed in the GFP channel using a confocal microscope, and fluorescent intensities were analyzed by the UN-SCAN-IT software. Data are presented as mean ± SD from three independent experiments. Significant differences of P

Techniques Used: Conditioned Place Preference, Microscopy, Software

Cell viability of the R9 / GFP delivery system in the presence of uptake modulators. ( A ) The MTT assay. The 6803 strain of cyanobacteria was treated with BG-11 medium as a negative control, or treated with 100% methanol as a positive control. In the presence of NEM, cells were treated with R9/GFP complexes in the presence of CytD, EIPA, or wortmannin (Wort), respectively, and analyzed by the MTT assay. Significant differences were determined at P
Figure Legend Snippet: Cell viability of the R9 / GFP delivery system in the presence of uptake modulators. ( A ) The MTT assay. The 6803 strain of cyanobacteria was treated with BG-11 medium as a negative control, or treated with 100% methanol as a positive control. In the presence of NEM, cells were treated with R9/GFP complexes in the presence of CytD, EIPA, or wortmannin (Wort), respectively, and analyzed by the MTT assay. Significant differences were determined at P

Techniques Used: MTT Assay, Negative Control, Positive Control

25) Product Images from "Internalization of the opioid growth factor, [Met5]-enkephalin, is dependent on clathrin-mediated endocytosis for downregulation of cell proliferation"

Article Title: Internalization of the opioid growth factor, [Met5]-enkephalin, is dependent on clathrin-mediated endocytosis for downregulation of cell proliferation

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

doi: 10.1152/ajpregu.00318.2010

RhoOGF uptake is impaired by inhibitors targeting different endocytic pathways. Cells were preincubated with serum-free medium for 30 min at 37°C, and treated with monodansylcadaverine (MDC) for 1 h, EIPA for 30 min, methyl-β-cyclodextrin (MβCD) for 30 min, or both MDC and EIPA together for 30 min; some cells remained untreated [control (Ctrl)]. Both RhoOGF (10 −5 M) and transferrin (Tf; 10 μg/ml) were added to cell cultures for 30 min in the presence of inhibitors. Cells were stained with Hoechst and analyzed by Epi-F (RhoOGF, Hoechst, or Tf) or DIC microscopy. Bar = 10 μm.
Figure Legend Snippet: RhoOGF uptake is impaired by inhibitors targeting different endocytic pathways. Cells were preincubated with serum-free medium for 30 min at 37°C, and treated with monodansylcadaverine (MDC) for 1 h, EIPA for 30 min, methyl-β-cyclodextrin (MβCD) for 30 min, or both MDC and EIPA together for 30 min; some cells remained untreated [control (Ctrl)]. Both RhoOGF (10 −5 M) and transferrin (Tf; 10 μg/ml) were added to cell cultures for 30 min in the presence of inhibitors. Cells were stained with Hoechst and analyzed by Epi-F (RhoOGF, Hoechst, or Tf) or DIC microscopy. Bar = 10 μm.

Techniques Used: Staining, Microscopy

26) Product Images from "Cuprous oxide nanoparticle-inhibited melanoma progress by targeting melanoma stem cells"

Article Title: Cuprous oxide nanoparticle-inhibited melanoma progress by targeting melanoma stem cells

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S130753

The CD271 +/high melanoma cells could take in more CONPs than CD271 −/low through clathrin-mediated endocytosis. Notes: ( A ) Wortmannin and chlorpromazine rather than dynasore, MβCD and EIPA could promote cell viability. Specific endocytosis inhibitors increased the cell viability of A375 cells treated by CONPs. After being pretreated with 5 kinds of endocytosis inhibitors respectively at different concentrations, A375 cells were cultivated in CONP medium (3.5 μg/mL) for 48 hours. Cells in control group were incubated in CONP medium without pretreatment of inhibitors. CCK8 assay was used to validate effect of inhibitor, n=3. ( B ) Chlorpromazine (5 μM) declined the content of Cu of A375 cells through inhibiting absorption of A375 cells. A375 cells were incubated in CONP medium (3.5 μg/mL) for 3, 6 and 9 hours after chlorpromazine treatment for 2 hours. Intracellular Cu means the total content of Cu in 10 7 A375 cells, n=3. ( C ) Chlorpromazine (5 μM) could significantly decline the content of Cu in CD271 +/high cells. After treating A375 as ( B ), A375 cells were separated into CD271 + /high cells and CD271 −/low cells by FACS and the intracellular Cu was tested by GF-AAS, n=3. ( D ) After cells were treated with CONPs (3.5 μg/mL) for 24 hours, one part of cells was harvested for GF-AAS test, whereas the other part of cells was transferred into CONP-free medium and harvested after 24 and 48 hours, respectively for GF-AAS test. ( E ) The expression of MITF and SOX10 decreased in CD271 +/high and CD271 −/low cells after CONP treatment. A375 cells were treated with CONPs (1.75 μg/mL) for 72 hours, collected and separated into CD271 +/high and CD271 −/low cells by FACS for Western blotting. The error bars represent ± standard deviation (* P
Figure Legend Snippet: The CD271 +/high melanoma cells could take in more CONPs than CD271 −/low through clathrin-mediated endocytosis. Notes: ( A ) Wortmannin and chlorpromazine rather than dynasore, MβCD and EIPA could promote cell viability. Specific endocytosis inhibitors increased the cell viability of A375 cells treated by CONPs. After being pretreated with 5 kinds of endocytosis inhibitors respectively at different concentrations, A375 cells were cultivated in CONP medium (3.5 μg/mL) for 48 hours. Cells in control group were incubated in CONP medium without pretreatment of inhibitors. CCK8 assay was used to validate effect of inhibitor, n=3. ( B ) Chlorpromazine (5 μM) declined the content of Cu of A375 cells through inhibiting absorption of A375 cells. A375 cells were incubated in CONP medium (3.5 μg/mL) for 3, 6 and 9 hours after chlorpromazine treatment for 2 hours. Intracellular Cu means the total content of Cu in 10 7 A375 cells, n=3. ( C ) Chlorpromazine (5 μM) could significantly decline the content of Cu in CD271 +/high cells. After treating A375 as ( B ), A375 cells were separated into CD271 + /high cells and CD271 −/low cells by FACS and the intracellular Cu was tested by GF-AAS, n=3. ( D ) After cells were treated with CONPs (3.5 μg/mL) for 24 hours, one part of cells was harvested for GF-AAS test, whereas the other part of cells was transferred into CONP-free medium and harvested after 24 and 48 hours, respectively for GF-AAS test. ( E ) The expression of MITF and SOX10 decreased in CD271 +/high and CD271 −/low cells after CONP treatment. A375 cells were treated with CONPs (1.75 μg/mL) for 72 hours, collected and separated into CD271 +/high and CD271 −/low cells by FACS for Western blotting. The error bars represent ± standard deviation (* P

Techniques Used: Incubation, CCK-8 Assay, FACS, Atomic Absorption Spectroscopy, Expressing, Western Blot, Standard Deviation

27) Product Images from "The invasiveness of human cervical cancer associated to the function of NaV1.6 channels is mediated by MMP-2 activity"

Article Title: The invasiveness of human cervical cancer associated to the function of NaV1.6 channels is mediated by MMP-2 activity

Journal: Scientific Reports

doi: 10.1038/s41598-018-31364-y

The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; Leupeptin, 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P
Figure Legend Snippet: The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; Leupeptin, 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P

Techniques Used: Activity Assay, Transfection, Staining

28) Product Images from "Host Cell Entry of Respiratory Syncytial Virus Involves Macropinocytosis Followed by Proteolytic Activation of the F Protein"

Article Title: Host Cell Entry of Respiratory Syncytial Virus Involves Macropinocytosis Followed by Proteolytic Activation of the F Protein

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003309

RSV F requires post endocytic activation. (A). HeLa cells were pretreated with dec-RVKR-CMK, α -PDX or leupeptin at indicated concentration for 1 h before experiment and inhibitors were continuously present during following steps of the experiment. Cells were infected with RSV or RSVΔGΔSH for 6 h before FACS analysis of GFP expressing cells. (B). HeLa cells were infected with RSV or RSVΔGΔSH for 1 h. Virus inoculum was replaced with medium containing 100 µM dec-RVKR-CMK and incubated for 6 before FACS analysis of GFP expressing cells. (C). Versene detached HeLa cells were pretreated with solvent (MOCK), dec-RVKR-CMK or EIPA at indicated concentrations and inhibitors were continuously present during following steps of the experiment. RSV-R18/DiOC or RSVΔGΔSH-R18/DiOC (moi ∼5) was bound to cells at 4°C. Unbound virus was removed and cells were incubated at 37°C for 2 h in the presence of inhibitor. Cells were fixed and the MFI of DiOC fluorescence measured by FACS and normalized to mock no inhibitor controls. (D). RSV F protein (574 aa) is proteolytically processed by furin like protease at the two sites (A aa-106 and B aa-136) to generate disulfide bonds linked F1+F2 and small peptide p27 (aa sequence depicted above). At the N-terminus of F1 is a FP (fusion peptide) and at the C-terminus TM (transmembrane domain), numbers indicate aa position and red underlines specify peptide sequences detected in mass spectrometry. (E). Proteomic analysis of HEp-2 cells and purified RSV particles. The N-terminal sequence of the p27 peptide (F protein) was quantified by a targeted mass spectrometry based on the selected reaction monitoring (SRM). Representative SRM peaks of peptides (left) FMNYTLNNAKKTNVTLSK 3+ and (right) ELPRFMNYTLNNAK 3+ peptides, corresponding to the aa 113–131 and 109–123 of F protein, respectively. Different SRM transitions for a peptide shown in different colors (see supporting information Table S1 ). The bar graphs show the results of the targeted peptide quantitation, presented as the sum of the areas of all the SRM peaks for a given peptide. Where no peptide peak was detectable, noise values were reported as a reference. RT retention time, and Cps counts per second. (F). HeLa cells were pretreated or MOCK treated with dec-RVKR-CMK, α-PDX, or leupeptin at indicated concentration. RSV (input control in the first line) was bound for 1 h in cold (B-binding) or after wash-away unbound virus was internalized for 1.5 h at 37°C before processing (I-internalized). Lysed cell samples were resolved by SDS-PAGE and blots were probed with anti-F1 antibody. (G). RSV was bound for 1 h in cold to HeLa cells; unbound virus wash away and cells were placed at 37°C for indicated times before, lysis, SDS-PAGE, and processing for western blot probed with anti-F1 antibody.
Figure Legend Snippet: RSV F requires post endocytic activation. (A). HeLa cells were pretreated with dec-RVKR-CMK, α -PDX or leupeptin at indicated concentration for 1 h before experiment and inhibitors were continuously present during following steps of the experiment. Cells were infected with RSV or RSVΔGΔSH for 6 h before FACS analysis of GFP expressing cells. (B). HeLa cells were infected with RSV or RSVΔGΔSH for 1 h. Virus inoculum was replaced with medium containing 100 µM dec-RVKR-CMK and incubated for 6 before FACS analysis of GFP expressing cells. (C). Versene detached HeLa cells were pretreated with solvent (MOCK), dec-RVKR-CMK or EIPA at indicated concentrations and inhibitors were continuously present during following steps of the experiment. RSV-R18/DiOC or RSVΔGΔSH-R18/DiOC (moi ∼5) was bound to cells at 4°C. Unbound virus was removed and cells were incubated at 37°C for 2 h in the presence of inhibitor. Cells were fixed and the MFI of DiOC fluorescence measured by FACS and normalized to mock no inhibitor controls. (D). RSV F protein (574 aa) is proteolytically processed by furin like protease at the two sites (A aa-106 and B aa-136) to generate disulfide bonds linked F1+F2 and small peptide p27 (aa sequence depicted above). At the N-terminus of F1 is a FP (fusion peptide) and at the C-terminus TM (transmembrane domain), numbers indicate aa position and red underlines specify peptide sequences detected in mass spectrometry. (E). Proteomic analysis of HEp-2 cells and purified RSV particles. The N-terminal sequence of the p27 peptide (F protein) was quantified by a targeted mass spectrometry based on the selected reaction monitoring (SRM). Representative SRM peaks of peptides (left) FMNYTLNNAKKTNVTLSK 3+ and (right) ELPRFMNYTLNNAK 3+ peptides, corresponding to the aa 113–131 and 109–123 of F protein, respectively. Different SRM transitions for a peptide shown in different colors (see supporting information Table S1 ). The bar graphs show the results of the targeted peptide quantitation, presented as the sum of the areas of all the SRM peaks for a given peptide. Where no peptide peak was detectable, noise values were reported as a reference. RT retention time, and Cps counts per second. (F). HeLa cells were pretreated or MOCK treated with dec-RVKR-CMK, α-PDX, or leupeptin at indicated concentration. RSV (input control in the first line) was bound for 1 h in cold (B-binding) or after wash-away unbound virus was internalized for 1.5 h at 37°C before processing (I-internalized). Lysed cell samples were resolved by SDS-PAGE and blots were probed with anti-F1 antibody. (G). RSV was bound for 1 h in cold to HeLa cells; unbound virus wash away and cells were placed at 37°C for indicated times before, lysis, SDS-PAGE, and processing for western blot probed with anti-F1 antibody.

Techniques Used: Activation Assay, Concentration Assay, Infection, FACS, Expressing, Incubation, Fluorescence, Sequencing, Mass Spectrometry, Purification, Quantitation Assay, Binding Assay, SDS Page, Lysis, Western Blot

RSV enters human bronchial epithelial cells by macropinocytosis. (A). Human bronchial epithelial cells (16HBE14o) were polarized for 9 days and then infected with rgRSV for 20 h. After fixation cells were stained with anti-ZO-1-AF594 antibody (red) and TO-PRO-3 nuclear dye (blue) to examine polarization of the cell monolayer. RSV infection was visualized by GFP expression (green). (B) Polarized 16HBE14o cells were pretreated with solvent (MOCK), low or high concentrations of dynasore (10, 20 µM), cytochalasin D (CytoD 1.5, 3 µM), jasplakinolide (Jas 0.5, 1 µM), IPA-3 (40, 80 µM), genistein (Gen 50, 100 µM), iressa (10, 20 µM), Ly294002, EIPA (40, 80 µM), and dec-RVKR-CMK (50, 100 µM). The cells were infected with RSV for 20 h in the presence of the inhibitors, fixed and counterstained with DAPI. RSV infection was quantified by an image-based approach and normalized to mock infected controls.
Figure Legend Snippet: RSV enters human bronchial epithelial cells by macropinocytosis. (A). Human bronchial epithelial cells (16HBE14o) were polarized for 9 days and then infected with rgRSV for 20 h. After fixation cells were stained with anti-ZO-1-AF594 antibody (red) and TO-PRO-3 nuclear dye (blue) to examine polarization of the cell monolayer. RSV infection was visualized by GFP expression (green). (B) Polarized 16HBE14o cells were pretreated with solvent (MOCK), low or high concentrations of dynasore (10, 20 µM), cytochalasin D (CytoD 1.5, 3 µM), jasplakinolide (Jas 0.5, 1 µM), IPA-3 (40, 80 µM), genistein (Gen 50, 100 µM), iressa (10, 20 µM), Ly294002, EIPA (40, 80 µM), and dec-RVKR-CMK (50, 100 µM). The cells were infected with RSV for 20 h in the presence of the inhibitors, fixed and counterstained with DAPI. RSV infection was quantified by an image-based approach and normalized to mock infected controls.

Techniques Used: Infection, Staining, Expressing, Indirect Immunoperoxidase Assay

29) Product Images from "Ebolavirus Is Internalized into Host Cells via Macropinocytosis in a Viral Glycoprotein-Dependent Manner"

Article Title: Ebolavirus Is Internalized into Host Cells via Macropinocytosis in a Viral Glycoprotein-Dependent Manner

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1001121

Macropinocytosis-associated events occur during Ebola virion internalization. (A) The effect of the internalization of DiI-labeled Ebola VLPs on dextran uptake. Vero cells were incubated with 0.5 mg/ml Alexa Fluor 647-Dex Mw 10K in the absence or presence of Ebola VLPs for 60 min at 37°C. The uptake of Alexa Fluor 647-Dex Mw 10K was analyzed by using flow cytometry. The effect of EIPA pretreatment was assessed in parallel. Each experiment was performed in triplicate and the results are presented as the mean ± SD. (B) Co-localization of internalized DiI-labeled Ebola VLPs and Dex Mw 10K. DiI-Ebola VLPs were adsorbed to Vero cells for 30 min on ice. The cells were cultured in the presence of 0.5 mg/ml Alexa Fluor 647-Dex Mw 10K for 10 min at 37°C. Co-localization of DiI-virions (red) and Alexa Fluor-Dex Mw 10K (green) was analyzed by using confocal laser scanning microscope. Co-localized virions are shown by arrows. Outlines of individual cells were drawn. Scale bar, 10 µm. (C) Effect of a dominant-negative form of Rac1 on the internalization of DiI-labeled Ebola virions. The eGFP-fused, wild-type Rac1 (wtRac1, upper panels) or the dominant-negative form of Rac1 (dnRac1, lower panels) was expressed in Vero cells. DiI-labeled Ebola VLPs were adsorbed to the cells for 30 min on ice. After incubation for 2 h at 37°C, surface-bound virions were removed by trypsin and the internalization of DiI-virions was analyzed by using confocal laser scanning microscope. Expression of dnRac1 interfered with the internalization of Alexa Fluor 647-Dex Mw 10K (blue; lower middle panel), attesting to its functionality. Scale bars, 10 µm. (D) Quantitative analysis of the internalization of DiI-labeled Ebola virions in wtRac1 or dnRac1-expressed Vero cells. The internalized DiI-virions were measured in 10 individual wtRac1 or dnRac1-expressed cells. Each experiment was performed in triplicate and the results are presented as the mean ± SD. (E) Effect of PKC inhibitors on the internalization of DiI-labeled Ebola virions. Vero cells were treated with DMSO or staurosporine (Stauro) for 30 min at 37°C. Labeled Ebola VLPs were adsorbed to the cells for 30 min on ice and incubated for 2 h at 37°C in the absence or presence of inhibitor. Surface-bound virions were removed by trypsin and the internalization of DiI-virions was analyzed by using confocal laser scanning microscope. The internalized DiI-virions were analyzed in 10 individual DMSO- or staurosporine-treated cells (red bars). The efficiency of Alexa Fluor-Dex Mw 10K uptake in inhibitor-treated cells was measured by using flow cytometry (blue bars). Each experiment was performed in triplicate and relative uptake efficiencies are presented as the mean ± SD (red bars). Staurosporine treatment interfered with the internalization of Alexa Fluor 633-Tf (blue bars), attesting to its functionality. (F) The down-regulation of Cdc42 and Pak1 by siRNA. The efficiencies of Cdc42 and Pak1 knock-down were assessed by use of RT-PCR. Total cellular RNA was isolated from siRNA-transfected Vero cells 48 h post-transfection by using the TRI reagent (Sigma-Aldrich) according to the manufacturer's instructions. cDNA synthesis was performed with Molony murine leukemia virus RTase using a random hexamer (Invitrogen) according to the manufacturer's protocol. PCR was carried out for 25–30 cycles consisting of a DNA denaturing step for 30 s at 94°C, annealing for 30 s at 55°C, and extension for 1 min at 72°C by use of Taq DNA polymerase (Promega). Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as an endogenous control. The oligonucleotides used for amplification of individual genes are shown in Table S1 . (G) Effect of down-regulation of Cdc42 and Pak1 on the internalization of DiI-labeled Ebola virions. Vero cells were transfected with control (Cont) non-targeting siRNA or siRNA to down-regulate Cdc42 and Pak1 expression. Labeled Ebola VLPs were adsorbed to the siRNA-transfected cells for 30 min on ice, 48 h post-transfection. After incubation for 2 h at 37°C, surface-bound virions were removed by trypsin for 5 min at 37°C and the internalization of Ebola VLPs was analyzed by using confocal laser scanning microscope, and the number of DiI-virions in 10 individual siRNA-transfected cells was measured. The efficiency of Alexa Fluor-Dex Mw 10K uptake in siRNA-transfected cells was measured by use of flow cytometry (blue bars). Each experiment was performed in triplicate and the relative uptake efficiencies are presented as the mean ± SD. (H) The internalization of Ebola virions is associated with plasma membrane ruffling. DiI-EbolaΔVP30 virions were adsorbed to eGFP-actin-expressing Vero cells for 30 min on ice. The cells were then incubated at 37°C and time-lapse images were acquired at 15-second intervals over a period of 10 min by using confocal laser scanning microscope. Still frames at the indicated times (sec) after the temperature shift to 37°C are shown. Scale bar, 10 µm.
Figure Legend Snippet: Macropinocytosis-associated events occur during Ebola virion internalization. (A) The effect of the internalization of DiI-labeled Ebola VLPs on dextran uptake. Vero cells were incubated with 0.5 mg/ml Alexa Fluor 647-Dex Mw 10K in the absence or presence of Ebola VLPs for 60 min at 37°C. The uptake of Alexa Fluor 647-Dex Mw 10K was analyzed by using flow cytometry. The effect of EIPA pretreatment was assessed in parallel. Each experiment was performed in triplicate and the results are presented as the mean ± SD. (B) Co-localization of internalized DiI-labeled Ebola VLPs and Dex Mw 10K. DiI-Ebola VLPs were adsorbed to Vero cells for 30 min on ice. The cells were cultured in the presence of 0.5 mg/ml Alexa Fluor 647-Dex Mw 10K for 10 min at 37°C. Co-localization of DiI-virions (red) and Alexa Fluor-Dex Mw 10K (green) was analyzed by using confocal laser scanning microscope. Co-localized virions are shown by arrows. Outlines of individual cells were drawn. Scale bar, 10 µm. (C) Effect of a dominant-negative form of Rac1 on the internalization of DiI-labeled Ebola virions. The eGFP-fused, wild-type Rac1 (wtRac1, upper panels) or the dominant-negative form of Rac1 (dnRac1, lower panels) was expressed in Vero cells. DiI-labeled Ebola VLPs were adsorbed to the cells for 30 min on ice. After incubation for 2 h at 37°C, surface-bound virions were removed by trypsin and the internalization of DiI-virions was analyzed by using confocal laser scanning microscope. Expression of dnRac1 interfered with the internalization of Alexa Fluor 647-Dex Mw 10K (blue; lower middle panel), attesting to its functionality. Scale bars, 10 µm. (D) Quantitative analysis of the internalization of DiI-labeled Ebola virions in wtRac1 or dnRac1-expressed Vero cells. The internalized DiI-virions were measured in 10 individual wtRac1 or dnRac1-expressed cells. Each experiment was performed in triplicate and the results are presented as the mean ± SD. (E) Effect of PKC inhibitors on the internalization of DiI-labeled Ebola virions. Vero cells were treated with DMSO or staurosporine (Stauro) for 30 min at 37°C. Labeled Ebola VLPs were adsorbed to the cells for 30 min on ice and incubated for 2 h at 37°C in the absence or presence of inhibitor. Surface-bound virions were removed by trypsin and the internalization of DiI-virions was analyzed by using confocal laser scanning microscope. The internalized DiI-virions were analyzed in 10 individual DMSO- or staurosporine-treated cells (red bars). The efficiency of Alexa Fluor-Dex Mw 10K uptake in inhibitor-treated cells was measured by using flow cytometry (blue bars). Each experiment was performed in triplicate and relative uptake efficiencies are presented as the mean ± SD (red bars). Staurosporine treatment interfered with the internalization of Alexa Fluor 633-Tf (blue bars), attesting to its functionality. (F) The down-regulation of Cdc42 and Pak1 by siRNA. The efficiencies of Cdc42 and Pak1 knock-down were assessed by use of RT-PCR. Total cellular RNA was isolated from siRNA-transfected Vero cells 48 h post-transfection by using the TRI reagent (Sigma-Aldrich) according to the manufacturer's instructions. cDNA synthesis was performed with Molony murine leukemia virus RTase using a random hexamer (Invitrogen) according to the manufacturer's protocol. PCR was carried out for 25–30 cycles consisting of a DNA denaturing step for 30 s at 94°C, annealing for 30 s at 55°C, and extension for 1 min at 72°C by use of Taq DNA polymerase (Promega). Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as an endogenous control. The oligonucleotides used for amplification of individual genes are shown in Table S1 . (G) Effect of down-regulation of Cdc42 and Pak1 on the internalization of DiI-labeled Ebola virions. Vero cells were transfected with control (Cont) non-targeting siRNA or siRNA to down-regulate Cdc42 and Pak1 expression. Labeled Ebola VLPs were adsorbed to the siRNA-transfected cells for 30 min on ice, 48 h post-transfection. After incubation for 2 h at 37°C, surface-bound virions were removed by trypsin for 5 min at 37°C and the internalization of Ebola VLPs was analyzed by using confocal laser scanning microscope, and the number of DiI-virions in 10 individual siRNA-transfected cells was measured. The efficiency of Alexa Fluor-Dex Mw 10K uptake in siRNA-transfected cells was measured by use of flow cytometry (blue bars). Each experiment was performed in triplicate and the relative uptake efficiencies are presented as the mean ± SD. (H) The internalization of Ebola virions is associated with plasma membrane ruffling. DiI-EbolaΔVP30 virions were adsorbed to eGFP-actin-expressing Vero cells for 30 min on ice. The cells were then incubated at 37°C and time-lapse images were acquired at 15-second intervals over a period of 10 min by using confocal laser scanning microscope. Still frames at the indicated times (sec) after the temperature shift to 37°C are shown. Scale bar, 10 µm.

Techniques Used: Labeling, Incubation, Flow Cytometry, Cytometry, Cell Culture, Laser-Scanning Microscopy, Dominant Negative Mutation, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Transfection, Random Hexamer Labeling, Polymerase Chain Reaction, Amplification, Size-exclusion Chromatography

Effect of macropinocytosis inhibitors on the co-localization of DiI-labeled viral particles with Rab7-positive vesicles. Vero cells expressing eGFP-Rab7 were pretreated with cytochalasin D (CytoD), wortmannin (Wort), LY294002, or EIPA for 30 min at 37°C as described in the Materials and Methods . DiI-EbolaΔVP30 virions, DiI-Ebola VLPs and DiI-influenza virus were adsorbed to the cells for 30 min on ice. The cells were then incubated at 37°C in the presence of inhibitors for 2 h. As a control, DMSO-treated cells were incubated with labeled EBOV particles. Representative images of the co-localization of DiI-EbolaΔVP30 virions with eGFP-Rab7 acquired 2 h after the temperature shift are shown (A). DiI-labeled EbolaΔVP30 virions that co-localize with eGFP-Rab7-positive vesicles are indicated by arrows. Scale bars, 10 µm. (B) shows a graphic representation of the data. The number of DiI-labeled EbolaΔVP30 virions (blue bars), Ebola VLPs (yellow bars) and influenza virions (red bars) co-localized with eGFP-Rab7-positive vesicles was measured in 10 individual cells and the percentage of co-localization in the total DiI-virions is shown for each time point. Each experiment was carried out in triplicate and the results are presented as the mean ± SD.
Figure Legend Snippet: Effect of macropinocytosis inhibitors on the co-localization of DiI-labeled viral particles with Rab7-positive vesicles. Vero cells expressing eGFP-Rab7 were pretreated with cytochalasin D (CytoD), wortmannin (Wort), LY294002, or EIPA for 30 min at 37°C as described in the Materials and Methods . DiI-EbolaΔVP30 virions, DiI-Ebola VLPs and DiI-influenza virus were adsorbed to the cells for 30 min on ice. The cells were then incubated at 37°C in the presence of inhibitors for 2 h. As a control, DMSO-treated cells were incubated with labeled EBOV particles. Representative images of the co-localization of DiI-EbolaΔVP30 virions with eGFP-Rab7 acquired 2 h after the temperature shift are shown (A). DiI-labeled EbolaΔVP30 virions that co-localize with eGFP-Rab7-positive vesicles are indicated by arrows. Scale bars, 10 µm. (B) shows a graphic representation of the data. The number of DiI-labeled EbolaΔVP30 virions (blue bars), Ebola VLPs (yellow bars) and influenza virions (red bars) co-localized with eGFP-Rab7-positive vesicles was measured in 10 individual cells and the percentage of co-localization in the total DiI-virions is shown for each time point. Each experiment was carried out in triplicate and the results are presented as the mean ± SD.

Techniques Used: Labeling, Expressing, Incubation

30) Product Images from "Distinct surveillance pathway for immunopathology during acute infection via autophagy and SR-BI"

Article Title: Distinct surveillance pathway for immunopathology during acute infection via autophagy and SR-BI

Journal: Scientific Reports

doi: 10.1038/srep34440

Local increases in membrane cholesterol levels regulate macropinocytic uptake of apoptotic cells and bacteria. ( a ) SR-BI-dependent uptake of macropinocytosis marker dextran. Internalization of 70 kDa dextran by WT (left) and SRBI −/− macrophages (right). Effect of EIPA (μM). n = 3, ten images per experiment. ( b ) SR-BI-induced engulfment of labeled apoptotic neutrophils by WT and SRBI −/− macrophages (left). Influence of MβCD and EIPA (μM). The fluoresence intensity refers to the internalized apoptotic cells. n = 3, ten images per experiment. ( c ) Colocalization of engulfed apoptotic neutrophils with dextran in macrophages. Confocal microscopy images. Scale bar, 5 μm. ( d ) Uptake of dead Listeria by WT (left) and SRBI −/− macrophages (right). Effect of EIPA (μM). n = 3, ten images per experiment. ( e ) Colocalization of incorporated dead bacteria with dextran in macrophages. Confocal microscopy images. Scale bar, 5 μm. ( f ) Influence of SR-BI on engulfment of labeled Listeria by macrophages (left). Influence of MβCD and EIPA (μM). n = 3 and n = 6 (middle panel), ten images per experiment. *P
Figure Legend Snippet: Local increases in membrane cholesterol levels regulate macropinocytic uptake of apoptotic cells and bacteria. ( a ) SR-BI-dependent uptake of macropinocytosis marker dextran. Internalization of 70 kDa dextran by WT (left) and SRBI −/− macrophages (right). Effect of EIPA (μM). n = 3, ten images per experiment. ( b ) SR-BI-induced engulfment of labeled apoptotic neutrophils by WT and SRBI −/− macrophages (left). Influence of MβCD and EIPA (μM). The fluoresence intensity refers to the internalized apoptotic cells. n = 3, ten images per experiment. ( c ) Colocalization of engulfed apoptotic neutrophils with dextran in macrophages. Confocal microscopy images. Scale bar, 5 μm. ( d ) Uptake of dead Listeria by WT (left) and SRBI −/− macrophages (right). Effect of EIPA (μM). n = 3, ten images per experiment. ( e ) Colocalization of incorporated dead bacteria with dextran in macrophages. Confocal microscopy images. Scale bar, 5 μm. ( f ) Influence of SR-BI on engulfment of labeled Listeria by macrophages (left). Influence of MβCD and EIPA (μM). n = 3 and n = 6 (middle panel), ten images per experiment. *P

Techniques Used: Marker, Labeling, Confocal Microscopy

31) Product Images from "Cell Line Models for Human Cytomegalovirus Latency Faithfully Mimic Viral Entry by Macropinocytosis and Endocytosis"

Article Title: Cell Line Models for Human Cytomegalovirus Latency Faithfully Mimic Viral Entry by Macropinocytosis and Endocytosis

Journal: Journal of Virology

doi: 10.1128/JVI.01021-19

Endocytosis inhibitors reduce HCMV latency-associated transcription. (A) NT2 cells were pretreated with EIPA (50 μM), dynasore (75 μM), chlorpromazine (25 μM), bafilomycin A1 (1 nM), or DMSO for 30 min at 37°C prior to infection. The cells were chilled at 4°C for 1 h and then mock infected or infected with HCMV (AD169; MOI = 10) for 1 h in the presence of inhibitors before shifting to 37°C (or not) for 1 h to allow entry. Extracellular virions were washed away with heparin, and the cells were incubated for 8 h in the absence of inhibitors. Harvested RNA was analyzed by quantitative reverse transcription-PCR for the HCMV B2.7 transcript. The average from three independent biological replicates relative to the DMSO control is displayed. Error bars represent standard deviations. P values were determined by Student's t test. ns, not significant ( P > 0.1); *, P ≤ 0.05; **, P
Figure Legend Snippet: Endocytosis inhibitors reduce HCMV latency-associated transcription. (A) NT2 cells were pretreated with EIPA (50 μM), dynasore (75 μM), chlorpromazine (25 μM), bafilomycin A1 (1 nM), or DMSO for 30 min at 37°C prior to infection. The cells were chilled at 4°C for 1 h and then mock infected or infected with HCMV (AD169; MOI = 10) for 1 h in the presence of inhibitors before shifting to 37°C (or not) for 1 h to allow entry. Extracellular virions were washed away with heparin, and the cells were incubated for 8 h in the absence of inhibitors. Harvested RNA was analyzed by quantitative reverse transcription-PCR for the HCMV B2.7 transcript. The average from three independent biological replicates relative to the DMSO control is displayed. Error bars represent standard deviations. P values were determined by Student's t test. ns, not significant ( P > 0.1); *, P ≤ 0.05; **, P

Techniques Used: Infection, Incubation, Polymerase Chain Reaction

32) Product Images from "An investigation into the relationship between small intestinal fluid secretion and systemic arterial blood pressure in the anesthetized rat"

Article Title: An investigation into the relationship between small intestinal fluid secretion and systemic arterial blood pressure in the anesthetized rat

Journal: Physiological Reports

doi: 10.14814/phy2.12407

Arterial blood pressure traces from four different animals during jejunal perfusions with 154 mmol L −1 choline chloride and EIPA showing: (A) Control recording without I.V. infusion. (B) Slow infusion of 50 μ g VIP I.V. (VIP). (C) Slow infusion of 300 μ g acetyl- β -methylcholine I.V. (MC). (D) I.V. injection of 0.5 mg Phentolamine (Phen). The dashed horizontal line denotes 0 mmHg. The disturbance in trace D was caused by flushing of the arterial cannula.
Figure Legend Snippet: Arterial blood pressure traces from four different animals during jejunal perfusions with 154 mmol L −1 choline chloride and EIPA showing: (A) Control recording without I.V. infusion. (B) Slow infusion of 50 μ g VIP I.V. (VIP). (C) Slow infusion of 300 μ g acetyl- β -methylcholine I.V. (MC). (D) I.V. injection of 0.5 mg Phentolamine (Phen). The dashed horizontal line denotes 0 mmHg. The disturbance in trace D was caused by flushing of the arterial cannula.

Techniques Used: Injection

Transverse section of jejunum with lumen at the top after jejunal perfusion experiment with 154 mmol L −1 choline chloride and EIPA in which I.V. bolus injection of 1 mg phentolamine reduced the DBP to 18 mmHg for 1 h. This was followed by slow infusion of 10 μ g AVP to restore the DBP to 70 mmHg for 1 h. Hematoxylin, eosin and alcian blue staining.
Figure Legend Snippet: Transverse section of jejunum with lumen at the top after jejunal perfusion experiment with 154 mmol L −1 choline chloride and EIPA in which I.V. bolus injection of 1 mg phentolamine reduced the DBP to 18 mmHg for 1 h. This was followed by slow infusion of 10 μ g AVP to restore the DBP to 70 mmHg for 1 h. Hematoxylin, eosin and alcian blue staining.

Techniques Used: Injection, Staining

Arterial blood pressure traces from a sequence of procedures in the same animal during jejunal perfusions with 154 mmol L −1 choline chloride and EIPA: (A) Control recording without I.V. infusion with an absorption rate of −8.8 μ L cm −1 h −1 at a DBP of 118 mmHg. (B) Slow coinfusion of 50 μ g VIP I.V. and 3 μ g AVP I.V. with a secretion rate of +17.8 μ L cm −1 h −1 at a DBP of 112 mmHg. (C) Slow infusion of 50 μ g VIP I.V. with secretion rate of +26.0 μ L cm −1 h −1 at a DBP of 62 mmHg. D. Control recording without I.V. infusion with an absorption rate of −5.0 μ L cm −1 h −1 at a DBP of 104 mmHg. In B, the VIP infusion caused an initial dip in blood pressure which was restored to normal with the commencement of AVP infusion. In C, the marked fall in blood pressure marks the start of VIP infusion. The dashed horizontal line denotes 0 mmHg.
Figure Legend Snippet: Arterial blood pressure traces from a sequence of procedures in the same animal during jejunal perfusions with 154 mmol L −1 choline chloride and EIPA: (A) Control recording without I.V. infusion with an absorption rate of −8.8 μ L cm −1 h −1 at a DBP of 118 mmHg. (B) Slow coinfusion of 50 μ g VIP I.V. and 3 μ g AVP I.V. with a secretion rate of +17.8 μ L cm −1 h −1 at a DBP of 112 mmHg. (C) Slow infusion of 50 μ g VIP I.V. with secretion rate of +26.0 μ L cm −1 h −1 at a DBP of 62 mmHg. D. Control recording without I.V. infusion with an absorption rate of −5.0 μ L cm −1 h −1 at a DBP of 104 mmHg. In B, the VIP infusion caused an initial dip in blood pressure which was restored to normal with the commencement of AVP infusion. In C, the marked fall in blood pressure marks the start of VIP infusion. The dashed horizontal line denotes 0 mmHg.

Techniques Used: Sequencing

33) Product Images from "Extracellular Conformational Changes in the Capsid of Human Papillomaviruses Contribute to Asynchronous Uptake into Host Cells"

Article Title: Extracellular Conformational Changes in the Capsid of Human Papillomaviruses Contribute to Asynchronous Uptake into Host Cells

Journal: Journal of Virology

doi: 10.1128/JVI.02106-17

FPC-HPV16 and HPV16 enter host cells by the same entry pathway. HeLa cells were infected with HPV16 (black), FPC-HPV16 (white), or VSV (gray) in the presence of the indicated concentrations of gefitinib (A), EIPA (B), dynasore (C), cytochalasin D (D), jasplakinolide (E), NH 4 Cl (F), cyclosporine (G), or aphidicolin (H). Depicted are infection values relative to solvent-treated controls in percentages ± SD.
Figure Legend Snippet: FPC-HPV16 and HPV16 enter host cells by the same entry pathway. HeLa cells were infected with HPV16 (black), FPC-HPV16 (white), or VSV (gray) in the presence of the indicated concentrations of gefitinib (A), EIPA (B), dynasore (C), cytochalasin D (D), jasplakinolide (E), NH 4 Cl (F), cyclosporine (G), or aphidicolin (H). Depicted are infection values relative to solvent-treated controls in percentages ± SD.

Techniques Used: Infection

34) Product Images from "The invasiveness of human cervical cancer associated to the function of NaV1.6 channels is mediated by MMP-2 activity"

Article Title: The invasiveness of human cervical cancer associated to the function of NaV1.6 channels is mediated by MMP-2 activity

Journal: Scientific Reports

doi: 10.1038/s41598-018-31364-y

The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; Leupeptin, 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P
Figure Legend Snippet: The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; Leupeptin, 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P

Techniques Used: Activity Assay, Transfection, Staining

35) Product Images from "ADAP2 Is an Interferon Stimulated Gene That Restricts RNA Virus Entry"

Article Title: ADAP2 Is an Interferon Stimulated Gene That Restricts RNA Virus Entry

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005150

ADAP2 induces macropinocytosis and associates with macropinosomes. (A), Uptake of fluorescently-labelled 70kD dextran (green) in U2OS cells transfected with DsRed-ADAP2. Approximately 48hrs following transfection, U2OS cells were incubated with dextran (0.1 mg/ml) for 45 min. White areas denote areas of colocalization. (B), Confocal micrographs of U2OS cells transfected with wild-type (WT), constitutively-active (Q111L), or dominant-negative (T66N) EGFP-Rab34 and DsRed-ADAP2. Areas of colocalization appear as yellow. White boxes denote zoomed images shown at bottom left. (C) Pearson correlation coefficient to assess the colocalization between ADAP2 and wild-type, T66N, or Q111L Rab34. Each point represents a unique cell that coexpressed both constructs. Line indicates the mean. (D), Selected frames (captured every 10min) from time-lapse movies of U2OS cells expressing EGFP-Rab34 WT and DsRed-ADAP2. See S4 Movie for complete movie. Newly formed Rab34-enriched membrane ruffles associated with DsRed-ADAP2 are denoted by white arrows. (E, F), Quantification of the number of ADAP2+ vesicles (E) or the sizes of ADAP2 + vesicles (F) in U2OS cells transfected with the indicated Rab34 constructs. (G), U2OS cells transfected with DsRed-ADAP2 were treated with EIPA (102 μM) for 1hr ~48hrs post-transfection. Cells were fixed and the number of ADAP2+ vesicles calculated (graph at left). At right, representative confocal micrograph of mock- or EIPA-treated cells. DAPI-stained nuclei are shown in blue and DsRed-ADAP2 is in red. Data in (E-F) are shown as mean ± standard deviation and are from at least 20 independent vesicles from at least three independent fields. In (D) and (E), *p
Figure Legend Snippet: ADAP2 induces macropinocytosis and associates with macropinosomes. (A), Uptake of fluorescently-labelled 70kD dextran (green) in U2OS cells transfected with DsRed-ADAP2. Approximately 48hrs following transfection, U2OS cells were incubated with dextran (0.1 mg/ml) for 45 min. White areas denote areas of colocalization. (B), Confocal micrographs of U2OS cells transfected with wild-type (WT), constitutively-active (Q111L), or dominant-negative (T66N) EGFP-Rab34 and DsRed-ADAP2. Areas of colocalization appear as yellow. White boxes denote zoomed images shown at bottom left. (C) Pearson correlation coefficient to assess the colocalization between ADAP2 and wild-type, T66N, or Q111L Rab34. Each point represents a unique cell that coexpressed both constructs. Line indicates the mean. (D), Selected frames (captured every 10min) from time-lapse movies of U2OS cells expressing EGFP-Rab34 WT and DsRed-ADAP2. See S4 Movie for complete movie. Newly formed Rab34-enriched membrane ruffles associated with DsRed-ADAP2 are denoted by white arrows. (E, F), Quantification of the number of ADAP2+ vesicles (E) or the sizes of ADAP2 + vesicles (F) in U2OS cells transfected with the indicated Rab34 constructs. (G), U2OS cells transfected with DsRed-ADAP2 were treated with EIPA (102 μM) for 1hr ~48hrs post-transfection. Cells were fixed and the number of ADAP2+ vesicles calculated (graph at left). At right, representative confocal micrograph of mock- or EIPA-treated cells. DAPI-stained nuclei are shown in blue and DsRed-ADAP2 is in red. Data in (E-F) are shown as mean ± standard deviation and are from at least 20 independent vesicles from at least three independent fields. In (D) and (E), *p

Techniques Used: Transfection, Incubation, Dominant Negative Mutation, Construct, Expressing, Staining, Standard Deviation

36) Product Images from "Coronaviruses Induce Entry-Independent, Continuous Macropinocytosis"

Article Title: Coronaviruses Induce Entry-Independent, Continuous Macropinocytosis

Journal: mBio

doi: 10.1128/mBio.01340-14

CoV-induced macropinocytosis is dependent on EGFR activation. (A) Gefitinib was added to cells for 12 h, and toxicity was assessed with CellTiter-Glo. (B, C) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell in DMEM or in DMEM supplemented with DMSO or gefitinib at 1.5 hpi. Cells were fixed at 8 hpi. Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. The percentage of cells with internalized nanoparticles (B) and the number of nuclei per syncytium (C) were determined. (D, E) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell in DMEM or in DMEM supplemented with DMSO or EIPA at 1.5 hpi. Cells were fixed at 8 hpi, stained, and imaged. The percentage of infected cells involved in syncytia (D) and the number of nuclei per syncytium (E) were determined. Data are means ± standard deviations of triplicates. n = ≥30 fields per replicate. Statistical significance was assessed by one-way ANOVA with Dunnett’s post hoc test. ***, P
Figure Legend Snippet: CoV-induced macropinocytosis is dependent on EGFR activation. (A) Gefitinib was added to cells for 12 h, and toxicity was assessed with CellTiter-Glo. (B, C) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell in DMEM or in DMEM supplemented with DMSO or gefitinib at 1.5 hpi. Cells were fixed at 8 hpi. Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. The percentage of cells with internalized nanoparticles (B) and the number of nuclei per syncytium (C) were determined. (D, E) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell in DMEM or in DMEM supplemented with DMSO or EIPA at 1.5 hpi. Cells were fixed at 8 hpi, stained, and imaged. The percentage of infected cells involved in syncytia (D) and the number of nuclei per syncytium (E) were determined. Data are means ± standard deviations of triplicates. n = ≥30 fields per replicate. Statistical significance was assessed by one-way ANOVA with Dunnett’s post hoc test. ***, P

Techniques Used: Activation Assay, Infection, Staining

Inhibition of macropinocytosis impairs MHV replication. (A) Cells were reverse transfected for 68 h and infected with MHV for 12 h. The titers of supernatant samples were determined via plaque assay. Data are represented as the means ± the standard errors of the means of two replicates performed in duplicate. (B) The 12-h toxicity of CdtA was assessed with CellTiter-Glo. (C) Cells were treated with HEPES buffer or 10 µM CdtA for 2 h prior to infection with MHV A59 at an MOI of 1 PFU/cell. The viral titer was measured at 10 hpi. Data are represented as the mean ± the standard errors of the mean of an experiment done in triplicate. (D, E) Cells were infected with MHV at an MOI of 1 PFU/cell. At the times indicated postinfection, 0.4% DMSO or 10, 20, or 40 µM EIPA was added. The viral titer was measured at 12 hpi. EIPA was added at 6 hpi at 10, 20, or 40 µM (D) or at 2, 4, 6, or 8 hpi at 40 µM (E). Data are represented as the mean ± the standard error of the mean of two replicates performed in duplicate. (F, G) Cells were infected with MHV-FFL2 at an MOI of 1 PFU/cell. At 8 hpi, 0.4% DMSO or 40 µM EIPA was added. Supernatant was collected at 10 hpi, and the viral titer was determined. Cells were collected in luciferase lysis buffer and assessed for luminescence (F) or in DMEM and subjected to three rounds of freezing and thawing before the titer was determined (G). Data are represented as the mean ± the standard error of the mean of two replicates performed in duplicate. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test. *, P
Figure Legend Snippet: Inhibition of macropinocytosis impairs MHV replication. (A) Cells were reverse transfected for 68 h and infected with MHV for 12 h. The titers of supernatant samples were determined via plaque assay. Data are represented as the means ± the standard errors of the means of two replicates performed in duplicate. (B) The 12-h toxicity of CdtA was assessed with CellTiter-Glo. (C) Cells were treated with HEPES buffer or 10 µM CdtA for 2 h prior to infection with MHV A59 at an MOI of 1 PFU/cell. The viral titer was measured at 10 hpi. Data are represented as the mean ± the standard errors of the mean of an experiment done in triplicate. (D, E) Cells were infected with MHV at an MOI of 1 PFU/cell. At the times indicated postinfection, 0.4% DMSO or 10, 20, or 40 µM EIPA was added. The viral titer was measured at 12 hpi. EIPA was added at 6 hpi at 10, 20, or 40 µM (D) or at 2, 4, 6, or 8 hpi at 40 µM (E). Data are represented as the mean ± the standard error of the mean of two replicates performed in duplicate. (F, G) Cells were infected with MHV-FFL2 at an MOI of 1 PFU/cell. At 8 hpi, 0.4% DMSO or 40 µM EIPA was added. Supernatant was collected at 10 hpi, and the viral titer was determined. Cells were collected in luciferase lysis buffer and assessed for luminescence (F) or in DMEM and subjected to three rounds of freezing and thawing before the titer was determined (G). Data are represented as the mean ± the standard error of the mean of two replicates performed in duplicate. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test. *, P

Techniques Used: Inhibition, Transfection, Infection, Plaque Assay, Luciferase, Lysis

37) Product Images from "Transcription Factor Eb Is Required for Macropinocytosis-Mediated Growth Recovery of Nutrient-Deprived Kras-Mutant Cells"

Article Title: Transcription Factor Eb Is Required for Macropinocytosis-Mediated Growth Recovery of Nutrient-Deprived Kras-Mutant Cells

Journal: Nutrients

doi: 10.3390/nu10111638

Transcription factor EB (TFEB) plays a role in macropinocytosis-mediated recovery of mTORC1 activity and cell growth in KRAS-mutant cells. ( A ) Representative Western blot showing mTORC1 activation in KRAS-mutant cells. Cells were transfected with scrambled siRNA or si TFEB for 24 h, and then maintained in leucine-free medium for 24 h with or without 3% BSA. ( B ) Quantitative densitometric data of phospho/total S6K abundance shown in ( A ). The intensity of each band was measured using ImageJ software. ( C ) Cells were transfected with scrambled siRNA or si TFEB for 24 h, and then maintained in leucine-free medium with or without 3% BSA or 10 μM EIPA for 72 h. Cell proliferation was measured using a CCK-8 assay. Data were normalized with control siRNA-transfected cells in leucine-replete media and expressed as means ±SEM of three independent experiments. n.s., not significant. * p
Figure Legend Snippet: Transcription factor EB (TFEB) plays a role in macropinocytosis-mediated recovery of mTORC1 activity and cell growth in KRAS-mutant cells. ( A ) Representative Western blot showing mTORC1 activation in KRAS-mutant cells. Cells were transfected with scrambled siRNA or si TFEB for 24 h, and then maintained in leucine-free medium for 24 h with or without 3% BSA. ( B ) Quantitative densitometric data of phospho/total S6K abundance shown in ( A ). The intensity of each band was measured using ImageJ software. ( C ) Cells were transfected with scrambled siRNA or si TFEB for 24 h, and then maintained in leucine-free medium with or without 3% BSA or 10 μM EIPA for 72 h. Cell proliferation was measured using a CCK-8 assay. Data were normalized with control siRNA-transfected cells in leucine-replete media and expressed as means ±SEM of three independent experiments. n.s., not significant. * p

Techniques Used: Activity Assay, Mutagenesis, Western Blot, Activation Assay, Transfection, Software, CCK-8 Assay

38) Product Images from "Human SLC4A11-C functions as a DIDS-stimulatable H+(OH−) permeation pathway: partial correction of R109H mutant transport"

Article Title: Human SLC4A11-C functions as a DIDS-stimulatable H+(OH−) permeation pathway: partial correction of R109H mutant transport

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00271.2014

A–E : lack of effect of changes in extracellular Na + on intracellular pH (pH i ) and SLC4A11-C-mediated H + (OH − ) flux under K + -valinomycin voltage-clamped conditions in the presence of 5-( N -ethyl- N -isopropyl)amiloride (EIPA). Na + removal/readdition experiments were performed with 110 mM K + - and valinomycin-containing solutions. Na + removal/readdition in the presence of Cl − : mock-transfected cells ( A ) and SLC4A11-C-expressing cells ( C ). Na + removal/readdition in the absence of Cl − : mock-transfected cells ( B ) and SLC4A11-C expressing cells ( D ). Na + removal/readdition in the presence of borate (10 mM): SLC4A11-C-exressing cells ( E ).
Figure Legend Snippet: A–E : lack of effect of changes in extracellular Na + on intracellular pH (pH i ) and SLC4A11-C-mediated H + (OH − ) flux under K + -valinomycin voltage-clamped conditions in the presence of 5-( N -ethyl- N -isopropyl)amiloride (EIPA). Na + removal/readdition experiments were performed with 110 mM K + - and valinomycin-containing solutions. Na + removal/readdition in the presence of Cl − : mock-transfected cells ( A ) and SLC4A11-C-expressing cells ( C ). Na + removal/readdition in the absence of Cl − : mock-transfected cells ( B ) and SLC4A11-C expressing cells ( D ). Na + removal/readdition in the presence of borate (10 mM): SLC4A11-C-exressing cells ( E ).

Techniques Used: Transfection, Expressing

Related Articles

Negative Control:

Article Title: Transcytosis route mediates rapid delivery of intact antibodies to draining lymph nodes
Article Snippet: .. The following endocytosis inhibitors and controls were used: clathrin inhibitor Pitstop 2 ( ) (Abcam) and its specific negative control (Pitstop 2 control, Abcam) both at 75 μM; the clathrin inhibitor monodansylcadaverine ( ) (MilliporeSigma; 100 μM) and its DMSO vehicle control; the macropinocytosis inhibitor EIPA ( ) [5-( N -ethyl- N -isopropyl amiloride, MilliporeSigma; 100 μM] and its DMSO vehicle control; the macropinocytosis inhibitor imipramine ( ) (MilliporeSigma, 15 μM) and its PBS vehicle control; the dynamin inhibitor Dyngo-4a ( ) (MilliporeSigma, 400 μM) and its DMSO vehicle control; and the dynamin inhibitor Dynole 34-2 (39 (Abcam) and its specific control Dynole 31-2 (Abcam), both at 75 μM. .. In addition, we applied adrenomedullin (Phoenix Pharmaceuticals; 100 nM), which stabilizes interendothelial junctions , and its vehicle control (PBS) using the same protocol.

Cell Culture:

Article Title: Visualization of Macropinocytosis in Prostate Fibroblasts
Article Snippet: .. Fetal Bovine Serum (Atlanta Biologicals, catalog number: S11550) DMEM/F12 medium (Fisher Scientific, catalog number: SH300404) Tetramethylrhodamine-conjugated high molecular weight dextran (TMR-dextran, 75 kDa), lysine fixable (Invitrogen Molecular Probes, catalog number: D1818) DQ-BSA (Thermo Fisher Scientific, Invitrogen™, catalog number: ) LysoTracker® Green DND-26 (Life Technologies, catalog number: L7526) 5-(N-ethyl-N-isopropyl) amiloride (EIPA) (Invitrogen Molecular Probes, catalog number: e-3111) Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories, catalog number: H- 1200) Formaldehyde solution, ACS reagent grade, 37% (vol/vol) (Sigma, catalog number: 252549) NuSerum (Fisher Scientific, catalog number: 355500) Insulin, human recombinant, zinc solution (Invitrogen, catalog number: 12585–014) Sodium chloride (NaCl) (Fisher Biosciences, catalog number: BP358–1) Dimethyl sulfoxide (DMSO) (Fisher Biosciences, catalog number: D128–1) Sodium dihydrogen phosphate (NaH2 PO4 ) (Fisher Biosciences, catalog number: ICN19550080) Sodium Phosphate, Dibasic, Anhydrous, Na2 HPO4 (Fisher Biosciences, catalog number: AC424375000) 10−8 M testosterone (Nacalai Tesque, catalog number: 32811–61) Poly-L-Lysine (Sigma, catalog number: P4707) EIPA (5-(N-ethyl-N-isopropyl) amiloride) (Sigma, catalog number: A3085) Sterile distilled water Stromal complete medium (see ) PBS 1x, pH 7.4 (see ) Dextran stock solution (see ) EIPA stock solution (see ) LysoTracker green DND-26 (see ) DQ-Green BSA stock solution (see ) Dextran cell culture incubation medium (see ) Fixation buffer (see ) .. Pipette-aid Tissue culture hood Centrifuge with adaptors for 15 ml conical tubes Humidified cell culture incubator set to 37 °C and 5% CO2 Confocal Laser Scanning Microscope (Leica, Germany) Fine-point forceps 4 °C refrigerator −20 °C refrigerator

Incubation:

Article Title: Visualization of Macropinocytosis in Prostate Fibroblasts
Article Snippet: .. Fetal Bovine Serum (Atlanta Biologicals, catalog number: S11550) DMEM/F12 medium (Fisher Scientific, catalog number: SH300404) Tetramethylrhodamine-conjugated high molecular weight dextran (TMR-dextran, 75 kDa), lysine fixable (Invitrogen Molecular Probes, catalog number: D1818) DQ-BSA (Thermo Fisher Scientific, Invitrogen™, catalog number: ) LysoTracker® Green DND-26 (Life Technologies, catalog number: L7526) 5-(N-ethyl-N-isopropyl) amiloride (EIPA) (Invitrogen Molecular Probes, catalog number: e-3111) Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories, catalog number: H- 1200) Formaldehyde solution, ACS reagent grade, 37% (vol/vol) (Sigma, catalog number: 252549) NuSerum (Fisher Scientific, catalog number: 355500) Insulin, human recombinant, zinc solution (Invitrogen, catalog number: 12585–014) Sodium chloride (NaCl) (Fisher Biosciences, catalog number: BP358–1) Dimethyl sulfoxide (DMSO) (Fisher Biosciences, catalog number: D128–1) Sodium dihydrogen phosphate (NaH2 PO4 ) (Fisher Biosciences, catalog number: ICN19550080) Sodium Phosphate, Dibasic, Anhydrous, Na2 HPO4 (Fisher Biosciences, catalog number: AC424375000) 10−8 M testosterone (Nacalai Tesque, catalog number: 32811–61) Poly-L-Lysine (Sigma, catalog number: P4707) EIPA (5-(N-ethyl-N-isopropyl) amiloride) (Sigma, catalog number: A3085) Sterile distilled water Stromal complete medium (see ) PBS 1x, pH 7.4 (see ) Dextran stock solution (see ) EIPA stock solution (see ) LysoTracker green DND-26 (see ) DQ-Green BSA stock solution (see ) Dextran cell culture incubation medium (see ) Fixation buffer (see ) .. Pipette-aid Tissue culture hood Centrifuge with adaptors for 15 ml conical tubes Humidified cell culture incubator set to 37 °C and 5% CO2 Confocal Laser Scanning Microscope (Leica, Germany) Fine-point forceps 4 °C refrigerator −20 °C refrigerator

other:

Article Title: Benzimidazolones enhance the function of epithelial Na+ transport
Article Snippet: Amiloride (A-7410, Sigma, Chemical Company, St. Louis, MO), amphotericin B (A-4888, Sigma), benzamil (B-2417, Sigma), CFTRinh 172 (3430, Tocris, Bristol, UK), chlorzoxazone (C-4397, Sigma), DC-EBIO (5,6-dichloro-1-ethyl-1,3-dihydro-2-benzimidazolone) (1422, Tocris), 1-EBIO (1-ethyl-1,3-dihydro-2-benzimidazolone) (SML0034, Sigma), EIPA (5-( N -Ethyl- N -isopropyl)amiloride) (A-3085, Sigma), N -methyl- d -glucamine (NMDG+ , M-2004, Sigma), ouabain (O-3125, Sigma).

Article Title: Flow cytometry analysis reveals a decrease in intracellular sodium during sperm capacitation
Article Snippet: Chemicals were obtained from the following sources: bovine serum albumin (BSA; fatty acid-free), dibutyryl-cAMP (Bt2cAMP), 3-isobutyl-1-methylxanthine (IBMX), 4-bromo-calcium ionophore A23187, amiloride hydrochloride hydrate, 5-( N -ethyl- N -isopropyl) amiloride (EIPA), sodium gluconate and carbonyl cyanide m -chlorophenylhydrazone (CCCP) were from Sigma (St Louis, MO); H-89 was from Cayman Chemical Company (Ann Arbor, MI); rabbit monoclonal anti-phosphorylated PKA substrate (clone 100G7E) was purchased from Cell Signaling (Danvers, MA); anti-phosphorylated tyrosine (Y- P ) monoclonal antibody (clone 4G10) was from Upstate Biotechnology (Lake Placid, NY); horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA) and GE Life Sciences, respectively; propidium iodide and CoroNaRed sodium fluorescent probes were from Invitrogen.

Recombinant:

Article Title: Visualization of Macropinocytosis in Prostate Fibroblasts
Article Snippet: .. Fetal Bovine Serum (Atlanta Biologicals, catalog number: S11550) DMEM/F12 medium (Fisher Scientific, catalog number: SH300404) Tetramethylrhodamine-conjugated high molecular weight dextran (TMR-dextran, 75 kDa), lysine fixable (Invitrogen Molecular Probes, catalog number: D1818) DQ-BSA (Thermo Fisher Scientific, Invitrogen™, catalog number: ) LysoTracker® Green DND-26 (Life Technologies, catalog number: L7526) 5-(N-ethyl-N-isopropyl) amiloride (EIPA) (Invitrogen Molecular Probes, catalog number: e-3111) Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories, catalog number: H- 1200) Formaldehyde solution, ACS reagent grade, 37% (vol/vol) (Sigma, catalog number: 252549) NuSerum (Fisher Scientific, catalog number: 355500) Insulin, human recombinant, zinc solution (Invitrogen, catalog number: 12585–014) Sodium chloride (NaCl) (Fisher Biosciences, catalog number: BP358–1) Dimethyl sulfoxide (DMSO) (Fisher Biosciences, catalog number: D128–1) Sodium dihydrogen phosphate (NaH2 PO4 ) (Fisher Biosciences, catalog number: ICN19550080) Sodium Phosphate, Dibasic, Anhydrous, Na2 HPO4 (Fisher Biosciences, catalog number: AC424375000) 10−8 M testosterone (Nacalai Tesque, catalog number: 32811–61) Poly-L-Lysine (Sigma, catalog number: P4707) EIPA (5-(N-ethyl-N-isopropyl) amiloride) (Sigma, catalog number: A3085) Sterile distilled water Stromal complete medium (see ) PBS 1x, pH 7.4 (see ) Dextran stock solution (see ) EIPA stock solution (see ) LysoTracker green DND-26 (see ) DQ-Green BSA stock solution (see ) Dextran cell culture incubation medium (see ) Fixation buffer (see ) .. Pipette-aid Tissue culture hood Centrifuge with adaptors for 15 ml conical tubes Humidified cell culture incubator set to 37 °C and 5% CO2 Confocal Laser Scanning Microscope (Leica, Germany) Fine-point forceps 4 °C refrigerator −20 °C refrigerator

Molecular Weight:

Article Title: Visualization of Macropinocytosis in Prostate Fibroblasts
Article Snippet: .. Fetal Bovine Serum (Atlanta Biologicals, catalog number: S11550) DMEM/F12 medium (Fisher Scientific, catalog number: SH300404) Tetramethylrhodamine-conjugated high molecular weight dextran (TMR-dextran, 75 kDa), lysine fixable (Invitrogen Molecular Probes, catalog number: D1818) DQ-BSA (Thermo Fisher Scientific, Invitrogen™, catalog number: ) LysoTracker® Green DND-26 (Life Technologies, catalog number: L7526) 5-(N-ethyl-N-isopropyl) amiloride (EIPA) (Invitrogen Molecular Probes, catalog number: e-3111) Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories, catalog number: H- 1200) Formaldehyde solution, ACS reagent grade, 37% (vol/vol) (Sigma, catalog number: 252549) NuSerum (Fisher Scientific, catalog number: 355500) Insulin, human recombinant, zinc solution (Invitrogen, catalog number: 12585–014) Sodium chloride (NaCl) (Fisher Biosciences, catalog number: BP358–1) Dimethyl sulfoxide (DMSO) (Fisher Biosciences, catalog number: D128–1) Sodium dihydrogen phosphate (NaH2 PO4 ) (Fisher Biosciences, catalog number: ICN19550080) Sodium Phosphate, Dibasic, Anhydrous, Na2 HPO4 (Fisher Biosciences, catalog number: AC424375000) 10−8 M testosterone (Nacalai Tesque, catalog number: 32811–61) Poly-L-Lysine (Sigma, catalog number: P4707) EIPA (5-(N-ethyl-N-isopropyl) amiloride) (Sigma, catalog number: A3085) Sterile distilled water Stromal complete medium (see ) PBS 1x, pH 7.4 (see ) Dextran stock solution (see ) EIPA stock solution (see ) LysoTracker green DND-26 (see ) DQ-Green BSA stock solution (see ) Dextran cell culture incubation medium (see ) Fixation buffer (see ) .. Pipette-aid Tissue culture hood Centrifuge with adaptors for 15 ml conical tubes Humidified cell culture incubator set to 37 °C and 5% CO2 Confocal Laser Scanning Microscope (Leica, Germany) Fine-point forceps 4 °C refrigerator −20 °C refrigerator

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  • 99
    Millipore macropinocytosis inhibitor eipa
    Analyses of the role of endocytosis routes and paracellular permeability in antibody transfer to the draining LNs. ( A ) Confocal and flow cytometric analyses of draining LNs of WT and Cav1 −/− mice after s.c. administration of the indicated fluorochrome-conjugated antibodies (1 μg, t = 5 min, n = 4). The cells were stained ex vivo for CD3 for flow cytometric analysis. Scale bars: 20 μm. ( B ) Experimental outline for topical application of endocytosis inhibitors (and controls) to surgically exposed LNs. ( C – G ) Analyses of transcytosis of the indicated s.c. administered fluorochrome-conjugated antibodies (5 μg each, t = 5 min) after topical pretreatment of the draining LNs with ( C – F ) endocytosis inhibitors or ( G ) a LEC junctional stabilizer. ( C ) Pitstop 2 (a clathrin inhibitor) and a Pitstop 2 negative control; ( D ) monodansylcadaverine (MDC) (a clathrin inhibitor) and vehicle; ( E ) <t>EIPA</t> (a <t>macropinocytosis</t> inhibitor) and vehicle; ( F ) imipramine (a macropinocytosis inhibitor) and vehicle; and ( G ) adrenomedullin and vehicle. The cells were stained ex vivo for CD3. In the bar graphs, each dot represents 1 LN, and data are the mean ± SD. Statistical significance was determined by Mann-Whitney U test.
    Macropinocytosis Inhibitor Eipa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore sodium proton exchanger inhibitor eipa
    Inhibitors against Pak1, <t>PKC,</t> sodium/proton exchanger, and actin reduce Ad35-eGFP transduction. HeLa-ATCC and HK-2 cells were preincubated with the indicated concentrations of drugs for 30 min and infected with Ad35-eGFP for 8 h. Cells were analyzed on a Safire2 plate reader, and eGFP intensities were normalized to the DAPI signal of the cell nuclei, representing cell numbers. Results for <t>EIPA</t> are shown in panel A, NH 4 Cl in B, bafilomycin (Baf) in C, jasplakinolide (Jas) and cytochalasin D (CytD) in D, the PKC inhibitor Gö6976 in E, and the Pak1 inhibitor IPA-3 and its inactive derivative PIR3.5 in F and G for both Ad35-eGFP and Ad5-eGFP, respectively. Panels H to K show results with macropinocytic interference in Wi-38 cells transduced with Ad35-eGFP or Ad5-eGFP. Note the absence of significant cell toxicity, as indicated by cell number measurements shown with gray line graphs, with 100% representing noninfected non-drug-treated conditions.
    Sodium Proton Exchanger Inhibitor Eipa, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore se met eipa protein
    Inhibitors against Pak1, <t>PKC,</t> sodium/proton exchanger, and actin reduce Ad35-eGFP transduction. HeLa-ATCC and HK-2 cells were preincubated with the indicated concentrations of drugs for 30 min and infected with Ad35-eGFP for 8 h. Cells were analyzed on a Safire2 plate reader, and eGFP intensities were normalized to the DAPI signal of the cell nuclei, representing cell numbers. Results for <t>EIPA</t> are shown in panel A, NH 4 Cl in B, bafilomycin (Baf) in C, jasplakinolide (Jas) and cytochalasin D (CytD) in D, the PKC inhibitor Gö6976 in E, and the Pak1 inhibitor IPA-3 and its inactive derivative PIR3.5 in F and G for both Ad35-eGFP and Ad5-eGFP, respectively. Panels H to K show results with macropinocytic interference in Wi-38 cells transduced with Ad35-eGFP or Ad5-eGFP. Note the absence of significant cell toxicity, as indicated by cell number measurements shown with gray line graphs, with 100% representing noninfected non-drug-treated conditions.
    Se Met Eipa Protein, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Analyses of the role of endocytosis routes and paracellular permeability in antibody transfer to the draining LNs. ( A ) Confocal and flow cytometric analyses of draining LNs of WT and Cav1 −/− mice after s.c. administration of the indicated fluorochrome-conjugated antibodies (1 μg, t = 5 min, n = 4). The cells were stained ex vivo for CD3 for flow cytometric analysis. Scale bars: 20 μm. ( B ) Experimental outline for topical application of endocytosis inhibitors (and controls) to surgically exposed LNs. ( C – G ) Analyses of transcytosis of the indicated s.c. administered fluorochrome-conjugated antibodies (5 μg each, t = 5 min) after topical pretreatment of the draining LNs with ( C – F ) endocytosis inhibitors or ( G ) a LEC junctional stabilizer. ( C ) Pitstop 2 (a clathrin inhibitor) and a Pitstop 2 negative control; ( D ) monodansylcadaverine (MDC) (a clathrin inhibitor) and vehicle; ( E ) EIPA (a macropinocytosis inhibitor) and vehicle; ( F ) imipramine (a macropinocytosis inhibitor) and vehicle; and ( G ) adrenomedullin and vehicle. The cells were stained ex vivo for CD3. In the bar graphs, each dot represents 1 LN, and data are the mean ± SD. Statistical significance was determined by Mann-Whitney U test.

    Journal: The Journal of Clinical Investigation

    Article Title: Transcytosis route mediates rapid delivery of intact antibodies to draining lymph nodes

    doi: 10.1172/JCI125740

    Figure Lengend Snippet: Analyses of the role of endocytosis routes and paracellular permeability in antibody transfer to the draining LNs. ( A ) Confocal and flow cytometric analyses of draining LNs of WT and Cav1 −/− mice after s.c. administration of the indicated fluorochrome-conjugated antibodies (1 μg, t = 5 min, n = 4). The cells were stained ex vivo for CD3 for flow cytometric analysis. Scale bars: 20 μm. ( B ) Experimental outline for topical application of endocytosis inhibitors (and controls) to surgically exposed LNs. ( C – G ) Analyses of transcytosis of the indicated s.c. administered fluorochrome-conjugated antibodies (5 μg each, t = 5 min) after topical pretreatment of the draining LNs with ( C – F ) endocytosis inhibitors or ( G ) a LEC junctional stabilizer. ( C ) Pitstop 2 (a clathrin inhibitor) and a Pitstop 2 negative control; ( D ) monodansylcadaverine (MDC) (a clathrin inhibitor) and vehicle; ( E ) EIPA (a macropinocytosis inhibitor) and vehicle; ( F ) imipramine (a macropinocytosis inhibitor) and vehicle; and ( G ) adrenomedullin and vehicle. The cells were stained ex vivo for CD3. In the bar graphs, each dot represents 1 LN, and data are the mean ± SD. Statistical significance was determined by Mann-Whitney U test.

    Article Snippet: The following endocytosis inhibitors and controls were used: clathrin inhibitor Pitstop 2 ( ) (Abcam) and its specific negative control (Pitstop 2 control, Abcam) both at 75 μM; the clathrin inhibitor monodansylcadaverine ( ) (MilliporeSigma; 100 μM) and its DMSO vehicle control; the macropinocytosis inhibitor EIPA ( ) [5-( N -ethyl- N -isopropyl amiloride, MilliporeSigma; 100 μM] and its DMSO vehicle control; the macropinocytosis inhibitor imipramine ( ) (MilliporeSigma, 15 μM) and its PBS vehicle control; the dynamin inhibitor Dyngo-4a ( ) (MilliporeSigma, 400 μM) and its DMSO vehicle control; and the dynamin inhibitor Dynole 34-2 (39 (Abcam) and its specific control Dynole 31-2 (Abcam), both at 75 μM.

    Techniques: Permeability, Flow Cytometry, Mouse Assay, Staining, Ex Vivo, Negative Control, MANN-WHITNEY

    Inhibitors against Pak1, PKC, sodium/proton exchanger, and actin reduce Ad35-eGFP transduction. HeLa-ATCC and HK-2 cells were preincubated with the indicated concentrations of drugs for 30 min and infected with Ad35-eGFP for 8 h. Cells were analyzed on a Safire2 plate reader, and eGFP intensities were normalized to the DAPI signal of the cell nuclei, representing cell numbers. Results for EIPA are shown in panel A, NH 4 Cl in B, bafilomycin (Baf) in C, jasplakinolide (Jas) and cytochalasin D (CytD) in D, the PKC inhibitor Gö6976 in E, and the Pak1 inhibitor IPA-3 and its inactive derivative PIR3.5 in F and G for both Ad35-eGFP and Ad5-eGFP, respectively. Panels H to K show results with macropinocytic interference in Wi-38 cells transduced with Ad35-eGFP or Ad5-eGFP. Note the absence of significant cell toxicity, as indicated by cell number measurements shown with gray line graphs, with 100% representing noninfected non-drug-treated conditions.

    Journal: Journal of Virology

    Article Title: Macropinocytotic Uptake and Infection of Human Epithelial Cells with Species B2 Adenovirus Type 35 ▿Macropinocytotic Uptake and Infection of Human Epithelial Cells with Species B2 Adenovirus Type 35 ▿ †

    doi: 10.1128/JVI.02494-09

    Figure Lengend Snippet: Inhibitors against Pak1, PKC, sodium/proton exchanger, and actin reduce Ad35-eGFP transduction. HeLa-ATCC and HK-2 cells were preincubated with the indicated concentrations of drugs for 30 min and infected with Ad35-eGFP for 8 h. Cells were analyzed on a Safire2 plate reader, and eGFP intensities were normalized to the DAPI signal of the cell nuclei, representing cell numbers. Results for EIPA are shown in panel A, NH 4 Cl in B, bafilomycin (Baf) in C, jasplakinolide (Jas) and cytochalasin D (CytD) in D, the PKC inhibitor Gö6976 in E, and the Pak1 inhibitor IPA-3 and its inactive derivative PIR3.5 in F and G for both Ad35-eGFP and Ad5-eGFP, respectively. Panels H to K show results with macropinocytic interference in Wi-38 cells transduced with Ad35-eGFP or Ad5-eGFP. Note the absence of significant cell toxicity, as indicated by cell number measurements shown with gray line graphs, with 100% representing noninfected non-drug-treated conditions.

    Article Snippet: The PKC inhibitor Gö6976, the sodium/proton exchanger inhibitor EIPA, cytochalasin D, and jasplakinolide were from Calbiochem.

    Techniques: Transduction, Infection, Indirect Immunoperoxidase Assay