Structured Review

TaKaRa lentiviral encoding fip2
<t>FIP2</t> controls E . coli induced IFN-β mRNA induction and secretion. ( A ) Immunoblots showing the phosphorylation patterns of TBK1, IRF3, IκBα and p38MAPK in FIP2 silenced THP-1 cells stimulated with E . coli bioparticles or LPS (100 ng/ml). Data are representative of three independent experiments. ( B ) Quantification of phosphorylation patterns of the proteins shown in the immunoblots presented in (A). ( C ) ELISA quantification IFN-β and TNF secretion in THP-1 cells treated with NS RNA or FIP2 siRNA and stimulated as indicated. ( D ) Quantification of E . coli -stimulated IFN-β and TNF mRNAs in THP-1 cells with <t>lentiviral</t> overexpression of FIP2. ( E ) Quantification of Poly I:C and LPS stimulated IFN-β mRNA induction in cells treated with NS RNA or FIP2 siRNA after 4 hours of stimulation. Poly I:C (5 μg/ml) was transfected using Lipofectamine® 2000. Data are representative of three independent experiments.
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Images

1) Product Images from "The TLR4 adaptor TRAM controls the phagocytosis of Gram-negative bacteria by interacting with the Rab11-family interacting protein 2"

Article Title: The TLR4 adaptor TRAM controls the phagocytosis of Gram-negative bacteria by interacting with the Rab11-family interacting protein 2

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007684

FIP2 controls E . coli induced IFN-β mRNA induction and secretion. ( A ) Immunoblots showing the phosphorylation patterns of TBK1, IRF3, IκBα and p38MAPK in FIP2 silenced THP-1 cells stimulated with E . coli bioparticles or LPS (100 ng/ml). Data are representative of three independent experiments. ( B ) Quantification of phosphorylation patterns of the proteins shown in the immunoblots presented in (A). ( C ) ELISA quantification IFN-β and TNF secretion in THP-1 cells treated with NS RNA or FIP2 siRNA and stimulated as indicated. ( D ) Quantification of E . coli -stimulated IFN-β and TNF mRNAs in THP-1 cells with lentiviral overexpression of FIP2. ( E ) Quantification of Poly I:C and LPS stimulated IFN-β mRNA induction in cells treated with NS RNA or FIP2 siRNA after 4 hours of stimulation. Poly I:C (5 μg/ml) was transfected using Lipofectamine® 2000. Data are representative of three independent experiments.
Figure Legend Snippet: FIP2 controls E . coli induced IFN-β mRNA induction and secretion. ( A ) Immunoblots showing the phosphorylation patterns of TBK1, IRF3, IκBα and p38MAPK in FIP2 silenced THP-1 cells stimulated with E . coli bioparticles or LPS (100 ng/ml). Data are representative of three independent experiments. ( B ) Quantification of phosphorylation patterns of the proteins shown in the immunoblots presented in (A). ( C ) ELISA quantification IFN-β and TNF secretion in THP-1 cells treated with NS RNA or FIP2 siRNA and stimulated as indicated. ( D ) Quantification of E . coli -stimulated IFN-β and TNF mRNAs in THP-1 cells with lentiviral overexpression of FIP2. ( E ) Quantification of Poly I:C and LPS stimulated IFN-β mRNA induction in cells treated with NS RNA or FIP2 siRNA after 4 hours of stimulation. Poly I:C (5 μg/ml) was transfected using Lipofectamine® 2000. Data are representative of three independent experiments.

Techniques Used: Western Blot, Enzyme-linked Immunosorbent Assay, Over Expression, Transfection

2) Product Images from "Bexarotene-Activated Retinoid X Receptors Regulate Neuronal Differentiation and Dendritic Complexity"

Article Title: Bexarotene-Activated Retinoid X Receptors Regulate Neuronal Differentiation and Dendritic Complexity

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1001-15.2015

Bexarotene enhances neurite branching and affects dendrite complexity in primary neurons. Primary rat neurons were infected with GFP expressing lentivirus at DIV0 and treated with bexarotene or vehicle at DIV4 for 24 h. Sholl analysis was performed 6
Figure Legend Snippet: Bexarotene enhances neurite branching and affects dendrite complexity in primary neurons. Primary rat neurons were infected with GFP expressing lentivirus at DIV0 and treated with bexarotene or vehicle at DIV4 for 24 h. Sholl analysis was performed 6

Techniques Used: Infection, Expressing

3) Product Images from "A Pitx2-MicroRNA Pathway Modulates Cell Proliferation in Myoblasts and Skeletal-Muscle Satellite Cells and Promotes Their Commitment to a Myogenic Cell Fate"

Article Title: A Pitx2-MicroRNA Pathway Modulates Cell Proliferation in Myoblasts and Skeletal-Muscle Satellite Cells and Promotes Their Commitment to a Myogenic Cell Fate

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00536-15

(A) qRT-PCR analyses showing Pitx2c overexpression after transfection with two different doses of the CMV- Pitx2c plasmid (400 and 800 ng/ml). (B) mRNA expression levels of Pitx2c and Pitx3 after treatment with siRNA against Pitx2 in Sol8 myoblasts.
Figure Legend Snippet: (A) qRT-PCR analyses showing Pitx2c overexpression after transfection with two different doses of the CMV- Pitx2c plasmid (400 and 800 ng/ml). (B) mRNA expression levels of Pitx2c and Pitx3 after treatment with siRNA against Pitx2 in Sol8 myoblasts.

Techniques Used: Quantitative RT-PCR, Over Expression, Transfection, Plasmid Preparation, Expressing

(A) Representative images of EPq cells transfected with a lentivirus-Pitx2c-ZsGreen vector (LVX-Pitx2c). (B) qRT-PCR for Pitx2c expression in EPq and EPa cells transfected with the LVX-Pitx2c vector with respect to cells transfected with the empty LVX-ZsGreen lentiviral vector (LVX). (C) Cyclin D1 and cyclin D2 gene expression in EPq and EPa Pitx2c-overexpressing cells with respect to control cells. (D) Representative images of immunohistochemical analyses for Ki67-positve cells in EPq cells transfected with the lentivirus-Pitx2c-ZsGreen vector (LVX) compared to cells transfected with the empty LVX-ZsGreen lentiviral vector (LVX-Pitx2c). (E) Percentage of Ki67 + cells in EPq cells transfected with the lentivirus-Pitx2c-ZsGreen vector with respect to cells transfected with the empty LVX-ZsGreen lentiviral vector.
Figure Legend Snippet: (A) Representative images of EPq cells transfected with a lentivirus-Pitx2c-ZsGreen vector (LVX-Pitx2c). (B) qRT-PCR for Pitx2c expression in EPq and EPa cells transfected with the LVX-Pitx2c vector with respect to cells transfected with the empty LVX-ZsGreen lentiviral vector (LVX). (C) Cyclin D1 and cyclin D2 gene expression in EPq and EPa Pitx2c-overexpressing cells with respect to control cells. (D) Representative images of immunohistochemical analyses for Ki67-positve cells in EPq cells transfected with the lentivirus-Pitx2c-ZsGreen vector (LVX) compared to cells transfected with the empty LVX-ZsGreen lentiviral vector (LVX-Pitx2c). (E) Percentage of Ki67 + cells in EPq cells transfected with the lentivirus-Pitx2c-ZsGreen vector with respect to cells transfected with the empty LVX-ZsGreen lentiviral vector.

Techniques Used: Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing, Immunohistochemistry

(A) Expression profiles for miR-15b, miR-23b, miR-106b, and miR-503 in EPq and EPa Pitx2c -overexpressing cells with respect to control (CN) cells. (B) Relative expression levels of of Pitx2c as well as of miR-15b, miR-23b, miR-106b, and miR-503 during myogenic progression. (C and D) miR-15b, miR-23b, miR-106b, and miR-503 overexpression in EPa cells leads to cyclin D1 and cyclin D2 gene downregulation.
Figure Legend Snippet: (A) Expression profiles for miR-15b, miR-23b, miR-106b, and miR-503 in EPq and EPa Pitx2c -overexpressing cells with respect to control (CN) cells. (B) Relative expression levels of of Pitx2c as well as of miR-15b, miR-23b, miR-106b, and miR-503 during myogenic progression. (C and D) miR-15b, miR-23b, miR-106b, and miR-503 overexpression in EPa cells leads to cyclin D1 and cyclin D2 gene downregulation.

Techniques Used: Expressing, Over Expression

(A) Hierarchical clustering of statistically significant microRNA microarray expression profiles with the Sol8 cell line transfected with 400 ng/ml and 800 ng/ml of the CMV- Pitx2c plasmid 24 h after transfection. The color range (−2 to 2) is related to Z-scored expression values. (B) Expression profiles of statistically significant miRNAs by qRT-PCR in Sol8 Pitx2c -transfected cells at 400 and 800 ng/ml of the Pitx2c plasmid compared to controls. (C) Expression profiles of the statistically significant miRNAs by qRT-PCR in Pitx2c -silenced Sol8 cells (siRNA against Pitx2c) compared to controls.
Figure Legend Snippet: (A) Hierarchical clustering of statistically significant microRNA microarray expression profiles with the Sol8 cell line transfected with 400 ng/ml and 800 ng/ml of the CMV- Pitx2c plasmid 24 h after transfection. The color range (−2 to 2) is related to Z-scored expression values. (B) Expression profiles of statistically significant miRNAs by qRT-PCR in Sol8 Pitx2c -transfected cells at 400 and 800 ng/ml of the Pitx2c plasmid compared to controls. (C) Expression profiles of the statistically significant miRNAs by qRT-PCR in Pitx2c -silenced Sol8 cells (siRNA against Pitx2c) compared to controls.

Techniques Used: Microarray, Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR

(A) Pitx2c overexpression is maintained after pre-miR-106 transfection in EPq cells. (B and C) Pre-miR-106b transfection in EPq cells overexpressing Pitx2c (B) rescues Myf5 expression at basal levels (control cells) (C).
Figure Legend Snippet: (A) Pitx2c overexpression is maintained after pre-miR-106 transfection in EPq cells. (B and C) Pre-miR-106b transfection in EPq cells overexpressing Pitx2c (B) rescues Myf5 expression at basal levels (control cells) (C).

Techniques Used: Over Expression, Transfection, Expressing

(A) Pitx2c overexpression is maintained after pre-miR-106 transfection in EPq cells. (B and C) Pre-miR-106b transfection in EPq cells overexpressing Pitx2c (B) rescues cyclin D1 and cyclin D2 gene expression at the basal levels (control cells) (C).
Figure Legend Snippet: (A) Pitx2c overexpression is maintained after pre-miR-106 transfection in EPq cells. (B and C) Pre-miR-106b transfection in EPq cells overexpressing Pitx2c (B) rescues cyclin D1 and cyclin D2 gene expression at the basal levels (control cells) (C).

Techniques Used: Over Expression, Transfection, Expressing

(A) Representative images of early-plated relatively quiescent/early-activated satellite cells (EPq), early-plated long-term-activated satellite cells (EPa), and EPa-derived differentiating fusing-myoblast cultures. (B) mRNA expression levels of the cyclin D2, Myf5 , and Pax7 genes in EPq and EPa cells. (C) mRNA expression levels of Pitx2c in EPq cells and EPa cells and myoblasts.
Figure Legend Snippet: (A) Representative images of early-plated relatively quiescent/early-activated satellite cells (EPq), early-plated long-term-activated satellite cells (EPa), and EPa-derived differentiating fusing-myoblast cultures. (B) mRNA expression levels of the cyclin D2, Myf5 , and Pax7 genes in EPq and EPa cells. (C) mRNA expression levels of Pitx2c in EPq cells and EPa cells and myoblasts.

Techniques Used: Derivative Assay, Expressing

(A) Myf5 expression profile in EPq Pitx2c -overexpressing cells. (B) Representative images of immunohistochemical analyses for Myf5-positive cells in EPq cells transfected with the lentivirus- Pitx2c -ZsGreen vector (LVX) compared to cells transfected with the empty LVX-ZsGreen lentiviral vector (LVX-Pitx2c). (C) Percentage of Myf5 + cells in EPq cells transfected with the lentivirus-Pitx2c-ZsGreen vector (LVX-control) with respect to cells transfected with the empty LVX-ZsGreen lentiviral vector (LVX-Pitx2). (D and E) miR-106b overexpression leads to Myf5 upregulation in EPq cells. (F) Normalized luciferase activity of the 3′-UTR Myf5 luciferase reporter (wild-type Myf5 3′ UTR) with an empty plasmid (vector) or pre-miR-106b shows the loss of luciferase activity with expression of miR-106b. There was no loss of luciferase activity when the miR-106b seed sequence was mutated.
Figure Legend Snippet: (A) Myf5 expression profile in EPq Pitx2c -overexpressing cells. (B) Representative images of immunohistochemical analyses for Myf5-positive cells in EPq cells transfected with the lentivirus- Pitx2c -ZsGreen vector (LVX) compared to cells transfected with the empty LVX-ZsGreen lentiviral vector (LVX-Pitx2c). (C) Percentage of Myf5 + cells in EPq cells transfected with the lentivirus-Pitx2c-ZsGreen vector (LVX-control) with respect to cells transfected with the empty LVX-ZsGreen lentiviral vector (LVX-Pitx2). (D and E) miR-106b overexpression leads to Myf5 upregulation in EPq cells. (F) Normalized luciferase activity of the 3′-UTR Myf5 luciferase reporter (wild-type Myf5 3′ UTR) with an empty plasmid (vector) or pre-miR-106b shows the loss of luciferase activity with expression of miR-106b. There was no loss of luciferase activity when the miR-106b seed sequence was mutated.

Techniques Used: Expressing, Immunohistochemistry, Transfection, Plasmid Preparation, Over Expression, Luciferase, Activity Assay, Sequencing

(A) qRT-PCR for Pitx2c expression in Pax3-cre +/− /Pitx2 +/− heterozygote adult mice. (B) qRT-PCR for Pitx2c expression in Pax3-cre +/− /Pitx2 +/− heterozygote and Pax3-cre +/− /Pitx2 −/− homozygote neonates.
Figure Legend Snippet: (A) qRT-PCR for Pitx2c expression in Pax3-cre +/− /Pitx2 +/− heterozygote adult mice. (B) qRT-PCR for Pitx2c expression in Pax3-cre +/− /Pitx2 +/− heterozygote and Pax3-cre +/− /Pitx2 −/− homozygote neonates.

Techniques Used: Quantitative RT-PCR, Expressing, Mouse Assay

4) Product Images from "Retinoic Acid Induces Neurogenesis by Activating Both Retinoic Acid Receptors (RARs) and Peroxisome Proliferator-activated Receptor ?/? (PPAR?/?) *"

Article Title: Retinoic Acid Induces Neurogenesis by Activating Both Retinoic Acid Receptors (RARs) and Peroxisome Proliferator-activated Receptor ?/? (PPAR?/?) *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.410381

CRABP-II/FABP5 ratio shifts during RA-induced neuronal differentiation. P19 cells were induced to differentiate as described under “Experimental Procedures.” RA was added on day 0. a, immunoblots demonstrating expression of Oct3/4, β3-tubulin,
Figure Legend Snippet: CRABP-II/FABP5 ratio shifts during RA-induced neuronal differentiation. P19 cells were induced to differentiate as described under “Experimental Procedures.” RA was added on day 0. a, immunoblots demonstrating expression of Oct3/4, β3-tubulin,

Techniques Used: Western Blot, Expressing

RA signaling through the PPARβ/δ/FABP5 pathway promotes differentiation of neuronal progenitor cells to mature neurons. P19 cells and corresponding cells with stable reduced levels of PPARβ/δ or FABP5 were induced to differentiate.
Figure Legend Snippet: RA signaling through the PPARβ/δ/FABP5 pathway promotes differentiation of neuronal progenitor cells to mature neurons. P19 cells and corresponding cells with stable reduced levels of PPARβ/δ or FABP5 were induced to differentiate.

Techniques Used:

Model for the involvement of the RA in neuronal differentiation. RA promotes differentiation of stem cells into neural progenitor cells by activating the CRABP-II/RAR pathway. The alternative RA path, mediated by FABP5 and PPARβ/δ, can
Figure Legend Snippet: Model for the involvement of the RA in neuronal differentiation. RA promotes differentiation of stem cells into neural progenitor cells by activating the CRABP-II/RAR pathway. The alternative RA path, mediated by FABP5 and PPARβ/δ, can

Techniques Used:

SIRT1 and Ajuba mediate inhibition of P19 cell differentiation to neuronal progenitors by the PPARβ/δ/FABP5 pathway. a, immunoblots demonstrating overexpression of SIRT1 or Ajuba in P19 cells. b, cells ectopically expressing GFP, Ajuba,
Figure Legend Snippet: SIRT1 and Ajuba mediate inhibition of P19 cell differentiation to neuronal progenitors by the PPARβ/δ/FABP5 pathway. a, immunoblots demonstrating overexpression of SIRT1 or Ajuba in P19 cells. b, cells ectopically expressing GFP, Ajuba,

Techniques Used: Inhibition, Cell Differentiation, Western Blot, Over Expression, Expressing

Localization of FABP5 in mouse brain and effects of its ablation on neuronal markers in hippocampus. a and b, location of FABP5 in mouse brain, analyzed by confocal fluorescence microscopy ( a, bar, 50 μ m ) and by immunoblots ( b ). OB, olfactory
Figure Legend Snippet: Localization of FABP5 in mouse brain and effects of its ablation on neuronal markers in hippocampus. a and b, location of FABP5 in mouse brain, analyzed by confocal fluorescence microscopy ( a, bar, 50 μ m ) and by immunoblots ( b ). OB, olfactory

Techniques Used: Fluorescence, Microscopy, Western Blot

Hippocampi of FABP5 −/− display lower expression of mature neuronal markers. a, immunoblots of denoted proteins in lysates of hippocampus of three WT and three FABP5 −/− mice. b–e, quantitation of immunoblots in
Figure Legend Snippet: Hippocampi of FABP5 −/− display lower expression of mature neuronal markers. a, immunoblots of denoted proteins in lysates of hippocampus of three WT and three FABP5 −/− mice. b–e, quantitation of immunoblots in

Techniques Used: Expressing, Western Blot, Mouse Assay, Quantitation Assay

PDK1 mediates promotion of progenitor cells to mature neurons by the PPARβ/δ/FABP5 pathway. a, P19 cells were treated with GW0742 (20 n m , 4 h) and PDK1 mRNA assessed by Q-PCR. **, p
Figure Legend Snippet: PDK1 mediates promotion of progenitor cells to mature neurons by the PPARβ/δ/FABP5 pathway. a, P19 cells were treated with GW0742 (20 n m , 4 h) and PDK1 mRNA assessed by Q-PCR. **, p

Techniques Used: Polymerase Chain Reaction

PPARβ/δ/FABP5 pathway inhibits RA-induced differentiation of P19 cells to neuronal progenitors. a and b, P19 cell lines with reduced expression of PPARβ/δ or FABP5 were established by stable transfection of corresponding
Figure Legend Snippet: PPARβ/δ/FABP5 pathway inhibits RA-induced differentiation of P19 cells to neuronal progenitors. a and b, P19 cell lines with reduced expression of PPARβ/δ or FABP5 were established by stable transfection of corresponding

Techniques Used: Expressing, Stable Transfection

5) Product Images from "A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia"

Article Title: A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia

Journal: BMC Cancer

doi: 10.1186/s12885-018-4097-z

Knockdown of HOXB3 and CDCA3 partially mimics the anti-leukemia activity by miR-375. a HL-60 and THP1 cells were transduced with special shRNA for HOXB 3 or a control shRNA (sh-NC). HOXB3 and CDCA3 expressions were detected by western blot. b Viable cell number by the trypan-blue exclusion assay was counted in HL-60 and THP1 cells, which were transduced with special shRNA for HOXB3 or sh-NC for the indicated times. * P
Figure Legend Snippet: Knockdown of HOXB3 and CDCA3 partially mimics the anti-leukemia activity by miR-375. a HL-60 and THP1 cells were transduced with special shRNA for HOXB 3 or a control shRNA (sh-NC). HOXB3 and CDCA3 expressions were detected by western blot. b Viable cell number by the trypan-blue exclusion assay was counted in HL-60 and THP1 cells, which were transduced with special shRNA for HOXB3 or sh-NC for the indicated times. * P

Techniques Used: Activity Assay, Transduction, shRNA, Western Blot, Trypan Blue Exclusion Assay

HOXB3 enhances the expression of DNMT3B to bind in pre-miR-375 promoter. a HL-60 and THP1 cells were transduced with special shRNA for HOXB3 (sh-HOXB3) or sh-NC. HOXB3 and DNMT3B expressions were detected by western blot. b HOXB3 and DNMT3B expressions were detected in HL-60 and THP1 cells, which were transduced with overexpression vector LVX-HOXB3 or LVX-NC. c DNMT3B expression was detected in HL-60 and THP1 cells transduced with special shRNA targeting DNMT3B (sh-DNMT3B) or sh-NC. d MiR-375 expression was measured in HL-60 and THP1 cells transduced with sh-DNMT3B or sh-NC. * P
Figure Legend Snippet: HOXB3 enhances the expression of DNMT3B to bind in pre-miR-375 promoter. a HL-60 and THP1 cells were transduced with special shRNA for HOXB3 (sh-HOXB3) or sh-NC. HOXB3 and DNMT3B expressions were detected by western blot. b HOXB3 and DNMT3B expressions were detected in HL-60 and THP1 cells, which were transduced with overexpression vector LVX-HOXB3 or LVX-NC. c DNMT3B expression was detected in HL-60 and THP1 cells transduced with special shRNA targeting DNMT3B (sh-DNMT3B) or sh-NC. d MiR-375 expression was measured in HL-60 and THP1 cells transduced with sh-DNMT3B or sh-NC. * P

Techniques Used: Expressing, Transduction, shRNA, Western Blot, Over Expression, Plasmid Preparation

The anti-leukemia effects of miR-375 in vivo. About 1х10 7 viable HL-60 cells transduced with MSCV-miR-375 or MSCV-NC were injected subcutaneously into right flank of each nude mouse. a A photograph of xenografted tumors in mice xenografted by HL-60 cells, which were transduced with MSCV-miR-375 or MSCV-NC. b Volumes of all xenografted tumors were measured when the experiment was terminated at 42 days after tumor cell inoculation. c Net weights of all xenografted tumors were measured at the termination of the experiment. d The protein expression of HOXB3 was detected in xenografted tumor lysates from HL-60 cells transduced with MSCV-miR-375 or MSCV-NC. e THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 and GFP-positive cells were sorted by flow cytometry. The GFP-positive cells were further transduced with MSCV-miR-375 or MSCV-NC and treated with puromycin for 1 week. Then, THP1-GFP-miR-375 or THP1-GFP-NC were xenografted into NSG mice. Peripheral blood cells were extracted from mice when the mice became moribund and GFP-positive cells were analyzed by flow cytometry. * P
Figure Legend Snippet: The anti-leukemia effects of miR-375 in vivo. About 1х10 7 viable HL-60 cells transduced with MSCV-miR-375 or MSCV-NC were injected subcutaneously into right flank of each nude mouse. a A photograph of xenografted tumors in mice xenografted by HL-60 cells, which were transduced with MSCV-miR-375 or MSCV-NC. b Volumes of all xenografted tumors were measured when the experiment was terminated at 42 days after tumor cell inoculation. c Net weights of all xenografted tumors were measured at the termination of the experiment. d The protein expression of HOXB3 was detected in xenografted tumor lysates from HL-60 cells transduced with MSCV-miR-375 or MSCV-NC. e THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 and GFP-positive cells were sorted by flow cytometry. The GFP-positive cells were further transduced with MSCV-miR-375 or MSCV-NC and treated with puromycin for 1 week. Then, THP1-GFP-miR-375 or THP1-GFP-NC were xenografted into NSG mice. Peripheral blood cells were extracted from mice when the mice became moribund and GFP-positive cells were analyzed by flow cytometry. * P

Techniques Used: In Vivo, Transduction, Injection, Mouse Assay, Expressing, Plasmid Preparation, Flow Cytometry, Cytometry

Decreased expression of miR-375 in AML patients predicts poor clinical outcome. a MiR-375 expressions were detected by qRT-PCR in several leukemic cell lines including NB4, HL-60, Kasumi-1, HEL, THP1, U937, and K562, 102 primary blasts from AML patients, and 20 normal controls (CD34 + cells, NC) by qRT-PCR. The lowest expression of miR-375 in one AML blast was set to 1.0 and then all other specimens were normalized by this lowest specimen. Housekeeping gene U6 is used as a reference. b MiR-375 expressions were detected in primary AML blasts according to FAB subtype (M1–M5). ( c and d ) The median expression of miR-375 was used as the cutoff. Kaplan-Meier overall survival curve ( c ) and disease-free survival curve ( d ) were indicated according to miR-375 expression level
Figure Legend Snippet: Decreased expression of miR-375 in AML patients predicts poor clinical outcome. a MiR-375 expressions were detected by qRT-PCR in several leukemic cell lines including NB4, HL-60, Kasumi-1, HEL, THP1, U937, and K562, 102 primary blasts from AML patients, and 20 normal controls (CD34 + cells, NC) by qRT-PCR. The lowest expression of miR-375 in one AML blast was set to 1.0 and then all other specimens were normalized by this lowest specimen. Housekeeping gene U6 is used as a reference. b MiR-375 expressions were detected in primary AML blasts according to FAB subtype (M1–M5). ( c and d ) The median expression of miR-375 was used as the cutoff. Kaplan-Meier overall survival curve ( c ) and disease-free survival curve ( d ) were indicated according to miR-375 expression level

Techniques Used: Expressing, Quantitative RT-PCR

DNA hypermethylation results in the low expression of miR-375. a – c The methylation status of miR-375 was analyzed by MSP in 7 leukemic cell lines ( a ), 40 primary AML blasts ( b ), and 20 normal controls ( c ). B: Blank; P: positive control of methylated DNA. Bands of ‘M’ or ‘U’ are PCR products amplified by methylation-specific or unmethylation-specific primers, respectively. Shown are the representative figures for primary AML blasts and normal controls. d A CpG map of the sequenced region was analyzed by MethPrimer software. e Bisulfite genomic sequencing was performed to detect the methylation status of the DNA sequences at − 260 bp − + 136 bp in the pre-miR-375 gene upstream region in HL-60, THP1, and one normal control (NC#1). Five PCR products were shown for each sample. Each row of circles represents the sequence of an individual clone. Black circles and empty circles represent methylated and unmethylated CpG dinucleotides, respectively (Left). Shown was the summary of frequencies of methylated CpG dinucleotides detected in HL-60, THP1, and one NC by bisulfite genomic sequencing (Right). f The expression of miR-375 was detected in HL-60 and THP1 cells treated with 5 μM AZA for 2 and 4 days. * P
Figure Legend Snippet: DNA hypermethylation results in the low expression of miR-375. a – c The methylation status of miR-375 was analyzed by MSP in 7 leukemic cell lines ( a ), 40 primary AML blasts ( b ), and 20 normal controls ( c ). B: Blank; P: positive control of methylated DNA. Bands of ‘M’ or ‘U’ are PCR products amplified by methylation-specific or unmethylation-specific primers, respectively. Shown are the representative figures for primary AML blasts and normal controls. d A CpG map of the sequenced region was analyzed by MethPrimer software. e Bisulfite genomic sequencing was performed to detect the methylation status of the DNA sequences at − 260 bp − + 136 bp in the pre-miR-375 gene upstream region in HL-60, THP1, and one normal control (NC#1). Five PCR products were shown for each sample. Each row of circles represents the sequence of an individual clone. Black circles and empty circles represent methylated and unmethylated CpG dinucleotides, respectively (Left). Shown was the summary of frequencies of methylated CpG dinucleotides detected in HL-60, THP1, and one NC by bisulfite genomic sequencing (Right). f The expression of miR-375 was detected in HL-60 and THP1 cells treated with 5 μM AZA for 2 and 4 days. * P

Techniques Used: Expressing, Methylation, Positive Control, Polymerase Chain Reaction, Amplification, Software, Genomic Sequencing, Sequencing

Ectopic expression of miR-375 inhibits cell growth and reduces colony formation. a Viable cell number was counted by the trypan-blue exclusion assay in HL-60 and THP1 cells, which were transduced with MSCV-miR-375 or MSCV-NC for the indicated times. * P
Figure Legend Snippet: Ectopic expression of miR-375 inhibits cell growth and reduces colony formation. a Viable cell number was counted by the trypan-blue exclusion assay in HL-60 and THP1 cells, which were transduced with MSCV-miR-375 or MSCV-NC for the indicated times. * P

Techniques Used: Expressing, Trypan Blue Exclusion Assay, Transduction

MiR-375 targets HOXB3 via binding 3′-UTR of HOXB3 . a Schematic of putative binding sites for miR-375 in 3′-UTR of HOXB3 . b 293 T cells were transfected with wide-type pMIR-HOXB3UTR (WT), pMIR-HOXB3UTR (Mut), pMIR-NC, and pRL-SV40 containing Renilla luciferase gene for 24 h, followed by the transfection with miR-375 mimic or Scramble for another 24 h. Firefly and Renilla luciferase activities were both detected and histograms showed that the Firefly luciferase activities were normalized to Renilla luciferase activities. c The expression of miR-375 was detected in HL-60 and THP1 cells transduced with MSCV-miR-375 or MSCV-NC. * P
Figure Legend Snippet: MiR-375 targets HOXB3 via binding 3′-UTR of HOXB3 . a Schematic of putative binding sites for miR-375 in 3′-UTR of HOXB3 . b 293 T cells were transfected with wide-type pMIR-HOXB3UTR (WT), pMIR-HOXB3UTR (Mut), pMIR-NC, and pRL-SV40 containing Renilla luciferase gene for 24 h, followed by the transfection with miR-375 mimic or Scramble for another 24 h. Firefly and Renilla luciferase activities were both detected and histograms showed that the Firefly luciferase activities were normalized to Renilla luciferase activities. c The expression of miR-375 was detected in HL-60 and THP1 cells transduced with MSCV-miR-375 or MSCV-NC. * P

Techniques Used: Binding Assay, Transfection, Luciferase, Expressing, Transduction

6) Product Images from "Notch Activation Differentially Regulates Renal Progenitors Proliferation and Differentiation Toward the Podocyte Lineage in Glomerular Disorders"

Article Title: Notch Activation Differentially Regulates Renal Progenitors Proliferation and Differentiation Toward the Podocyte Lineage in Glomerular Disorders

Journal: Stem Cells (Dayton, Ohio)

doi: 10.1002/stem.492

Notch downregulation is necessary to achieve differentiation of human renal progenitors into the podocyte lineage. ( A ): Sustained Notch protein expression in renal progenitors as obtained following infection with a vector leading to the N1ICD, N2ICD, or N3ICD and the GFP to be simultaneously coexpressed. Cells infected with the empty vector (mock) express GFP but do not express the NICDs. Uninfected cells are shown for comparison. One representative of 10 independent experiments is shown. ( B ): Downregulation of the Notch pathway activity following differentiation of renal progenitors toward the podocyte lineage is rescued by infection with vectors expressing N1ICD, N2ICD, or N3ICD as demonstrated by coinfection with a reporter vector for the RBP-J transcriptional response element. Results are expressed as mean ± SEM fold increase of luciferase activity in renal progenitors coinfected with a reporter vector for the RBP-J and the vectors expressing N1ICD, N2ICD, or N3ICD in comparison with the empty vector before (control) or after their differentiation toward the podocyte lineage (VRADD) as obtained in at least four independent experiments. b versus a , c , d , e , f , g , h : p
Figure Legend Snippet: Notch downregulation is necessary to achieve differentiation of human renal progenitors into the podocyte lineage. ( A ): Sustained Notch protein expression in renal progenitors as obtained following infection with a vector leading to the N1ICD, N2ICD, or N3ICD and the GFP to be simultaneously coexpressed. Cells infected with the empty vector (mock) express GFP but do not express the NICDs. Uninfected cells are shown for comparison. One representative of 10 independent experiments is shown. ( B ): Downregulation of the Notch pathway activity following differentiation of renal progenitors toward the podocyte lineage is rescued by infection with vectors expressing N1ICD, N2ICD, or N3ICD as demonstrated by coinfection with a reporter vector for the RBP-J transcriptional response element. Results are expressed as mean ± SEM fold increase of luciferase activity in renal progenitors coinfected with a reporter vector for the RBP-J and the vectors expressing N1ICD, N2ICD, or N3ICD in comparison with the empty vector before (control) or after their differentiation toward the podocyte lineage (VRADD) as obtained in at least four independent experiments. b versus a , c , d , e , f , g , h : p

Techniques Used: Expressing, Infection, Plasmid Preparation, Activity Assay, Luciferase

7) Product Images from "Pannexin3 inhibits TNF‐α‐induced inflammatory response by suppressing NF‐κB signalling pathway in human dental pulp cells"

Article Title: Pannexin3 inhibits TNF‐α‐induced inflammatory response by suppressing NF‐κB signalling pathway in human dental pulp cells

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.12988

Panx3 regulates NF ‐κB signalling pathway. ( A, B ) NF ‐κB transcriptional activities in HDPC /Panx3, HDPC /Plvx, HDPC /sh RNA and HDPC /Ctrl groups were measured using dual‐luciferase reporter system. * P
Figure Legend Snippet: Panx3 regulates NF ‐κB signalling pathway. ( A, B ) NF ‐κB transcriptional activities in HDPC /Panx3, HDPC /Plvx, HDPC /sh RNA and HDPC /Ctrl groups were measured using dual‐luciferase reporter system. * P

Techniques Used: Luciferase

Panx3 regulates TNF ‐α‐induced inflammatory cytokine expression in HDPC s. qRT ‐ PCR ( A, B ) and ELISA ( C ) analysis of IL ‐1β and IL ‐6 expression in HDPC /Panx3 and HDPC /Plvx cells upon TNF ‐α stimulation for 24 hrs. qRT ‐ PCR ( D, E ) and ELISA ( F ) analysis of IL ‐1β and IL ‐6 expression in HDPC /sh RNA and HDPC /Ctrl cells upon TNF ‐α stimulation for 24 hrs. * P
Figure Legend Snippet: Panx3 regulates TNF ‐α‐induced inflammatory cytokine expression in HDPC s. qRT ‐ PCR ( A, B ) and ELISA ( C ) analysis of IL ‐1β and IL ‐6 expression in HDPC /Panx3 and HDPC /Plvx cells upon TNF ‐α stimulation for 24 hrs. qRT ‐ PCR ( D, E ) and ELISA ( F ) analysis of IL ‐1β and IL ‐6 expression in HDPC /sh RNA and HDPC /Ctrl cells upon TNF ‐α stimulation for 24 hrs. * P

Techniques Used: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Schematic illustration of the role of Panx3 in dental pulp inflammation. TNF ‐α could down‐regulate the expression of Panx3 via activating proteasome pathway, meanwhile the NF ‐κB might balance the effect. In addition, Panx3 could suppress NF ‐κB signalling pathway, leading to inhibition of TNF ‐α‐induced inflammatory response. The black lines indicate that the interactions have been clearly established in previous studies. The purple lines indicate the connection established in this study. MG 132, a proteasome inhibitor; BAY 11‐7082, a NF ‐κB inhibitor.
Figure Legend Snippet: Schematic illustration of the role of Panx3 in dental pulp inflammation. TNF ‐α could down‐regulate the expression of Panx3 via activating proteasome pathway, meanwhile the NF ‐κB might balance the effect. In addition, Panx3 could suppress NF ‐κB signalling pathway, leading to inhibition of TNF ‐α‐induced inflammatory response. The black lines indicate that the interactions have been clearly established in previous studies. The purple lines indicate the connection established in this study. MG 132, a proteasome inhibitor; BAY 11‐7082, a NF ‐κB inhibitor.

Techniques Used: Expressing, Inhibition

MG 132 not BAY 11‐7082 rescued the TNF ‐α‐induced down‐regulation of Panx3. Cells were pre‐treated with 2 μM BAY 11‐7082, 1 μM MG 132 or DMSO for 30 min. before TNF ‐α treatment. qRT ‐ PCR ( A, C ) and Western blot analysis ( B, D ) were then performed to determine the Panx3 expression. * P
Figure Legend Snippet: MG 132 not BAY 11‐7082 rescued the TNF ‐α‐induced down‐regulation of Panx3. Cells were pre‐treated with 2 μM BAY 11‐7082, 1 μM MG 132 or DMSO for 30 min. before TNF ‐α treatment. qRT ‐ PCR ( A, C ) and Western blot analysis ( B, D ) were then performed to determine the Panx3 expression. * P

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

TNF ‐α repressed Panx3 expression at the mRNA and protein levels in a concentration‐dependent manner in HDPC s. The mRNA and protein expression levels of Panx3 in HDPC s stimulated with TNF ‐α (0–10 ng/ml) for 24 hrs were detected by qRT ‐ PCR ( A ) and Western blot ( B ). GAPDH served as an internal control. ( C ) Immunofluorescence of Panx3 in HDPC s with or without TNF ‐α (10 ng/ml) treatment for 24 hrs. Scale bar = 200 μm. Data were expressed as mean ± SEM . * P
Figure Legend Snippet: TNF ‐α repressed Panx3 expression at the mRNA and protein levels in a concentration‐dependent manner in HDPC s. The mRNA and protein expression levels of Panx3 in HDPC s stimulated with TNF ‐α (0–10 ng/ml) for 24 hrs were detected by qRT ‐ PCR ( A ) and Western blot ( B ). GAPDH served as an internal control. ( C ) Immunofluorescence of Panx3 in HDPC s with or without TNF ‐α (10 ng/ml) treatment for 24 hrs. Scale bar = 200 μm. Data were expressed as mean ± SEM . * P

Techniques Used: Expressing, Concentration Assay, Quantitative RT-PCR, Western Blot, Immunofluorescence

The expression of Panx3 was decreased in inflamed human ( A ) and rat dental pulpitis tissue (B). The dental pulp tissue sections were stained with HE , anti‐Panx3 antibody or anti‐ TNF ‐α antibody. HE ‐stained sections of normal and inflamed dental pulp tissue verified the progression of inflammation. Immunohistochemistry performed on dental pulp tissues showed the presence of Panx3 in normal tissue, but decreased in the inflamed pulp tissue. In contrast to Panx3 expression, TNF ‐α was increased in inflamed dental pulp tissue. Cell nuclei were visualized with haematoxylin. Scale bars are stamped in the images, respectively.
Figure Legend Snippet: The expression of Panx3 was decreased in inflamed human ( A ) and rat dental pulpitis tissue (B). The dental pulp tissue sections were stained with HE , anti‐Panx3 antibody or anti‐ TNF ‐α antibody. HE ‐stained sections of normal and inflamed dental pulp tissue verified the progression of inflammation. Immunohistochemistry performed on dental pulp tissues showed the presence of Panx3 in normal tissue, but decreased in the inflamed pulp tissue. In contrast to Panx3 expression, TNF ‐α was increased in inflamed dental pulp tissue. Cell nuclei were visualized with haematoxylin. Scale bars are stamped in the images, respectively.

Techniques Used: Expressing, Staining, Immunohistochemistry

Lentiviral‐mediated expression of Panx3 in HDPC s. ( A, B ) Cell images of HDPC s/Panx3, HDPC /Plvx, HDPC /sh RNA and HCPD /Ctrl groups were photographed in normal light (lower panels) and under a fluorescence microscope (upper panels). Protein and mRNA expression of Panx3 were determined by Western blot ( C, D ) and qRT ‐ PCR ( E, F ) analysis. * P
Figure Legend Snippet: Lentiviral‐mediated expression of Panx3 in HDPC s. ( A, B ) Cell images of HDPC s/Panx3, HDPC /Plvx, HDPC /sh RNA and HCPD /Ctrl groups were photographed in normal light (lower panels) and under a fluorescence microscope (upper panels). Protein and mRNA expression of Panx3 were determined by Western blot ( C, D ) and qRT ‐ PCR ( E, F ) analysis. * P

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

8) Product Images from "Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program"

Article Title: Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program

Journal: Acta Neuropathologica

doi: 10.1007/s00401-019-02062-4

NSUN5 epigenetic loss is associated with depletion of global protein synthesis and the emergence of a stress-response translational program. a NSUN5 unmethylated glioma cell lines DBTRG-05MG, MO59J, and CAS-1 show higher overall protein synthesis assessed by OP-Puro under oxidative stress (100 mM H 2 O 2 ) than the NSUN5 methylated cells (A172, LN229 and KS-1). b Restoration of NSUN5 function by transfection in epigenetically inactive LN299 cells increases overall protein synthesis under oxidative stress (100 mM H 2 O 2 ) assessed by OP-Puro. Enhancement of global protein synthesis upon NSUN5 recovery in LN299 cells is also observed by the [3H] leucine incorporation assay. c Similar results were obtained upon nutrient deprivation. d Comparison of the total RNA (RNA-seq) and ribosome-protected RNA (Ribo-seq) deep-sequencing profiles to identify those RNAs with enhanced translational efficiency in NSUN5 deficient cells. 1987 RNAs that did not change in the RNA-seq of LN229 cells upon NSUN5-transfection were upregulated in the Ribo-seq of empty-vector-transfected cells indicating enhanced translational efficiency. e NSUN5 affects both CAP-dependent and CAP-independent translation according to the use of a reporter plasmid encoding for Firefly (IRES) and Renilla (CAP) luciferases. f Gene set enrichment analysis (GSEA) of the RNAs with increased translational efficiency in NSUN5 deficient cells (hypergeometric test with a FDR adjusted P value
Figure Legend Snippet: NSUN5 epigenetic loss is associated with depletion of global protein synthesis and the emergence of a stress-response translational program. a NSUN5 unmethylated glioma cell lines DBTRG-05MG, MO59J, and CAS-1 show higher overall protein synthesis assessed by OP-Puro under oxidative stress (100 mM H 2 O 2 ) than the NSUN5 methylated cells (A172, LN229 and KS-1). b Restoration of NSUN5 function by transfection in epigenetically inactive LN299 cells increases overall protein synthesis under oxidative stress (100 mM H 2 O 2 ) assessed by OP-Puro. Enhancement of global protein synthesis upon NSUN5 recovery in LN299 cells is also observed by the [3H] leucine incorporation assay. c Similar results were obtained upon nutrient deprivation. d Comparison of the total RNA (RNA-seq) and ribosome-protected RNA (Ribo-seq) deep-sequencing profiles to identify those RNAs with enhanced translational efficiency in NSUN5 deficient cells. 1987 RNAs that did not change in the RNA-seq of LN229 cells upon NSUN5-transfection were upregulated in the Ribo-seq of empty-vector-transfected cells indicating enhanced translational efficiency. e NSUN5 affects both CAP-dependent and CAP-independent translation according to the use of a reporter plasmid encoding for Firefly (IRES) and Renilla (CAP) luciferases. f Gene set enrichment analysis (GSEA) of the RNAs with increased translational efficiency in NSUN5 deficient cells (hypergeometric test with a FDR adjusted P value

Techniques Used: Methylation, Transfection, RNA Sequencing Assay, Sequencing, Plasmid Preparation

Forest plots of the multivariable Cox regression analysis for clinical outcome in the glioma cohorts studied for NSUN5 methylation status taking into account different prognostic factors. P values ( P ) correspond to hazard ratios (HR), with a 95% of confidence interval (95%CI), associated with OS. Co-variables with associated P value under 0.05 were considered as independent prognostic factor (* P
Figure Legend Snippet: Forest plots of the multivariable Cox regression analysis for clinical outcome in the glioma cohorts studied for NSUN5 methylation status taking into account different prognostic factors. P values ( P ) correspond to hazard ratios (HR), with a 95% of confidence interval (95%CI), associated with OS. Co-variables with associated P value under 0.05 were considered as independent prognostic factor (* P

Techniques Used: Methylation

NSUN5 epigenetic silencing activates stress-related protein and confers growth inhibition sensitivity to NQO1-targeting molecules. a Validation of NSUN5 translational regulation of the identified stress-related target NQO1 expression at the RNA level determined by RNA-seq counts (left) and real-time quantitative PCR (middle) do not change upon NSUN5 transfection, but NQO1 expression decreased at the protein level (right) upon NSUN5 restoration. b qPCR shows enrichment of the NQO1 transcript in the polysome fraction of the empty-vector LN229 cells in comparison to NSUN5 transfected-LN229 cells. c NQO1 expression levels in glioma cell lines determined by western blot according to NSUN5 methylation status. d IC50 determination using the SRB assay in the glioma cell lines grouped by NSUN5 methylation status. Black dashed curves represent the 95% confidence band for each group. Glioma cells harboring NSUN5 methylation-associated NQO1 overexpression (A172, LN229 and KS-1) show increased sensitivity to deoxynyboquinone (DNQ) and IB-DNQ in comparison to NSUN5 unmethylated cells (DBTRG-05MG, MO59 J, and CAS-1). Drug-response curves were generated using GraphPad Prism software and analyses were performed with the drc R package. For each cell line and the drug, we fit a four-parameter generalized log-logistic model. Comparison of the IC50 values calculated from the slopes were obtained by means of a z test ( P
Figure Legend Snippet: NSUN5 epigenetic silencing activates stress-related protein and confers growth inhibition sensitivity to NQO1-targeting molecules. a Validation of NSUN5 translational regulation of the identified stress-related target NQO1 expression at the RNA level determined by RNA-seq counts (left) and real-time quantitative PCR (middle) do not change upon NSUN5 transfection, but NQO1 expression decreased at the protein level (right) upon NSUN5 restoration. b qPCR shows enrichment of the NQO1 transcript in the polysome fraction of the empty-vector LN229 cells in comparison to NSUN5 transfected-LN229 cells. c NQO1 expression levels in glioma cell lines determined by western blot according to NSUN5 methylation status. d IC50 determination using the SRB assay in the glioma cell lines grouped by NSUN5 methylation status. Black dashed curves represent the 95% confidence band for each group. Glioma cells harboring NSUN5 methylation-associated NQO1 overexpression (A172, LN229 and KS-1) show increased sensitivity to deoxynyboquinone (DNQ) and IB-DNQ in comparison to NSUN5 unmethylated cells (DBTRG-05MG, MO59 J, and CAS-1). Drug-response curves were generated using GraphPad Prism software and analyses were performed with the drc R package. For each cell line and the drug, we fit a four-parameter generalized log-logistic model. Comparison of the IC50 values calculated from the slopes were obtained by means of a z test ( P

Techniques Used: Inhibition, Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Western Blot, Methylation, Sulforhodamine B Assay, Over Expression, Generated, Software

NSUN5 epigenetic inactivation occurs in human primary gliomas in association with good clinical outcome. a Percentage of NSUN5 methylation in the TCGA data set of primary tumors according to cancer type (top) and according to the cellular grade of the glioma (down). b NSUN5 promoter CpG island methylation is associated with the loss of the transcript among all cellular grades in primary glioma, in low-grade glioma and in glioblastoma. c Kaplan–Meier analysis of overall survival (OS) across overall glioma grades and in low- and high-grade glioma with respect to NSUN5 methylation status. Significance of the log-rank test is shown. Results of the univariate Cox regression analysis are represented by the hazards ratio (HR) and 95% confidence interval (CI). d Kaplan–Meier analysis of OS in IDH1 wild-type gliomas according to NSUN5 methylation status. e Kaplan–Meier analysis of OS in gliomas without 1p/19q deletion according to NSUN5 methylation status. f Kaplan–Meier analysis of OS in gliomas with unmethylated MGMT according to NSUN5 methylation status. For all graphs, the P value corresponds to the Log-Rank test. Cox regression univariate analysis is represented by HR with a 95% CI
Figure Legend Snippet: NSUN5 epigenetic inactivation occurs in human primary gliomas in association with good clinical outcome. a Percentage of NSUN5 methylation in the TCGA data set of primary tumors according to cancer type (top) and according to the cellular grade of the glioma (down). b NSUN5 promoter CpG island methylation is associated with the loss of the transcript among all cellular grades in primary glioma, in low-grade glioma and in glioblastoma. c Kaplan–Meier analysis of overall survival (OS) across overall glioma grades and in low- and high-grade glioma with respect to NSUN5 methylation status. Significance of the log-rank test is shown. Results of the univariate Cox regression analysis are represented by the hazards ratio (HR) and 95% confidence interval (CI). d Kaplan–Meier analysis of OS in IDH1 wild-type gliomas according to NSUN5 methylation status. e Kaplan–Meier analysis of OS in gliomas without 1p/19q deletion according to NSUN5 methylation status. f Kaplan–Meier analysis of OS in gliomas with unmethylated MGMT according to NSUN5 methylation status. For all graphs, the P value corresponds to the Log-Rank test. Cox regression univariate analysis is represented by HR with a 95% CI

Techniques Used: Methylation

Transcriptional silencing of NSUN5 by promoter CpG island hypermethylation in human glioma cells. a Percentage of NSUN5 methylation in the Sanger panel of cancer cell lines by tumor type. b NSUN5 methylation is associated with loss of the transcript in the glioma cell lines from Sanger ( n = 48). Correlation analysis between methylation beta values and expression Z -score are shown. The P value corresponding to Spearman’s rank correlation test and the associated rho coefficient are indicated in the figure. c Bisulfite genomic sequencing of NSUN5 promoter CpG Island in glioma cells lines and brain white matter. CpG dinucleotides are represented as short vertical lines and the transcription start site (TSS) is represented as a long black arrow. Single clones are shown for each sample. Presence of an unmethylated or methylated cytosine is indicated by a white or black square, respectively, and percentage of methylation is indicated on the right. d DNA methylation profile of the CpG island promoter for the NSUN5 gene analyzed by the 450 K DNA methylation microarray. Single CpG absolute methylation levels (0–1) are shown. Green, unmethylated; red, methylated. Data from the studied six glioma cell lines, brain white matter and nineteen normal brain samples are shown. e NSUN5 expression levels in glioma cell lines determined by real-time PCR (data shown represent mean ± S.D. of biological triplicates) and western blot. f Expression of the NSUN5 RNA transcript and protein was restored in the A172, LN229 and KS-1 cells by treatment with the demethylating drug 5-aza-2′-deoxycytidine (AZA). Data shown represent the mean ± S.D. of biological triplicates and P values were obtained by the Mann–Whitney test. ** P
Figure Legend Snippet: Transcriptional silencing of NSUN5 by promoter CpG island hypermethylation in human glioma cells. a Percentage of NSUN5 methylation in the Sanger panel of cancer cell lines by tumor type. b NSUN5 methylation is associated with loss of the transcript in the glioma cell lines from Sanger ( n = 48). Correlation analysis between methylation beta values and expression Z -score are shown. The P value corresponding to Spearman’s rank correlation test and the associated rho coefficient are indicated in the figure. c Bisulfite genomic sequencing of NSUN5 promoter CpG Island in glioma cells lines and brain white matter. CpG dinucleotides are represented as short vertical lines and the transcription start site (TSS) is represented as a long black arrow. Single clones are shown for each sample. Presence of an unmethylated or methylated cytosine is indicated by a white or black square, respectively, and percentage of methylation is indicated on the right. d DNA methylation profile of the CpG island promoter for the NSUN5 gene analyzed by the 450 K DNA methylation microarray. Single CpG absolute methylation levels (0–1) are shown. Green, unmethylated; red, methylated. Data from the studied six glioma cell lines, brain white matter and nineteen normal brain samples are shown. e NSUN5 expression levels in glioma cell lines determined by real-time PCR (data shown represent mean ± S.D. of biological triplicates) and western blot. f Expression of the NSUN5 RNA transcript and protein was restored in the A172, LN229 and KS-1 cells by treatment with the demethylating drug 5-aza-2′-deoxycytidine (AZA). Data shown represent the mean ± S.D. of biological triplicates and P values were obtained by the Mann–Whitney test. ** P

Techniques Used: Methylation, Expressing, Genomic Sequencing, Clone Assay, DNA Methylation Assay, Microarray, Real-time Polymerase Chain Reaction, Western Blot, MANN-WHITNEY

Restoration of NSUN5 impairs glioma tumor growth in vivo. a Western blot to show efficient restoration of NSUN5 protein expression upon stable transfection in A172 and LN299 glioma cells and efficient depletion of NSUN5 protein expression in NSUN5-shRNA DBTRG-05MG glioma cells. EV empty vector. An equal number of the indicated A172 and LN299 cells populations were stereotactically inoculated into the brain of athymic mice. The size of the tumors was estimated at 10 and 17 days post-inoculation (DPI) by the quantification of luciferase activity in the tumor cells. b Scatter plots showing the individual size of the indicated LN229 and A172 tumors after 10 and 17 DPI. c Representative images of the luciferase signal from mice inoculated with the indicated LN229 and A172 tumors after 17 DPI. d LN229-EV and LN229-NSUN5 cells were injected in the left or right flank of 10 mice, respectively. Tumor volume measured over time ( left panel ) and tumor weight upon sacrifice ( right panel ) are shown. P values obtained by Student’s t test. Error bars show means ± s.d. e Scramble and NSUN5-shRNA-depleted DBTRG-05MG cells were injected in the left or right flank of 10 mice, respectively. Tumor volume measured over time (left panel) and tumor weight upon sacrifice (right panel) are shown. P values obtained by Student’s t test. Error bars show means ± s.d
Figure Legend Snippet: Restoration of NSUN5 impairs glioma tumor growth in vivo. a Western blot to show efficient restoration of NSUN5 protein expression upon stable transfection in A172 and LN299 glioma cells and efficient depletion of NSUN5 protein expression in NSUN5-shRNA DBTRG-05MG glioma cells. EV empty vector. An equal number of the indicated A172 and LN299 cells populations were stereotactically inoculated into the brain of athymic mice. The size of the tumors was estimated at 10 and 17 days post-inoculation (DPI) by the quantification of luciferase activity in the tumor cells. b Scatter plots showing the individual size of the indicated LN229 and A172 tumors after 10 and 17 DPI. c Representative images of the luciferase signal from mice inoculated with the indicated LN229 and A172 tumors after 17 DPI. d LN229-EV and LN229-NSUN5 cells were injected in the left or right flank of 10 mice, respectively. Tumor volume measured over time ( left panel ) and tumor weight upon sacrifice ( right panel ) are shown. P values obtained by Student’s t test. Error bars show means ± s.d. e Scramble and NSUN5-shRNA-depleted DBTRG-05MG cells were injected in the left or right flank of 10 mice, respectively. Tumor volume measured over time (left panel) and tumor weight upon sacrifice (right panel) are shown. P values obtained by Student’s t test. Error bars show means ± s.d

Techniques Used: In Vivo, Western Blot, Expressing, Stable Transfection, shRNA, Plasmid Preparation, Mouse Assay, Luciferase, Activity Assay, Injection

NSUN5 epigenetic inactivation abrogates the methylation of the C3782 position of human 28S rRNA. a Top , RNA sequence alignment of the conserved human 28S rRNA C3782 position (black square) in the corresponding 26S, 25S and 28S rRNAs orthologues from C. elegans , S. cerevisiae and M. musculus . Below , Protein sequence alignment of human NSUN5 with its recognized rRNA 5-methylcytosine RNA-methyltransferase orthologues in C. Elegans , S. Cerevisiae and M. musculus . Highlighted in black and grey the identical and physicochemically similar (scoring > 0.5 in the Gonnet PAM 250 matrix) residues, respectively. The aligned region includes the RNA-methyltransferase domain. b NSUN5 interaction with 28S rRNA. Total extracts from LN229 cells, either transfected with empty vector (EV) or NSUN5 were immunoprecipitated with an anti-Flag antibody ( left panel ), followed by analysis of the retrieved RNA by quantitative RT-PCR ( right panel ). c RNA bisulfite sequencing of the 28S rRNA in glioma cells lines according to NSUN5 epigenetic status. Cytosines are represented as short vertical lines and the C3782 site is represented as a long black arrow. Single clones are shown for each sample. Presence of an unmethylated or methylated cytosine is indicated by a white or black square, respectively. d RNA bisulfite sequencing of the 28S rRNA in empty-vector (EV) and NSUN5-transfected LN229 and A172 glioma cells. e RNA bisulfite sequencing of the 28S rRNA in scramble and NSUN5-shRNA-depleted DBTRG-05MG and CAS-1 glioma cells. For CAS-1, western-blot to show efficient NSUN5 depletion is shown above
Figure Legend Snippet: NSUN5 epigenetic inactivation abrogates the methylation of the C3782 position of human 28S rRNA. a Top , RNA sequence alignment of the conserved human 28S rRNA C3782 position (black square) in the corresponding 26S, 25S and 28S rRNAs orthologues from C. elegans , S. cerevisiae and M. musculus . Below , Protein sequence alignment of human NSUN5 with its recognized rRNA 5-methylcytosine RNA-methyltransferase orthologues in C. Elegans , S. Cerevisiae and M. musculus . Highlighted in black and grey the identical and physicochemically similar (scoring > 0.5 in the Gonnet PAM 250 matrix) residues, respectively. The aligned region includes the RNA-methyltransferase domain. b NSUN5 interaction with 28S rRNA. Total extracts from LN229 cells, either transfected with empty vector (EV) or NSUN5 were immunoprecipitated with an anti-Flag antibody ( left panel ), followed by analysis of the retrieved RNA by quantitative RT-PCR ( right panel ). c RNA bisulfite sequencing of the 28S rRNA in glioma cells lines according to NSUN5 epigenetic status. Cytosines are represented as short vertical lines and the C3782 site is represented as a long black arrow. Single clones are shown for each sample. Presence of an unmethylated or methylated cytosine is indicated by a white or black square, respectively. d RNA bisulfite sequencing of the 28S rRNA in empty-vector (EV) and NSUN5-transfected LN229 and A172 glioma cells. e RNA bisulfite sequencing of the 28S rRNA in scramble and NSUN5-shRNA-depleted DBTRG-05MG and CAS-1 glioma cells. For CAS-1, western-blot to show efficient NSUN5 depletion is shown above

Techniques Used: Methylation, Sequencing, Transfection, Plasmid Preparation, Immunoprecipitation, Quantitative RT-PCR, Methylation Sequencing, Clone Assay, shRNA, Western Blot

9) Product Images from "A Pitx2-MicroRNA Pathway Modulates Cell Proliferation in Myoblasts and Skeletal-Muscle Satellite Cells and Promotes Their Commitment to a Myogenic Cell Fate"

Article Title: A Pitx2-MicroRNA Pathway Modulates Cell Proliferation in Myoblasts and Skeletal-Muscle Satellite Cells and Promotes Their Commitment to a Myogenic Cell Fate

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00536-15

(A) Representative images of EPq cells transfected with a lentivirus-Pitx2c-ZsGreen vector (LVX-Pitx2c). (B) qRT-PCR for Pitx2c expression in EPq and EPa cells transfected with the LVX-Pitx2c vector with respect to cells transfected with the empty LVX-ZsGreen lentiviral vector (LVX). (C) Cyclin D1 and cyclin D2 gene expression in EPq and EPa Pitx2c-overexpressing cells with respect to control cells. (D) Representative images of immunohistochemical analyses for Ki67-positve cells in EPq cells transfected with the lentivirus-Pitx2c-ZsGreen vector (LVX) compared to cells transfected with the empty LVX-ZsGreen lentiviral vector (LVX-Pitx2c). (E) Percentage of Ki67 + cells in EPq cells transfected with the lentivirus-Pitx2c-ZsGreen vector with respect to cells transfected with the empty LVX-ZsGreen lentiviral vector.
Figure Legend Snippet: (A) Representative images of EPq cells transfected with a lentivirus-Pitx2c-ZsGreen vector (LVX-Pitx2c). (B) qRT-PCR for Pitx2c expression in EPq and EPa cells transfected with the LVX-Pitx2c vector with respect to cells transfected with the empty LVX-ZsGreen lentiviral vector (LVX). (C) Cyclin D1 and cyclin D2 gene expression in EPq and EPa Pitx2c-overexpressing cells with respect to control cells. (D) Representative images of immunohistochemical analyses for Ki67-positve cells in EPq cells transfected with the lentivirus-Pitx2c-ZsGreen vector (LVX) compared to cells transfected with the empty LVX-ZsGreen lentiviral vector (LVX-Pitx2c). (E) Percentage of Ki67 + cells in EPq cells transfected with the lentivirus-Pitx2c-ZsGreen vector with respect to cells transfected with the empty LVX-ZsGreen lentiviral vector.

Techniques Used: Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing, Immunohistochemistry

(A) Myf5 expression profile in EPq Pitx2c -overexpressing cells. (B) Representative images of immunohistochemical analyses for Myf5-positive cells in EPq cells transfected with the lentivirus- Pitx2c -ZsGreen vector (LVX) compared to cells transfected with the empty LVX-ZsGreen lentiviral vector (LVX-Pitx2c). (C) Percentage of Myf5 + cells in EPq cells transfected with the lentivirus-Pitx2c-ZsGreen vector (LVX-control) with respect to cells transfected with the empty LVX-ZsGreen lentiviral vector (LVX-Pitx2). (D and E) miR-106b overexpression leads to Myf5 upregulation in EPq cells. (F) Normalized luciferase activity of the 3′-UTR Myf5 luciferase reporter (wild-type Myf5 3′ UTR) with an empty plasmid (vector) or pre-miR-106b shows the loss of luciferase activity with expression of miR-106b. There was no loss of luciferase activity when the miR-106b seed sequence was mutated.
Figure Legend Snippet: (A) Myf5 expression profile in EPq Pitx2c -overexpressing cells. (B) Representative images of immunohistochemical analyses for Myf5-positive cells in EPq cells transfected with the lentivirus- Pitx2c -ZsGreen vector (LVX) compared to cells transfected with the empty LVX-ZsGreen lentiviral vector (LVX-Pitx2c). (C) Percentage of Myf5 + cells in EPq cells transfected with the lentivirus-Pitx2c-ZsGreen vector (LVX-control) with respect to cells transfected with the empty LVX-ZsGreen lentiviral vector (LVX-Pitx2). (D and E) miR-106b overexpression leads to Myf5 upregulation in EPq cells. (F) Normalized luciferase activity of the 3′-UTR Myf5 luciferase reporter (wild-type Myf5 3′ UTR) with an empty plasmid (vector) or pre-miR-106b shows the loss of luciferase activity with expression of miR-106b. There was no loss of luciferase activity when the miR-106b seed sequence was mutated.

Techniques Used: Expressing, Immunohistochemistry, Transfection, Plasmid Preparation, Over Expression, Luciferase, Activity Assay, Sequencing

10) Product Images from "A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia"

Article Title: A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia

Journal: BMC Cancer

doi: 10.1186/s12885-018-4097-z

The anti-leukemia effects of miR-375 in vivo. About 1х10 7 viable HL-60 cells transduced with MSCV-miR-375 or MSCV-NC were injected subcutaneously into right flank of each nude mouse. a A photograph of xenografted tumors in mice xenografted by HL-60 cells, which were transduced with MSCV-miR-375 or MSCV-NC. b Volumes of all xenografted tumors were measured when the experiment was terminated at 42 days after tumor cell inoculation. c Net weights of all xenografted tumors were measured at the termination of the experiment. d The protein expression of HOXB3 was detected in xenografted tumor lysates from HL-60 cells transduced with MSCV-miR-375 or MSCV-NC. e THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 and GFP-positive cells were sorted by flow cytometry. The GFP-positive cells were further transduced with MSCV-miR-375 or MSCV-NC and treated with puromycin for 1 week. Then, THP1-GFP-miR-375 or THP1-GFP-NC were xenografted into NSG mice. Peripheral blood cells were extracted from mice when the mice became moribund and GFP-positive cells were analyzed by flow cytometry. * P
Figure Legend Snippet: The anti-leukemia effects of miR-375 in vivo. About 1х10 7 viable HL-60 cells transduced with MSCV-miR-375 or MSCV-NC were injected subcutaneously into right flank of each nude mouse. a A photograph of xenografted tumors in mice xenografted by HL-60 cells, which were transduced with MSCV-miR-375 or MSCV-NC. b Volumes of all xenografted tumors were measured when the experiment was terminated at 42 days after tumor cell inoculation. c Net weights of all xenografted tumors were measured at the termination of the experiment. d The protein expression of HOXB3 was detected in xenografted tumor lysates from HL-60 cells transduced with MSCV-miR-375 or MSCV-NC. e THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 and GFP-positive cells were sorted by flow cytometry. The GFP-positive cells were further transduced with MSCV-miR-375 or MSCV-NC and treated with puromycin for 1 week. Then, THP1-GFP-miR-375 or THP1-GFP-NC were xenografted into NSG mice. Peripheral blood cells were extracted from mice when the mice became moribund and GFP-positive cells were analyzed by flow cytometry. * P

Techniques Used: In Vivo, Transduction, Injection, Mouse Assay, Expressing, Plasmid Preparation, Flow Cytometry, Cytometry

11) Product Images from "A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia"

Article Title: A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia

Journal: BMC Cancer

doi: 10.1186/s12885-018-4097-z

The anti-leukemia effects of miR-375 in vivo. About 1х10 7 viable HL-60 cells transduced with MSCV-miR-375 or MSCV-NC were injected subcutaneously into right flank of each nude mouse. a A photograph of xenografted tumors in mice xenografted by HL-60 cells, which were transduced with MSCV-miR-375 or MSCV-NC. b Volumes of all xenografted tumors were measured when the experiment was terminated at 42 days after tumor cell inoculation. c Net weights of all xenografted tumors were measured at the termination of the experiment. d The protein expression of HOXB3 was detected in xenografted tumor lysates from HL-60 cells transduced with MSCV-miR-375 or MSCV-NC. e THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 and GFP-positive cells were sorted by flow cytometry. The GFP-positive cells were further transduced with MSCV-miR-375 or MSCV-NC and treated with puromycin for 1 week. Then, THP1-GFP-miR-375 or THP1-GFP-NC were xenografted into NSG mice. Peripheral blood cells were extracted from mice when the mice became moribund and GFP-positive cells were analyzed by flow cytometry. * P
Figure Legend Snippet: The anti-leukemia effects of miR-375 in vivo. About 1х10 7 viable HL-60 cells transduced with MSCV-miR-375 or MSCV-NC were injected subcutaneously into right flank of each nude mouse. a A photograph of xenografted tumors in mice xenografted by HL-60 cells, which were transduced with MSCV-miR-375 or MSCV-NC. b Volumes of all xenografted tumors were measured when the experiment was terminated at 42 days after tumor cell inoculation. c Net weights of all xenografted tumors were measured at the termination of the experiment. d The protein expression of HOXB3 was detected in xenografted tumor lysates from HL-60 cells transduced with MSCV-miR-375 or MSCV-NC. e THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 and GFP-positive cells were sorted by flow cytometry. The GFP-positive cells were further transduced with MSCV-miR-375 or MSCV-NC and treated with puromycin for 1 week. Then, THP1-GFP-miR-375 or THP1-GFP-NC were xenografted into NSG mice. Peripheral blood cells were extracted from mice when the mice became moribund and GFP-positive cells were analyzed by flow cytometry. * P

Techniques Used: In Vivo, Transduction, Injection, Mouse Assay, Expressing, Plasmid Preparation, Flow Cytometry, Cytometry

12) Product Images from "The feedback loop of LITAF and BCL6 is involved in regulating apoptosis in B cell non-Hodgkin's-lymphoma"

Article Title: The feedback loop of LITAF and BCL6 is involved in regulating apoptosis in B cell non-Hodgkin's-lymphoma

Journal: Oncotarget

doi: 10.18632/oncotarget.12680

Transcriptional regulation of BCL6 by LITAF A. qRT-PCR showing temporal regulation of BCL6 and its target genes by LITAF in Ramos and OCI-Ly6 cells 48 h post-infection. Control, cells with pLVX virus; LITAF-myc, cells with over-expressed LITAF. B. Effect of silencing LITAF on BCL6 , PRDM1 and c-Myc mRNA in OCI-Ly3 and Namalwa cells. LITAF-shR, shRNA against LITAF; CTRL-shR, scrambled shRNA. C. Graphic representation of the BCL6 gene highlighting the three potential LITAF binding sites. The sequences of A (−87 to +65), B (+66 to +206) and C (+358 to +488) of the BCL6 gene are shown containing the LITAF consensus sites (CTCCC) underlined. Sequence numbers are in reference to the + 1 nucleotide identified by previous study [ 43 ]. D. ChIP assay in OCI-Ly3 cells. PCR was performed with primers specific for three potential regulating regions (A, B and C) in the promoter of BCL6 , respectively. The PCR product was analyzed by agarose gel electrophoresis (left), and qRT-PCR results are expressed as fold enrichment calculated as the percentage of input for the specific antibody (LITAF) with respect to IgG control of three replicates (right). Marker, 100-bp ladder. E. Schematic representation of the wild-type and three mutant (CTCCC to CTAAA) BCL6 reporters used in the experiments. F. The luciferase activity of BCL6 reporter plasmid in cell lines including 293T, Ramos and OCI-Ly6 after over-expression of LITAF (upper); and OCI-Ly3 cells infected with shRNA for LITAF or control virus (lower). G. Luciferase activity of wild-type (WT) or mutant (Mut-A, Mut-B and Mut-C) BCL6 reporters in OCI-Ly6 cells transfected with LITAF. Mean±s.d. of three technical replicates were plotted. * P
Figure Legend Snippet: Transcriptional regulation of BCL6 by LITAF A. qRT-PCR showing temporal regulation of BCL6 and its target genes by LITAF in Ramos and OCI-Ly6 cells 48 h post-infection. Control, cells with pLVX virus; LITAF-myc, cells with over-expressed LITAF. B. Effect of silencing LITAF on BCL6 , PRDM1 and c-Myc mRNA in OCI-Ly3 and Namalwa cells. LITAF-shR, shRNA against LITAF; CTRL-shR, scrambled shRNA. C. Graphic representation of the BCL6 gene highlighting the three potential LITAF binding sites. The sequences of A (−87 to +65), B (+66 to +206) and C (+358 to +488) of the BCL6 gene are shown containing the LITAF consensus sites (CTCCC) underlined. Sequence numbers are in reference to the + 1 nucleotide identified by previous study [ 43 ]. D. ChIP assay in OCI-Ly3 cells. PCR was performed with primers specific for three potential regulating regions (A, B and C) in the promoter of BCL6 , respectively. The PCR product was analyzed by agarose gel electrophoresis (left), and qRT-PCR results are expressed as fold enrichment calculated as the percentage of input for the specific antibody (LITAF) with respect to IgG control of three replicates (right). Marker, 100-bp ladder. E. Schematic representation of the wild-type and three mutant (CTCCC to CTAAA) BCL6 reporters used in the experiments. F. The luciferase activity of BCL6 reporter plasmid in cell lines including 293T, Ramos and OCI-Ly6 after over-expression of LITAF (upper); and OCI-Ly3 cells infected with shRNA for LITAF or control virus (lower). G. Luciferase activity of wild-type (WT) or mutant (Mut-A, Mut-B and Mut-C) BCL6 reporters in OCI-Ly6 cells transfected with LITAF. Mean±s.d. of three technical replicates were plotted. * P

Techniques Used: Quantitative RT-PCR, Infection, shRNA, Binding Assay, Sequencing, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Mutagenesis, Luciferase, Activity Assay, Plasmid Preparation, Over Expression, Transfection

13) Product Images from "Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles"

Article Title: Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles

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

doi: 10.1073/pnas.1614955114

( A ) Schematic representation of the bicistronic lentiviral constructs for expressing HEV ORF2, ORF3, IAV M2, and IAV M2(A30P). ( B ) Representative flow cytometry plots demonstrating efficient ORF2 and ORF3, IAV M2, or M2(A30P) expression. HepG2C3A cells were transduced with LVX-ORF2-IRES-zsGreen and/or LEX-[ORF3 or M2 or M2(A30P)]-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of ORF2- and/or ORF3- and M2-expressing cells. ( C ) Schematic representation of SP6-driven constructs used for in vitro transcription of HEV ORF3, IAV M2, and IAV M2(A30P) mRNAs. ( D ) HEV ORF3, IAV M2, or M2(A30P) expressed in X. laevis oocytes localizes to the plasma membrane. Water-injected and HEV ORF3, IAV M2, or M2(A30P) mRNA-injected oocytes were immunolabeled with polyclonal ORF3 or M2 antibodies and analyzed by confocal microscopy.
Figure Legend Snippet: ( A ) Schematic representation of the bicistronic lentiviral constructs for expressing HEV ORF2, ORF3, IAV M2, and IAV M2(A30P). ( B ) Representative flow cytometry plots demonstrating efficient ORF2 and ORF3, IAV M2, or M2(A30P) expression. HepG2C3A cells were transduced with LVX-ORF2-IRES-zsGreen and/or LEX-[ORF3 or M2 or M2(A30P)]-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of ORF2- and/or ORF3- and M2-expressing cells. ( C ) Schematic representation of SP6-driven constructs used for in vitro transcription of HEV ORF3, IAV M2, and IAV M2(A30P) mRNAs. ( D ) HEV ORF3, IAV M2, or M2(A30P) expressed in X. laevis oocytes localizes to the plasma membrane. Water-injected and HEV ORF3, IAV M2, or M2(A30P) mRNA-injected oocytes were immunolabeled with polyclonal ORF3 or M2 antibodies and analyzed by confocal microscopy.

Techniques Used: Construct, Expressing, Flow Cytometry, Cytometry, Transduction, In Vitro, Injection, Immunolabeling, Confocal Microscopy

ORF2 and ORF3 are required for releasing viral particles to infect naïve HepG2C3A cells. ( A ) Schematic representation of the transcomplementation system for packaging HEV virions. ( B ) Representative flow cytometry plots demonstrating efficient ORF2 and ORF3 expression. HepG2C3A cells were transduced with pLVX-ORF2-IRES-zsGreen and/or pLEX-ORF3-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of ORF2 and/or ORF3 expressing cells. ( C ) Infection kinetics of transcomplemented HEV in HepG2C3A cells. Cell culture supernatants from naïve HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, were collected 5 d posttransfection with rHEVΔORF2/3[Gluc] RNA. Naïve HepG2C3A cells were incubated with these supernatants. After 12 h, cells were washed and Gaussia luciferase activity quantified in the cell culture supernatants at the indicated time points. ( D ) Five days following transfection of rHEVΔORF2/3[Gluc] RNA into HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, lysates were used to infect naïve HepG2C3A cells. Gluc activity was measured in the supernatants 4 d postinfection. Shown are averages and SDs of triplicate measurements of three independent experiments. * P
Figure Legend Snippet: ORF2 and ORF3 are required for releasing viral particles to infect naïve HepG2C3A cells. ( A ) Schematic representation of the transcomplementation system for packaging HEV virions. ( B ) Representative flow cytometry plots demonstrating efficient ORF2 and ORF3 expression. HepG2C3A cells were transduced with pLVX-ORF2-IRES-zsGreen and/or pLEX-ORF3-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of ORF2 and/or ORF3 expressing cells. ( C ) Infection kinetics of transcomplemented HEV in HepG2C3A cells. Cell culture supernatants from naïve HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, were collected 5 d posttransfection with rHEVΔORF2/3[Gluc] RNA. Naïve HepG2C3A cells were incubated with these supernatants. After 12 h, cells were washed and Gaussia luciferase activity quantified in the cell culture supernatants at the indicated time points. ( D ) Five days following transfection of rHEVΔORF2/3[Gluc] RNA into HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, lysates were used to infect naïve HepG2C3A cells. Gluc activity was measured in the supernatants 4 d postinfection. Shown are averages and SDs of triplicate measurements of three independent experiments. * P

Techniques Used: Flow Cytometry, Cytometry, Expressing, Transduction, Infection, Cell Culture, Incubation, Luciferase, Activity Assay, Transfection

14) Product Images from "A Mutant Tat Protein Provides Strong Protection from HIV-1 Infection in Human CD4+ T Cells"

Article Title: A Mutant Tat Protein Provides Strong Protection from HIV-1 Infection in Human CD4+ T Cells

Journal: Human Gene Therapy

doi: 10.1089/hum.2012.176

Transduction of Jurkat cells with retroviral VLPs conveying NB-EGFP or EGFP. (a) Schematic diagrams of the retroviral vector pGCsamEN (Chuah et al. ) carrying either the NB-EGFP or control EGFP gene cassette and an overview of the method used to
Figure Legend Snippet: Transduction of Jurkat cells with retroviral VLPs conveying NB-EGFP or EGFP. (a) Schematic diagrams of the retroviral vector pGCsamEN (Chuah et al. ) carrying either the NB-EGFP or control EGFP gene cassette and an overview of the method used to

Techniques Used: Transduction, Plasmid Preparation

15) Product Images from "Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription"

Article Title: Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription

Journal: mBio

doi: 10.1128/mBio.00769-18

Mapping the putative promoter region required for subgenomic RNA synthesis. A series of truncated rHEVΔORF2/3[Gluc] Pol- mutant constructs were generated, and the in vitro -transcribed RNA was transfected into HepG2C3A cells expressing ORF1. Two days posttransfection, supernatants were collected, and Gaussia luciferase activity was quantified. The data are presented as the percentage of Gaussia luciferase activity relative to that of the full-length rHEVΔORF2/3[Gluc] Pol-. The numbering denotes the positions of the Kernow C1/p6 viral genome. Values are means plus SD ( n = 3). Values that are significantly different by one-way ANOVA are indicated by asterisks as follows: *, P
Figure Legend Snippet: Mapping the putative promoter region required for subgenomic RNA synthesis. A series of truncated rHEVΔORF2/3[Gluc] Pol- mutant constructs were generated, and the in vitro -transcribed RNA was transfected into HepG2C3A cells expressing ORF1. Two days posttransfection, supernatants were collected, and Gaussia luciferase activity was quantified. The data are presented as the percentage of Gaussia luciferase activity relative to that of the full-length rHEVΔORF2/3[Gluc] Pol-. The numbering denotes the positions of the Kernow C1/p6 viral genome. Values are means plus SD ( n = 3). Values that are significantly different by one-way ANOVA are indicated by asterisks as follows: *, P

Techniques Used: Mutagenesis, Construct, Generated, In Vitro, Transfection, Expressing, Luciferase, Activity Assay

Identification of the minimal putative promoter region upstream of the TSS critical for subgenomic RNA synthesis. The truncated mutant viral RNA was transfected into HepG2C3A cells expressing ORF1. Two days posttransfection, supernatants were collected, and Gaussia luciferase activity was quantified. The data are presented as the percentage of Gaussia luciferase activity relative to that of the full-length rHEVΔORF2/3[Gluc] Pol-. The numbering denotes the positions of the Kernow C1/p6 viral genome. Values are means plus SD ( n = 3). *, P
Figure Legend Snippet: Identification of the minimal putative promoter region upstream of the TSS critical for subgenomic RNA synthesis. The truncated mutant viral RNA was transfected into HepG2C3A cells expressing ORF1. Two days posttransfection, supernatants were collected, and Gaussia luciferase activity was quantified. The data are presented as the percentage of Gaussia luciferase activity relative to that of the full-length rHEVΔORF2/3[Gluc] Pol-. The numbering denotes the positions of the Kernow C1/p6 viral genome. Values are means plus SD ( n = 3). *, P

Techniques Used: Mutagenesis, Transfection, Expressing, Luciferase, Activity Assay

Full-length genome mutated in the intragenomic promoter is significantly impaired in its ability to produce the infectious virus. (a) Schematic diagrams of the putative promoter (WT) and the SgP mutant (SgPmut). As for the promoter impaired mutant, synonymous mutations are introduced in the ORF1 coding region. (b and c) Transfection of in vitro -transcribed WT, SgPmut or GAD (Pol-) RNA of Kernow-C1/p6 (gt 3), TW6196E (gt 4) into Huh7.5 cells. Cell lysate supernatant was collected after 5 days transfection to infect naive Huh7.5 cells. Quantification of HEV RNA Kernow-C1/p6 (b) and TW6196E (c) 3 days following infection by quantitative RT-PCR. Values are means plus SD ( n = 4). *, P
Figure Legend Snippet: Full-length genome mutated in the intragenomic promoter is significantly impaired in its ability to produce the infectious virus. (a) Schematic diagrams of the putative promoter (WT) and the SgP mutant (SgPmut). As for the promoter impaired mutant, synonymous mutations are introduced in the ORF1 coding region. (b and c) Transfection of in vitro -transcribed WT, SgPmut or GAD (Pol-) RNA of Kernow-C1/p6 (gt 3), TW6196E (gt 4) into Huh7.5 cells. Cell lysate supernatant was collected after 5 days transfection to infect naive Huh7.5 cells. Quantification of HEV RNA Kernow-C1/p6 (b) and TW6196E (c) 3 days following infection by quantitative RT-PCR. Values are means plus SD ( n = 4). *, P

Techniques Used: Mutagenesis, Transfection, In Vitro, Infection, Quantitative RT-PCR

16) Product Images from "Ecrg4 Attenuates the Inflammatory Proliferative Response of Mucosal Epithelial Cells to Infection"

Article Title: Ecrg4 Attenuates the Inflammatory Proliferative Response of Mucosal Epithelial Cells to Infection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0061394

The effect of Ecrg4 expression on mucosal epithelial growth in vitro . Panel A: Fluorescent images of representative NTHi-infected ME explant outgrowth after transduction with Lenti-ZsGreen or (Panel B) lenti-ZsGreen+Ecrg4 and culture for 10 days. The NTHi-induced hyperplastic growth response is decreased by Ecrg4 gene expression in vitro suggesting that Ecrg4 is a regulatory component of the cellular response to inflammation. Explants of mucosa from MEs were harvested and cultured in vitro for 2 days then transduced. Panel C: Surface area quantification of control (uninfected) explant expansion showing no effect of transduction with ADEcrg4. Panel D: ADEcrg4 transduction dramatically decreases the growth of mucosal explants harvested from NTHi-infected MEs, when compared to those transduced with ADgfp or non-transduced infected explants. In panels C and D, n > 6 explants per group per time point, bars represent mean ± SEM with *P
Figure Legend Snippet: The effect of Ecrg4 expression on mucosal epithelial growth in vitro . Panel A: Fluorescent images of representative NTHi-infected ME explant outgrowth after transduction with Lenti-ZsGreen or (Panel B) lenti-ZsGreen+Ecrg4 and culture for 10 days. The NTHi-induced hyperplastic growth response is decreased by Ecrg4 gene expression in vitro suggesting that Ecrg4 is a regulatory component of the cellular response to inflammation. Explants of mucosa from MEs were harvested and cultured in vitro for 2 days then transduced. Panel C: Surface area quantification of control (uninfected) explant expansion showing no effect of transduction with ADEcrg4. Panel D: ADEcrg4 transduction dramatically decreases the growth of mucosal explants harvested from NTHi-infected MEs, when compared to those transduced with ADgfp or non-transduced infected explants. In panels C and D, n > 6 explants per group per time point, bars represent mean ± SEM with *P

Techniques Used: Expressing, In Vitro, Infection, Transduction, Cell Culture

17) Product Images from "SLAMF1 is required for TLR4-mediated TRAM-TRIF–dependent signaling in human macrophages"

Article Title: SLAMF1 is required for TLR4-mediated TRAM-TRIF–dependent signaling in human macrophages

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201707027

TRAM acts as a bridge between the SLAMF1 and TLR4 signaling complex. (A and B) Coprecipitations of SLAMF1 Flag with TLR4 Cherry (A) or TRIF HA (B) with or without TRAM YFP overexpression. (C) Coprecipitation of TLR4 Flag with SLAMF1 with or without TRAM YFP overexpression. (D) TLR4 Flag interaction with TRAM YFP and TRIF HA with or without SLAMF1 coexpression. (E) Coprecipitation of SLAMF1 with or without TRIF HA in the presence of TRAM YFP by TLR4 Flag . Indicated constructs were transfected to HEK293T cells. pDuo-CD14/MD-2 vector was cotransfected to all wells (A and C–E). Anti-Flag agarose was used for IPs. At least three independent experiments were performed. Molecular weight is given in kilodaltons. WB, Western blot; WCL, whole-cell lysate.
Figure Legend Snippet: TRAM acts as a bridge between the SLAMF1 and TLR4 signaling complex. (A and B) Coprecipitations of SLAMF1 Flag with TLR4 Cherry (A) or TRIF HA (B) with or without TRAM YFP overexpression. (C) Coprecipitation of TLR4 Flag with SLAMF1 with or without TRAM YFP overexpression. (D) TLR4 Flag interaction with TRAM YFP and TRIF HA with or without SLAMF1 coexpression. (E) Coprecipitation of SLAMF1 with or without TRIF HA in the presence of TRAM YFP by TLR4 Flag . Indicated constructs were transfected to HEK293T cells. pDuo-CD14/MD-2 vector was cotransfected to all wells (A and C–E). Anti-Flag agarose was used for IPs. At least three independent experiments were performed. Molecular weight is given in kilodaltons. WB, Western blot; WCL, whole-cell lysate.

Techniques Used: Over Expression, Construct, Transfection, Plasmid Preparation, Molecular Weight, Western Blot

Knockdown of SLAMF1 in macrophages results in strongly reduced TLR4-mediated IFNβ mRNA expression and protein secretion as well as some decrease of TNF, IL-6, and CXCL10 secretion. (A and B) Quantification of SLAMF1 , IFNβ , and TNF mRNA expression by qPCR in THP-1 cells (A) and macrophages (B) treated by 100 ng/ml ultrapure K12 LPS. (C and D) IFNβ and TNF secretion levels by THP-1 cells (C) and macrophages (D) in response to LPS (4 and 6 h) assessed by ELISA. (E and F) Secretion levels of IL-1β, IL-6, IL-8, and CXCL-10 (6 h LPS) analyzed by multiplex assays. Data are presented as means with SD for combined data from three independent experiments (A, C, and E), for three biological replicates from one of six donors (B and D), or one of three donors (F). *, P
Figure Legend Snippet: Knockdown of SLAMF1 in macrophages results in strongly reduced TLR4-mediated IFNβ mRNA expression and protein secretion as well as some decrease of TNF, IL-6, and CXCL10 secretion. (A and B) Quantification of SLAMF1 , IFNβ , and TNF mRNA expression by qPCR in THP-1 cells (A) and macrophages (B) treated by 100 ng/ml ultrapure K12 LPS. (C and D) IFNβ and TNF secretion levels by THP-1 cells (C) and macrophages (D) in response to LPS (4 and 6 h) assessed by ELISA. (E and F) Secretion levels of IL-1β, IL-6, IL-8, and CXCL-10 (6 h LPS) analyzed by multiplex assays. Data are presented as means with SD for combined data from three independent experiments (A, C, and E), for three biological replicates from one of six donors (B and D), or one of three donors (F). *, P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Multiplex Assay

SLAMF1 interacts with all class I Rab11 FIPs. (A) Anti-Flag IPs for Rab11a Flag with EGFP-tagged Rab11FIPs (1–5) and SLAMF1. (B) Schematic figure for class I and class II Rab11 FIPs domain structure. C2, phospholipid-binding C2 domain; EF, EF-hand domain; PRR, proline-rich region; RBD, Rab11 binding domain. (C) Homologous protein sequence in class I FIPs, which follow the C2 domain. Identical amino acids in all three class I FIPs are highlighted. (D) Coprecipitation of SLAMF1 Flag with FIP2 EGFP WT or FIP2 deletion mutant lacking the C2 domain (ΔC2). (E and F) Coprecipitation of untagged SLAMF1 with FIP2 Flag (1–512 aa) and Flag-tagged FIP2 deletion mutants in anti-Flag IPs in the absence (E) or presence (F) of overexpressed Rab11 CFP . (G) Quantification of coprecipitations in E and F between SLAMF1 and FIP2 Flag variants correlated with the amount of Flag-tagged protein on the blot and Flag-tagged protein sizes. Error bars represent means ± SD for three independent experiments. (H) Coprecipitation of FIP2 Flag with SLAMF1 and Rab11a WT, Rab11a Q70L mutant (QL), or Rab11a S25N mutant (SN). (I) Coprecipitation of SLAMF1 Flag deletion mutants with FIP2 EGFP . Molecular weight is given in kilodaltons. WB, Western blot; WCL, whole-cell lysate. (J) Scheme for FIP2- and TRAM-interacting domains in SLAMF1ct. The results are representative of at least three independent experiments.
Figure Legend Snippet: SLAMF1 interacts with all class I Rab11 FIPs. (A) Anti-Flag IPs for Rab11a Flag with EGFP-tagged Rab11FIPs (1–5) and SLAMF1. (B) Schematic figure for class I and class II Rab11 FIPs domain structure. C2, phospholipid-binding C2 domain; EF, EF-hand domain; PRR, proline-rich region; RBD, Rab11 binding domain. (C) Homologous protein sequence in class I FIPs, which follow the C2 domain. Identical amino acids in all three class I FIPs are highlighted. (D) Coprecipitation of SLAMF1 Flag with FIP2 EGFP WT or FIP2 deletion mutant lacking the C2 domain (ΔC2). (E and F) Coprecipitation of untagged SLAMF1 with FIP2 Flag (1–512 aa) and Flag-tagged FIP2 deletion mutants in anti-Flag IPs in the absence (E) or presence (F) of overexpressed Rab11 CFP . (G) Quantification of coprecipitations in E and F between SLAMF1 and FIP2 Flag variants correlated with the amount of Flag-tagged protein on the blot and Flag-tagged protein sizes. Error bars represent means ± SD for three independent experiments. (H) Coprecipitation of FIP2 Flag with SLAMF1 and Rab11a WT, Rab11a Q70L mutant (QL), or Rab11a S25N mutant (SN). (I) Coprecipitation of SLAMF1 Flag deletion mutants with FIP2 EGFP . Molecular weight is given in kilodaltons. WB, Western blot; WCL, whole-cell lysate. (J) Scheme for FIP2- and TRAM-interacting domains in SLAMF1ct. The results are representative of at least three independent experiments.

Techniques Used: Binding Assay, Sequencing, Mutagenesis, Molecular Weight, Western Blot

SLAMF1 silencing in macrophages impairs TLR4-mediated phosphorylation of TBK1, IRF3, and TAK1. Western blotting of lysate macrophages treated with a control nonsilencing oligonucleotide or SLAMF1 -specific siRNA oligonucleotides and stimulated with 100 ng/ml LPS. The antibodies used are indicated in the figure. An antibody toward SLAMF1 was used to control for SLAMF1 silencing, and GAPDH was used as an equal loading control. Same GAPDH controls are presented for pTBK1, total TBK1, and phospho-p38MAPK, for total IRF3 and total TAK1, and for pTAK1 and pIκBα because they were probed on the same membranes. Western blots are representative of one of five donors. Molecular weight is given in kilodaltons. Graphs (right) show quantifications of protein levels relative to GAPDH levels obtained with Odyssey software.
Figure Legend Snippet: SLAMF1 silencing in macrophages impairs TLR4-mediated phosphorylation of TBK1, IRF3, and TAK1. Western blotting of lysate macrophages treated with a control nonsilencing oligonucleotide or SLAMF1 -specific siRNA oligonucleotides and stimulated with 100 ng/ml LPS. The antibodies used are indicated in the figure. An antibody toward SLAMF1 was used to control for SLAMF1 silencing, and GAPDH was used as an equal loading control. Same GAPDH controls are presented for pTBK1, total TBK1, and phospho-p38MAPK, for total IRF3 and total TAK1, and for pTAK1 and pIκBα because they were probed on the same membranes. Western blots are representative of one of five donors. Molecular weight is given in kilodaltons. Graphs (right) show quantifications of protein levels relative to GAPDH levels obtained with Odyssey software.

Techniques Used: Western Blot, Molecular Weight, Software

SLAMF1 regulates TRAM recruitment to E. coli phagosomes. (A) SLAMF1 costaining with TRAM, EEA1, or LAMP1 in primary macrophages coincubated with E. coli pHrodo particles for indicated time points. SLAMF1 (green), E. coli (blue), and TRAM, EEA1, or LAMP1 (red) are shown. The data shown are representative of one out of four donors. Bars, 10 μm. (B and C) TRAM and SLAMF1 MIs on E. coli phagosomes upon SLAMF1 silencing (B) or simultaneous Rab11a and Rab11b silencing (C) in primary human macrophages quantified from xyz images. The scatter plots are presented as median values of TRAM voxel intensity, and numbers of phagosomes are shown at the top. The nonparametric Mann-Whitney test was used to evaluate statistical significance. *, P
Figure Legend Snippet: SLAMF1 regulates TRAM recruitment to E. coli phagosomes. (A) SLAMF1 costaining with TRAM, EEA1, or LAMP1 in primary macrophages coincubated with E. coli pHrodo particles for indicated time points. SLAMF1 (green), E. coli (blue), and TRAM, EEA1, or LAMP1 (red) are shown. The data shown are representative of one out of four donors. Bars, 10 μm. (B and C) TRAM and SLAMF1 MIs on E. coli phagosomes upon SLAMF1 silencing (B) or simultaneous Rab11a and Rab11b silencing (C) in primary human macrophages quantified from xyz images. The scatter plots are presented as median values of TRAM voxel intensity, and numbers of phagosomes are shown at the top. The nonparametric Mann-Whitney test was used to evaluate statistical significance. *, P

Techniques Used: MANN-WHITNEY

Lentiviral transduction of SLAMF1 in macrophages results in the increase of IRF3 and TBK1 phosphorylation in response to LPS and upregulation of IFNβ and TNF expression. (A) Quantification of SLAMF1 , IFNβ , and TNF mRNA expression by qPCR in macrophages transduced by Flag-tagged SLAMF1 coding or control virus and treated by LPS. The qPCR data are presented as means and SD for three biological replicates of one of three experiments. Significance was calculated by two-tailed t tests. *, P
Figure Legend Snippet: Lentiviral transduction of SLAMF1 in macrophages results in the increase of IRF3 and TBK1 phosphorylation in response to LPS and upregulation of IFNβ and TNF expression. (A) Quantification of SLAMF1 , IFNβ , and TNF mRNA expression by qPCR in macrophages transduced by Flag-tagged SLAMF1 coding or control virus and treated by LPS. The qPCR data are presented as means and SD for three biological replicates of one of three experiments. Significance was calculated by two-tailed t tests. *, P

Techniques Used: Transduction, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

SLAMF1 is enriched in the Rab11-positive ERCs in unstimulated macrophages, and SLAMF1 expression is induced by LPS and several other TLR ligands in primary human monocytes and macrophages. (A) Monocytes, macrophages, and differentiated THP-1 cells stained with antibodies against SLAMF1 (green) and GM130 (red) and imaged by confocal microscopy. (B) 3D model of cis-Golgi (GM130) and SLAMF1 in THP-1 cells. Z stacks from the GM130 and SLAMF1 channels were obtained using high-resolution confocal microscopy followed by 3D modeling in IMARIS software. (C) Macrophages stained for SLAMF1 and Rab11 (ERC marker). Representative image. Overlapping pixels for SLAMF1 and Rab11 are shown in the white overlap. tM1 = 0.683 ± 0.08 (mean with SD) for z stacks of ERCs as ROIs (30 ROIs analyzed per donor) where tM1 was the Manders’s colocalization coefficient with thresholds calculated in the Coloc 2 Fiji plugin with anti-SLAMF1 staining as first channel. (D) Macrophages costained for SLAMF1 and EEA1. (E) Macrophages costained for SLAMF1 and LAMP1. Colocalization accessed for z stacks for at least 30 cells for each experiment (four total) showing no colocalization for markers in both D and E. (F) Flow cytometry analysis of SLAMF1 surface expression by primary macrophages and differentiated THP-1 cells. Cells were costained for SLAMF1 and CD14 and gated for CD14-positive cells (primary cells) or stained for SLAMF1 (THP-1 cells). (G) Flow cytometry analysis of SLAMF1 surface expression by human macrophages stimulated by ultrapure K12 LPS (100 ng/ml) for 2, 4, and 6 h. (H) Western blot analysis of lysates from primary human macrophages stimulated by LPS for 2, 4, and 6 h. Graphs present mean values for three biological replicates with SD. Molecular weight is given in kilodaltons. (I and J ) Quantification of SLAMF1 mRNA expression by qPCR in monocytes (I) and macrophages (J) stimulated by TLRs’ ligands FSL-1 (20 ng/ml), K12 LPS (100 ng/ml), and CL075 (1 μg/ml; both I and J) as well as R848 (1 μg/ml), Pam3Cys (P3C; 1 μg/ml), or K12 E. coli particles (20/cell; I only). Results are presented as means with SD. Statistical significance between groups was evaluated by a two-tailed t test. *, P
Figure Legend Snippet: SLAMF1 is enriched in the Rab11-positive ERCs in unstimulated macrophages, and SLAMF1 expression is induced by LPS and several other TLR ligands in primary human monocytes and macrophages. (A) Monocytes, macrophages, and differentiated THP-1 cells stained with antibodies against SLAMF1 (green) and GM130 (red) and imaged by confocal microscopy. (B) 3D model of cis-Golgi (GM130) and SLAMF1 in THP-1 cells. Z stacks from the GM130 and SLAMF1 channels were obtained using high-resolution confocal microscopy followed by 3D modeling in IMARIS software. (C) Macrophages stained for SLAMF1 and Rab11 (ERC marker). Representative image. Overlapping pixels for SLAMF1 and Rab11 are shown in the white overlap. tM1 = 0.683 ± 0.08 (mean with SD) for z stacks of ERCs as ROIs (30 ROIs analyzed per donor) where tM1 was the Manders’s colocalization coefficient with thresholds calculated in the Coloc 2 Fiji plugin with anti-SLAMF1 staining as first channel. (D) Macrophages costained for SLAMF1 and EEA1. (E) Macrophages costained for SLAMF1 and LAMP1. Colocalization accessed for z stacks for at least 30 cells for each experiment (four total) showing no colocalization for markers in both D and E. (F) Flow cytometry analysis of SLAMF1 surface expression by primary macrophages and differentiated THP-1 cells. Cells were costained for SLAMF1 and CD14 and gated for CD14-positive cells (primary cells) or stained for SLAMF1 (THP-1 cells). (G) Flow cytometry analysis of SLAMF1 surface expression by human macrophages stimulated by ultrapure K12 LPS (100 ng/ml) for 2, 4, and 6 h. (H) Western blot analysis of lysates from primary human macrophages stimulated by LPS for 2, 4, and 6 h. Graphs present mean values for three biological replicates with SD. Molecular weight is given in kilodaltons. (I and J ) Quantification of SLAMF1 mRNA expression by qPCR in monocytes (I) and macrophages (J) stimulated by TLRs’ ligands FSL-1 (20 ng/ml), K12 LPS (100 ng/ml), and CL075 (1 μg/ml; both I and J) as well as R848 (1 μg/ml), Pam3Cys (P3C; 1 μg/ml), or K12 E. coli particles (20/cell; I only). Results are presented as means with SD. Statistical significance between groups was evaluated by a two-tailed t test. *, P

Techniques Used: Expressing, Staining, Confocal Microscopy, Software, Marker, Flow Cytometry, Cytometry, Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Two Tailed Test

SLAMF1 interacts with TRAM protein. (A) Endogenous IPs using specific anti-SLAMF1 mAbs from macrophages stimulated by LPS. (B) Endogenous IPs using anti-TRAM polyclonal antibodies from macrophages stimulated by LPS. (C) TRAM Flag -precipitated SLAMF1 and SLAMF1ct was needed for interaction with TRAM. (D) Coprecipitation of TRAM deletion mutants: TIR domain (68–235), short TRAM TIR domain (68–176 aa), and N-terminal (1–68 aa) or C-terminal (158–235 aa) domains with SLAMF1 protein. (E) Coprecipitation of TRAM deletion mutants containing the N-terminal part of TRAM TIR domain with SLAMF1. (F) Coprecipitation of SLAMF1 Flag deletion mutants with TRAM YFP . (G) Coprecipitation of human SLAMF1 Flag with human TRAM YFP and of mouse SLAMF1 Flag with mouse TRAM EGFP . Black dashed lines indicate that intervening lanes have been spliced out. (H) Human SLAMF1 cytoplasmic tail coprecipitation with TRAM YFP with or without amino acid substitutions at 321–324. Graphs under C–F summarize the IPs’ results. Indicated constructs were transfected to HEK293T cells, and anti-Flag agarose was used for the IPs. For endogenous IPs, specific SLAMF1 or TRAM antibodies were covalently coupled to beads. At least three independent experiments were carried out for anti-Flag IPs, and five independent experiments were carried out for the endogenous IPs, and one representative experiment is shown for each. Molecular weight is given in kilodaltons. WB, Western blot; WCL, whole-cell lysate.
Figure Legend Snippet: SLAMF1 interacts with TRAM protein. (A) Endogenous IPs using specific anti-SLAMF1 mAbs from macrophages stimulated by LPS. (B) Endogenous IPs using anti-TRAM polyclonal antibodies from macrophages stimulated by LPS. (C) TRAM Flag -precipitated SLAMF1 and SLAMF1ct was needed for interaction with TRAM. (D) Coprecipitation of TRAM deletion mutants: TIR domain (68–235), short TRAM TIR domain (68–176 aa), and N-terminal (1–68 aa) or C-terminal (158–235 aa) domains with SLAMF1 protein. (E) Coprecipitation of TRAM deletion mutants containing the N-terminal part of TRAM TIR domain with SLAMF1. (F) Coprecipitation of SLAMF1 Flag deletion mutants with TRAM YFP . (G) Coprecipitation of human SLAMF1 Flag with human TRAM YFP and of mouse SLAMF1 Flag with mouse TRAM EGFP . Black dashed lines indicate that intervening lanes have been spliced out. (H) Human SLAMF1 cytoplasmic tail coprecipitation with TRAM YFP with or without amino acid substitutions at 321–324. Graphs under C–F summarize the IPs’ results. Indicated constructs were transfected to HEK293T cells, and anti-Flag agarose was used for the IPs. For endogenous IPs, specific SLAMF1 or TRAM antibodies were covalently coupled to beads. At least three independent experiments were carried out for anti-Flag IPs, and five independent experiments were carried out for the endogenous IPs, and one representative experiment is shown for each. Molecular weight is given in kilodaltons. WB, Western blot; WCL, whole-cell lysate.

Techniques Used: Construct, Transfection, Molecular Weight, Western Blot

18) Product Images from "Notch Activation Differentially Regulates Renal Progenitors Proliferation and Differentiation Toward the Podocyte Lineage in Glomerular Disorders"

Article Title: Notch Activation Differentially Regulates Renal Progenitors Proliferation and Differentiation Toward the Podocyte Lineage in Glomerular Disorders

Journal: Stem Cells (Dayton, Ohio)

doi: 10.1002/stem.492

Regulation of cell cycle progression, mitosis, cell death, and cytoskeleton organization in N3ICD-infected human renal progenitors before and after differentiation toward the podocyte lineage. ( A, B ): Cell cycle analysis performed on ( A ) mock- and ( B ) N3ICD-infected renal progenitors. One representative of four independent experiments is shown. ( C, D ): Cell cycle analysis performed on ( C ) mock- and ( D ) N3ICD-infected renal progenitors after their differentiation toward the podocyte lineage. One representative of four independent experiments is shown. ( E, F ): FACS analysis of apoptosis/necrosis in mock- and N3ICD-infected renal progenitors as assessed by annexin-V and propidium iodide (PI) staining. One representative of four independent experiments is shown. ( G, H ): FACS analysis of apoptosis/necrosis in mock- and N3ICD-infected renal progenitors after their differentiation toward the podocyte lineage as assessed by annexin-V and PI staining reveals an increase in the percentage of PI/annexin V positive cells in N3ICD-infected cells. One representative of four independent experiments is shown. ( I, J ): Above: H3-Ser10 (red) and tubulin (blue) staining of mock- and N3ICD-infected undifferentiated renal progenitors reveals normal mitoses. For mock-infected cells, a representative metaphase is shown, while for N3ICD-infected cells, a representative anaphase is shown. One representative of six independent experiments is shown. Scale bar = 10 μm. Below: Phalloidin staining (red) of mock- and N3ICD-infected undifferentiated renal progenitors. Topro-3 (blue) counterstains nuclei. One representative of six independent experiments is shown. Scale bar = 50 μm. ( K, L ): Above: H3-Ser10 (red) and tubulin (blue) staining of renal progenitors infected with vector expressing N3ICD ( L ) after their differentiation toward the podocyte lineage reveals aberrant mitoses characterized by micro/multinucleation (red) and abnormal spindle distribution (blue) in comparison with those infected with an empty vector (mock, [ K ]). One representative of six independent experiments is shown. Scale bar = 10 μm. Below: Phalloidin staining (red) of mock- ( K ) and N3ICD-infected ( L ) renal progenitors after their differentiation toward the podocyte lineage reveals F-actin filaments distributed as stress-like bundles along the axis of the cells in mock-infected podocytes and redistribution of F-actin fibers to the periphery of the cells in podocytes infected with N3ICD. Topro-3 (blue) counterstains nuclei. One representative of six independent experiments is shown. Scale bar = 50 μm. ( M, N ): Assessment by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) of Aurora kinase B ( M ), p21 Cip1/WAF-1 and p27 Kip1 ( N ) mRNA expression in mock- and N3ICD-infected undifferentiated renal progenitors. Results are expressed as mean ± SEM of triplicate assessments in seven separate experiments. ( O, P ): Assessment by real-time quantitative RT-PCR of Aurora kinase B ( O ) and p27 Kip1 , p21 Cip1/WAF-1 ( P ) mRNA expression in mock- and N3ICD-infected renal progenitors after their differentiation toward the podocyte lineage. Results are expressed as mean ± SEM of triplicate assessments in seven separate experiments. Abbreviation: FACS, fluorescence activated cell sorting.
Figure Legend Snippet: Regulation of cell cycle progression, mitosis, cell death, and cytoskeleton organization in N3ICD-infected human renal progenitors before and after differentiation toward the podocyte lineage. ( A, B ): Cell cycle analysis performed on ( A ) mock- and ( B ) N3ICD-infected renal progenitors. One representative of four independent experiments is shown. ( C, D ): Cell cycle analysis performed on ( C ) mock- and ( D ) N3ICD-infected renal progenitors after their differentiation toward the podocyte lineage. One representative of four independent experiments is shown. ( E, F ): FACS analysis of apoptosis/necrosis in mock- and N3ICD-infected renal progenitors as assessed by annexin-V and propidium iodide (PI) staining. One representative of four independent experiments is shown. ( G, H ): FACS analysis of apoptosis/necrosis in mock- and N3ICD-infected renal progenitors after their differentiation toward the podocyte lineage as assessed by annexin-V and PI staining reveals an increase in the percentage of PI/annexin V positive cells in N3ICD-infected cells. One representative of four independent experiments is shown. ( I, J ): Above: H3-Ser10 (red) and tubulin (blue) staining of mock- and N3ICD-infected undifferentiated renal progenitors reveals normal mitoses. For mock-infected cells, a representative metaphase is shown, while for N3ICD-infected cells, a representative anaphase is shown. One representative of six independent experiments is shown. Scale bar = 10 μm. Below: Phalloidin staining (red) of mock- and N3ICD-infected undifferentiated renal progenitors. Topro-3 (blue) counterstains nuclei. One representative of six independent experiments is shown. Scale bar = 50 μm. ( K, L ): Above: H3-Ser10 (red) and tubulin (blue) staining of renal progenitors infected with vector expressing N3ICD ( L ) after their differentiation toward the podocyte lineage reveals aberrant mitoses characterized by micro/multinucleation (red) and abnormal spindle distribution (blue) in comparison with those infected with an empty vector (mock, [ K ]). One representative of six independent experiments is shown. Scale bar = 10 μm. Below: Phalloidin staining (red) of mock- ( K ) and N3ICD-infected ( L ) renal progenitors after their differentiation toward the podocyte lineage reveals F-actin filaments distributed as stress-like bundles along the axis of the cells in mock-infected podocytes and redistribution of F-actin fibers to the periphery of the cells in podocytes infected with N3ICD. Topro-3 (blue) counterstains nuclei. One representative of six independent experiments is shown. Scale bar = 50 μm. ( M, N ): Assessment by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) of Aurora kinase B ( M ), p21 Cip1/WAF-1 and p27 Kip1 ( N ) mRNA expression in mock- and N3ICD-infected undifferentiated renal progenitors. Results are expressed as mean ± SEM of triplicate assessments in seven separate experiments. ( O, P ): Assessment by real-time quantitative RT-PCR of Aurora kinase B ( O ) and p27 Kip1 , p21 Cip1/WAF-1 ( P ) mRNA expression in mock- and N3ICD-infected renal progenitors after their differentiation toward the podocyte lineage. Results are expressed as mean ± SEM of triplicate assessments in seven separate experiments. Abbreviation: FACS, fluorescence activated cell sorting.

Techniques Used: Infection, Cell Cycle Assay, FACS, Staining, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Fluorescence

19) Product Images from "Reprogramming Human Endothelial to Hematopoietic Cells Requires Vascular Induction"

Article Title: Reprogramming Human Endothelial to Hematopoietic Cells Requires Vascular Induction

Journal: Nature

doi: 10.1038/nature13547

Conditional expression of FGRS is sufficient for optimal generation of rEC-hMPPs with multilineage potential, including T-Cell lymphoid cells A to C: Conditional expression of mouse inducible FGRS factors activates endogenous human FGRS in HUVECs sustaining functional hematopoietic cell fate of rEC-hMPPs. A . To test whether FGRS-induced reprogramming triggered expression of endogenous FGRS genes 24 , HUVECs were transduced with lentivirus expressing FGRS-Tet-On and a trans-activator, and grown on E4EC-vascular niche for 18–22 days (n=4) i n the presence of doxycycline. Doxycycline was removed from the culture medium after 18–22 days to shut off the expression of mouse FGRS and cells were cultured for additional 7–10 days. Human CD45 + CD34 + cells were FACS isolated for CFC assay and whole-transcriptome deep sequencing (RNA-seq). CFC assay revealed emergence of hematopoietic colonies with cells expressing human CD235, CD11b, CD83, and CD14. B . Comparison of transcriptional profiles of the human FGRS expression in human HUVECs, hCD45 + rEC-hMPPs programmed using inducible mouse FGRS, CD45 + CD34 + rEC-hMPPs, 22 weeks post-transplantation, hDMEC-derived CD45 + CD34 + rEC − hMPPs after 15 weeks post-secondary engraftment and naïve CD34 + Lin + cells purified from cord blood. C . Analysis of whole-transcriptome RNA-Seq of rEC-hMPPs derived using inducible mouse FGRS (n=3). All RNA-Seq reads were aligned against human and mouse FGRS sequences. RNA-Seq reads that align to human FGRS sequences – “Map to Human”, RNA-Seq reads that align to mouse FGRS sequences – “Map to Mouse”, and RNA-Seq reads that align to mouse FGRS sequences without a possibility to align to human sequences – “Map to mouse Only”. D to E: Optimizing differentiation of rEC-hMPPs into lymphoid progeny D . The number of T-lymphoid progeny of engrafted rEC-hMPP was negligibly small, raising the possibility that constitutive SPI1 expression prevents rEC-hMPP from differentiating into T-cells 39 , 40 . To test this, HUVECs were transduced with lentiviral vectors expressing GFP and that constitutively express FGR-TFs with a Tet-inducible SPI1 (FGR+ SPI1 -Tet-On construct) for 3 days followed by replating for E4EC-induction. After 27 days of FGR and doxycycline-induced SPI1 expression on E4ECs, GFP + CD45 + hematopoietic-like colonies emerged. Then, doxycycline was withdrawn and the reprogrammed cells were cultured serum-free with Delta-like-4 expressing OP9-stroma (OP9-DLL4) supplemented with IL-7, IL-11, and IL-2. There is an increase of the number of GFP + CD45 + cells emerging during reprogramming of HUVECs by FGR+ SPI1 -Tet-On construct and E4EC-induction. E. rEC-hMPPs differentiate into CD3 + , CD19 + and CD14 + hematopoietic cells in the absence of exogenous expression of SPI1 . After 3 weeks, the numbers of the myeloid and lymphoid cells were quantified by flow cytometry. We were able to reliably detect a small fraction of CD3 + cells (0.16±0.01%; n=3), a larger number of CD19 + (1.17±0.13%; n=3) and CD14 + (16.46±1.02%; n=3) cells. Thus, generation of lymphoid cells from rEC-hMPPs could be optimized by transient expression of TFs.
Figure Legend Snippet: Conditional expression of FGRS is sufficient for optimal generation of rEC-hMPPs with multilineage potential, including T-Cell lymphoid cells A to C: Conditional expression of mouse inducible FGRS factors activates endogenous human FGRS in HUVECs sustaining functional hematopoietic cell fate of rEC-hMPPs. A . To test whether FGRS-induced reprogramming triggered expression of endogenous FGRS genes 24 , HUVECs were transduced with lentivirus expressing FGRS-Tet-On and a trans-activator, and grown on E4EC-vascular niche for 18–22 days (n=4) i n the presence of doxycycline. Doxycycline was removed from the culture medium after 18–22 days to shut off the expression of mouse FGRS and cells were cultured for additional 7–10 days. Human CD45 + CD34 + cells were FACS isolated for CFC assay and whole-transcriptome deep sequencing (RNA-seq). CFC assay revealed emergence of hematopoietic colonies with cells expressing human CD235, CD11b, CD83, and CD14. B . Comparison of transcriptional profiles of the human FGRS expression in human HUVECs, hCD45 + rEC-hMPPs programmed using inducible mouse FGRS, CD45 + CD34 + rEC-hMPPs, 22 weeks post-transplantation, hDMEC-derived CD45 + CD34 + rEC − hMPPs after 15 weeks post-secondary engraftment and naïve CD34 + Lin + cells purified from cord blood. C . Analysis of whole-transcriptome RNA-Seq of rEC-hMPPs derived using inducible mouse FGRS (n=3). All RNA-Seq reads were aligned against human and mouse FGRS sequences. RNA-Seq reads that align to human FGRS sequences – “Map to Human”, RNA-Seq reads that align to mouse FGRS sequences – “Map to Mouse”, and RNA-Seq reads that align to mouse FGRS sequences without a possibility to align to human sequences – “Map to mouse Only”. D to E: Optimizing differentiation of rEC-hMPPs into lymphoid progeny D . The number of T-lymphoid progeny of engrafted rEC-hMPP was negligibly small, raising the possibility that constitutive SPI1 expression prevents rEC-hMPP from differentiating into T-cells 39 , 40 . To test this, HUVECs were transduced with lentiviral vectors expressing GFP and that constitutively express FGR-TFs with a Tet-inducible SPI1 (FGR+ SPI1 -Tet-On construct) for 3 days followed by replating for E4EC-induction. After 27 days of FGR and doxycycline-induced SPI1 expression on E4ECs, GFP + CD45 + hematopoietic-like colonies emerged. Then, doxycycline was withdrawn and the reprogrammed cells were cultured serum-free with Delta-like-4 expressing OP9-stroma (OP9-DLL4) supplemented with IL-7, IL-11, and IL-2. There is an increase of the number of GFP + CD45 + cells emerging during reprogramming of HUVECs by FGR+ SPI1 -Tet-On construct and E4EC-induction. E. rEC-hMPPs differentiate into CD3 + , CD19 + and CD14 + hematopoietic cells in the absence of exogenous expression of SPI1 . After 3 weeks, the numbers of the myeloid and lymphoid cells were quantified by flow cytometry. We were able to reliably detect a small fraction of CD3 + cells (0.16±0.01%; n=3), a larger number of CD19 + (1.17±0.13%; n=3) and CD14 + (16.46±1.02%; n=3) cells. Thus, generation of lymphoid cells from rEC-hMPPs could be optimized by transient expression of TFs.

Techniques Used: Expressing, Functional Assay, Transduction, Cell Culture, FACS, Isolation, Sequencing, RNA Sequencing Assay, Transplantation Assay, Derivative Assay, Purification, Construct, Flow Cytometry, Cytometry

20) Product Images from "The TLR4 adaptor TRAM controls the phagocytosis of Gram-negative bacteria by interacting with the Rab11-family interacting protein 2"

Article Title: The TLR4 adaptor TRAM controls the phagocytosis of Gram-negative bacteria by interacting with the Rab11-family interacting protein 2

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007684

FIP2 controls E . coli induced IFN-β mRNA induction and secretion. ( A ) Immunoblots showing the phosphorylation patterns of TBK1, IRF3, IκBα and p38MAPK in FIP2 silenced THP-1 cells stimulated with E . coli bioparticles or LPS (100 ng/ml). Data are representative of three independent experiments. ( B ) Quantification of phosphorylation patterns of the proteins shown in the immunoblots presented in (A). ( C ) ELISA quantification IFN-β and TNF secretion in THP-1 cells treated with NS RNA or FIP2 siRNA and stimulated as indicated. ( D ) Quantification of E . coli -stimulated IFN-β and TNF mRNAs in THP-1 cells with lentiviral overexpression of FIP2. ( E ) Quantification of Poly I:C and LPS stimulated IFN-β mRNA induction in cells treated with NS RNA or FIP2 siRNA after 4 hours of stimulation. Poly I:C (5 μg/ml) was transfected using Lipofectamine® 2000. Data are representative of three independent experiments.
Figure Legend Snippet: FIP2 controls E . coli induced IFN-β mRNA induction and secretion. ( A ) Immunoblots showing the phosphorylation patterns of TBK1, IRF3, IκBα and p38MAPK in FIP2 silenced THP-1 cells stimulated with E . coli bioparticles or LPS (100 ng/ml). Data are representative of three independent experiments. ( B ) Quantification of phosphorylation patterns of the proteins shown in the immunoblots presented in (A). ( C ) ELISA quantification IFN-β and TNF secretion in THP-1 cells treated with NS RNA or FIP2 siRNA and stimulated as indicated. ( D ) Quantification of E . coli -stimulated IFN-β and TNF mRNAs in THP-1 cells with lentiviral overexpression of FIP2. ( E ) Quantification of Poly I:C and LPS stimulated IFN-β mRNA induction in cells treated with NS RNA or FIP2 siRNA after 4 hours of stimulation. Poly I:C (5 μg/ml) was transfected using Lipofectamine® 2000. Data are representative of three independent experiments.

Techniques Used: Western Blot, Enzyme-linked Immunosorbent Assay, Over Expression, Transfection

21) Product Images from "Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch"

Article Title: Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch

Journal: Cell

doi: 10.1016/j.cell.2018.03.016

Functional rescue analysis identifies BCL11A isoform XL and domains required for transcriptional repression (A) Schematic of functional rescue analysis in BCL11A enhancer or exon knockout mouse erythroleukemia (MEL) cells by ectopic expression of various BCL11A isoforms or domain mutants. Expression of BCL11A-XL, but no other known isoforms, restored the transcriptional repression of the embryonic εy- and βh1-globin genes, whereas the adult βmajor-globin remained unaffected. Results are mean ± SEM of three experiments and analyzed by two-sided t -test. * P
Figure Legend Snippet: Functional rescue analysis identifies BCL11A isoform XL and domains required for transcriptional repression (A) Schematic of functional rescue analysis in BCL11A enhancer or exon knockout mouse erythroleukemia (MEL) cells by ectopic expression of various BCL11A isoforms or domain mutants. Expression of BCL11A-XL, but no other known isoforms, restored the transcriptional repression of the embryonic εy- and βh1-globin genes, whereas the adult βmajor-globin remained unaffected. Results are mean ± SEM of three experiments and analyzed by two-sided t -test. * P

Techniques Used: Functional Assay, Knock-Out, Expressing

22) Product Images from "BCL6 modulation of acute lymphoblastic leukemia response to chemotherapy"

Article Title: BCL6 modulation of acute lymphoblastic leukemia response to chemotherapy

Journal: Oncotarget

doi: 10.18632/oncotarget.8273

Modulation of BCL6 alters cell cycle progression and proliferation of ALL cells A. - B. Cell density and viability of REH, Sup-B15, and Nalm-27 following exposure to the small molecule BCL6 inhibitor 79-6 (125μM) relative to DMSO controls as shown by trypan blue exclusion cell counts. C. Proliferation index of 79-6 treatment of REH, Sup-B15 and Nalm-27 ALL cells compared to DMSO controls using a CSFE cell retention dye flow cytometry analysis. D. Propidium iodide (PI) DNA staining for cell cycle assessment of REH, Sup-B15 and Nalm-27 treated with 79-6 compared to DMSO controls. E. Cell density of shRNA knockdown of BCL6 (KD1 and KD3) (left panel) and BCL6 overexpression (BCL6 OX) (right panel) of REH cells over time compared to vector controls as evaluated by trypan blue exclusion counts. F. Cell cycle analysis of BCL6 knockdown (left panel) and BCL6 overexpression (right panel) in REH cells using PI staining. (* = p
Figure Legend Snippet: Modulation of BCL6 alters cell cycle progression and proliferation of ALL cells A. - B. Cell density and viability of REH, Sup-B15, and Nalm-27 following exposure to the small molecule BCL6 inhibitor 79-6 (125μM) relative to DMSO controls as shown by trypan blue exclusion cell counts. C. Proliferation index of 79-6 treatment of REH, Sup-B15 and Nalm-27 ALL cells compared to DMSO controls using a CSFE cell retention dye flow cytometry analysis. D. Propidium iodide (PI) DNA staining for cell cycle assessment of REH, Sup-B15 and Nalm-27 treated with 79-6 compared to DMSO controls. E. Cell density of shRNA knockdown of BCL6 (KD1 and KD3) (left panel) and BCL6 overexpression (BCL6 OX) (right panel) of REH cells over time compared to vector controls as evaluated by trypan blue exclusion counts. F. Cell cycle analysis of BCL6 knockdown (left panel) and BCL6 overexpression (right panel) in REH cells using PI staining. (* = p

Techniques Used: Flow Cytometry, Cytometry, Staining, shRNA, Over Expression, Plasmid Preparation, Cell Cycle Assay

In vivo sensitivity to Ara-C is increased by BCL6 overexpression or pre-treatment with caffeine A. Schematic of NSG mouse experiment to determine GFP+ ALL burden in the femurs of NSG mice. B. Box plot representation of median percentage of GFP+ REH ALL cells relative to total mononuclear cells recovered from femurs of NOD-SCID Gamma (NSG) mice infected with REH vector control ( n = 5) or REH BCL6 overexpression ( n = 4) ALL cells following three consecutive days of Ara-C treatment. C. Schematic of NSG mouse experiment to determine event free survival of mice pre-treated with BCL6 modulating drugs MG-132 or caffeine. D. Event free survival of NSG mice following treatment with Ara-C ( n = 5), MG-132 + Ara-C ( n = 6), or caffeine + Ara-C ( n = 6) (* = p
Figure Legend Snippet: In vivo sensitivity to Ara-C is increased by BCL6 overexpression or pre-treatment with caffeine A. Schematic of NSG mouse experiment to determine GFP+ ALL burden in the femurs of NSG mice. B. Box plot representation of median percentage of GFP+ REH ALL cells relative to total mononuclear cells recovered from femurs of NOD-SCID Gamma (NSG) mice infected with REH vector control ( n = 5) or REH BCL6 overexpression ( n = 4) ALL cells following three consecutive days of Ara-C treatment. C. Schematic of NSG mouse experiment to determine event free survival of mice pre-treated with BCL6 modulating drugs MG-132 or caffeine. D. Event free survival of NSG mice following treatment with Ara-C ( n = 5), MG-132 + Ara-C ( n = 6), or caffeine + Ara-C ( n = 6) (* = p

Techniques Used: In Vivo, Acetylene Reduction Assay, Over Expression, Mouse Assay, Infection, Plasmid Preparation

Co-culture with BMSC or HOB reduces BCL6 in ALL cells A. BCL6 protein in REH and Nalm-27 ALL cells when co-cultured with BMSC or HOB cells relative to media (M) controls as shown by western blot analysis. B. Flow cytometry analysis of REH and Nalm-27 ALL cell BCL6 protein levels when removed from the PD population compared to cells in media alone as shown by median florescence intensity (MFI). C. MFI of Patient 1 (P1) and Patient 2 (P2) when in physical contact with BMSC or HOB compared to those in media alone (ND = not detected). D. Confocal microscopy images of REH and Nalm-27 for BCL6 (yellow) and DAPI (Blue) in cells cultured in media alone compare to those recovered from the PD population of BMSC or HOB co-culture. E. P1 and P2 BCL6 confocal staining of media alone cells relative to those in contact with BMSC or HOB. Scale bar = 10μm.
Figure Legend Snippet: Co-culture with BMSC or HOB reduces BCL6 in ALL cells A. BCL6 protein in REH and Nalm-27 ALL cells when co-cultured with BMSC or HOB cells relative to media (M) controls as shown by western blot analysis. B. Flow cytometry analysis of REH and Nalm-27 ALL cell BCL6 protein levels when removed from the PD population compared to cells in media alone as shown by median florescence intensity (MFI). C. MFI of Patient 1 (P1) and Patient 2 (P2) when in physical contact with BMSC or HOB compared to those in media alone (ND = not detected). D. Confocal microscopy images of REH and Nalm-27 for BCL6 (yellow) and DAPI (Blue) in cells cultured in media alone compare to those recovered from the PD population of BMSC or HOB co-culture. E. P1 and P2 BCL6 confocal staining of media alone cells relative to those in contact with BMSC or HOB. Scale bar = 10μm.

Techniques Used: Co-Culture Assay, Cell Culture, Western Blot, Flow Cytometry, Cytometry, Confocal Microscopy, Staining

BCL6 modulates the cell cycle regulating protein cyclin D3 A. Western blot analysis of protein abundance of BCL6 and cyclin D3 in REH and Nalm-27 cells in media alone compared to PD cells recovered from BMSC or HOB co-culture. B. Comparison of REH BCL6 knockdown and overexpression to vector controls for BCL6 and cyclin D3 protein levels by western blot. C. Protein analysis by western blot of cyclin D3 in REH and Nalm-27 cells when exposed to 79-6.
Figure Legend Snippet: BCL6 modulates the cell cycle regulating protein cyclin D3 A. Western blot analysis of protein abundance of BCL6 and cyclin D3 in REH and Nalm-27 cells in media alone compared to PD cells recovered from BMSC or HOB co-culture. B. Comparison of REH BCL6 knockdown and overexpression to vector controls for BCL6 and cyclin D3 protein levels by western blot. C. Protein analysis by western blot of cyclin D3 in REH and Nalm-27 cells when exposed to 79-6.

Techniques Used: Western Blot, Co-Culture Assay, Over Expression, Plasmid Preparation

Forced expression of BCL6 sensitizes PD ALL cells to chemotherapy exposure A. Viability comparison of REH vector control, BCL6 overexpression, or BCL6 overexpression cells pre-treated with 79-6 (125μM) following exposure to three chemotherapy drugs (Ara-C [1 μM], MTX [50 μM], VCR [25 μM]). (* = p
Figure Legend Snippet: Forced expression of BCL6 sensitizes PD ALL cells to chemotherapy exposure A. Viability comparison of REH vector control, BCL6 overexpression, or BCL6 overexpression cells pre-treated with 79-6 (125μM) following exposure to three chemotherapy drugs (Ara-C [1 μM], MTX [50 μM], VCR [25 μM]). (* = p

Techniques Used: Expressing, Plasmid Preparation, Over Expression, Acetylene Reduction Assay

23) Product Images from "A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia"

Article Title: A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia

Journal: BMC Cancer

doi: 10.1186/s12885-018-4097-z

An illustration of miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry. a The decreased expression of miR-375 due to DNA hypermethylation results in the high expression of HOXB3, contributing to cell proliferation and colony formation through increasing the expression of CDCA3. Moreover, HOXB3 enhances and recruits DNMT3B to bind in pre-miR-375 promoter, leading to further DNA hypermethylation and subsequent downregulation of miR-375 in AML cells, in turn
Figure Legend Snippet: An illustration of miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry. a The decreased expression of miR-375 due to DNA hypermethylation results in the high expression of HOXB3, contributing to cell proliferation and colony formation through increasing the expression of CDCA3. Moreover, HOXB3 enhances and recruits DNMT3B to bind in pre-miR-375 promoter, leading to further DNA hypermethylation and subsequent downregulation of miR-375 in AML cells, in turn

Techniques Used: Expressing

HOXB3 enhances the expression of DNMT3B to bind in pre-miR-375 promoter. a HL-60 and THP1 cells were transduced with special shRNA for HOXB3 (sh-HOXB3) or sh-NC. HOXB3 and DNMT3B expressions were detected by western blot. b HOXB3 and DNMT3B expressions were detected in HL-60 and THP1 cells, which were transduced with overexpression vector LVX-HOXB3 or LVX-NC. c DNMT3B expression was detected in HL-60 and THP1 cells transduced with special shRNA targeting DNMT3B (sh-DNMT3B) or sh-NC. d MiR-375 expression was measured in HL-60 and THP1 cells transduced with sh-DNMT3B or sh-NC. * P
Figure Legend Snippet: HOXB3 enhances the expression of DNMT3B to bind in pre-miR-375 promoter. a HL-60 and THP1 cells were transduced with special shRNA for HOXB3 (sh-HOXB3) or sh-NC. HOXB3 and DNMT3B expressions were detected by western blot. b HOXB3 and DNMT3B expressions were detected in HL-60 and THP1 cells, which were transduced with overexpression vector LVX-HOXB3 or LVX-NC. c DNMT3B expression was detected in HL-60 and THP1 cells transduced with special shRNA targeting DNMT3B (sh-DNMT3B) or sh-NC. d MiR-375 expression was measured in HL-60 and THP1 cells transduced with sh-DNMT3B or sh-NC. * P

Techniques Used: Expressing, Transduction, shRNA, Western Blot, Over Expression, Plasmid Preparation

24) Product Images from "A Mutant Tat Protein Provides Strong Protection from HIV-1 Infection in Human CD4+ T Cells"

Article Title: A Mutant Tat Protein Provides Strong Protection from HIV-1 Infection in Human CD4+ T Cells

Journal: Human Gene Therapy

doi: 10.1089/hum.2012.176

Transduction of human CD4 + primary cells with MLV-A-based retroviral VLPs conveying NB-ZsGreen1 or ZsGreen1. (a) Schematic of the methods used to generate CD4 + primary cells transductants. (b) NB-ZsGreen1 or ZsGreen1 VLPs were used to transduce activated
Figure Legend Snippet: Transduction of human CD4 + primary cells with MLV-A-based retroviral VLPs conveying NB-ZsGreen1 or ZsGreen1. (a) Schematic of the methods used to generate CD4 + primary cells transductants. (b) NB-ZsGreen1 or ZsGreen1 VLPs were used to transduce activated

Techniques Used: Transduction

HIV replication is inhibited in activated human CD4 + primary cells expressing NB-ZsGreen1. (a) The two CD4 + populations described in were infected with HIV-1 89.6 containing 20 ng of CAp24 and grown for 21 days. Cell-free supernatant was
Figure Legend Snippet: HIV replication is inhibited in activated human CD4 + primary cells expressing NB-ZsGreen1. (a) The two CD4 + populations described in were infected with HIV-1 89.6 containing 20 ng of CAp24 and grown for 21 days. Cell-free supernatant was

Techniques Used: Expressing, Infection

25) Product Images from "CCR6 Is a Prognostic Marker for Overall Survival in Patients with Colorectal Cancer, and Its Overexpression Enhances Metastasis In Vivo"

Article Title: CCR6 Is a Prognostic Marker for Overall Survival in Patients with Colorectal Cancer, and Its Overexpression Enhances Metastasis In Vivo

Journal: PLoS ONE

doi: 10.1371/journal.pone.0101137

CCR6 is Upregulated in Primary Human CRC Samples. (A) Immunohistochemical staining of CCR6 in primary CRC derived from 191 CRC patients with clinical stage I–IV. (B) Digital image analysis was performed to count staining intensity of CCR6 area fraction (CCR6-AF) values of paired para-tumor/tumor samples in each clinical stage, Wilcoxon test.
Figure Legend Snippet: CCR6 is Upregulated in Primary Human CRC Samples. (A) Immunohistochemical staining of CCR6 in primary CRC derived from 191 CRC patients with clinical stage I–IV. (B) Digital image analysis was performed to count staining intensity of CCR6 area fraction (CCR6-AF) values of paired para-tumor/tumor samples in each clinical stage, Wilcoxon test.

Techniques Used: Immunohistochemistry, Staining, Derivative Assay

Survival Analysis of CRC Patients with Low versus High Expression of CCR6. (A) Kaplan-Meier curves of CRC patients with low versus high expression of CCR6 (n = 191, p
Figure Legend Snippet: Survival Analysis of CRC Patients with Low versus High Expression of CCR6. (A) Kaplan-Meier curves of CRC patients with low versus high expression of CCR6 (n = 191, p

Techniques Used: Expressing

Inhibition of Mouse CRC Progression by Targeting Tumor-expressing CCR6. (A) Western blotting analysis of CCR6 in murine CMT93 colorectal tumor cell line and CRC tissue derived from CCR6 −/− mice grafted with CMT93 at day10. (B) Statistical analysis of tumor weight in each group treated with IgG or anti-CCR6. (C) Western blotting of CCR6 in murine CT26 colorectal tumor cell line and CRC tissue derived from Balb/c mice grafted with CT26 at day 10. (D) Statistical analysis of tumor weight in each group treated with IgG or anti-CCR6. * p
Figure Legend Snippet: Inhibition of Mouse CRC Progression by Targeting Tumor-expressing CCR6. (A) Western blotting analysis of CCR6 in murine CMT93 colorectal tumor cell line and CRC tissue derived from CCR6 −/− mice grafted with CMT93 at day10. (B) Statistical analysis of tumor weight in each group treated with IgG or anti-CCR6. (C) Western blotting of CCR6 in murine CT26 colorectal tumor cell line and CRC tissue derived from Balb/c mice grafted with CT26 at day 10. (D) Statistical analysis of tumor weight in each group treated with IgG or anti-CCR6. * p

Techniques Used: Inhibition, Expressing, Western Blot, Derivative Assay, Mouse Assay

Signaling Pathway Involved in the Aggressiveness of HCT116 CCR6 . (A, B) Western blotting analysis of Erk1/2 or phospho-Erk1/2 and Akt, phosphorylated Akt (Ser473) or phosphorylated Akt (Ser308) in HCT116 CCR6 and HCT116 Ctr cells. Values were expressed as fold changes relative to HCT116 Ctr , and normalized to β-actin. (C) Upregulated (FXYD5 and SYK) or down-regulated genes (CDH1, KISS1 and TIMP2) in HCT116 CCR6 cells screening with a human tumor metastasis RT 2 profiler PCR Array. (D) Western blotting analysis of changed FXYD5, SYK, CDH1, KISS1 and TIMP2 genes in HCT116 CCR6 and HCT116 Ctr cells. Values were expressed as fold changes relative to HCT116 Ctr , and normalized to β-actin.
Figure Legend Snippet: Signaling Pathway Involved in the Aggressiveness of HCT116 CCR6 . (A, B) Western blotting analysis of Erk1/2 or phospho-Erk1/2 and Akt, phosphorylated Akt (Ser473) or phosphorylated Akt (Ser308) in HCT116 CCR6 and HCT116 Ctr cells. Values were expressed as fold changes relative to HCT116 Ctr , and normalized to β-actin. (C) Upregulated (FXYD5 and SYK) or down-regulated genes (CDH1, KISS1 and TIMP2) in HCT116 CCR6 cells screening with a human tumor metastasis RT 2 profiler PCR Array. (D) Western blotting analysis of changed FXYD5, SYK, CDH1, KISS1 and TIMP2 genes in HCT116 CCR6 and HCT116 Ctr cells. Values were expressed as fold changes relative to HCT116 Ctr , and normalized to β-actin.

Techniques Used: Western Blot, Polymerase Chain Reaction

Enhanced Proliferation and Migration of CRC cells with Overexpressed CCR6. (A) Western blotting analysis of ectopic expression of CCR6 in HCT116 Ctr and HCT116 CCR6 cells or Caco-2 Ctr and Caco-2 CCR6 or SW1116 Ctr and SW1116 CCR6 cells. β-actin served as a loading control. Values were expressed as fold changes relative to controls (Ctr), and normalized to β-actin. (B) Wound-healing assay for motility of HCT116 Ctr and HCT116 CCR6 or Caco-2 Ctr and Caco-2 CCR6 or SW1116 Ctr and SW1116 CCR6 cells. Representative pictures of one field at the beginning (t = 0) (upper panel) and at the end of the recording (t = 24 h) (lower panel) in each condition are shown. The relative cell migration in CCR6 and control groups are shown in the right panel. (C) Representative images of transwell migrated cells in stably transfected HCT116 Ctr , Caco-2 Ctr , SW1116 Ctr (upper panel) or HCT116 CCR6 , Caco-2 CCR6 , SW1116 CCR6 (lower panel) cells. Average number of migrated cells of HCT116 Ctr and HCT116 CCR6 or Caco-2 Ctr and Caco-2 CCR6 or SW1116 Ctr and SW1116 CCR6 cells are shown in the right panel. (D) Representative image of colony formation in HCT116 Ctr , Caco-2 Ctr , SW1116 Ctr (upper panel) or HCT116 CCR6 , Caco-2 CCR6 , SW1116 CCR6 cells (lower panel). Values represent mean from triplicate wells, ± S.D. * p
Figure Legend Snippet: Enhanced Proliferation and Migration of CRC cells with Overexpressed CCR6. (A) Western blotting analysis of ectopic expression of CCR6 in HCT116 Ctr and HCT116 CCR6 cells or Caco-2 Ctr and Caco-2 CCR6 or SW1116 Ctr and SW1116 CCR6 cells. β-actin served as a loading control. Values were expressed as fold changes relative to controls (Ctr), and normalized to β-actin. (B) Wound-healing assay for motility of HCT116 Ctr and HCT116 CCR6 or Caco-2 Ctr and Caco-2 CCR6 or SW1116 Ctr and SW1116 CCR6 cells. Representative pictures of one field at the beginning (t = 0) (upper panel) and at the end of the recording (t = 24 h) (lower panel) in each condition are shown. The relative cell migration in CCR6 and control groups are shown in the right panel. (C) Representative images of transwell migrated cells in stably transfected HCT116 Ctr , Caco-2 Ctr , SW1116 Ctr (upper panel) or HCT116 CCR6 , Caco-2 CCR6 , SW1116 CCR6 (lower panel) cells. Average number of migrated cells of HCT116 Ctr and HCT116 CCR6 or Caco-2 Ctr and Caco-2 CCR6 or SW1116 Ctr and SW1116 CCR6 cells are shown in the right panel. (D) Representative image of colony formation in HCT116 Ctr , Caco-2 Ctr , SW1116 Ctr (upper panel) or HCT116 CCR6 , Caco-2 CCR6 , SW1116 CCR6 cells (lower panel). Values represent mean from triplicate wells, ± S.D. * p

Techniques Used: Migration, Western Blot, Expressing, Wound Healing Assay, Stable Transfection, Transfection

Knockdown of CCR6 by shRNA Inhibits CRC Cell Migration in vitro . (A) Western blotting analysis of CCR6 levels in 7 cultured CRC cell lines. Values were expressed as fold changes relative to Caco-2, and normalized to β-actin. (B) Western blotting analysis of knockdown of CCR6 in SW480 and LoVo cells, β-actin served as a loading control. Values were expressed as fold changes relative to controls (ShCtr), and normalized to β-actin. (C) Wound-healing assays for motility of CCR6-silenced SW480 and LoVo cells and control cells. Representative pictures of one field at the beginning (t = 0 hr) (upper panel) and at the end of the recording (t = 24 hr) (lower panel) in each condition are shown. The relative cell migration in ShCtr and shCCR6 groups are shown in the right panel. (D) Representative images of transwell migrated cells in CCR6-silenced SW480 and LoVo cells (lower panel) or control cells (upper panel) cells. The numbers of migrated cells in ShCtr and shCCR6 groups are shown in the right panel. Values represent mean from triplicate wells, ± S.D. * p
Figure Legend Snippet: Knockdown of CCR6 by shRNA Inhibits CRC Cell Migration in vitro . (A) Western blotting analysis of CCR6 levels in 7 cultured CRC cell lines. Values were expressed as fold changes relative to Caco-2, and normalized to β-actin. (B) Western blotting analysis of knockdown of CCR6 in SW480 and LoVo cells, β-actin served as a loading control. Values were expressed as fold changes relative to controls (ShCtr), and normalized to β-actin. (C) Wound-healing assays for motility of CCR6-silenced SW480 and LoVo cells and control cells. Representative pictures of one field at the beginning (t = 0 hr) (upper panel) and at the end of the recording (t = 24 hr) (lower panel) in each condition are shown. The relative cell migration in ShCtr and shCCR6 groups are shown in the right panel. (D) Representative images of transwell migrated cells in CCR6-silenced SW480 and LoVo cells (lower panel) or control cells (upper panel) cells. The numbers of migrated cells in ShCtr and shCCR6 groups are shown in the right panel. Values represent mean from triplicate wells, ± S.D. * p

Techniques Used: shRNA, Migration, In Vitro, Western Blot, Cell Culture

Increased Metastasis of CRC Cells with Overexpressed CCR6 in vivo . (A) Number of metastatic nodules (indicated by white arrows) formed in the liver of BALB/c nude mice 5 weeks after spleen injection of HCT116 Ctr (upper panel) or HCT116 CCR6 (lower panel) cells (six mice per group). (B) In vivo metastasis assays of Luc-HCT116 Ctr and Luc-HCT116 CCR6 cells by tail vein injection. The whole body metastasis burden of xenografted animals was monitored at 6 weeks after CRC cell injection using the IVIS Imaging System. Statistical analysis of luciferase intensity from mice injected with Luc-HCT116 Ctr or Luc-HCT116 CCR6 cells was shown in the right panel. (C) Representative images of H E staining of lungs prepared from mice injected with Luc-HCT116 Ctr or Luc-HCT116 CCR6 cells at ×5 (left panel) and ×10 (right panel) magnification. Statistical analysis of the number or mitosis by mm 2 in each metastatic nodule in the five lung H E staining from mice injected with Luc-HCT116 Ctr or Luc-HCT116 CCR6 cells was shown in the right panel. * p
Figure Legend Snippet: Increased Metastasis of CRC Cells with Overexpressed CCR6 in vivo . (A) Number of metastatic nodules (indicated by white arrows) formed in the liver of BALB/c nude mice 5 weeks after spleen injection of HCT116 Ctr (upper panel) or HCT116 CCR6 (lower panel) cells (six mice per group). (B) In vivo metastasis assays of Luc-HCT116 Ctr and Luc-HCT116 CCR6 cells by tail vein injection. The whole body metastasis burden of xenografted animals was monitored at 6 weeks after CRC cell injection using the IVIS Imaging System. Statistical analysis of luciferase intensity from mice injected with Luc-HCT116 Ctr or Luc-HCT116 CCR6 cells was shown in the right panel. (C) Representative images of H E staining of lungs prepared from mice injected with Luc-HCT116 Ctr or Luc-HCT116 CCR6 cells at ×5 (left panel) and ×10 (right panel) magnification. Statistical analysis of the number or mitosis by mm 2 in each metastatic nodule in the five lung H E staining from mice injected with Luc-HCT116 Ctr or Luc-HCT116 CCR6 cells was shown in the right panel. * p

Techniques Used: In Vivo, Mouse Assay, Injection, Imaging, Luciferase, Staining

26) Product Images from "Forced co-expression of IL-21 and IL-7 in whole-cell cancer vaccines promotes antitumor immunity"

Article Title: Forced co-expression of IL-21 and IL-7 in whole-cell cancer vaccines promotes antitumor immunity

Journal: Scientific Reports

doi: 10.1038/srep32351

Establishment of vaccine cell lines by lentiviral transduction. ( A ) Lentiviral constructs used in this study. LTR, long terminal repeat. Ψ, packaging signal. RRE, Rev response element. cPPT, central polypurine tract. CMVp, CMV promoter. IRES, internal ribosome entry site. Bsd, blasticidin resistance gene. WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. Sp, Signal peptide of IL-7. FA, furin cleavage site and P2A peptide. ( B ) Western blot analysis of secreted IL-21 and IL-7 in vaccine cell-conditioned media. Note that the two bands of IL-21 in the 21/7 CM lane represented two possible cleavage products: cleavage at furin site resulted in IL-21 + 4AA, while cleavage at P2A site resulted in IL-21 + 25AA. CM, conditioned medium. AA, amino acids. ( C ) Western blot analysis of STAT proteins activated in response to secreted IL-21 and IL-7. SC, splenocytes. MC, medium control. Full-length blots are presented in Supplementary Figure S1 .
Figure Legend Snippet: Establishment of vaccine cell lines by lentiviral transduction. ( A ) Lentiviral constructs used in this study. LTR, long terminal repeat. Ψ, packaging signal. RRE, Rev response element. cPPT, central polypurine tract. CMVp, CMV promoter. IRES, internal ribosome entry site. Bsd, blasticidin resistance gene. WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. Sp, Signal peptide of IL-7. FA, furin cleavage site and P2A peptide. ( B ) Western blot analysis of secreted IL-21 and IL-7 in vaccine cell-conditioned media. Note that the two bands of IL-21 in the 21/7 CM lane represented two possible cleavage products: cleavage at furin site resulted in IL-21 + 4AA, while cleavage at P2A site resulted in IL-21 + 25AA. CM, conditioned medium. AA, amino acids. ( C ) Western blot analysis of STAT proteins activated in response to secreted IL-21 and IL-7. SC, splenocytes. MC, medium control. Full-length blots are presented in Supplementary Figure S1 .

Techniques Used: Transduction, Construct, Western Blot

27) Product Images from "Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles"

Article Title: Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles

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

doi: 10.1073/pnas.1614955114

( A ) Schematic representation of the bicistronic lentiviral constructs for expressing HEV ORF2, ORF3, IAV M2, and IAV M2(A30P). ( B ) Representative flow cytometry plots demonstrating efficient ORF2 and ORF3, IAV M2, or M2(A30P) expression. HepG2C3A cells were transduced with LVX-ORF2-IRES-zsGreen and/or LEX-[ORF3 or M2 or M2(A30P)]-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of ORF2- and/or ORF3- and M2-expressing cells. ( C ) Schematic representation of SP6-driven constructs used for in vitro transcription of HEV ORF3, IAV M2, and IAV M2(A30P) mRNAs. ( D ) HEV ORF3, IAV M2, or M2(A30P) expressed in X. laevis oocytes localizes to the plasma membrane. Water-injected and HEV ORF3, IAV M2, or M2(A30P) mRNA-injected oocytes were immunolabeled with polyclonal ORF3 or M2 antibodies and analyzed by confocal microscopy.
Figure Legend Snippet: ( A ) Schematic representation of the bicistronic lentiviral constructs for expressing HEV ORF2, ORF3, IAV M2, and IAV M2(A30P). ( B ) Representative flow cytometry plots demonstrating efficient ORF2 and ORF3, IAV M2, or M2(A30P) expression. HepG2C3A cells were transduced with LVX-ORF2-IRES-zsGreen and/or LEX-[ORF3 or M2 or M2(A30P)]-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of ORF2- and/or ORF3- and M2-expressing cells. ( C ) Schematic representation of SP6-driven constructs used for in vitro transcription of HEV ORF3, IAV M2, and IAV M2(A30P) mRNAs. ( D ) HEV ORF3, IAV M2, or M2(A30P) expressed in X. laevis oocytes localizes to the plasma membrane. Water-injected and HEV ORF3, IAV M2, or M2(A30P) mRNA-injected oocytes were immunolabeled with polyclonal ORF3 or M2 antibodies and analyzed by confocal microscopy.

Techniques Used: Construct, Expressing, Flow Cytometry, Cytometry, Transduction, In Vitro, Injection, Immunolabeling, Confocal Microscopy

ORF2 and ORF3 are required for releasing viral particles to infect naïve HepG2C3A cells. ( A ) Schematic representation of the transcomplementation system for packaging HEV virions. ( B ) Representative flow cytometry plots demonstrating efficient ORF2 and ORF3 expression. HepG2C3A cells were transduced with pLVX-ORF2-IRES-zsGreen and/or pLEX-ORF3-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of ORF2 and/or ORF3 expressing cells. ( C ) Infection kinetics of transcomplemented HEV in HepG2C3A cells. Cell culture supernatants from naïve HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, were collected 5 d posttransfection with rHEVΔORF2/3[Gluc] RNA. Naïve HepG2C3A cells were incubated with these supernatants. After 12 h, cells were washed and Gaussia luciferase activity quantified in the cell culture supernatants at the indicated time points. ( D ) Five days following transfection of rHEVΔORF2/3[Gluc] RNA into HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, lysates were used to infect naïve HepG2C3A cells. Gluc activity was measured in the supernatants 4 d postinfection. Shown are averages and SDs of triplicate measurements of three independent experiments. * P
Figure Legend Snippet: ORF2 and ORF3 are required for releasing viral particles to infect naïve HepG2C3A cells. ( A ) Schematic representation of the transcomplementation system for packaging HEV virions. ( B ) Representative flow cytometry plots demonstrating efficient ORF2 and ORF3 expression. HepG2C3A cells were transduced with pLVX-ORF2-IRES-zsGreen and/or pLEX-ORF3-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of ORF2 and/or ORF3 expressing cells. ( C ) Infection kinetics of transcomplemented HEV in HepG2C3A cells. Cell culture supernatants from naïve HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, were collected 5 d posttransfection with rHEVΔORF2/3[Gluc] RNA. Naïve HepG2C3A cells were incubated with these supernatants. After 12 h, cells were washed and Gaussia luciferase activity quantified in the cell culture supernatants at the indicated time points. ( D ) Five days following transfection of rHEVΔORF2/3[Gluc] RNA into HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, lysates were used to infect naïve HepG2C3A cells. Gluc activity was measured in the supernatants 4 d postinfection. Shown are averages and SDs of triplicate measurements of three independent experiments. * P

Techniques Used: Flow Cytometry, Cytometry, Expressing, Transduction, Infection, Cell Culture, Incubation, Luciferase, Activity Assay, Transfection

28) Product Images from "Alternative splicing regulates the expression of G9A and SUV39H2 methyltransferases, and dramatically changes SUV39H2 functions"

Article Title: Alternative splicing regulates the expression of G9A and SUV39H2 methyltransferases, and dramatically changes SUV39H2 functions

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv013

Protein stability and sub-nuclear localization of SUV39H2 isoforms are regulated by alternative inclusion of exon 3. ( A ) Expression in HEK 293T cells of ectopic tagged G9A and SUV39H2 protein isoforms. Cells were transduced with the pLVX vector containing Flag-V5 tags and SUV39H2 or G9A cDNA to express exogenous protein isoforms ( FV SUV39H2 or FV G9A). Cells transduced with pLVX empty vector were used as controls (Ctl). The ectopic proteins were revealed by western blot using the V5 antibody. ( B ) and ( C ) Stability analysis of each G9A and SUV39H2 protein isoform. FV G9A and FV SUV39H2 isoforms were assessed by western blot in HeLa cells treated with cycloheximide (CHX) (B) or MG132 (C) for the indicated times in hours (h). The signal detected with V5 antibody was normalized to histone H3 levels. ( D ) Localization of FV G9A and FV SUV39H2 isoforms in HeLa cells. Exogenous proteins were revealed with V5 antibody (red), while DNA was counterstained with DAPI (blue). ( E ) and ( F ) Analysis of FV G9A and FV SUV39H2 and the endogenous SUV39H2 isoforms after fractionation of HeLa cells. (E) Scheme displaying the cell fractionation procedure. (F) Protein isoforms were detected in whole cell extracts (WCE) and fractions by western blot using the V5 or SUV39H2 antibodies. Endogenous tubulin, HP1α and H3 proteins were analyzed as markers of the cell fractions.
Figure Legend Snippet: Protein stability and sub-nuclear localization of SUV39H2 isoforms are regulated by alternative inclusion of exon 3. ( A ) Expression in HEK 293T cells of ectopic tagged G9A and SUV39H2 protein isoforms. Cells were transduced with the pLVX vector containing Flag-V5 tags and SUV39H2 or G9A cDNA to express exogenous protein isoforms ( FV SUV39H2 or FV G9A). Cells transduced with pLVX empty vector were used as controls (Ctl). The ectopic proteins were revealed by western blot using the V5 antibody. ( B ) and ( C ) Stability analysis of each G9A and SUV39H2 protein isoform. FV G9A and FV SUV39H2 isoforms were assessed by western blot in HeLa cells treated with cycloheximide (CHX) (B) or MG132 (C) for the indicated times in hours (h). The signal detected with V5 antibody was normalized to histone H3 levels. ( D ) Localization of FV G9A and FV SUV39H2 isoforms in HeLa cells. Exogenous proteins were revealed with V5 antibody (red), while DNA was counterstained with DAPI (blue). ( E ) and ( F ) Analysis of FV G9A and FV SUV39H2 and the endogenous SUV39H2 isoforms after fractionation of HeLa cells. (E) Scheme displaying the cell fractionation procedure. (F) Protein isoforms were detected in whole cell extracts (WCE) and fractions by western blot using the V5 or SUV39H2 antibodies. Endogenous tubulin, HP1α and H3 proteins were analyzed as markers of the cell fractions.

Techniques Used: Expressing, Transduction, Plasmid Preparation, CTL Assay, Western Blot, Fractionation, Cell Fractionation

29) Product Images from "A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia"

Article Title: A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia

Journal: BMC Cancer

doi: 10.1186/s12885-018-4097-z

Knockdown of HOXB3 and CDCA3 partially mimics the anti-leukemia activity by miR-375. a HL-60 and THP1 cells were transduced with special shRNA for HOXB 3 or a control shRNA (sh-NC). HOXB3 and CDCA3 expressions were detected by western blot. b Viable cell number by the trypan-blue exclusion assay was counted in HL-60 and THP1 cells, which were transduced with special shRNA for HOXB3 or sh-NC for the indicated times. * P
Figure Legend Snippet: Knockdown of HOXB3 and CDCA3 partially mimics the anti-leukemia activity by miR-375. a HL-60 and THP1 cells were transduced with special shRNA for HOXB 3 or a control shRNA (sh-NC). HOXB3 and CDCA3 expressions were detected by western blot. b Viable cell number by the trypan-blue exclusion assay was counted in HL-60 and THP1 cells, which were transduced with special shRNA for HOXB3 or sh-NC for the indicated times. * P

Techniques Used: Activity Assay, Transduction, shRNA, Western Blot, Trypan Blue Exclusion Assay

An illustration of miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry. a The decreased expression of miR-375 due to DNA hypermethylation results in the high expression of HOXB3, contributing to cell proliferation and colony formation through increasing the expression of CDCA3. Moreover, HOXB3 enhances and recruits DNMT3B to bind in pre-miR-375 promoter, leading to further DNA hypermethylation and subsequent downregulation of miR-375 in AML cells, in turn
Figure Legend Snippet: An illustration of miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry. a The decreased expression of miR-375 due to DNA hypermethylation results in the high expression of HOXB3, contributing to cell proliferation and colony formation through increasing the expression of CDCA3. Moreover, HOXB3 enhances and recruits DNMT3B to bind in pre-miR-375 promoter, leading to further DNA hypermethylation and subsequent downregulation of miR-375 in AML cells, in turn

Techniques Used: Expressing

HOXB3 enhances the expression of DNMT3B to bind in pre-miR-375 promoter. a HL-60 and THP1 cells were transduced with special shRNA for HOXB3 (sh-HOXB3) or sh-NC. HOXB3 and DNMT3B expressions were detected by western blot. b HOXB3 and DNMT3B expressions were detected in HL-60 and THP1 cells, which were transduced with overexpression vector LVX-HOXB3 or LVX-NC. c DNMT3B expression was detected in HL-60 and THP1 cells transduced with special shRNA targeting DNMT3B (sh-DNMT3B) or sh-NC. d MiR-375 expression was measured in HL-60 and THP1 cells transduced with sh-DNMT3B or sh-NC. * P
Figure Legend Snippet: HOXB3 enhances the expression of DNMT3B to bind in pre-miR-375 promoter. a HL-60 and THP1 cells were transduced with special shRNA for HOXB3 (sh-HOXB3) or sh-NC. HOXB3 and DNMT3B expressions were detected by western blot. b HOXB3 and DNMT3B expressions were detected in HL-60 and THP1 cells, which were transduced with overexpression vector LVX-HOXB3 or LVX-NC. c DNMT3B expression was detected in HL-60 and THP1 cells transduced with special shRNA targeting DNMT3B (sh-DNMT3B) or sh-NC. d MiR-375 expression was measured in HL-60 and THP1 cells transduced with sh-DNMT3B or sh-NC. * P

Techniques Used: Expressing, Transduction, shRNA, Western Blot, Over Expression, Plasmid Preparation

The anti-leukemia effects of miR-375 in vivo. About 1х10 7 viable HL-60 cells transduced with MSCV-miR-375 or MSCV-NC were injected subcutaneously into right flank of each nude mouse. a A photograph of xenografted tumors in mice xenografted by HL-60 cells, which were transduced with MSCV-miR-375 or MSCV-NC. b Volumes of all xenografted tumors were measured when the experiment was terminated at 42 days after tumor cell inoculation. c Net weights of all xenografted tumors were measured at the termination of the experiment. d The protein expression of HOXB3 was detected in xenografted tumor lysates from HL-60 cells transduced with MSCV-miR-375 or MSCV-NC. e THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 and GFP-positive cells were sorted by flow cytometry. The GFP-positive cells were further transduced with MSCV-miR-375 or MSCV-NC and treated with puromycin for 1 week. Then, THP1-GFP-miR-375 or THP1-GFP-NC were xenografted into NSG mice. Peripheral blood cells were extracted from mice when the mice became moribund and GFP-positive cells were analyzed by flow cytometry. * P
Figure Legend Snippet: The anti-leukemia effects of miR-375 in vivo. About 1х10 7 viable HL-60 cells transduced with MSCV-miR-375 or MSCV-NC were injected subcutaneously into right flank of each nude mouse. a A photograph of xenografted tumors in mice xenografted by HL-60 cells, which were transduced with MSCV-miR-375 or MSCV-NC. b Volumes of all xenografted tumors were measured when the experiment was terminated at 42 days after tumor cell inoculation. c Net weights of all xenografted tumors were measured at the termination of the experiment. d The protein expression of HOXB3 was detected in xenografted tumor lysates from HL-60 cells transduced with MSCV-miR-375 or MSCV-NC. e THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 and GFP-positive cells were sorted by flow cytometry. The GFP-positive cells were further transduced with MSCV-miR-375 or MSCV-NC and treated with puromycin for 1 week. Then, THP1-GFP-miR-375 or THP1-GFP-NC were xenografted into NSG mice. Peripheral blood cells were extracted from mice when the mice became moribund and GFP-positive cells were analyzed by flow cytometry. * P

Techniques Used: In Vivo, Transduction, Injection, Mouse Assay, Expressing, Plasmid Preparation, Flow Cytometry, Cytometry

MiR-375 targets HOXB3 via binding 3′-UTR of HOXB3 . a Schematic of putative binding sites for miR-375 in 3′-UTR of HOXB3 . b 293 T cells were transfected with wide-type pMIR-HOXB3UTR (WT), pMIR-HOXB3UTR (Mut), pMIR-NC, and pRL-SV40 containing Renilla luciferase gene for 24 h, followed by the transfection with miR-375 mimic or Scramble for another 24 h. Firefly and Renilla luciferase activities were both detected and histograms showed that the Firefly luciferase activities were normalized to Renilla luciferase activities. c The expression of miR-375 was detected in HL-60 and THP1 cells transduced with MSCV-miR-375 or MSCV-NC. * P
Figure Legend Snippet: MiR-375 targets HOXB3 via binding 3′-UTR of HOXB3 . a Schematic of putative binding sites for miR-375 in 3′-UTR of HOXB3 . b 293 T cells were transfected with wide-type pMIR-HOXB3UTR (WT), pMIR-HOXB3UTR (Mut), pMIR-NC, and pRL-SV40 containing Renilla luciferase gene for 24 h, followed by the transfection with miR-375 mimic or Scramble for another 24 h. Firefly and Renilla luciferase activities were both detected and histograms showed that the Firefly luciferase activities were normalized to Renilla luciferase activities. c The expression of miR-375 was detected in HL-60 and THP1 cells transduced with MSCV-miR-375 or MSCV-NC. * P

Techniques Used: Binding Assay, Transfection, Luciferase, Expressing, Transduction

30) Product Images from "Selection of an optimal promoter for gene transfer in normal B cells"

Article Title: Selection of an optimal promoter for gene transfer in normal B cells

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2017.6974

Constructs created and used for sorting transduced cells. HIV-SFFV-mRFP-WPRE vector was used to create bicistronic lentiviral vectors with genes encoding fluorescent reporter proteins (GFP or RFP), as well as XBP-1 and BiP. GFP, green fluorescent protein; RFP, red fluorescent protein; IRES, internal ribosome entry site; SFFV, spleen focus-forming virus; MCS, multiple cloning site; XBP-1, X-box binding protein 1; BiP, binding immunoglobulin protein.
Figure Legend Snippet: Constructs created and used for sorting transduced cells. HIV-SFFV-mRFP-WPRE vector was used to create bicistronic lentiviral vectors with genes encoding fluorescent reporter proteins (GFP or RFP), as well as XBP-1 and BiP. GFP, green fluorescent protein; RFP, red fluorescent protein; IRES, internal ribosome entry site; SFFV, spleen focus-forming virus; MCS, multiple cloning site; XBP-1, X-box binding protein 1; BiP, binding immunoglobulin protein.

Techniques Used: Construct, Plasmid Preparation, Clone Assay, Binding Assay

Lentiviral modification of normal B cells and Raji cells. (A) Three vectors (pLVX ZsGreen1, pLVTHM and pGIPZ) were used for lentiviral modification of (B) normal B cells or (C) Raji cells. The percentage of GFP-positive cells was determined using flow cytometry. Co-cultures of normal B cells and feeder cells were additionally stained with anti-CD20 antibody to distinguish B cells from feeder cells. GFP, green fluorescent protein; CMV, cytomegalovirus; EF1α, elongation factor 1 alpha; CD20, cluster of differentiation 20; PE, phycoerythrin.
Figure Legend Snippet: Lentiviral modification of normal B cells and Raji cells. (A) Three vectors (pLVX ZsGreen1, pLVTHM and pGIPZ) were used for lentiviral modification of (B) normal B cells or (C) Raji cells. The percentage of GFP-positive cells was determined using flow cytometry. Co-cultures of normal B cells and feeder cells were additionally stained with anti-CD20 antibody to distinguish B cells from feeder cells. GFP, green fluorescent protein; CMV, cytomegalovirus; EF1α, elongation factor 1 alpha; CD20, cluster of differentiation 20; PE, phycoerythrin.

Techniques Used: Modification, Flow Cytometry, Cytometry, Staining

31) Product Images from "Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program"

Article Title: Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program

Journal: Acta Neuropathologica

doi: 10.1007/s00401-019-02062-4

NSUN5 epigenetic loss is associated with depletion of global protein synthesis and the emergence of a stress-response translational program. a NSUN5 unmethylated glioma cell lines DBTRG-05MG, MO59J, and CAS-1 show higher overall protein synthesis assessed by OP-Puro under oxidative stress (100 mM H 2 O 2 ) than the NSUN5 methylated cells (A172, LN229 and KS-1). b Restoration of NSUN5 function by transfection in epigenetically inactive LN299 cells increases overall protein synthesis under oxidative stress (100 mM H 2 O 2 ) assessed by OP-Puro. Enhancement of global protein synthesis upon NSUN5 recovery in LN299 cells is also observed by the [3H] leucine incorporation assay. c Similar results were obtained upon nutrient deprivation. d Comparison of the total RNA (RNA-seq) and ribosome-protected RNA (Ribo-seq) deep-sequencing profiles to identify those RNAs with enhanced translational efficiency in NSUN5 deficient cells. 1987 RNAs that did not change in the RNA-seq of LN229 cells upon NSUN5-transfection were upregulated in the Ribo-seq of empty-vector-transfected cells indicating enhanced translational efficiency. e NSUN5 affects both CAP-dependent and CAP-independent translation according to the use of a reporter plasmid encoding for Firefly (IRES) and Renilla (CAP) luciferases. f Gene set enrichment analysis (GSEA) of the RNAs with increased translational efficiency in NSUN5 deficient cells (hypergeometric test with a FDR adjusted P value
Figure Legend Snippet: NSUN5 epigenetic loss is associated with depletion of global protein synthesis and the emergence of a stress-response translational program. a NSUN5 unmethylated glioma cell lines DBTRG-05MG, MO59J, and CAS-1 show higher overall protein synthesis assessed by OP-Puro under oxidative stress (100 mM H 2 O 2 ) than the NSUN5 methylated cells (A172, LN229 and KS-1). b Restoration of NSUN5 function by transfection in epigenetically inactive LN299 cells increases overall protein synthesis under oxidative stress (100 mM H 2 O 2 ) assessed by OP-Puro. Enhancement of global protein synthesis upon NSUN5 recovery in LN299 cells is also observed by the [3H] leucine incorporation assay. c Similar results were obtained upon nutrient deprivation. d Comparison of the total RNA (RNA-seq) and ribosome-protected RNA (Ribo-seq) deep-sequencing profiles to identify those RNAs with enhanced translational efficiency in NSUN5 deficient cells. 1987 RNAs that did not change in the RNA-seq of LN229 cells upon NSUN5-transfection were upregulated in the Ribo-seq of empty-vector-transfected cells indicating enhanced translational efficiency. e NSUN5 affects both CAP-dependent and CAP-independent translation according to the use of a reporter plasmid encoding for Firefly (IRES) and Renilla (CAP) luciferases. f Gene set enrichment analysis (GSEA) of the RNAs with increased translational efficiency in NSUN5 deficient cells (hypergeometric test with a FDR adjusted P value

Techniques Used: Methylation, Transfection, RNA Sequencing Assay, Sequencing, Plasmid Preparation

NSUN5 epigenetic silencing activates stress-related protein and confers growth inhibition sensitivity to NQO1-targeting molecules. a Validation of NSUN5 translational regulation of the identified stress-related target NQO1 expression at the RNA level determined by RNA-seq counts (left) and real-time quantitative PCR (middle) do not change upon NSUN5 transfection, but NQO1 expression decreased at the protein level (right) upon NSUN5 restoration. b qPCR shows enrichment of the NQO1 transcript in the polysome fraction of the empty-vector LN229 cells in comparison to NSUN5 transfected-LN229 cells. c NQO1 expression levels in glioma cell lines determined by western blot according to NSUN5 methylation status. d IC50 determination using the SRB assay in the glioma cell lines grouped by NSUN5 methylation status. Black dashed curves represent the 95% confidence band for each group. Glioma cells harboring NSUN5 methylation-associated NQO1 overexpression (A172, LN229 and KS-1) show increased sensitivity to deoxynyboquinone (DNQ) and IB-DNQ in comparison to NSUN5 unmethylated cells (DBTRG-05MG, MO59 J, and CAS-1). Drug-response curves were generated using GraphPad Prism software and analyses were performed with the drc R package. For each cell line and the drug, we fit a four-parameter generalized log-logistic model. Comparison of the IC50 values calculated from the slopes were obtained by means of a z test ( P
Figure Legend Snippet: NSUN5 epigenetic silencing activates stress-related protein and confers growth inhibition sensitivity to NQO1-targeting molecules. a Validation of NSUN5 translational regulation of the identified stress-related target NQO1 expression at the RNA level determined by RNA-seq counts (left) and real-time quantitative PCR (middle) do not change upon NSUN5 transfection, but NQO1 expression decreased at the protein level (right) upon NSUN5 restoration. b qPCR shows enrichment of the NQO1 transcript in the polysome fraction of the empty-vector LN229 cells in comparison to NSUN5 transfected-LN229 cells. c NQO1 expression levels in glioma cell lines determined by western blot according to NSUN5 methylation status. d IC50 determination using the SRB assay in the glioma cell lines grouped by NSUN5 methylation status. Black dashed curves represent the 95% confidence band for each group. Glioma cells harboring NSUN5 methylation-associated NQO1 overexpression (A172, LN229 and KS-1) show increased sensitivity to deoxynyboquinone (DNQ) and IB-DNQ in comparison to NSUN5 unmethylated cells (DBTRG-05MG, MO59 J, and CAS-1). Drug-response curves were generated using GraphPad Prism software and analyses were performed with the drc R package. For each cell line and the drug, we fit a four-parameter generalized log-logistic model. Comparison of the IC50 values calculated from the slopes were obtained by means of a z test ( P

Techniques Used: Inhibition, Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Western Blot, Methylation, Sulforhodamine B Assay, Over Expression, Generated, Software

Restoration of NSUN5 impairs glioma tumor growth in vivo. a Western blot to show efficient restoration of NSUN5 protein expression upon stable transfection in A172 and LN299 glioma cells and efficient depletion of NSUN5 protein expression in NSUN5-shRNA DBTRG-05MG glioma cells. EV empty vector. An equal number of the indicated A172 and LN299 cells populations were stereotactically inoculated into the brain of athymic mice. The size of the tumors was estimated at 10 and 17 days post-inoculation (DPI) by the quantification of luciferase activity in the tumor cells. b Scatter plots showing the individual size of the indicated LN229 and A172 tumors after 10 and 17 DPI. c Representative images of the luciferase signal from mice inoculated with the indicated LN229 and A172 tumors after 17 DPI. d LN229-EV and LN229-NSUN5 cells were injected in the left or right flank of 10 mice, respectively. Tumor volume measured over time ( left panel ) and tumor weight upon sacrifice ( right panel ) are shown. P values obtained by Student’s t test. Error bars show means ± s.d. e Scramble and NSUN5-shRNA-depleted DBTRG-05MG cells were injected in the left or right flank of 10 mice, respectively. Tumor volume measured over time (left panel) and tumor weight upon sacrifice (right panel) are shown. P values obtained by Student’s t test. Error bars show means ± s.d
Figure Legend Snippet: Restoration of NSUN5 impairs glioma tumor growth in vivo. a Western blot to show efficient restoration of NSUN5 protein expression upon stable transfection in A172 and LN299 glioma cells and efficient depletion of NSUN5 protein expression in NSUN5-shRNA DBTRG-05MG glioma cells. EV empty vector. An equal number of the indicated A172 and LN299 cells populations were stereotactically inoculated into the brain of athymic mice. The size of the tumors was estimated at 10 and 17 days post-inoculation (DPI) by the quantification of luciferase activity in the tumor cells. b Scatter plots showing the individual size of the indicated LN229 and A172 tumors after 10 and 17 DPI. c Representative images of the luciferase signal from mice inoculated with the indicated LN229 and A172 tumors after 17 DPI. d LN229-EV and LN229-NSUN5 cells were injected in the left or right flank of 10 mice, respectively. Tumor volume measured over time ( left panel ) and tumor weight upon sacrifice ( right panel ) are shown. P values obtained by Student’s t test. Error bars show means ± s.d. e Scramble and NSUN5-shRNA-depleted DBTRG-05MG cells were injected in the left or right flank of 10 mice, respectively. Tumor volume measured over time (left panel) and tumor weight upon sacrifice (right panel) are shown. P values obtained by Student’s t test. Error bars show means ± s.d

Techniques Used: In Vivo, Western Blot, Expressing, Stable Transfection, shRNA, Plasmid Preparation, Mouse Assay, Luciferase, Activity Assay, Injection

32) Product Images from "Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation"

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

Journal: eLife

doi: 10.7554/eLife.40167

Loss of Yap1 substantially increases apoptosis during ESC differentiation. ( A ) Lactate dehydrogenase (LDH) assay of WT and Yap1 KO ESCs in ±LIF. Cells were treated with either Z-VAD-FMK (Z-VAD), necrostatin-1, DMSO, or no treatment. Values were normalized to wells that had been lysed completely. ( B ) LDH assay measuring cell death after Yap1 KO in three different ESC lines during differentiation (72 hr) or self-renewal. ( C ) LDH assay measuring cell death in Yap1 KO, WT, and three different stable FLAG-Bio (FB) Yap1 overexpression cell lines during differentiation (72 hr). ( D ) Representative brightfield and fluorescence microscopy images of WT and Yap1 KO ESCs incubated with NucView 488 Casp3 substrate at the indicated times after LIF withdrawal. ( E ) Representative flow cytometry density plots of WT and Yap1 KO ESCs detecting fluorescent signal from annexin-V (conjugated to CF594) and NucView 488 reagent during differentiation (60 hr). ( F ) Fold enrichment of annexin-V and active Casp3-positive Yap1 KO vs. WT ESCs according to flow cytometry. ( G ) Immunoblot of Casp9, Casp8, Casp3, cleaved Casp3, and cleaved Parp1 in WT and Yap1 KO cells during differentiation. β-actin was used as a loading control. ( H ) Luminescent assay of caspase activity in Yap1 KO vs. WT ESCs in ±LIF media. ( I ) LDH assay of WT and Yap1 KO cells ± KD of Casp9 during differentiation (72 hr). All data are expressed as mean ±standard deviation (n = 4 independent samples for LDH assays and n = 3 for other experiments). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.
Figure Legend Snippet: Loss of Yap1 substantially increases apoptosis during ESC differentiation. ( A ) Lactate dehydrogenase (LDH) assay of WT and Yap1 KO ESCs in ±LIF. Cells were treated with either Z-VAD-FMK (Z-VAD), necrostatin-1, DMSO, or no treatment. Values were normalized to wells that had been lysed completely. ( B ) LDH assay measuring cell death after Yap1 KO in three different ESC lines during differentiation (72 hr) or self-renewal. ( C ) LDH assay measuring cell death in Yap1 KO, WT, and three different stable FLAG-Bio (FB) Yap1 overexpression cell lines during differentiation (72 hr). ( D ) Representative brightfield and fluorescence microscopy images of WT and Yap1 KO ESCs incubated with NucView 488 Casp3 substrate at the indicated times after LIF withdrawal. ( E ) Representative flow cytometry density plots of WT and Yap1 KO ESCs detecting fluorescent signal from annexin-V (conjugated to CF594) and NucView 488 reagent during differentiation (60 hr). ( F ) Fold enrichment of annexin-V and active Casp3-positive Yap1 KO vs. WT ESCs according to flow cytometry. ( G ) Immunoblot of Casp9, Casp8, Casp3, cleaved Casp3, and cleaved Parp1 in WT and Yap1 KO cells during differentiation. β-actin was used as a loading control. ( H ) Luminescent assay of caspase activity in Yap1 KO vs. WT ESCs in ±LIF media. ( I ) LDH assay of WT and Yap1 KO cells ± KD of Casp9 during differentiation (72 hr). All data are expressed as mean ±standard deviation (n = 4 independent samples for LDH assays and n = 3 for other experiments). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

Techniques Used: Lactate Dehydrogenase Assay, Over Expression, Fluorescence, Microscopy, Incubation, Flow Cytometry, Cytometry, Luminescence Assay, Activity Assay, Standard Deviation, Two Tailed Test

33) Product Images from "CHRFAM7A alters Binding to the Neuronal alpha-7 Nicotinic Acetylcholine Receptor"

Article Title: CHRFAM7A alters Binding to the Neuronal alpha-7 Nicotinic Acetylcholine Receptor

Journal: Neuroscience letters

doi: 10.1016/j.neulet.2018.10.010

CHRFAM7A expression decreases ligand binding to the α7nAchR in PC12 cells. Rat PC12 cells were transduced with human CHRFAM7A to quantify changes in α-BTX binding using flow cytometry. A. Gating strategy to identify the GFP + CHRFAM7A + population in vector and CHRFAM7A-transduced cells. B. Representative histogram demonstrating decreased α-BTX binding in CHRFAM7A transduced PC12 cells compared to vector. C. Cell preincubated with the α7nAchR ligand nicotine (50μM) to prevent α-BTX binding to functional α7nAch receptors. D. Quantification of all flow cytometry experiments showing that the introduction of uniquely human CHRFAM7A decreases α-BTX binding to PC12 cells. * p
Figure Legend Snippet: CHRFAM7A expression decreases ligand binding to the α7nAchR in PC12 cells. Rat PC12 cells were transduced with human CHRFAM7A to quantify changes in α-BTX binding using flow cytometry. A. Gating strategy to identify the GFP + CHRFAM7A + population in vector and CHRFAM7A-transduced cells. B. Representative histogram demonstrating decreased α-BTX binding in CHRFAM7A transduced PC12 cells compared to vector. C. Cell preincubated with the α7nAchR ligand nicotine (50μM) to prevent α-BTX binding to functional α7nAch receptors. D. Quantification of all flow cytometry experiments showing that the introduction of uniquely human CHRFAM7A decreases α-BTX binding to PC12 cells. * p

Techniques Used: Expressing, Ligand Binding Assay, Transduction, Binding Assay, Flow Cytometry, Cytometry, Plasmid Preparation, Functional Assay

Forced expression of CHRFAM7A alters α-bungarotoxin (α-BTX) binding. Rat PC12 cells were transduced with CHRFAM7A to assess the effect of the uniquely human gene on ligand binding to the α7nAchR. A. PCR confirmed the presence of CHRFAM7A in transduced cells with no expression seen in vector PC12 cells. B. PCR demonstrated that CHRNA7, the α7nAchR gene, was expressed in both vector and CHRFAM7A transduced cells. C. Immunoblotting demonstrated that CHFAM7A transduced PC12 cells were functional and produced a protein with molecular weight consistent with CHRFAM7A. D. PC12 cells were stained with α-BTX (red) to assess binding to the α7nAchR. Cells were co-stained with DAPI (blue) to identify the cell nucleus. E. Fluorescence was measured using an Omega Fluorimeter to quantify changes in α-BTX binding between vector and CHRFAM7A transduced cells. Decreased staining for α-BTX was seen in CHRFAM7A transduced cells compared to vector. * p
Figure Legend Snippet: Forced expression of CHRFAM7A alters α-bungarotoxin (α-BTX) binding. Rat PC12 cells were transduced with CHRFAM7A to assess the effect of the uniquely human gene on ligand binding to the α7nAchR. A. PCR confirmed the presence of CHRFAM7A in transduced cells with no expression seen in vector PC12 cells. B. PCR demonstrated that CHRNA7, the α7nAchR gene, was expressed in both vector and CHRFAM7A transduced cells. C. Immunoblotting demonstrated that CHFAM7A transduced PC12 cells were functional and produced a protein with molecular weight consistent with CHRFAM7A. D. PC12 cells were stained with α-BTX (red) to assess binding to the α7nAchR. Cells were co-stained with DAPI (blue) to identify the cell nucleus. E. Fluorescence was measured using an Omega Fluorimeter to quantify changes in α-BTX binding between vector and CHRFAM7A transduced cells. Decreased staining for α-BTX was seen in CHRFAM7A transduced cells compared to vector. * p

Techniques Used: Expressing, Binding Assay, Transduction, Ligand Binding Assay, Polymerase Chain Reaction, Plasmid Preparation, Functional Assay, Produced, Molecular Weight, Staining, Fluorescence

34) Product Images from "Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription"

Article Title: Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription

Journal: mBio

doi: 10.1128/mBio.00769-18

HEV ORF1 is able to function in trans to replicate HEV RNA. (a) Schematic representation of the ORF1 transcomplementation system. (b) Representative flow cytometry plots demonstrating efficient ORF1 expression. HepG2C3A cells were transduced with pLVX-ORF1-IRES-zsGreen (wild type [wt] or GAD mutant) or not transduced. Flow cytometric analysis was performed 3 days following transduction to quantify the frequencies of ORF1-expressing cells. FSC, forward scatter. (c) Replication kinetics of HEV RNA in ORF1 transcomplemented HepG2C3A cells. Cell culture supernatants from naive HepG2C3A cells, or HepG2C3A cells transduced with HEV ORF1 or its GAD mutant, were collected at the indicated time points posttransfection with rHEVΔORF2/3[Gluc] Pol- RNA or RNA from its mutants, and Gaussia luciferase (Gluc) activity was quantified. Values are means plus standard deviations (SD) (error bars) ( n = 3). Values that are significantly different ( P
Figure Legend Snippet: HEV ORF1 is able to function in trans to replicate HEV RNA. (a) Schematic representation of the ORF1 transcomplementation system. (b) Representative flow cytometry plots demonstrating efficient ORF1 expression. HepG2C3A cells were transduced with pLVX-ORF1-IRES-zsGreen (wild type [wt] or GAD mutant) or not transduced. Flow cytometric analysis was performed 3 days following transduction to quantify the frequencies of ORF1-expressing cells. FSC, forward scatter. (c) Replication kinetics of HEV RNA in ORF1 transcomplemented HepG2C3A cells. Cell culture supernatants from naive HepG2C3A cells, or HepG2C3A cells transduced with HEV ORF1 or its GAD mutant, were collected at the indicated time points posttransfection with rHEVΔORF2/3[Gluc] Pol- RNA or RNA from its mutants, and Gaussia luciferase (Gluc) activity was quantified. Values are means plus standard deviations (SD) (error bars) ( n = 3). Values that are significantly different ( P

Techniques Used: Flow Cytometry, Cytometry, Expressing, Transduction, Mutagenesis, Cell Culture, Luciferase, Activity Assay

35) Product Images from "A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia"

Article Title: A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia

Journal: BMC Cancer

doi: 10.1186/s12885-018-4097-z

Knockdown of HOXB3 and CDCA3 partially mimics the anti-leukemia activity by miR-375. a HL-60 and THP1 cells were transduced with special shRNA for HOXB 3 or a control shRNA (sh-NC). HOXB3 and CDCA3 expressions were detected by western blot. b Viable cell number by the trypan-blue exclusion assay was counted in HL-60 and THP1 cells, which were transduced with special shRNA for HOXB3 or sh-NC for the indicated times. * P
Figure Legend Snippet: Knockdown of HOXB3 and CDCA3 partially mimics the anti-leukemia activity by miR-375. a HL-60 and THP1 cells were transduced with special shRNA for HOXB 3 or a control shRNA (sh-NC). HOXB3 and CDCA3 expressions were detected by western blot. b Viable cell number by the trypan-blue exclusion assay was counted in HL-60 and THP1 cells, which were transduced with special shRNA for HOXB3 or sh-NC for the indicated times. * P

Techniques Used: Activity Assay, Transduction, shRNA, Western Blot, Trypan Blue Exclusion Assay

An illustration of miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry. a The decreased expression of miR-375 due to DNA hypermethylation results in the high expression of HOXB3, contributing to cell proliferation and colony formation through increasing the expression of CDCA3. Moreover, HOXB3 enhances and recruits DNMT3B to bind in pre-miR-375 promoter, leading to further DNA hypermethylation and subsequent downregulation of miR-375 in AML cells, in turn
Figure Legend Snippet: An illustration of miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry. a The decreased expression of miR-375 due to DNA hypermethylation results in the high expression of HOXB3, contributing to cell proliferation and colony formation through increasing the expression of CDCA3. Moreover, HOXB3 enhances and recruits DNMT3B to bind in pre-miR-375 promoter, leading to further DNA hypermethylation and subsequent downregulation of miR-375 in AML cells, in turn

Techniques Used: Expressing

HOXB3 enhances the expression of DNMT3B to bind in pre-miR-375 promoter. a HL-60 and THP1 cells were transduced with special shRNA for HOXB3 (sh-HOXB3) or sh-NC. HOXB3 and DNMT3B expressions were detected by western blot. b HOXB3 and DNMT3B expressions were detected in HL-60 and THP1 cells, which were transduced with overexpression vector LVX-HOXB3 or LVX-NC. c DNMT3B expression was detected in HL-60 and THP1 cells transduced with special shRNA targeting DNMT3B (sh-DNMT3B) or sh-NC. d MiR-375 expression was measured in HL-60 and THP1 cells transduced with sh-DNMT3B or sh-NC. * P
Figure Legend Snippet: HOXB3 enhances the expression of DNMT3B to bind in pre-miR-375 promoter. a HL-60 and THP1 cells were transduced with special shRNA for HOXB3 (sh-HOXB3) or sh-NC. HOXB3 and DNMT3B expressions were detected by western blot. b HOXB3 and DNMT3B expressions were detected in HL-60 and THP1 cells, which were transduced with overexpression vector LVX-HOXB3 or LVX-NC. c DNMT3B expression was detected in HL-60 and THP1 cells transduced with special shRNA targeting DNMT3B (sh-DNMT3B) or sh-NC. d MiR-375 expression was measured in HL-60 and THP1 cells transduced with sh-DNMT3B or sh-NC. * P

Techniques Used: Expressing, Transduction, shRNA, Western Blot, Over Expression, Plasmid Preparation

The anti-leukemia effects of miR-375 in vivo. About 1х10 7 viable HL-60 cells transduced with MSCV-miR-375 or MSCV-NC were injected subcutaneously into right flank of each nude mouse. a A photograph of xenografted tumors in mice xenografted by HL-60 cells, which were transduced with MSCV-miR-375 or MSCV-NC. b Volumes of all xenografted tumors were measured when the experiment was terminated at 42 days after tumor cell inoculation. c Net weights of all xenografted tumors were measured at the termination of the experiment. d The protein expression of HOXB3 was detected in xenografted tumor lysates from HL-60 cells transduced with MSCV-miR-375 or MSCV-NC. e THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 and GFP-positive cells were sorted by flow cytometry. The GFP-positive cells were further transduced with MSCV-miR-375 or MSCV-NC and treated with puromycin for 1 week. Then, THP1-GFP-miR-375 or THP1-GFP-NC were xenografted into NSG mice. Peripheral blood cells were extracted from mice when the mice became moribund and GFP-positive cells were analyzed by flow cytometry. * P
Figure Legend Snippet: The anti-leukemia effects of miR-375 in vivo. About 1х10 7 viable HL-60 cells transduced with MSCV-miR-375 or MSCV-NC were injected subcutaneously into right flank of each nude mouse. a A photograph of xenografted tumors in mice xenografted by HL-60 cells, which were transduced with MSCV-miR-375 or MSCV-NC. b Volumes of all xenografted tumors were measured when the experiment was terminated at 42 days after tumor cell inoculation. c Net weights of all xenografted tumors were measured at the termination of the experiment. d The protein expression of HOXB3 was detected in xenografted tumor lysates from HL-60 cells transduced with MSCV-miR-375 or MSCV-NC. e THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 and GFP-positive cells were sorted by flow cytometry. The GFP-positive cells were further transduced with MSCV-miR-375 or MSCV-NC and treated with puromycin for 1 week. Then, THP1-GFP-miR-375 or THP1-GFP-NC were xenografted into NSG mice. Peripheral blood cells were extracted from mice when the mice became moribund and GFP-positive cells were analyzed by flow cytometry. * P

Techniques Used: In Vivo, Transduction, Injection, Mouse Assay, Expressing, Plasmid Preparation, Flow Cytometry, Cytometry

MiR-375 targets HOXB3 via binding 3′-UTR of HOXB3 . a Schematic of putative binding sites for miR-375 in 3′-UTR of HOXB3 . b 293 T cells were transfected with wide-type pMIR-HOXB3UTR (WT), pMIR-HOXB3UTR (Mut), pMIR-NC, and pRL-SV40 containing Renilla luciferase gene for 24 h, followed by the transfection with miR-375 mimic or Scramble for another 24 h. Firefly and Renilla luciferase activities were both detected and histograms showed that the Firefly luciferase activities were normalized to Renilla luciferase activities. c The expression of miR-375 was detected in HL-60 and THP1 cells transduced with MSCV-miR-375 or MSCV-NC. * P
Figure Legend Snippet: MiR-375 targets HOXB3 via binding 3′-UTR of HOXB3 . a Schematic of putative binding sites for miR-375 in 3′-UTR of HOXB3 . b 293 T cells were transfected with wide-type pMIR-HOXB3UTR (WT), pMIR-HOXB3UTR (Mut), pMIR-NC, and pRL-SV40 containing Renilla luciferase gene for 24 h, followed by the transfection with miR-375 mimic or Scramble for another 24 h. Firefly and Renilla luciferase activities were both detected and histograms showed that the Firefly luciferase activities were normalized to Renilla luciferase activities. c The expression of miR-375 was detected in HL-60 and THP1 cells transduced with MSCV-miR-375 or MSCV-NC. * P

Techniques Used: Binding Assay, Transfection, Luciferase, Expressing, Transduction

36) Product Images from "SLAMF1 is required for TLR4-mediated TRAM-TRIF–dependent signaling in human macrophages"

Article Title: SLAMF1 is required for TLR4-mediated TRAM-TRIF–dependent signaling in human macrophages

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201707027

TRAM acts as a bridge between the SLAMF1 and TLR4 signaling complex. (A and B) Coprecipitations of SLAMF1 Flag with TLR4 Cherry (A) or TRIF HA (B) with or without TRAM YFP overexpression. (C) Coprecipitation of TLR4 Flag with SLAMF1 with or without TRAM YFP overexpression. (D) TLR4 Flag interaction with TRAM YFP and TRIF HA with or without SLAMF1 coexpression. (E) Coprecipitation of SLAMF1 with or without TRIF HA in the presence of TRAM YFP by TLR4 Flag . Indicated constructs were transfected to HEK293T cells. pDuo-CD14/MD-2 vector was cotransfected to all wells (A and C–E). Anti-Flag agarose was used for IPs. At least three independent experiments were performed. Molecular weight is given in kilodaltons. WB, Western blot; WCL, whole-cell lysate.
Figure Legend Snippet: TRAM acts as a bridge between the SLAMF1 and TLR4 signaling complex. (A and B) Coprecipitations of SLAMF1 Flag with TLR4 Cherry (A) or TRIF HA (B) with or without TRAM YFP overexpression. (C) Coprecipitation of TLR4 Flag with SLAMF1 with or without TRAM YFP overexpression. (D) TLR4 Flag interaction with TRAM YFP and TRIF HA with or without SLAMF1 coexpression. (E) Coprecipitation of SLAMF1 with or without TRIF HA in the presence of TRAM YFP by TLR4 Flag . Indicated constructs were transfected to HEK293T cells. pDuo-CD14/MD-2 vector was cotransfected to all wells (A and C–E). Anti-Flag agarose was used for IPs. At least three independent experiments were performed. Molecular weight is given in kilodaltons. WB, Western blot; WCL, whole-cell lysate.

Techniques Used: Over Expression, Construct, Transfection, Plasmid Preparation, Molecular Weight, Western Blot

TRAM and SLAMF1 are essential for the killing of E. coli by human macrophages. (A) Flow cytometry analysis of dihydrorhodamine 123 (DHR-123) fluorescence to access ROS activation in control siRNA or SLAMF1 siRNA human macrophages upon stimulation by E. coli red pHrodo particles. One of three experiments shown. (B and C) Bacterial killing assays by SLAMF1-silenced and control THP-1 cells (B) as well as TRAM KO and control THP-1 cells (C) infected with a DH5α strain at MOI 40. (D and E) Western blot analysis of pAkt (S473) and pIRF3 (S396) levels induced by E. coli particles in THP-1 WT and TRAM KO cells (D) as well as SLAMF1-silenced or control oligonucleotide–treated cells (E). Graphs (right) on Western blotting show quantification of protein levels relative to β-tubulin obtained with Odyssey software. (F) Western blot showing phospho-(S396) IRF3 and phospho-(S473) Akt levels in lysates of THP-1 cells coincubated with E. coli particles for 1 h in the presence or absence of TBK1-IKKε inhibitor (MRT67307), pan-Akt allosteric inhibitor (MK2206), or DMSO. Molecular weight is given in kilodaltons. (G) Bacterial killing assays by THP-1 cells with DMSO (
Figure Legend Snippet: TRAM and SLAMF1 are essential for the killing of E. coli by human macrophages. (A) Flow cytometry analysis of dihydrorhodamine 123 (DHR-123) fluorescence to access ROS activation in control siRNA or SLAMF1 siRNA human macrophages upon stimulation by E. coli red pHrodo particles. One of three experiments shown. (B and C) Bacterial killing assays by SLAMF1-silenced and control THP-1 cells (B) as well as TRAM KO and control THP-1 cells (C) infected with a DH5α strain at MOI 40. (D and E) Western blot analysis of pAkt (S473) and pIRF3 (S396) levels induced by E. coli particles in THP-1 WT and TRAM KO cells (D) as well as SLAMF1-silenced or control oligonucleotide–treated cells (E). Graphs (right) on Western blotting show quantification of protein levels relative to β-tubulin obtained with Odyssey software. (F) Western blot showing phospho-(S396) IRF3 and phospho-(S473) Akt levels in lysates of THP-1 cells coincubated with E. coli particles for 1 h in the presence or absence of TBK1-IKKε inhibitor (MRT67307), pan-Akt allosteric inhibitor (MK2206), or DMSO. Molecular weight is given in kilodaltons. (G) Bacterial killing assays by THP-1 cells with DMSO (

Techniques Used: Flow Cytometry, Cytometry, Fluorescence, Activation Assay, Infection, Western Blot, Software, Molecular Weight

Knockdown of SLAMF1 in macrophages results in strongly reduced TLR4-mediated IFNβ mRNA expression and protein secretion as well as some decrease of TNF, IL-6, and CXCL10 secretion. (A and B) Quantification of SLAMF1 , IFNβ , and TNF mRNA expression by qPCR in THP-1 cells (A) and macrophages (B) treated by 100 ng/ml ultrapure K12 LPS. (C and D) IFNβ and TNF secretion levels by THP-1 cells (C) and macrophages (D) in response to LPS (4 and 6 h) assessed by ELISA. (E and F) Secretion levels of IL-1β, IL-6, IL-8, and CXCL-10 (6 h LPS) analyzed by multiplex assays. Data are presented as means with SD for combined data from three independent experiments (A, C, and E), for three biological replicates from one of six donors (B and D), or one of three donors (F). *, P
Figure Legend Snippet: Knockdown of SLAMF1 in macrophages results in strongly reduced TLR4-mediated IFNβ mRNA expression and protein secretion as well as some decrease of TNF, IL-6, and CXCL10 secretion. (A and B) Quantification of SLAMF1 , IFNβ , and TNF mRNA expression by qPCR in THP-1 cells (A) and macrophages (B) treated by 100 ng/ml ultrapure K12 LPS. (C and D) IFNβ and TNF secretion levels by THP-1 cells (C) and macrophages (D) in response to LPS (4 and 6 h) assessed by ELISA. (E and F) Secretion levels of IL-1β, IL-6, IL-8, and CXCL-10 (6 h LPS) analyzed by multiplex assays. Data are presented as means with SD for combined data from three independent experiments (A, C, and E), for three biological replicates from one of six donors (B and D), or one of three donors (F). *, P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Multiplex Assay

SLAMF1 interacts with all class I Rab11 FIPs. (A) Anti-Flag IPs for Rab11a Flag with EGFP-tagged Rab11FIPs (1–5) and SLAMF1. (B) Schematic figure for class I and class II Rab11 FIPs domain structure. C2, phospholipid-binding C2 domain; EF, EF-hand domain; PRR, proline-rich region; RBD, Rab11 binding domain. (C) Homologous protein sequence in class I FIPs, which follow the C2 domain. Identical amino acids in all three class I FIPs are highlighted. (D) Coprecipitation of SLAMF1 Flag with FIP2 EGFP WT or FIP2 deletion mutant lacking the C2 domain (ΔC2). (E and F) Coprecipitation of untagged SLAMF1 with FIP2 Flag (1–512 aa) and Flag-tagged FIP2 deletion mutants in anti-Flag IPs in the absence (E) or presence (F) of overexpressed Rab11 CFP . (G) Quantification of coprecipitations in E and F between SLAMF1 and FIP2 Flag variants correlated with the amount of Flag-tagged protein on the blot and Flag-tagged protein sizes. Error bars represent means ± SD for three independent experiments. (H) Coprecipitation of FIP2 Flag with SLAMF1 and Rab11a WT, Rab11a Q70L mutant (QL), or Rab11a S25N mutant (SN). (I) Coprecipitation of SLAMF1 Flag deletion mutants with FIP2 EGFP . Molecular weight is given in kilodaltons. WB, Western blot; WCL, whole-cell lysate. (J) Scheme for FIP2- and TRAM-interacting domains in SLAMF1ct. The results are representative of at least three independent experiments.
Figure Legend Snippet: SLAMF1 interacts with all class I Rab11 FIPs. (A) Anti-Flag IPs for Rab11a Flag with EGFP-tagged Rab11FIPs (1–5) and SLAMF1. (B) Schematic figure for class I and class II Rab11 FIPs domain structure. C2, phospholipid-binding C2 domain; EF, EF-hand domain; PRR, proline-rich region; RBD, Rab11 binding domain. (C) Homologous protein sequence in class I FIPs, which follow the C2 domain. Identical amino acids in all three class I FIPs are highlighted. (D) Coprecipitation of SLAMF1 Flag with FIP2 EGFP WT or FIP2 deletion mutant lacking the C2 domain (ΔC2). (E and F) Coprecipitation of untagged SLAMF1 with FIP2 Flag (1–512 aa) and Flag-tagged FIP2 deletion mutants in anti-Flag IPs in the absence (E) or presence (F) of overexpressed Rab11 CFP . (G) Quantification of coprecipitations in E and F between SLAMF1 and FIP2 Flag variants correlated with the amount of Flag-tagged protein on the blot and Flag-tagged protein sizes. Error bars represent means ± SD for three independent experiments. (H) Coprecipitation of FIP2 Flag with SLAMF1 and Rab11a WT, Rab11a Q70L mutant (QL), or Rab11a S25N mutant (SN). (I) Coprecipitation of SLAMF1 Flag deletion mutants with FIP2 EGFP . Molecular weight is given in kilodaltons. WB, Western blot; WCL, whole-cell lysate. (J) Scheme for FIP2- and TRAM-interacting domains in SLAMF1ct. The results are representative of at least three independent experiments.

Techniques Used: Binding Assay, Sequencing, Mutagenesis, Molecular Weight, Western Blot

SLAMF1 regulates TRAM recruitment to E. coli phagosomes. (A) SLAMF1 costaining with TRAM, EEA1, or LAMP1 in primary macrophages coincubated with E. coli pHrodo particles for indicated time points. SLAMF1 (green), E. coli (blue), and TRAM, EEA1, or LAMP1 (red) are shown. The data shown are representative of one out of four donors. Bars, 10 μm. (B and C) TRAM and SLAMF1 MIs on E. coli phagosomes upon SLAMF1 silencing (B) or simultaneous Rab11a and Rab11b silencing (C) in primary human macrophages quantified from xyz images. The scatter plots are presented as median values of TRAM voxel intensity, and numbers of phagosomes are shown at the top. The nonparametric Mann-Whitney test was used to evaluate statistical significance. *, P
Figure Legend Snippet: SLAMF1 regulates TRAM recruitment to E. coli phagosomes. (A) SLAMF1 costaining with TRAM, EEA1, or LAMP1 in primary macrophages coincubated with E. coli pHrodo particles for indicated time points. SLAMF1 (green), E. coli (blue), and TRAM, EEA1, or LAMP1 (red) are shown. The data shown are representative of one out of four donors. Bars, 10 μm. (B and C) TRAM and SLAMF1 MIs on E. coli phagosomes upon SLAMF1 silencing (B) or simultaneous Rab11a and Rab11b silencing (C) in primary human macrophages quantified from xyz images. The scatter plots are presented as median values of TRAM voxel intensity, and numbers of phagosomes are shown at the top. The nonparametric Mann-Whitney test was used to evaluate statistical significance. *, P

Techniques Used: MANN-WHITNEY

SLAMF1 silencing in macrophages impairs TLR4-mediated phosphorylation of TBK1, IRF3, and TAK1. Western blotting of lysate macrophages treated with a control nonsilencing oligonucleotide or SLAMF1 -specific siRNA oligonucleotides and stimulated with 100 ng/ml LPS. The antibodies used are indicated in the figure. An antibody toward SLAMF1 was used to control for SLAMF1 silencing, and GAPDH was used as an equal loading control. Same GAPDH controls are presented for pTBK1, total TBK1, and phospho-p38MAPK, for total IRF3 and total TAK1, and for pTAK1 and pIκBα because they were probed on the same membranes. Western blots are representative of one of five donors. Molecular weight is given in kilodaltons. Graphs (right) show quantifications of protein levels relative to GAPDH levels obtained with Odyssey software.
Figure Legend Snippet: SLAMF1 silencing in macrophages impairs TLR4-mediated phosphorylation of TBK1, IRF3, and TAK1. Western blotting of lysate macrophages treated with a control nonsilencing oligonucleotide or SLAMF1 -specific siRNA oligonucleotides and stimulated with 100 ng/ml LPS. The antibodies used are indicated in the figure. An antibody toward SLAMF1 was used to control for SLAMF1 silencing, and GAPDH was used as an equal loading control. Same GAPDH controls are presented for pTBK1, total TBK1, and phospho-p38MAPK, for total IRF3 and total TAK1, and for pTAK1 and pIκBα because they were probed on the same membranes. Western blots are representative of one of five donors. Molecular weight is given in kilodaltons. Graphs (right) show quantifications of protein levels relative to GAPDH levels obtained with Odyssey software.

Techniques Used: Western Blot, Molecular Weight, Software

Lentiviral transduction of SLAMF1 in macrophages results in the increase of IRF3 and TBK1 phosphorylation in response to LPS and upregulation of IFNβ and TNF expression. (A) Quantification of SLAMF1 , IFNβ , and TNF mRNA expression by qPCR in macrophages transduced by Flag-tagged SLAMF1 coding or control virus and treated by LPS. The qPCR data are presented as means and SD for three biological replicates of one of three experiments. Significance was calculated by two-tailed t tests. *, P
Figure Legend Snippet: Lentiviral transduction of SLAMF1 in macrophages results in the increase of IRF3 and TBK1 phosphorylation in response to LPS and upregulation of IFNβ and TNF expression. (A) Quantification of SLAMF1 , IFNβ , and TNF mRNA expression by qPCR in macrophages transduced by Flag-tagged SLAMF1 coding or control virus and treated by LPS. The qPCR data are presented as means and SD for three biological replicates of one of three experiments. Significance was calculated by two-tailed t tests. *, P

Techniques Used: Transduction, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

SLAMF1 is enriched in the Rab11-positive ERCs in unstimulated macrophages, and SLAMF1 expression is induced by LPS and several other TLR ligands in primary human monocytes and macrophages. (A) Monocytes, macrophages, and differentiated THP-1 cells stained with antibodies against SLAMF1 (green) and GM130 (red) and imaged by confocal microscopy. (B) 3D model of cis-Golgi (GM130) and SLAMF1 in THP-1 cells. Z stacks from the GM130 and SLAMF1 channels were obtained using high-resolution confocal microscopy followed by 3D modeling in IMARIS software. (C) Macrophages stained for SLAMF1 and Rab11 (ERC marker). Representative image. Overlapping pixels for SLAMF1 and Rab11 are shown in the white overlap. tM1 = 0.683 ± 0.08 (mean with SD) for z stacks of ERCs as ROIs (30 ROIs analyzed per donor) where tM1 was the Manders’s colocalization coefficient with thresholds calculated in the Coloc 2 Fiji plugin with anti-SLAMF1 staining as first channel. (D) Macrophages costained for SLAMF1 and EEA1. (E) Macrophages costained for SLAMF1 and LAMP1. Colocalization accessed for z stacks for at least 30 cells for each experiment (four total) showing no colocalization for markers in both D and E. (F) Flow cytometry analysis of SLAMF1 surface expression by primary macrophages and differentiated THP-1 cells. Cells were costained for SLAMF1 and CD14 and gated for CD14-positive cells (primary cells) or stained for SLAMF1 (THP-1 cells). (G) Flow cytometry analysis of SLAMF1 surface expression by human macrophages stimulated by ultrapure K12 LPS (100 ng/ml) for 2, 4, and 6 h. (H) Western blot analysis of lysates from primary human macrophages stimulated by LPS for 2, 4, and 6 h. Graphs present mean values for three biological replicates with SD. Molecular weight is given in kilodaltons. (I and J ) Quantification of SLAMF1 mRNA expression by qPCR in monocytes (I) and macrophages (J) stimulated by TLRs’ ligands FSL-1 (20 ng/ml), K12 LPS (100 ng/ml), and CL075 (1 μg/ml; both I and J) as well as R848 (1 μg/ml), Pam3Cys (P3C; 1 μg/ml), or K12 E. coli particles (20/cell; I only). Results are presented as means with SD. Statistical significance between groups was evaluated by a two-tailed t test. *, P
Figure Legend Snippet: SLAMF1 is enriched in the Rab11-positive ERCs in unstimulated macrophages, and SLAMF1 expression is induced by LPS and several other TLR ligands in primary human monocytes and macrophages. (A) Monocytes, macrophages, and differentiated THP-1 cells stained with antibodies against SLAMF1 (green) and GM130 (red) and imaged by confocal microscopy. (B) 3D model of cis-Golgi (GM130) and SLAMF1 in THP-1 cells. Z stacks from the GM130 and SLAMF1 channels were obtained using high-resolution confocal microscopy followed by 3D modeling in IMARIS software. (C) Macrophages stained for SLAMF1 and Rab11 (ERC marker). Representative image. Overlapping pixels for SLAMF1 and Rab11 are shown in the white overlap. tM1 = 0.683 ± 0.08 (mean with SD) for z stacks of ERCs as ROIs (30 ROIs analyzed per donor) where tM1 was the Manders’s colocalization coefficient with thresholds calculated in the Coloc 2 Fiji plugin with anti-SLAMF1 staining as first channel. (D) Macrophages costained for SLAMF1 and EEA1. (E) Macrophages costained for SLAMF1 and LAMP1. Colocalization accessed for z stacks for at least 30 cells for each experiment (four total) showing no colocalization for markers in both D and E. (F) Flow cytometry analysis of SLAMF1 surface expression by primary macrophages and differentiated THP-1 cells. Cells were costained for SLAMF1 and CD14 and gated for CD14-positive cells (primary cells) or stained for SLAMF1 (THP-1 cells). (G) Flow cytometry analysis of SLAMF1 surface expression by human macrophages stimulated by ultrapure K12 LPS (100 ng/ml) for 2, 4, and 6 h. (H) Western blot analysis of lysates from primary human macrophages stimulated by LPS for 2, 4, and 6 h. Graphs present mean values for three biological replicates with SD. Molecular weight is given in kilodaltons. (I and J ) Quantification of SLAMF1 mRNA expression by qPCR in monocytes (I) and macrophages (J) stimulated by TLRs’ ligands FSL-1 (20 ng/ml), K12 LPS (100 ng/ml), and CL075 (1 μg/ml; both I and J) as well as R848 (1 μg/ml), Pam3Cys (P3C; 1 μg/ml), or K12 E. coli particles (20/cell; I only). Results are presented as means with SD. Statistical significance between groups was evaluated by a two-tailed t test. *, P

Techniques Used: Expressing, Staining, Confocal Microscopy, Software, Marker, Flow Cytometry, Cytometry, Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Two Tailed Test

SLAMF1 interacts with TRAM protein. (A) Endogenous IPs using specific anti-SLAMF1 mAbs from macrophages stimulated by LPS. (B) Endogenous IPs using anti-TRAM polyclonal antibodies from macrophages stimulated by LPS. (C) TRAM Flag -precipitated SLAMF1 and SLAMF1ct was needed for interaction with TRAM. (D) Coprecipitation of TRAM deletion mutants: TIR domain (68–235), short TRAM TIR domain (68–176 aa), and N-terminal (1–68 aa) or C-terminal (158–235 aa) domains with SLAMF1 protein. (E) Coprecipitation of TRAM deletion mutants containing the N-terminal part of TRAM TIR domain with SLAMF1. (F) Coprecipitation of SLAMF1 Flag deletion mutants with TRAM YFP . (G) Coprecipitation of human SLAMF1 Flag with human TRAM YFP and of mouse SLAMF1 Flag with mouse TRAM EGFP . Black dashed lines indicate that intervening lanes have been spliced out. (H) Human SLAMF1 cytoplasmic tail coprecipitation with TRAM YFP with or without amino acid substitutions at 321–324. Graphs under C–F summarize the IPs’ results. Indicated constructs were transfected to HEK293T cells, and anti-Flag agarose was used for the IPs. For endogenous IPs, specific SLAMF1 or TRAM antibodies were covalently coupled to beads. At least three independent experiments were carried out for anti-Flag IPs, and five independent experiments were carried out for the endogenous IPs, and one representative experiment is shown for each. Molecular weight is given in kilodaltons. WB, Western blot; WCL, whole-cell lysate.
Figure Legend Snippet: SLAMF1 interacts with TRAM protein. (A) Endogenous IPs using specific anti-SLAMF1 mAbs from macrophages stimulated by LPS. (B) Endogenous IPs using anti-TRAM polyclonal antibodies from macrophages stimulated by LPS. (C) TRAM Flag -precipitated SLAMF1 and SLAMF1ct was needed for interaction with TRAM. (D) Coprecipitation of TRAM deletion mutants: TIR domain (68–235), short TRAM TIR domain (68–176 aa), and N-terminal (1–68 aa) or C-terminal (158–235 aa) domains with SLAMF1 protein. (E) Coprecipitation of TRAM deletion mutants containing the N-terminal part of TRAM TIR domain with SLAMF1. (F) Coprecipitation of SLAMF1 Flag deletion mutants with TRAM YFP . (G) Coprecipitation of human SLAMF1 Flag with human TRAM YFP and of mouse SLAMF1 Flag with mouse TRAM EGFP . Black dashed lines indicate that intervening lanes have been spliced out. (H) Human SLAMF1 cytoplasmic tail coprecipitation with TRAM YFP with or without amino acid substitutions at 321–324. Graphs under C–F summarize the IPs’ results. Indicated constructs were transfected to HEK293T cells, and anti-Flag agarose was used for the IPs. For endogenous IPs, specific SLAMF1 or TRAM antibodies were covalently coupled to beads. At least three independent experiments were carried out for anti-Flag IPs, and five independent experiments were carried out for the endogenous IPs, and one representative experiment is shown for each. Molecular weight is given in kilodaltons. WB, Western blot; WCL, whole-cell lysate.

Techniques Used: Construct, Transfection, Molecular Weight, Western Blot

37) Product Images from "Lipocalin2 suppresses metastasis of colorectal cancer by attenuating NF-κB-dependent activation of snail and epithelial mesenchymal transition"

Article Title: Lipocalin2 suppresses metastasis of colorectal cancer by attenuating NF-κB-dependent activation of snail and epithelial mesenchymal transition

Journal: Molecular Cancer

doi: 10.1186/s12943-016-0564-9

LCN2 suppresses the NF-κB/snail signaling pathway and attenuates NF-κB promoter activity. a Western blots detection of p65 expression in LCN2-overexpressing and LCN2-knockdown cells. b Western blots detection, quantification of relative mRNA expression of snail expression in corresponding cells. c Immunofluorescence of snail expression and location in the indicated cells. d , e Western blots detection of EMT markers and snail changes ( d ) and quantification of relative mRNA expression of EMT markers ( e ) after treatment with LMB (20nM for 10 h) and Bay11-7082 (50 μM for 3 h) in corresponding cells. The grey value ratio of Vimentin/GAPDH was shown. f Survival analysis (Kaplan-Meier method, log-rank test) of combined LCN2 and NF-κB expression in CRC patients ( n = 126; group 1 ( n = 21, 3 died) vs group 2 ( n = 48, 17 died), P = .083; group 1 vs group 3 ( n = 27, 10 died), P = .088; group 1 vs group 4 ( n = 30, 13 died), P = .042). Values shown in real-time PCR assay are the mean ± SD from at least three independent experiments. (N, nuclear; P-phosphorylated). * P
Figure Legend Snippet: LCN2 suppresses the NF-κB/snail signaling pathway and attenuates NF-κB promoter activity. a Western blots detection of p65 expression in LCN2-overexpressing and LCN2-knockdown cells. b Western blots detection, quantification of relative mRNA expression of snail expression in corresponding cells. c Immunofluorescence of snail expression and location in the indicated cells. d , e Western blots detection of EMT markers and snail changes ( d ) and quantification of relative mRNA expression of EMT markers ( e ) after treatment with LMB (20nM for 10 h) and Bay11-7082 (50 μM for 3 h) in corresponding cells. The grey value ratio of Vimentin/GAPDH was shown. f Survival analysis (Kaplan-Meier method, log-rank test) of combined LCN2 and NF-κB expression in CRC patients ( n = 126; group 1 ( n = 21, 3 died) vs group 2 ( n = 48, 17 died), P = .083; group 1 vs group 3 ( n = 27, 10 died), P = .088; group 1 vs group 4 ( n = 30, 13 died), P = .042). Values shown in real-time PCR assay are the mean ± SD from at least three independent experiments. (N, nuclear; P-phosphorylated). * P

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

EMT changes depend on the LCN2/NF-κB/Snail pathway. a , b Changes of EMT markers measured by western blots and real-time PCR after enhancing NF-ĸB activity with LMB (40nM for 6 h), and quantification of relative mRNA expression of EMT markers in SW620-LCN2 cells ( a ) and suppressing NF-κB activity with specific siRNA (50nM for 48 h), and quantification of relative mRNA expression of EMT markers in SW480-sh-LCN2 cells ( b ), The grey value ratios of the corresponding proteins/GAPDH were shown. Values shown in real-time PCR assay are the mean ± SD from at least three independent experiments. (N, nuclear; C, cytoplasm; P, phosphorylated; NC, negative control). * P
Figure Legend Snippet: EMT changes depend on the LCN2/NF-κB/Snail pathway. a , b Changes of EMT markers measured by western blots and real-time PCR after enhancing NF-ĸB activity with LMB (40nM for 6 h), and quantification of relative mRNA expression of EMT markers in SW620-LCN2 cells ( a ) and suppressing NF-κB activity with specific siRNA (50nM for 48 h), and quantification of relative mRNA expression of EMT markers in SW480-sh-LCN2 cells ( b ), The grey value ratios of the corresponding proteins/GAPDH were shown. Values shown in real-time PCR assay are the mean ± SD from at least three independent experiments. (N, nuclear; C, cytoplasm; P, phosphorylated; NC, negative control). * P

Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Activity Assay, Expressing, Negative Control

LCN2 inhibits EMT characteristics in vitro and in vivo. a Western blots and real-time PCR analysis of EMT markers on LCN2 overexpression in SW620 or knockdown in SW480 cells, and quantification of relative mRNA expression of EMT markers and key proteins of corresponding cells (mean ± SD from at least three independent experiments). b Immunofluorescent staining of E-cadherin and Vimentin in corresponding cells. c Western blots and real-time PCR analysis of EMT characteristics in mouse xenografts (mean ± SD from at least three independent experiments). * P
Figure Legend Snippet: LCN2 inhibits EMT characteristics in vitro and in vivo. a Western blots and real-time PCR analysis of EMT markers on LCN2 overexpression in SW620 or knockdown in SW480 cells, and quantification of relative mRNA expression of EMT markers and key proteins of corresponding cells (mean ± SD from at least three independent experiments). b Immunofluorescent staining of E-cadherin and Vimentin in corresponding cells. c Western blots and real-time PCR analysis of EMT characteristics in mouse xenografts (mean ± SD from at least three independent experiments). * P

Techniques Used: In Vitro, In Vivo, Western Blot, Real-time Polymerase Chain Reaction, Over Expression, Expressing, Staining

LCN2 expression in CRC cells and tissues. a Reverse transcription-polymerase chain reaction (RT-PCR) and western blots of LCN2 expression in the indicated CRC cell lines. b LCN2 expression ( upper ), and quantification of LCN2 positive rates ( lower ) in CRC primary lesions and metastatic lymph node lesions (*** P
Figure Legend Snippet: LCN2 expression in CRC cells and tissues. a Reverse transcription-polymerase chain reaction (RT-PCR) and western blots of LCN2 expression in the indicated CRC cell lines. b LCN2 expression ( upper ), and quantification of LCN2 positive rates ( lower ) in CRC primary lesions and metastatic lymph node lesions (*** P

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

Negative effects of LCN2 on proliferation and tumorigenicity. a LCN2 expression confirmed by western blots and real-time PCR in the indicated cell. (s: supernatant) and quantification of relative mRNA expression of LCN2 expression of corresponding cells. b Cell proliferation measured by the CCK8 assay, and quantification of OD value at 450 nm of corresponding cells with microplate reader (5000 cells/well, mean ± SD from at least three independent experiments). (C,D) Colony-formation assays on soft agar ( c ) and plate ( d ) with corresponding cells and quantification of the fold change of average clone diameter of eight fields (for soft agar colony-formation assay) and relative counts based on the colony diameter (for plate colony-formation assay, SW480-SHB/sh-LCN2: diameter ≧5 cm; SW620-OB/LCN2: diameter ≧2 cm) (mean ± SD from at least three independent experiments. Scale bars, 100 μm). e Tumor weights ( g ) and volumes (mm 3 ) of mouse xenografts formed with corresponding cells. SW620-OB (5 × 10 6 ), SW620-LCN2 (5 × 10 6 ), SW480-SHB (1 × 10 7 ), and SW480-sh-LCN2 (1 × 10 7 ) cells in 100 μl PBS were inoculated subcutaneously into the left shoulders of 4 to 5-week-old BALB/c nude mice. Tumor length and width were measured with calipers, and tumor volumes were calculated according to the equation: V (mm 3 ) = ½ [width 2 (mm 2 ) × length (mm)]. Growth curves were plotted based on mean tumor volume within each experimental group at the indicated time points. Tumor growth was observed for 27 days (for SW620-OB and SW620-LCN2 cells) and 16 days (for SW480-SHB and SW480-sh-LCN2 cells), respectively. * P
Figure Legend Snippet: Negative effects of LCN2 on proliferation and tumorigenicity. a LCN2 expression confirmed by western blots and real-time PCR in the indicated cell. (s: supernatant) and quantification of relative mRNA expression of LCN2 expression of corresponding cells. b Cell proliferation measured by the CCK8 assay, and quantification of OD value at 450 nm of corresponding cells with microplate reader (5000 cells/well, mean ± SD from at least three independent experiments). (C,D) Colony-formation assays on soft agar ( c ) and plate ( d ) with corresponding cells and quantification of the fold change of average clone diameter of eight fields (for soft agar colony-formation assay) and relative counts based on the colony diameter (for plate colony-formation assay, SW480-SHB/sh-LCN2: diameter ≧5 cm; SW620-OB/LCN2: diameter ≧2 cm) (mean ± SD from at least three independent experiments. Scale bars, 100 μm). e Tumor weights ( g ) and volumes (mm 3 ) of mouse xenografts formed with corresponding cells. SW620-OB (5 × 10 6 ), SW620-LCN2 (5 × 10 6 ), SW480-SHB (1 × 10 7 ), and SW480-sh-LCN2 (1 × 10 7 ) cells in 100 μl PBS were inoculated subcutaneously into the left shoulders of 4 to 5-week-old BALB/c nude mice. Tumor length and width were measured with calipers, and tumor volumes were calculated according to the equation: V (mm 3 ) = ½ [width 2 (mm 2 ) × length (mm)]. Growth curves were plotted based on mean tumor volume within each experimental group at the indicated time points. Tumor growth was observed for 27 days (for SW620-OB and SW620-LCN2 cells) and 16 days (for SW480-SHB and SW480-sh-LCN2 cells), respectively. * P

Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, CCK-8 Assay, Soft Agar Assay, Colony Assay, Mouse Assay

LCN2-mediated inhibition of metastasis/invasion in CRC. a , b Migration assay of corresponding cells and quantification of cell migration was measured with microplate reader at 570 nm of OD value. ( n = 3, mean ± SD, scale bars, 100 μm). (C,D) Wound-closure assays of LCN2-overexpressing and LCN2-knockdown cells and corresponding vector control cells and quantification of the fold change of average migrated distance (48 h/0 h) of wound healing assays ( n = 3, mean ± SD, scale bars, 100 μm). e , f Invasion assay of corresponding cells and quantification of cell invasion was measured with microplate reader at 570 nm of OD value. ( n = 3, mean ± SD, scale bars, 100 μm). g Metastatic tumor nodules in the lungs of nude mice after injecting LCN2-overexpressing cells and control cells and quantification of the number of lung metastatic foci per section ( n = 5 mice/group, P = .043). h Hematoxylin and Eosin (H.E.) staining of lung metastases of nude mice with corresponding cells (scale bars, 500 μm). * P
Figure Legend Snippet: LCN2-mediated inhibition of metastasis/invasion in CRC. a , b Migration assay of corresponding cells and quantification of cell migration was measured with microplate reader at 570 nm of OD value. ( n = 3, mean ± SD, scale bars, 100 μm). (C,D) Wound-closure assays of LCN2-overexpressing and LCN2-knockdown cells and corresponding vector control cells and quantification of the fold change of average migrated distance (48 h/0 h) of wound healing assays ( n = 3, mean ± SD, scale bars, 100 μm). e , f Invasion assay of corresponding cells and quantification of cell invasion was measured with microplate reader at 570 nm of OD value. ( n = 3, mean ± SD, scale bars, 100 μm). g Metastatic tumor nodules in the lungs of nude mice after injecting LCN2-overexpressing cells and control cells and quantification of the number of lung metastatic foci per section ( n = 5 mice/group, P = .043). h Hematoxylin and Eosin (H.E.) staining of lung metastases of nude mice with corresponding cells (scale bars, 500 μm). * P

Techniques Used: Inhibition, Migration, Plasmid Preparation, Invasion Assay, Mouse Assay, Staining

EMT changes depend on the LCN2/NF-κB/Snail pathway. a Changes of EMT markers measured by western blots and real-time PCR after restoring snail expression, and quantification of relative mRNA expression of EMT markers in SW620-LCN2 cells. Snail protein (55KD) is a fusion protein with green fluorescence protein (GFP). The grey value ratios of the corresponding proteins/GAPDH were shown. b Migration assay and quantification of migration after overexpression of snail in SW620-LCN2 cells (OD values were measured by a microplate reader at 570 nm). Values shown in real-time PCR assay are the mean ± SD from at least three independent experiments. (N, nuclear; C, cytoplasm; P, phosphorylated; over, overexpression). * P
Figure Legend Snippet: EMT changes depend on the LCN2/NF-κB/Snail pathway. a Changes of EMT markers measured by western blots and real-time PCR after restoring snail expression, and quantification of relative mRNA expression of EMT markers in SW620-LCN2 cells. Snail protein (55KD) is a fusion protein with green fluorescence protein (GFP). The grey value ratios of the corresponding proteins/GAPDH were shown. b Migration assay and quantification of migration after overexpression of snail in SW620-LCN2 cells (OD values were measured by a microplate reader at 570 nm). Values shown in real-time PCR assay are the mean ± SD from at least three independent experiments. (N, nuclear; C, cytoplasm; P, phosphorylated; over, overexpression). * P

Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Expressing, Fluorescence, Migration, Over Expression

38) Product Images from "Subcellular Distribution of HDAC1 in Neurotoxic Conditions Is Dependent on Serine Phosphorylation"

Article Title: Subcellular Distribution of HDAC1 in Neurotoxic Conditions Is Dependent on Serine Phosphorylation

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.3000-16.2017

Mutation of phosphorylation sites induce HDAC1 nuclear export. A , Schematic representation of HDAC1 protein sequence displaying its domains and PTM sites. A, Acetylation; C, carbonylation; P, phosphorylation; NES, nuclear export signal. B , Primary cultures of rat hippocampal neurons transfected with FLAG-tagged HDAC1 constructs containing different mutations on PTM sites were immunostained against FLAG (green) and NFH (red) and counterstained with DAPI (blue). Scale bar, 20 μm. C , Quantification of subcellular localization of FLAG signal from B showing that only mutation of phosphorylation sites (S 421, 423 →A) induced localization of recombinant HDAC1 to the neuronal cytosolic processes. Error bars indicate mean ± SEM. D , Quantification of neurons with SMI32 + processes over total DAPI + cells after lentivirus-mediated overexpression of either WT or the nonphosphorylated mutant HDAC1 HDAC1 SA(2×). NA, Uninfected naive neurons. Error bars indicate mean ± SEM. Data were analyzed with one-way ANOVA with Bonferroni post hoc test, p > 0.05 (NS).
Figure Legend Snippet: Mutation of phosphorylation sites induce HDAC1 nuclear export. A , Schematic representation of HDAC1 protein sequence displaying its domains and PTM sites. A, Acetylation; C, carbonylation; P, phosphorylation; NES, nuclear export signal. B , Primary cultures of rat hippocampal neurons transfected with FLAG-tagged HDAC1 constructs containing different mutations on PTM sites were immunostained against FLAG (green) and NFH (red) and counterstained with DAPI (blue). Scale bar, 20 μm. C , Quantification of subcellular localization of FLAG signal from B showing that only mutation of phosphorylation sites (S 421, 423 →A) induced localization of recombinant HDAC1 to the neuronal cytosolic processes. Error bars indicate mean ± SEM. D , Quantification of neurons with SMI32 + processes over total DAPI + cells after lentivirus-mediated overexpression of either WT or the nonphosphorylated mutant HDAC1 HDAC1 SA(2×). NA, Uninfected naive neurons. Error bars indicate mean ± SEM. Data were analyzed with one-way ANOVA with Bonferroni post hoc test, p > 0.05 (NS).

Techniques Used: Mutagenesis, Sequencing, Transfection, Construct, Recombinant, Over Expression

Glutamate- and TNFα-induced HDAC1 translocation coincides with HDAC1 dephosphorylation. A , HEK293 cells naive (uninfected, NA) or after overexpression of FLAG-tagged WT or SA HDAC1. Total proteins were extracted, immunoprecipitated with anti-FLAG antibody, and probed with indicated antibodies. B , Western blot analysis of phosphorylated S 421, 423 -HDAC1 (P-HDAC1) and total HDAC1 from rat hippocampal neurons (12 DIV) treated with Glut/TNFα for 30 min to 3 h. Quantification from three independent experiments and P-HDAC1 levels referred to total HDAC1. Data represent average ± SEM. Data were analyzed with one-way ANOVA with Bonferroni post hoc test, * p
Figure Legend Snippet: Glutamate- and TNFα-induced HDAC1 translocation coincides with HDAC1 dephosphorylation. A , HEK293 cells naive (uninfected, NA) or after overexpression of FLAG-tagged WT or SA HDAC1. Total proteins were extracted, immunoprecipitated with anti-FLAG antibody, and probed with indicated antibodies. B , Western blot analysis of phosphorylated S 421, 423 -HDAC1 (P-HDAC1) and total HDAC1 from rat hippocampal neurons (12 DIV) treated with Glut/TNFα for 30 min to 3 h. Quantification from three independent experiments and P-HDAC1 levels referred to total HDAC1. Data represent average ± SEM. Data were analyzed with one-way ANOVA with Bonferroni post hoc test, * p

Techniques Used: Translocation Assay, De-Phosphorylation Assay, Over Expression, Immunoprecipitation, Western Blot

39) Product Images from "Notch Activation Differentially Regulates Renal Progenitors Proliferation and Differentiation Toward the Podocyte Lineage in Glomerular Disorders"

Article Title: Notch Activation Differentially Regulates Renal Progenitors Proliferation and Differentiation Toward the Podocyte Lineage in Glomerular Disorders

Journal: Stem Cells (Dayton, Ohio)

doi: 10.1002/stem.492

Regulation of cell cycle progression, mitosis, cell death, and cytoskeleton organization in N3ICD-infected human renal progenitors before and after differentiation toward the podocyte lineage. ( A, B ): Cell cycle analysis performed on ( A ) mock- and ( B ) N3ICD-infected renal progenitors. One representative of four independent experiments is shown. ( C, D ): Cell cycle analysis performed on ( C ) mock- and ( D ) N3ICD-infected renal progenitors after their differentiation toward the podocyte lineage. One representative of four independent experiments is shown. ( E, F ): FACS analysis of apoptosis/necrosis in mock- and N3ICD-infected renal progenitors as assessed by annexin-V and propidium iodide (PI) staining. One representative of four independent experiments is shown. ( G, H ): FACS analysis of apoptosis/necrosis in mock- and N3ICD-infected renal progenitors after their differentiation toward the podocyte lineage as assessed by annexin-V and PI staining reveals an increase in the percentage of PI/annexin V positive cells in N3ICD-infected cells. One representative of four independent experiments is shown. ( I, J ): Above: H3-Ser10 (red) and tubulin (blue) staining of mock- and N3ICD-infected undifferentiated renal progenitors reveals normal mitoses. For mock-infected cells, a representative metaphase is shown, while for N3ICD-infected cells, a representative anaphase is shown. One representative of six independent experiments is shown. Scale bar = 10 μm. Below: Phalloidin staining (red) of mock- and N3ICD-infected undifferentiated renal progenitors. Topro-3 (blue) counterstains nuclei. One representative of six independent experiments is shown. Scale bar = 50 μm. ( K, L ): Above: H3-Ser10 (red) and tubulin (blue) staining of renal progenitors infected with vector expressing N3ICD ( L ) after their differentiation toward the podocyte lineage reveals aberrant mitoses characterized by micro/multinucleation (red) and abnormal spindle distribution (blue) in comparison with those infected with an empty vector (mock, [ K ]). One representative of six independent experiments is shown. Scale bar = 10 μm. Below: Phalloidin staining (red) of mock- ( K ) and N3ICD-infected ( L ) renal progenitors after their differentiation toward the podocyte lineage reveals F-actin filaments distributed as stress-like bundles along the axis of the cells in mock-infected podocytes and redistribution of F-actin fibers to the periphery of the cells in podocytes infected with N3ICD. Topro-3 (blue) counterstains nuclei. One representative of six independent experiments is shown. Scale bar = 50 μm. ( M, N ): Assessment by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) of Aurora kinase B ( M ), p21 Cip1/WAF-1 and p27 Kip1 ( N ) mRNA expression in mock- and N3ICD-infected undifferentiated renal progenitors. Results are expressed as mean ± SEM of triplicate assessments in seven separate experiments. ( O, P ): Assessment by real-time quantitative RT-PCR of Aurora kinase B ( O ) and p27 Kip1 , p21 Cip1/WAF-1 ( P ) mRNA expression in mock- and N3ICD-infected renal progenitors after their differentiation toward the podocyte lineage. Results are expressed as mean ± SEM of triplicate assessments in seven separate experiments. Abbreviation: FACS, fluorescence activated cell sorting.
Figure Legend Snippet: Regulation of cell cycle progression, mitosis, cell death, and cytoskeleton organization in N3ICD-infected human renal progenitors before and after differentiation toward the podocyte lineage. ( A, B ): Cell cycle analysis performed on ( A ) mock- and ( B ) N3ICD-infected renal progenitors. One representative of four independent experiments is shown. ( C, D ): Cell cycle analysis performed on ( C ) mock- and ( D ) N3ICD-infected renal progenitors after their differentiation toward the podocyte lineage. One representative of four independent experiments is shown. ( E, F ): FACS analysis of apoptosis/necrosis in mock- and N3ICD-infected renal progenitors as assessed by annexin-V and propidium iodide (PI) staining. One representative of four independent experiments is shown. ( G, H ): FACS analysis of apoptosis/necrosis in mock- and N3ICD-infected renal progenitors after their differentiation toward the podocyte lineage as assessed by annexin-V and PI staining reveals an increase in the percentage of PI/annexin V positive cells in N3ICD-infected cells. One representative of four independent experiments is shown. ( I, J ): Above: H3-Ser10 (red) and tubulin (blue) staining of mock- and N3ICD-infected undifferentiated renal progenitors reveals normal mitoses. For mock-infected cells, a representative metaphase is shown, while for N3ICD-infected cells, a representative anaphase is shown. One representative of six independent experiments is shown. Scale bar = 10 μm. Below: Phalloidin staining (red) of mock- and N3ICD-infected undifferentiated renal progenitors. Topro-3 (blue) counterstains nuclei. One representative of six independent experiments is shown. Scale bar = 50 μm. ( K, L ): Above: H3-Ser10 (red) and tubulin (blue) staining of renal progenitors infected with vector expressing N3ICD ( L ) after their differentiation toward the podocyte lineage reveals aberrant mitoses characterized by micro/multinucleation (red) and abnormal spindle distribution (blue) in comparison with those infected with an empty vector (mock, [ K ]). One representative of six independent experiments is shown. Scale bar = 10 μm. Below: Phalloidin staining (red) of mock- ( K ) and N3ICD-infected ( L ) renal progenitors after their differentiation toward the podocyte lineage reveals F-actin filaments distributed as stress-like bundles along the axis of the cells in mock-infected podocytes and redistribution of F-actin fibers to the periphery of the cells in podocytes infected with N3ICD. Topro-3 (blue) counterstains nuclei. One representative of six independent experiments is shown. Scale bar = 50 μm. ( M, N ): Assessment by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) of Aurora kinase B ( M ), p21 Cip1/WAF-1 and p27 Kip1 ( N ) mRNA expression in mock- and N3ICD-infected undifferentiated renal progenitors. Results are expressed as mean ± SEM of triplicate assessments in seven separate experiments. ( O, P ): Assessment by real-time quantitative RT-PCR of Aurora kinase B ( O ) and p27 Kip1 , p21 Cip1/WAF-1 ( P ) mRNA expression in mock- and N3ICD-infected renal progenitors after their differentiation toward the podocyte lineage. Results are expressed as mean ± SEM of triplicate assessments in seven separate experiments. Abbreviation: FACS, fluorescence activated cell sorting.

Techniques Used: Infection, Cell Cycle Assay, FACS, Staining, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Fluorescence

Notch downregulation is necessary to achieve differentiation of human renal progenitors into the podocyte lineage. ( A ): Sustained Notch protein expression in renal progenitors as obtained following infection with a vector leading to the N1ICD, N2ICD, or N3ICD and the GFP to be simultaneously coexpressed. Cells infected with the empty vector (mock) express GFP but do not express the NICDs. Uninfected cells are shown for comparison. One representative of 10 independent experiments is shown. ( B ): Downregulation of the Notch pathway activity following differentiation of renal progenitors toward the podocyte lineage is rescued by infection with vectors expressing N1ICD, N2ICD, or N3ICD as demonstrated by coinfection with a reporter vector for the RBP-J transcriptional response element. Results are expressed as mean ± SEM fold increase of luciferase activity in renal progenitors coinfected with a reporter vector for the RBP-J and the vectors expressing N1ICD, N2ICD, or N3ICD in comparison with the empty vector before (control) or after their differentiation toward the podocyte lineage (VRADD) as obtained in at least four independent experiments. b versus a , c , d , e , f , g , h : p
Figure Legend Snippet: Notch downregulation is necessary to achieve differentiation of human renal progenitors into the podocyte lineage. ( A ): Sustained Notch protein expression in renal progenitors as obtained following infection with a vector leading to the N1ICD, N2ICD, or N3ICD and the GFP to be simultaneously coexpressed. Cells infected with the empty vector (mock) express GFP but do not express the NICDs. Uninfected cells are shown for comparison. One representative of 10 independent experiments is shown. ( B ): Downregulation of the Notch pathway activity following differentiation of renal progenitors toward the podocyte lineage is rescued by infection with vectors expressing N1ICD, N2ICD, or N3ICD as demonstrated by coinfection with a reporter vector for the RBP-J transcriptional response element. Results are expressed as mean ± SEM fold increase of luciferase activity in renal progenitors coinfected with a reporter vector for the RBP-J and the vectors expressing N1ICD, N2ICD, or N3ICD in comparison with the empty vector before (control) or after their differentiation toward the podocyte lineage (VRADD) as obtained in at least four independent experiments. b versus a , c , d , e , f , g , h : p

Techniques Used: Expressing, Infection, Plasmid Preparation, Activity Assay, Luciferase

40) Product Images from "Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles"

Article Title: Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles

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

doi: 10.1073/pnas.1614955114

ORF2 and ORF3 are required for releasing viral particles to infect naïve HepG2C3A cells. ( A ) Schematic representation of the transcomplementation system for packaging HEV virions. ( B ) Representative flow cytometry plots demonstrating efficient ORF2 and ORF3 expression. HepG2C3A cells were transduced with pLVX-ORF2-IRES-zsGreen and/or pLEX-ORF3-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of ORF2 and/or ORF3 expressing cells. ( C ) Infection kinetics of transcomplemented HEV in HepG2C3A cells. Cell culture supernatants from naïve HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, were collected 5 d posttransfection with rHEVΔORF2/3[Gluc] RNA. Naïve HepG2C3A cells were incubated with these supernatants. After 12 h, cells were washed and Gaussia luciferase activity quantified in the cell culture supernatants at the indicated time points. ( D ) Five days following transfection of rHEVΔORF2/3[Gluc] RNA into HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, lysates were used to infect naïve HepG2C3A cells. Gluc activity was measured in the supernatants 4 d postinfection. Shown are averages and SDs of triplicate measurements of three independent experiments. * P
Figure Legend Snippet: ORF2 and ORF3 are required for releasing viral particles to infect naïve HepG2C3A cells. ( A ) Schematic representation of the transcomplementation system for packaging HEV virions. ( B ) Representative flow cytometry plots demonstrating efficient ORF2 and ORF3 expression. HepG2C3A cells were transduced with pLVX-ORF2-IRES-zsGreen and/or pLEX-ORF3-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of ORF2 and/or ORF3 expressing cells. ( C ) Infection kinetics of transcomplemented HEV in HepG2C3A cells. Cell culture supernatants from naïve HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, were collected 5 d posttransfection with rHEVΔORF2/3[Gluc] RNA. Naïve HepG2C3A cells were incubated with these supernatants. After 12 h, cells were washed and Gaussia luciferase activity quantified in the cell culture supernatants at the indicated time points. ( D ) Five days following transfection of rHEVΔORF2/3[Gluc] RNA into HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, lysates were used to infect naïve HepG2C3A cells. Gluc activity was measured in the supernatants 4 d postinfection. Shown are averages and SDs of triplicate measurements of three independent experiments. * P

Techniques Used: Flow Cytometry, Cytometry, Expressing, Transduction, Infection, Cell Culture, Incubation, Luciferase, Activity Assay, Transfection

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Clone Assay:

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Amplification:

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Article Snippet: .. To construct a lentiviral construct that encodes Kernow C1/p6 ORF2 or Kernow C1/p6 ORF3, the Kernow C1/p6 ORF2 or Kernow C1/p6 ORF3 cDNA was amplified by PCR from the HEV Kernow C1/p6 construct (a kind gift from Suzanne Emerson, NIH, Bethesda, MD) and then cloned into pLVX-IRES-zsGreen1 or pLEX-IRES-mCherry vectors using the In-Fusion HD Cloning Kit (Clontech). .. To construct the pLEX-IAV M2-IRES-mCherry vector, cDNA encoding Influenza A virus M2 (A/Puerto Rico/8/34/Mount Sinai/Wi(H1N1)) was synthesized by IDT with gBlock, and then cloned into pLEX-IRES-mCherry vectors using In-Fusion HD Cloning Kit (Clontech).

Article Title: Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription
Article Snippet: .. To construct lentiviral constructs encoding ORF1 of Kernow C1/p6 (GenBank accession number JQ679013 ), the Kernow C1/p6 ORF1 cDNA was amplified by PCR from a plasmid encoding the full-length (FL) infectious HEV clone Kernow C1/p6 (a kind gift from Suzanne Emerson, NIH) and then cloned into pLVX-IRES-zsGreen1 vector using an In-Fusion HD cloning kit (Clontech, Mountain View, CA, USA). .. The GAD mutant of ORF1 inactivating the polymerase was generated by QuikChange (Stratagene) site-directed mutagenesis.

Mouse Assay:

Article Title: A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia
Article Snippet: .. Engraftment of NOD/SCID-IL2Rγ mice (NSG) THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 (Clontech), followed by sorting to obtain GFP-positive cells. .. GFP-positive THP1 cells were transduced with MSCV-miR-375 or MSCV-NC, followed by puromycin selection for 1 week.

Expressing:

Article Title: A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia
Article Snippet: .. To produce plasmids expressing HOXB3 and DNMT3B, human HOXB3 and DNMT3B coding sequences were amplified by PCR and then cloned into lentivirus vector pLVX-IRES-ZsGreen1 (Clontech) and pMSCV-puro (Clontech), respectively. .. To construct pMIR-HOXB3 3′UTR plasmid, human HOXB3 3′UTR was amplified by PCR and cloned into pMIR-REPORT vector (Ambion, Dallas, TX, USA).

Article Title: Selection of an optimal promoter for gene transfer in normal B cells
Article Snippet: .. B cells activated with CD40L and IL-21 are resistant to transduction To determine the levels of transgene expression in B cells three bicistronic plasmids were used that encode various GFPs under the control of different promoters, namely pGIPZ [cytomegalovirus (CMV) promoter; turbo GFP, which is an improved variant of the green fluorescent protein CopGFP], pLVTHM [elongation factor 1 alpha (EF1α) promoter; GFP) and pLVX-IRES-ZsGreen1 (CMV promoter; ZsGreen1, which is a human codon-optimized variant of ZsGreen) ( ). .. Independent of the vectors used in the present study, a very low level of transgene expression was detected in B cells; expression did not exceed 10%, as assessed with flow cytometry 7 days post-transduction ( ).

Article Title: A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia
Article Snippet: .. To produce plasmids expressing HOXB3 and DNMT3B, human HOXB3 and DNMT3B coding sequences were amplified by PCR and then cloned into lentivirus vector pLVX-IRES-ZsGreen1 (Clontech) and pMSCV-puro (Clontech), respectively. .. To construct pMIR-HOXB3 3′UTR plasmid, human HOXB3 3′UTR was amplified by PCR and cloned into pMIR-REPORT vector (Ambion, Dallas, TX, USA).

Polymerase Chain Reaction:

Article Title: Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles
Article Snippet: .. To construct a lentiviral construct that encodes Kernow C1/p6 ORF2 or Kernow C1/p6 ORF3, the Kernow C1/p6 ORF2 or Kernow C1/p6 ORF3 cDNA was amplified by PCR from the HEV Kernow C1/p6 construct (a kind gift from Suzanne Emerson, NIH, Bethesda, MD) and then cloned into pLVX-IRES-zsGreen1 or pLEX-IRES-mCherry vectors using the In-Fusion HD Cloning Kit (Clontech). .. To construct the pLEX-IAV M2-IRES-mCherry vector, cDNA encoding Influenza A virus M2 (A/Puerto Rico/8/34/Mount Sinai/Wi(H1N1)) was synthesized by IDT with gBlock, and then cloned into pLEX-IRES-mCherry vectors using In-Fusion HD Cloning Kit (Clontech).

Article Title: A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia
Article Snippet: .. To produce plasmids expressing HOXB3 and DNMT3B, human HOXB3 and DNMT3B coding sequences were amplified by PCR and then cloned into lentivirus vector pLVX-IRES-ZsGreen1 (Clontech) and pMSCV-puro (Clontech), respectively. .. To construct pMIR-HOXB3 3′UTR plasmid, human HOXB3 3′UTR was amplified by PCR and cloned into pMIR-REPORT vector (Ambion, Dallas, TX, USA).

Article Title: A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia
Article Snippet: .. To produce plasmids expressing HOXB3 and DNMT3B, human HOXB3 and DNMT3B coding sequences were amplified by PCR and then cloned into lentivirus vector pLVX-IRES-ZsGreen1 (Clontech) and pMSCV-puro (Clontech), respectively. .. To construct pMIR-HOXB3 3′UTR plasmid, human HOXB3 3′UTR was amplified by PCR and cloned into pMIR-REPORT vector (Ambion, Dallas, TX, USA).

Article Title: Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription
Article Snippet: .. To construct lentiviral constructs encoding ORF1 of Kernow C1/p6 (GenBank accession number JQ679013 ), the Kernow C1/p6 ORF1 cDNA was amplified by PCR from a plasmid encoding the full-length (FL) infectious HEV clone Kernow C1/p6 (a kind gift from Suzanne Emerson, NIH) and then cloned into pLVX-IRES-zsGreen1 vector using an In-Fusion HD cloning kit (Clontech, Mountain View, CA, USA). .. The GAD mutant of ORF1 inactivating the polymerase was generated by QuikChange (Stratagene) site-directed mutagenesis.

Variant Assay:

Article Title: Selection of an optimal promoter for gene transfer in normal B cells
Article Snippet: .. B cells activated with CD40L and IL-21 are resistant to transduction To determine the levels of transgene expression in B cells three bicistronic plasmids were used that encode various GFPs under the control of different promoters, namely pGIPZ [cytomegalovirus (CMV) promoter; turbo GFP, which is an improved variant of the green fluorescent protein CopGFP], pLVTHM [elongation factor 1 alpha (EF1α) promoter; GFP) and pLVX-IRES-ZsGreen1 (CMV promoter; ZsGreen1, which is a human codon-optimized variant of ZsGreen) ( ). .. Independent of the vectors used in the present study, a very low level of transgene expression was detected in B cells; expression did not exceed 10%, as assessed with flow cytometry 7 days post-transduction ( ).

Plasmid Preparation:

Article Title: A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia
Article Snippet: .. To produce plasmids expressing HOXB3 and DNMT3B, human HOXB3 and DNMT3B coding sequences were amplified by PCR and then cloned into lentivirus vector pLVX-IRES-ZsGreen1 (Clontech) and pMSCV-puro (Clontech), respectively. .. To construct pMIR-HOXB3 3′UTR plasmid, human HOXB3 3′UTR was amplified by PCR and cloned into pMIR-REPORT vector (Ambion, Dallas, TX, USA).

Article Title: A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia
Article Snippet: .. Engraftment of NOD/SCID-IL2Rγ mice (NSG) THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 (Clontech), followed by sorting to obtain GFP-positive cells. .. GFP-positive THP1 cells were transduced with MSCV-miR-375 or MSCV-NC, followed by puromycin selection for 1 week.

Article Title: A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia
Article Snippet: .. To produce plasmids expressing HOXB3 and DNMT3B, human HOXB3 and DNMT3B coding sequences were amplified by PCR and then cloned into lentivirus vector pLVX-IRES-ZsGreen1 (Clontech) and pMSCV-puro (Clontech), respectively. .. To construct pMIR-HOXB3 3′UTR plasmid, human HOXB3 3′UTR was amplified by PCR and cloned into pMIR-REPORT vector (Ambion, Dallas, TX, USA).

Article Title: Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription
Article Snippet: .. To construct lentiviral constructs encoding ORF1 of Kernow C1/p6 (GenBank accession number JQ679013 ), the Kernow C1/p6 ORF1 cDNA was amplified by PCR from a plasmid encoding the full-length (FL) infectious HEV clone Kernow C1/p6 (a kind gift from Suzanne Emerson, NIH) and then cloned into pLVX-IRES-zsGreen1 vector using an In-Fusion HD cloning kit (Clontech, Mountain View, CA, USA). .. The GAD mutant of ORF1 inactivating the polymerase was generated by QuikChange (Stratagene) site-directed mutagenesis.

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    TaKaRa lentivirus vector plvx ires zsgreen1
    The anti-leukemia effects of miR-375 in vivo. About 1х10 7 viable HL-60 cells transduced with MSCV-miR-375 or MSCV-NC were injected subcutaneously into right flank of each nude mouse. a A photograph of xenografted tumors in mice xenografted by HL-60 cells, which were transduced with MSCV-miR-375 or MSCV-NC. b Volumes of all xenografted tumors were measured when the experiment was terminated at 42 days after tumor cell inoculation. c Net weights of all xenografted tumors were measured at the termination of the experiment. d The protein expression of HOXB3 was detected in xenografted tumor lysates from HL-60 cells transduced with MSCV-miR-375 or MSCV-NC. e THP1 cells were transduced with <t>lentivirus</t> vector <t>pLVX-IRES-ZsGreen1</t> and GFP-positive cells were sorted by flow cytometry. The GFP-positive cells were further transduced with MSCV-miR-375 or MSCV-NC and treated with puromycin for 1 week. Then, THP1-GFP-miR-375 or THP1-GFP-NC were xenografted into NSG mice. Peripheral blood cells were extracted from mice when the mice became moribund and GFP-positive cells were analyzed by flow cytometry. * P
    Lentivirus Vector Plvx Ires Zsgreen1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The anti-leukemia effects of miR-375 in vivo. About 1х10 7 viable HL-60 cells transduced with MSCV-miR-375 or MSCV-NC were injected subcutaneously into right flank of each nude mouse. a A photograph of xenografted tumors in mice xenografted by HL-60 cells, which were transduced with MSCV-miR-375 or MSCV-NC. b Volumes of all xenografted tumors were measured when the experiment was terminated at 42 days after tumor cell inoculation. c Net weights of all xenografted tumors were measured at the termination of the experiment. d The protein expression of HOXB3 was detected in xenografted tumor lysates from HL-60 cells transduced with MSCV-miR-375 or MSCV-NC. e THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 and GFP-positive cells were sorted by flow cytometry. The GFP-positive cells were further transduced with MSCV-miR-375 or MSCV-NC and treated with puromycin for 1 week. Then, THP1-GFP-miR-375 or THP1-GFP-NC were xenografted into NSG mice. Peripheral blood cells were extracted from mice when the mice became moribund and GFP-positive cells were analyzed by flow cytometry. * P

    Journal: BMC Cancer

    Article Title: A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia

    doi: 10.1186/s12885-018-4097-z

    Figure Lengend Snippet: The anti-leukemia effects of miR-375 in vivo. About 1х10 7 viable HL-60 cells transduced with MSCV-miR-375 or MSCV-NC were injected subcutaneously into right flank of each nude mouse. a A photograph of xenografted tumors in mice xenografted by HL-60 cells, which were transduced with MSCV-miR-375 or MSCV-NC. b Volumes of all xenografted tumors were measured when the experiment was terminated at 42 days after tumor cell inoculation. c Net weights of all xenografted tumors were measured at the termination of the experiment. d The protein expression of HOXB3 was detected in xenografted tumor lysates from HL-60 cells transduced with MSCV-miR-375 or MSCV-NC. e THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 and GFP-positive cells were sorted by flow cytometry. The GFP-positive cells were further transduced with MSCV-miR-375 or MSCV-NC and treated with puromycin for 1 week. Then, THP1-GFP-miR-375 or THP1-GFP-NC were xenografted into NSG mice. Peripheral blood cells were extracted from mice when the mice became moribund and GFP-positive cells were analyzed by flow cytometry. * P

    Article Snippet: To produce plasmids expressing HOXB3 and DNMT3B, human HOXB3 and DNMT3B coding sequences were amplified by PCR and then cloned into lentivirus vector pLVX-IRES-ZsGreen1 (Clontech) and pMSCV-puro (Clontech), respectively.

    Techniques: In Vivo, Transduction, Injection, Mouse Assay, Expressing, Plasmid Preparation, Flow Cytometry, Cytometry

    The anti-leukemia effects of miR-375 in vivo. About 1х10 7 viable HL-60 cells transduced with MSCV-miR-375 or MSCV-NC were injected subcutaneously into right flank of each nude mouse. a A photograph of xenografted tumors in mice xenografted by HL-60 cells, which were transduced with MSCV-miR-375 or MSCV-NC. b Volumes of all xenografted tumors were measured when the experiment was terminated at 42 days after tumor cell inoculation. c Net weights of all xenografted tumors were measured at the termination of the experiment. d The protein expression of HOXB3 was detected in xenografted tumor lysates from HL-60 cells transduced with MSCV-miR-375 or MSCV-NC. e THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 and GFP-positive cells were sorted by flow cytometry. The GFP-positive cells were further transduced with MSCV-miR-375 or MSCV-NC and treated with puromycin for 1 week. Then, THP1-GFP-miR-375 or THP1-GFP-NC were xenografted into NSG mice. Peripheral blood cells were extracted from mice when the mice became moribund and GFP-positive cells were analyzed by flow cytometry. * P

    Journal: BMC Cancer

    Article Title: A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia

    doi: 10.1186/s12885-018-4097-z

    Figure Lengend Snippet: The anti-leukemia effects of miR-375 in vivo. About 1х10 7 viable HL-60 cells transduced with MSCV-miR-375 or MSCV-NC were injected subcutaneously into right flank of each nude mouse. a A photograph of xenografted tumors in mice xenografted by HL-60 cells, which were transduced with MSCV-miR-375 or MSCV-NC. b Volumes of all xenografted tumors were measured when the experiment was terminated at 42 days after tumor cell inoculation. c Net weights of all xenografted tumors were measured at the termination of the experiment. d The protein expression of HOXB3 was detected in xenografted tumor lysates from HL-60 cells transduced with MSCV-miR-375 or MSCV-NC. e THP1 cells were transduced with lentivirus vector pLVX-IRES-ZsGreen1 and GFP-positive cells were sorted by flow cytometry. The GFP-positive cells were further transduced with MSCV-miR-375 or MSCV-NC and treated with puromycin for 1 week. Then, THP1-GFP-miR-375 or THP1-GFP-NC were xenografted into NSG mice. Peripheral blood cells were extracted from mice when the mice became moribund and GFP-positive cells were analyzed by flow cytometry. * P

    Article Snippet: To produce plasmids expressing HOXB3 and DNMT3B, human HOXB3 and DNMT3B coding sequences were amplified by PCR and then cloned into lentivirus vector pLVX-IRES-ZsGreen1 (Clontech) and pMSCV-puro (Clontech), respectively.

    Techniques: In Vivo, Transduction, Injection, Mouse Assay, Expressing, Plasmid Preparation, Flow Cytometry, Cytometry

    Transduction of human CD4 + primary cells with MLV-A-based retroviral VLPs conveying NB-ZsGreen1 or ZsGreen1. (a) Schematic of the methods used to generate CD4 + primary cells transductants. (b) NB-ZsGreen1 or ZsGreen1 VLPs were used to transduce activated

    Journal: Human Gene Therapy

    Article Title: A Mutant Tat Protein Provides Strong Protection from HIV-1 Infection in Human CD4+ T Cells

    doi: 10.1089/hum.2012.176

    Figure Lengend Snippet: Transduction of human CD4 + primary cells with MLV-A-based retroviral VLPs conveying NB-ZsGreen1 or ZsGreen1. (a) Schematic of the methods used to generate CD4 + primary cells transductants. (b) NB-ZsGreen1 or ZsGreen1 VLPs were used to transduce activated

    Article Snippet: Similarly, pGCsamEN-EGFP was made by PCR of an EGFP cassette from pCDN3.1-EGFP, using primers P2 and P3, and pGCsamEN-ZsGreen1 was made by PCR of ZsGreen1 from pLVX-IRES-ZsGreen1 (Clontech, Mountain View, CA), using primers P4 and P5.

    Techniques: Transduction

    HIV replication is inhibited in activated human CD4 + primary cells expressing NB-ZsGreen1. (a) The two CD4 + populations described in were infected with HIV-1 89.6 containing 20 ng of CAp24 and grown for 21 days. Cell-free supernatant was

    Journal: Human Gene Therapy

    Article Title: A Mutant Tat Protein Provides Strong Protection from HIV-1 Infection in Human CD4+ T Cells

    doi: 10.1089/hum.2012.176

    Figure Lengend Snippet: HIV replication is inhibited in activated human CD4 + primary cells expressing NB-ZsGreen1. (a) The two CD4 + populations described in were infected with HIV-1 89.6 containing 20 ng of CAp24 and grown for 21 days. Cell-free supernatant was

    Article Snippet: Similarly, pGCsamEN-EGFP was made by PCR of an EGFP cassette from pCDN3.1-EGFP, using primers P2 and P3, and pGCsamEN-ZsGreen1 was made by PCR of ZsGreen1 from pLVX-IRES-ZsGreen1 (Clontech, Mountain View, CA), using primers P4 and P5.

    Techniques: Expressing, Infection

    HEV ORF1 is able to function in trans to replicate HEV RNA. (a) Schematic representation of the ORF1 transcomplementation system. (b) Representative flow cytometry plots demonstrating efficient ORF1 expression. HepG2C3A cells were transduced with pLVX-ORF1-IRES-zsGreen (wild type [wt] or GAD mutant) or not transduced. Flow cytometric analysis was performed 3 days following transduction to quantify the frequencies of ORF1-expressing cells. FSC, forward scatter. (c) Replication kinetics of HEV RNA in ORF1 transcomplemented HepG2C3A cells. Cell culture supernatants from naive HepG2C3A cells, or HepG2C3A cells transduced with HEV ORF1 or its GAD mutant, were collected at the indicated time points posttransfection with rHEVΔORF2/3[Gluc] Pol- RNA or RNA from its mutants, and Gaussia luciferase (Gluc) activity was quantified. Values are means plus standard deviations (SD) (error bars) ( n = 3). Values that are significantly different ( P

    Journal: mBio

    Article Title: Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription

    doi: 10.1128/mBio.00769-18

    Figure Lengend Snippet: HEV ORF1 is able to function in trans to replicate HEV RNA. (a) Schematic representation of the ORF1 transcomplementation system. (b) Representative flow cytometry plots demonstrating efficient ORF1 expression. HepG2C3A cells were transduced with pLVX-ORF1-IRES-zsGreen (wild type [wt] or GAD mutant) or not transduced. Flow cytometric analysis was performed 3 days following transduction to quantify the frequencies of ORF1-expressing cells. FSC, forward scatter. (c) Replication kinetics of HEV RNA in ORF1 transcomplemented HepG2C3A cells. Cell culture supernatants from naive HepG2C3A cells, or HepG2C3A cells transduced with HEV ORF1 or its GAD mutant, were collected at the indicated time points posttransfection with rHEVΔORF2/3[Gluc] Pol- RNA or RNA from its mutants, and Gaussia luciferase (Gluc) activity was quantified. Values are means plus standard deviations (SD) (error bars) ( n = 3). Values that are significantly different ( P

    Article Snippet: To construct lentiviral constructs encoding ORF1 of Kernow C1/p6 (GenBank accession number JQ679013 ), the Kernow C1/p6 ORF1 cDNA was amplified by PCR from a plasmid encoding the full-length (FL) infectious HEV clone Kernow C1/p6 (a kind gift from Suzanne Emerson, NIH) and then cloned into pLVX-IRES-zsGreen1 vector using an In-Fusion HD cloning kit (Clontech, Mountain View, CA, USA).

    Techniques: Flow Cytometry, Cytometry, Expressing, Transduction, Mutagenesis, Cell Culture, Luciferase, Activity Assay