Structured Review

GeneTex gapdh
Diaph1 deletion improved calcium dynamics after I/R. (a) <t>SERCA2a/GAPDH</t> protein levels were increased by Diaph1 deletion in both mouse hearts after LAD/reperfusion (n = 10/group; *p
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Images

1) Product Images from "The Formin, DIAPH1, is a Key Modulator of Myocardial Ischemia/Reperfusion Injury"

Article Title: The Formin, DIAPH1, is a Key Modulator of Myocardial Ischemia/Reperfusion Injury

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2017.11.012

Diaph1 deletion improved calcium dynamics after I/R. (a) SERCA2a/GAPDH protein levels were increased by Diaph1 deletion in both mouse hearts after LAD/reperfusion (n = 10/group; *p
Figure Legend Snippet: Diaph1 deletion improved calcium dynamics after I/R. (a) SERCA2a/GAPDH protein levels were increased by Diaph1 deletion in both mouse hearts after LAD/reperfusion (n = 10/group; *p

Techniques Used:

2) Product Images from "Differential expression of myocardial heat shock proteins in rats acutely exposed to fluoride"

Article Title: Differential expression of myocardial heat shock proteins in rats acutely exposed to fluoride

Journal: Cell Stress & Chaperones

doi: 10.1007/s12192-017-0801-1

Effect of acute F − intoxication on myocardial protein expression of Hsf1 and Hsps in rats. Western blotting and its densitometric analysis of myocardial Hsf1 and Hsps (Hsp27, Hsp32, Hsp40, Hsp60, Hsp70, and Hsp90) in control and F − -treated rats. GAPDH was used as loading control. The values represent mean ± SD ( n = 3). *p
Figure Legend Snippet: Effect of acute F − intoxication on myocardial protein expression of Hsf1 and Hsps in rats. Western blotting and its densitometric analysis of myocardial Hsf1 and Hsps (Hsp27, Hsp32, Hsp40, Hsp60, Hsp70, and Hsp90) in control and F − -treated rats. GAPDH was used as loading control. The values represent mean ± SD ( n = 3). *p

Techniques Used: Expressing, Western Blot

Effect of acute F − intoxication on myocardial mRNA expression of Hsf1 and Hsps in rats. mRNA expression and its densitometric analysis of myocardial Hsf1 and Hsps (Hsp27, Hsp32, Hsp40, Hsp60, Hsp70, and Hsp90) in control and F − -treated rats. GAPDH was used as loading control. The values represent mean ± SD ( n = 3). *p
Figure Legend Snippet: Effect of acute F − intoxication on myocardial mRNA expression of Hsf1 and Hsps in rats. mRNA expression and its densitometric analysis of myocardial Hsf1 and Hsps (Hsp27, Hsp32, Hsp40, Hsp60, Hsp70, and Hsp90) in control and F − -treated rats. GAPDH was used as loading control. The values represent mean ± SD ( n = 3). *p

Techniques Used: Expressing

3) Product Images from "Arginine methylation of hnRNPK negatively modulates apoptosis upon DNA damage through local regulation of phosphorylation"

Article Title: Arginine methylation of hnRNPK negatively modulates apoptosis upon DNA damage through local regulation of phosphorylation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku705

Blockage of Arg296 and Arg299 methylation in hnRNPK promotes apoptosis in a p53-independent manner. ( a ) SaOS-2 cells were simultaneously infected with lentivirus carrying shRNA against endogenous hnRNPK and lentivirus carrying shRNA-resistant WT or 2RK mutant hnRNPKs. The efficiency of knockdown and ectopic expression was determined after measuring the protein levels of endogenous and exogenous hnRNPKs using hnRNPK and GAPDH antibodies. ( b ) The SaOS2-K-WT and SaOS2-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ for 24 h and subsequently treated with etoposide for 12 h. The cell lysates were collected and analyzed for Myc-PKCδ, GAPDH and cleaved caspase 3 expression levels using specific antibodies.
Figure Legend Snippet: Blockage of Arg296 and Arg299 methylation in hnRNPK promotes apoptosis in a p53-independent manner. ( a ) SaOS-2 cells were simultaneously infected with lentivirus carrying shRNA against endogenous hnRNPK and lentivirus carrying shRNA-resistant WT or 2RK mutant hnRNPKs. The efficiency of knockdown and ectopic expression was determined after measuring the protein levels of endogenous and exogenous hnRNPKs using hnRNPK and GAPDH antibodies. ( b ) The SaOS2-K-WT and SaOS2-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ for 24 h and subsequently treated with etoposide for 12 h. The cell lysates were collected and analyzed for Myc-PKCδ, GAPDH and cleaved caspase 3 expression levels using specific antibodies.

Techniques Used: Methylation, Infection, shRNA, Mutagenesis, Expressing, Transfection

Blockage of Arg299 and Arg296 methylation in hnRNPK promotes U2OS cell apoptosis upon DNA damage. ( a ) Establishment of stable cell lines carrying Arg296 and Arg299 methylation-defective hnRNPK. U2OS cells were simultaneously infected with lentivirus carrying shRNA against endogenous hnRNPK and lentivirus carrying shRNA-resistant WT or 2RK mutant hnRNPKs. The efficiency of knockdown and ectopic expression were determined according to the protein levels of endogenous and exogenous hnRNPKs, measured using hnRNPK and GAPDH antibodies. ( b ) U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ for 24 h and treated with etoposide for the indicated times. The cell lysates were collected, stained with propidium iodide and measured through FACS to calculate the percentages of sub-G1 cells. The data are shown as the mean value and SD from three independent experiments. ( c ) Under the same treatment as described above, the cells were collected at the indicated times and analyzed using a TUNEL assay to determine the percentages of apoptotic cells through FACS. ( d ) U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells under the same treatment at 12 h were collected and analyzed for the expression levels of Myc-PKCδ, GAPDH and cleaved caspase 3 using specific antibodies. Pretreatment with the PKCδ inhibitor rottlerin in U2OS-K-2RK cells prior to etoposide treatment was also performed. ( e ) Arg296 and Arg299 methylation-defective hnRNPK promotes apoptosis via both extrinsic and intrinsic pathways. U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ, followed by etoposide treatment for 12 h. The expression levels of Myc-PKCδ and GAPDH and cleaved caspases 3, 8 and 9 were measured using specific antibodies.
Figure Legend Snippet: Blockage of Arg299 and Arg296 methylation in hnRNPK promotes U2OS cell apoptosis upon DNA damage. ( a ) Establishment of stable cell lines carrying Arg296 and Arg299 methylation-defective hnRNPK. U2OS cells were simultaneously infected with lentivirus carrying shRNA against endogenous hnRNPK and lentivirus carrying shRNA-resistant WT or 2RK mutant hnRNPKs. The efficiency of knockdown and ectopic expression were determined according to the protein levels of endogenous and exogenous hnRNPKs, measured using hnRNPK and GAPDH antibodies. ( b ) U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ for 24 h and treated with etoposide for the indicated times. The cell lysates were collected, stained with propidium iodide and measured through FACS to calculate the percentages of sub-G1 cells. The data are shown as the mean value and SD from three independent experiments. ( c ) Under the same treatment as described above, the cells were collected at the indicated times and analyzed using a TUNEL assay to determine the percentages of apoptotic cells through FACS. ( d ) U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells under the same treatment at 12 h were collected and analyzed for the expression levels of Myc-PKCδ, GAPDH and cleaved caspase 3 using specific antibodies. Pretreatment with the PKCδ inhibitor rottlerin in U2OS-K-2RK cells prior to etoposide treatment was also performed. ( e ) Arg296 and Arg299 methylation-defective hnRNPK promotes apoptosis via both extrinsic and intrinsic pathways. U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ, followed by etoposide treatment for 12 h. The expression levels of Myc-PKCδ and GAPDH and cleaved caspases 3, 8 and 9 were measured using specific antibodies.

Techniques Used: Methylation, Stable Transfection, Infection, shRNA, Mutagenesis, Expressing, Transfection, Staining, FACS, TUNEL Assay

Supplementing with exogenous hnRNPK carrying R296/R299 methylation reduced U2OS-K-2RK cell apoptosis after DNA damage. The U2OS-K-2RK cells were transfected with shRNA-resistant WT or 3RK-2 mutant (R256K/ R258K/ R268K) hnRNPKs, followed by etoposide treatment for 12 h. The expression levels of hnRNPK, GAPDH and cleaved caspase 3 were determined using specific antibodies.
Figure Legend Snippet: Supplementing with exogenous hnRNPK carrying R296/R299 methylation reduced U2OS-K-2RK cell apoptosis after DNA damage. The U2OS-K-2RK cells were transfected with shRNA-resistant WT or 3RK-2 mutant (R256K/ R258K/ R268K) hnRNPKs, followed by etoposide treatment for 12 h. The expression levels of hnRNPK, GAPDH and cleaved caspase 3 were determined using specific antibodies.

Techniques Used: Methylation, Transfection, shRNA, Mutagenesis, Expressing

4) Product Images from "Proteomic Analysis of the Mammalian Katanin Family of Microtubule-severing Enzymes Defines Katanin p80 subunit B-like 1 (KATNBL1) as a Regulator of Mammalian Katanin Microtubule-severing *"

Article Title: Proteomic Analysis of the Mammalian Katanin Family of Microtubule-severing Enzymes Defines Katanin p80 subunit B-like 1 (KATNBL1) as a Regulator of Mammalian Katanin Microtubule-severing *

Journal: Molecular & Cellular Proteomics : MCP

doi: 10.1074/mcp.M115.056465

The Katanin-Katanin pairwise binding assays. A, B , In cell Katanin subunit pairwise binding reactions. LAP-tagged GFP-B1 ( A ) or GFP-BL1 ( B ) HeLa stable cell lines were transfected with HA-tagged A1, AL1, or AL2 subunits or the negative control (Ctrl) STARD9-START. HA-A1, HA-AL1, HA-AL2, or HA-Ctrl were immunoprecipitated from 140 μg of protein extracts. Six percent of the input and all of the immunoprecipitates were western blotted (WB) for the indicated GFP-tagged B subunits (using anti-GFP antibodies) and the HA-tagged A subunits or HA-Ctrl (using anti-HA antibodies). Note that the negative control HA-Ctrl does not bind to either Katanin B subunit. Additionally, the negative control GAPDH protein is not found in any of the immunoprecipitations. C, D , In vitro 35 S-radiolabeled Katanin subunit pairwise binding reactions. In vitro transcribed and translated 35 S-radiolabeled FLAG-tagged A subunits or negative control (Ctrl) STARD9-MD and HA-tagged B1 ( C ) or BL1 ( D ) subunits were used for pairwise in vitro binding reactions as indicated. HA-tagged Katanin B subunits were then immunoprecipitated and the radiolabeled Katanin A and B subunits and Ctrl in the immunoprecipitates were visualized by autoradiography. Note that FLAG-A1 and FLAG-AL1 co-precipitate with HA-B1 and HA-BL1, whereas FLAG-AL2 and the negative control FLAG-Ctrl do not. E , Summary of in cell and in vitro binding data. Arrows indicate the direction of the interaction, double arrows indicate that the interaction was recapitulated in reciprocal co-immunoprecipitations. See supplemental Fig. S1 for in cell and in vitro reciprocal Katanin subunit binding data.
Figure Legend Snippet: The Katanin-Katanin pairwise binding assays. A, B , In cell Katanin subunit pairwise binding reactions. LAP-tagged GFP-B1 ( A ) or GFP-BL1 ( B ) HeLa stable cell lines were transfected with HA-tagged A1, AL1, or AL2 subunits or the negative control (Ctrl) STARD9-START. HA-A1, HA-AL1, HA-AL2, or HA-Ctrl were immunoprecipitated from 140 μg of protein extracts. Six percent of the input and all of the immunoprecipitates were western blotted (WB) for the indicated GFP-tagged B subunits (using anti-GFP antibodies) and the HA-tagged A subunits or HA-Ctrl (using anti-HA antibodies). Note that the negative control HA-Ctrl does not bind to either Katanin B subunit. Additionally, the negative control GAPDH protein is not found in any of the immunoprecipitations. C, D , In vitro 35 S-radiolabeled Katanin subunit pairwise binding reactions. In vitro transcribed and translated 35 S-radiolabeled FLAG-tagged A subunits or negative control (Ctrl) STARD9-MD and HA-tagged B1 ( C ) or BL1 ( D ) subunits were used for pairwise in vitro binding reactions as indicated. HA-tagged Katanin B subunits were then immunoprecipitated and the radiolabeled Katanin A and B subunits and Ctrl in the immunoprecipitates were visualized by autoradiography. Note that FLAG-A1 and FLAG-AL1 co-precipitate with HA-B1 and HA-BL1, whereas FLAG-AL2 and the negative control FLAG-Ctrl do not. E , Summary of in cell and in vitro binding data. Arrows indicate the direction of the interaction, double arrows indicate that the interaction was recapitulated in reciprocal co-immunoprecipitations. See supplemental Fig. S1 for in cell and in vitro reciprocal Katanin subunit binding data.

Techniques Used: Binding Assay, Stable Transfection, Transfection, Negative Control, Immunoprecipitation, Western Blot, In Vitro, Autoradiography

5) Product Images from "The Formin, DIAPH1, is a Key Modulator of Myocardial Ischemia/Reperfusion Injury"

Article Title: The Formin, DIAPH1, is a Key Modulator of Myocardial Ischemia/Reperfusion Injury

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2017.11.012

Diaph1 deletion improved calcium dynamics after I/R. (a) SERCA2a/GAPDH protein levels were increased by Diaph1 deletion in both mouse hearts after LAD/reperfusion (n = 10/group; *p
Figure Legend Snippet: Diaph1 deletion improved calcium dynamics after I/R. (a) SERCA2a/GAPDH protein levels were increased by Diaph1 deletion in both mouse hearts after LAD/reperfusion (n = 10/group; *p

Techniques Used:

6) Product Images from "MicroRNA let-7g inhibits angiotensin II-induced endothelial senescence via the LOX-1-independent mechanism"

Article Title: MicroRNA let-7g inhibits angiotensin II-induced endothelial senescence via the LOX-1-independent mechanism

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2018.3416

Angiotensin II influences the expression of β-gal, SIRT1 and IGF1 pathway. The mRNA levels of (A) β-gal, (B) SIRT1, (C) LOX-1, (D) IGF1 and (E) IGF1R are presented. GAPDH was used as the internal control. Values are expressed as the mean ± standard error of the mean. * P
Figure Legend Snippet: Angiotensin II influences the expression of β-gal, SIRT1 and IGF1 pathway. The mRNA levels of (A) β-gal, (B) SIRT1, (C) LOX-1, (D) IGF1 and (E) IGF1R are presented. GAPDH was used as the internal control. Values are expressed as the mean ± standard error of the mean. * P

Techniques Used: Expressing

7) Product Images from "Delaying histone deacetylase response to injury accelerates conversion into repair Schwann cells and nerve regeneration"

Article Title: Delaying histone deacetylase response to injury accelerates conversion into repair Schwann cells and nerve regeneration

Journal: Nature Communications

doi: 10.1038/ncomms14272

Assembly of multifunctional protein complex. ( a – c ) Non-denaturing IP of Sox10, HDAC2, JMJD2C, KDM3A or ctrl (GFP or Flag) in unlesioned (no lesion) adult mouse sciatic nerve lysates or at 1 dpl or 12 dpl, and western blot of HDAC2 ( a ), KDM3A ( b ) or Sox10 ( c ). Membranes where Sox10 and JMJD2C IPs were run together were first blotted with the HDAC2 antibody ( a ) and were re-blotted with the Sox10 antibody ( c ). GAPDH western blots on lysates used for IP show the inputs (in a , only one input for no lesion IP KDM3A and ctrl that were done on the same lysate divided by two). Sample size: each IP was done three times, using nerves of three different animals. One nerve was used per IP.
Figure Legend Snippet: Assembly of multifunctional protein complex. ( a – c ) Non-denaturing IP of Sox10, HDAC2, JMJD2C, KDM3A or ctrl (GFP or Flag) in unlesioned (no lesion) adult mouse sciatic nerve lysates or at 1 dpl or 12 dpl, and western blot of HDAC2 ( a ), KDM3A ( b ) or Sox10 ( c ). Membranes where Sox10 and JMJD2C IPs were run together were first blotted with the HDAC2 antibody ( a ) and were re-blotted with the Sox10 antibody ( c ). GAPDH western blots on lysates used for IP show the inputs (in a , only one input for no lesion IP KDM3A and ctrl that were done on the same lysate divided by two). Sample size: each IP was done three times, using nerves of three different animals. One nerve was used per IP.

Techniques Used: Western Blot

Reduced Krox20 and P0 expressions in dKO. ( a ) Western blot of Krox20 in crushed (Cr) and contralateral (Co) nerves of control mice at 12 dpl and quantification normalized to GAPDH and compared with Co=1. ( b , f ) Western blots of Krox20 and Sox10 at 12 dpl ( b ) and of P0 at 1 mpl ( f ) showing reduced Krox20 (but unaffected Sox10) and P0 levels in dKO compared with Ctrl crushed nerves at 12 dpl and 1 mpl, respectively. Dashed lines—samples run on the same gel, but not on consecutive lanes. ( c ) Co-immunofluorescence of Krox20 (red) with CD68 (green, macrophages) and DAPI labelling (blue, nuclei) in cryosections of control and dKO sciatic nerves at 12 dpl. White arrows indicate SCs (CD68-negative cells with elongated nuclei, blue arrowheads indicate macrophages (CD68-positive cells). Krox20 ( d ) and P0 ( e ) in situ hybridizations in control and dKO nerves showing decreased transcript levels in dKO nerves. Scale bar, 10 μm. One-tailed (grey asterisk) or two-tailed (black asterisks) Student's t -tests, unpaired ( b ) or paired ( a , f ), P values: * P
Figure Legend Snippet: Reduced Krox20 and P0 expressions in dKO. ( a ) Western blot of Krox20 in crushed (Cr) and contralateral (Co) nerves of control mice at 12 dpl and quantification normalized to GAPDH and compared with Co=1. ( b , f ) Western blots of Krox20 and Sox10 at 12 dpl ( b ) and of P0 at 1 mpl ( f ) showing reduced Krox20 (but unaffected Sox10) and P0 levels in dKO compared with Ctrl crushed nerves at 12 dpl and 1 mpl, respectively. Dashed lines—samples run on the same gel, but not on consecutive lanes. ( c ) Co-immunofluorescence of Krox20 (red) with CD68 (green, macrophages) and DAPI labelling (blue, nuclei) in cryosections of control and dKO sciatic nerves at 12 dpl. White arrows indicate SCs (CD68-negative cells with elongated nuclei, blue arrowheads indicate macrophages (CD68-positive cells). Krox20 ( d ) and P0 ( e ) in situ hybridizations in control and dKO nerves showing decreased transcript levels in dKO nerves. Scale bar, 10 μm. One-tailed (grey asterisk) or two-tailed (black asterisks) Student's t -tests, unpaired ( b ) or paired ( a , f ), P values: * P

Techniques Used: Western Blot, Mouse Assay, Immunofluorescence, In Situ, One-tailed Test, Two Tailed Test

8) Product Images from "A Conserved Acidic-Cluster Motif in SERINC5 Confers Partial Resistance to Antagonism by HIV-1 Nef"

Article Title: A Conserved Acidic-Cluster Motif in SERINC5 Confers Partial Resistance to Antagonism by HIV-1 Nef

Journal: Journal of Virology

doi: 10.1128/JVI.01554-19

Deletion of the EDTEE sequence enhances the activity of diverse Nef proteins. (A) HEK293 cells were cotransfected with plasmid constructs encoding SERINC5-iHA (100 ng) and env - and nef -deficient HIV-1 provirus. Env expression was provided in trans by an NL4-3-derived construct (pVRE). FLAG-tagged Nef derived from NL4-3, SF2, or TF clones CH040 and SUMA and an HA-tagged minimally active glycoGag (HA-gg189) were also provided in trans (via 100 ng plasmid). Virions were harvested at 24 h posttransfection, partially purified by centrifugation through a sucrose cushion, and used to infect HeLa-TZM-bl luciferase indicator cells. Luciferase activity was measured at 48 h postinfection and normalized to that of the p24 antigen (relative light units [RLU]/concentration of p24 [in nanograms per milliliter] ratio). Data are expressed as percent infectivity relative to that for the no-added-SERINC control (100%) for each condition (with or without Nef or glycoGag). Error bars indicate the standard deviations from 3 independent experiments. P values derived from Student's t test are indicated. (B) Protein from whole-cell lysates was subject to SDS-PAGE and Western blotting. The membranes were probed with antibodies to detect SERINC5 (HA), glycoGag (gg189, HA), Nef, p55 (Gag), and GAPDH. The numbers to the left of the gel are molecular masses (in kilodaltons).
Figure Legend Snippet: Deletion of the EDTEE sequence enhances the activity of diverse Nef proteins. (A) HEK293 cells were cotransfected with plasmid constructs encoding SERINC5-iHA (100 ng) and env - and nef -deficient HIV-1 provirus. Env expression was provided in trans by an NL4-3-derived construct (pVRE). FLAG-tagged Nef derived from NL4-3, SF2, or TF clones CH040 and SUMA and an HA-tagged minimally active glycoGag (HA-gg189) were also provided in trans (via 100 ng plasmid). Virions were harvested at 24 h posttransfection, partially purified by centrifugation through a sucrose cushion, and used to infect HeLa-TZM-bl luciferase indicator cells. Luciferase activity was measured at 48 h postinfection and normalized to that of the p24 antigen (relative light units [RLU]/concentration of p24 [in nanograms per milliliter] ratio). Data are expressed as percent infectivity relative to that for the no-added-SERINC control (100%) for each condition (with or without Nef or glycoGag). Error bars indicate the standard deviations from 3 independent experiments. P values derived from Student's t test are indicated. (B) Protein from whole-cell lysates was subject to SDS-PAGE and Western blotting. The membranes were probed with antibodies to detect SERINC5 (HA), glycoGag (gg189, HA), Nef, p55 (Gag), and GAPDH. The numbers to the left of the gel are molecular masses (in kilodaltons).

Techniques Used: Sequencing, Activity Assay, Plasmid Preparation, Construct, Expressing, Derivative Assay, Clone Assay, Purification, Centrifugation, Luciferase, Concentration Assay, Infection, SDS Page, Western Blot

Deletion of the EDTEE sequence does not enhance the activity of MLV glycoGag as an antagonist of SERINC5. (A) HEK293 cells were cotransfected with plasmid constructs encoding the indicated amounts of SERINC5-iHA and either env - and nef -deficient (−Nef) or env -deficient (+Nef) HIV-1 provirus. Env expression was provided in trans by an NL4-3-derived construct (pVRE). An HA-tagged minimally active glycoGag (HA-gg189) was also provided in trans . Virions were harvested at 24 h posttransfection, partially purified by centrifugation through a sucrose cushion, and used to infect HeLa-TZM-bl luciferase indicator cells. Luciferase activity was measured at 48 h postinfection and normalized to that of the p24 antigen (relative light units [RLU]/concentration of p24 [in nanograms per milliliter] ratio). Data are expressed as percent infectivity relative to that for the no-added-SERINC control for each condition (with or without Nef or glycoGag). Error bars indicate the standard deviations from 2 independent experiments. (B) Comparison of relative infectivity of virions in the absence or the presence of 100 ng SERINC5-iHA ( P values derived from Student's t test are indicated). (C) Protein from whole-cell lysates was subject to SDS-PAGE and Western blotting. The membranes were probed with antibodies to detect SERINC5 (HA), glycoGag (gg189, HA), Nef, p55 (Gag), and GAPDH. The numbers to the left of the gel are molecular masses (in kilodaltons). No Nef/gg, expression of neither Nef nor gg189.
Figure Legend Snippet: Deletion of the EDTEE sequence does not enhance the activity of MLV glycoGag as an antagonist of SERINC5. (A) HEK293 cells were cotransfected with plasmid constructs encoding the indicated amounts of SERINC5-iHA and either env - and nef -deficient (−Nef) or env -deficient (+Nef) HIV-1 provirus. Env expression was provided in trans by an NL4-3-derived construct (pVRE). An HA-tagged minimally active glycoGag (HA-gg189) was also provided in trans . Virions were harvested at 24 h posttransfection, partially purified by centrifugation through a sucrose cushion, and used to infect HeLa-TZM-bl luciferase indicator cells. Luciferase activity was measured at 48 h postinfection and normalized to that of the p24 antigen (relative light units [RLU]/concentration of p24 [in nanograms per milliliter] ratio). Data are expressed as percent infectivity relative to that for the no-added-SERINC control for each condition (with or without Nef or glycoGag). Error bars indicate the standard deviations from 2 independent experiments. (B) Comparison of relative infectivity of virions in the absence or the presence of 100 ng SERINC5-iHA ( P values derived from Student's t test are indicated). (C) Protein from whole-cell lysates was subject to SDS-PAGE and Western blotting. The membranes were probed with antibodies to detect SERINC5 (HA), glycoGag (gg189, HA), Nef, p55 (Gag), and GAPDH. The numbers to the left of the gel are molecular masses (in kilodaltons). No Nef/gg, expression of neither Nef nor gg189.

Techniques Used: Sequencing, Activity Assay, Plasmid Preparation, Construct, Expressing, Derivative Assay, Purification, Centrifugation, Luciferase, Concentration Assay, Infection, SDS Page, Western Blot

9) Product Images from "S-Adenosyl-l-methionine-competitive inhibitors of the histone methyltransferase EZH2 induce autophagy and enhance drug sensitivity in cancer cells"

Article Title: S-Adenosyl-l-methionine-competitive inhibitors of the histone methyltransferase EZH2 induce autophagy and enhance drug sensitivity in cancer cells

Journal: Anti-Cancer Drugs

doi: 10.1097/CAD.0000000000000166

Effects of DZNep and GSK343 on the anticancer activity of sorafenib in HepG2 cells. (a) HepG2 cells were treated with 2.5–10 μmol/l sorafenib for 24 h. Whole-cell lysates were subjected to a western blot analysis using antibodies against EZH2, LC3B, or GAPDH. (b) HepG2 cells were treated with various doses of sorafenib in the absence and presence of 10 μmol/l DZNep or GSK343 for 72 h. Cell viability was analyzed using an MTT assay. * P
Figure Legend Snippet: Effects of DZNep and GSK343 on the anticancer activity of sorafenib in HepG2 cells. (a) HepG2 cells were treated with 2.5–10 μmol/l sorafenib for 24 h. Whole-cell lysates were subjected to a western blot analysis using antibodies against EZH2, LC3B, or GAPDH. (b) HepG2 cells were treated with various doses of sorafenib in the absence and presence of 10 μmol/l DZNep or GSK343 for 72 h. Cell viability was analyzed using an MTT assay. * P

Techniques Used: Activity Assay, Western Blot, MTT Assay

Effects of DZNep and GSK343 on the cell viability of MDA-MB-231 cells. (a) Chemical structures of DZNep and GSK343. (b) MDA-MB-231 cells were treated with different doses of DZNep or GSK343 for 72 h, and cell viability was analyzed using an MTT assay. (c) MDA-MB-231 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 72 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against H3K27-me3 or GAPDH. (d) MDA-MB-231 cells were treated with 10 and 20 μmol/l DZNep or GSK343, or 0.75 mol/l doxorubicin (DOXO) for 72 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against PARP1, caspase-3, LC3B, or GAPDH. DZNep, 3-deazaneplanocin A; EZH2, enhancer of zeste homolog 2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; SAH, S -adenosyl- l -homocysteine; SAM, S -adenosyl- l -methionine.
Figure Legend Snippet: Effects of DZNep and GSK343 on the cell viability of MDA-MB-231 cells. (a) Chemical structures of DZNep and GSK343. (b) MDA-MB-231 cells were treated with different doses of DZNep or GSK343 for 72 h, and cell viability was analyzed using an MTT assay. (c) MDA-MB-231 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 72 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against H3K27-me3 or GAPDH. (d) MDA-MB-231 cells were treated with 10 and 20 μmol/l DZNep or GSK343, or 0.75 mol/l doxorubicin (DOXO) for 72 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against PARP1, caspase-3, LC3B, or GAPDH. DZNep, 3-deazaneplanocin A; EZH2, enhancer of zeste homolog 2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; SAH, S -adenosyl- l -homocysteine; SAM, S -adenosyl- l -methionine.

Techniques Used: Multiple Displacement Amplification, MTT Assay, Western Blot

Effects of DZNep and GSK343 on autophagy of MDA-MB-231 cells. (a) MDA-MB-231 cells were treated with 10 and 20 μmol/l DZNep or GSK343 for 24 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B or GAPDH. (b) MDA-MB-231 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 24 h, and 20 nmol/l bafilomycin was added 4 h before cells were harvested. Whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B, p62, LAMP2, or GAPDH. (c) MDA-MB-231 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 24 h, and then stained by Cyto-ID autophagy detection reagent. The Cyto-ID fluorescence was analyzed by fluorescence microscopy. (d) MDA-MB-231 cells were treated with 10 μmol/l berberine (BBR), 2 μmol/l SAHA, 1 μmol/l doxorubicin (DOXO), 1 μmol/l taxol, 10 μmol/l VP-16, 10 μmol/l GSK343 (GSK), or 10 μmol/l UNC1999 (UNC) for 48 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B or β-actin. (e) The chemical structure of UNC1999. DZNep, 3-deazaneplanocin A.
Figure Legend Snippet: Effects of DZNep and GSK343 on autophagy of MDA-MB-231 cells. (a) MDA-MB-231 cells were treated with 10 and 20 μmol/l DZNep or GSK343 for 24 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B or GAPDH. (b) MDA-MB-231 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 24 h, and 20 nmol/l bafilomycin was added 4 h before cells were harvested. Whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B, p62, LAMP2, or GAPDH. (c) MDA-MB-231 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 24 h, and then stained by Cyto-ID autophagy detection reagent. The Cyto-ID fluorescence was analyzed by fluorescence microscopy. (d) MDA-MB-231 cells were treated with 10 μmol/l berberine (BBR), 2 μmol/l SAHA, 1 μmol/l doxorubicin (DOXO), 1 μmol/l taxol, 10 μmol/l VP-16, 10 μmol/l GSK343 (GSK), or 10 μmol/l UNC1999 (UNC) for 48 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B or β-actin. (e) The chemical structure of UNC1999. DZNep, 3-deazaneplanocin A.

Techniques Used: Multiple Displacement Amplification, Western Blot, Staining, Fluorescence, Microscopy

Effects of DZNep and GSK343 on the cell viability and autophagy of HepG2 and A549 cells. (a) HepG2 and A549 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 72 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against H3K27-me3 or GAPDH. (b) HepG2 and A549 cells were treated with different doses of DZNep or GSK343 for 72 h, and the cell viability was analyzed using an MTT assay. (c) HepG2 and A549 cells were treated with 10 and 20 μmol/l DZNep or GSK343 for 24 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B, GAPDH, or β-actin. (d) HepG2 and A549 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 24 h, and 20 nmol/l bafilomycin was added 4 h before cells were harvested. Whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B, GAPDH, or β-actin. DZNep, 3-deazaneplanocin A; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.
Figure Legend Snippet: Effects of DZNep and GSK343 on the cell viability and autophagy of HepG2 and A549 cells. (a) HepG2 and A549 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 72 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against H3K27-me3 or GAPDH. (b) HepG2 and A549 cells were treated with different doses of DZNep or GSK343 for 72 h, and the cell viability was analyzed using an MTT assay. (c) HepG2 and A549 cells were treated with 10 and 20 μmol/l DZNep or GSK343 for 24 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B, GAPDH, or β-actin. (d) HepG2 and A549 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 24 h, and 20 nmol/l bafilomycin was added 4 h before cells were harvested. Whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B, GAPDH, or β-actin. DZNep, 3-deazaneplanocin A; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

Techniques Used: Western Blot, MTT Assay

Effects of EZH2 knockdown on autophagy of cancer cells. MDA-MB-231, HepG2, and A549 cells were transiently transfected with EZH2 siRNA for 72 h, and then whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B, p62, or GAPDH. EZH2, enhancer of zeste homolog 2; siRNA, small interfering RNA.
Figure Legend Snippet: Effects of EZH2 knockdown on autophagy of cancer cells. MDA-MB-231, HepG2, and A549 cells were transiently transfected with EZH2 siRNA for 72 h, and then whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B, p62, or GAPDH. EZH2, enhancer of zeste homolog 2; siRNA, small interfering RNA.

Techniques Used: Multiple Displacement Amplification, Transfection, Western Blot, Small Interfering RNA

Effect of 3-MA on GSK343-induced autophagy and cytotoxicity of MDA-MB-231 cells. (a) MDA-MB-231 cells were pretreated with 2 and 5 mmol/l 3-MA for 1 h and then exposed to 10 μmol/l GSK343 for 24 h. Whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B or GAPDH. (b) MDA-MB-231 cells were pretreated with 2 mmol/l 3-MA for 1 h and then exposed to various doses of GSK343 for 72 h. Cell viability was analyzed using an MTT assay. Cell viability was analyzed using an MTT assay. * P
Figure Legend Snippet: Effect of 3-MA on GSK343-induced autophagy and cytotoxicity of MDA-MB-231 cells. (a) MDA-MB-231 cells were pretreated with 2 and 5 mmol/l 3-MA for 1 h and then exposed to 10 μmol/l GSK343 for 24 h. Whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B or GAPDH. (b) MDA-MB-231 cells were pretreated with 2 mmol/l 3-MA for 1 h and then exposed to various doses of GSK343 for 72 h. Cell viability was analyzed using an MTT assay. Cell viability was analyzed using an MTT assay. * P

Techniques Used: Multiple Displacement Amplification, Western Blot, MTT Assay

10) Product Images from "An in vivo functional genomics screen of nuclear receptors and their co-regulators identifies FOXA1 as an essential gene in lung tumorigenesis"

Article Title: An in vivo functional genomics screen of nuclear receptors and their co-regulators identifies FOXA1 as an essential gene in lung tumorigenesis

Journal: Neoplasia (New York, N.Y.)

doi: 10.1016/j.neo.2020.04.005

Intersection of transcriptomic and cistromic characterizations of FOXA1 function identifies transcriptional targets in 14q-amplified NSCLCs. (A) Overlap of MSigDB Hallmark gene sets with genes that are positively regulated by FOXA1 (downregulated after FOXA1 knockdown in H1781; top panel) and negatively regulated by FOXA1 (upregulated after FOXA1 knockdown in H1781; bottom panel). (B) Intersection between differentially expressed genes after knockdown of FOXA1 and FOXA1 targets as identified by Chip-Seq in H1819 and H1781 identifies 29 direct FOXA1 targets. Heatmap values in the first two columns are log2 fold changes in genes with matched directional expression changes in H1819-shFOXA1 and H1781-shFOXA1 that were also identified as FOXA1 targets. Genes identified as FOXA1 targets or both FOXA1/NKX2-1 by Chip-seq analysis are indicated with a purple box. (C) QPCR results showing differential expression of IGFBP3 and THBS1 in response to knockdown of FOXA1. RQ = relative quantification, normalized to GAPDH, calculated from three technical replicates. Error bars indicate the upper and lower limits of the RQ value.
Figure Legend Snippet: Intersection of transcriptomic and cistromic characterizations of FOXA1 function identifies transcriptional targets in 14q-amplified NSCLCs. (A) Overlap of MSigDB Hallmark gene sets with genes that are positively regulated by FOXA1 (downregulated after FOXA1 knockdown in H1781; top panel) and negatively regulated by FOXA1 (upregulated after FOXA1 knockdown in H1781; bottom panel). (B) Intersection between differentially expressed genes after knockdown of FOXA1 and FOXA1 targets as identified by Chip-Seq in H1819 and H1781 identifies 29 direct FOXA1 targets. Heatmap values in the first two columns are log2 fold changes in genes with matched directional expression changes in H1819-shFOXA1 and H1781-shFOXA1 that were also identified as FOXA1 targets. Genes identified as FOXA1 targets or both FOXA1/NKX2-1 by Chip-seq analysis are indicated with a purple box. (C) QPCR results showing differential expression of IGFBP3 and THBS1 in response to knockdown of FOXA1. RQ = relative quantification, normalized to GAPDH, calculated from three technical replicates. Error bars indicate the upper and lower limits of the RQ value.

Techniques Used: Amplification, Chromatin Immunoprecipitation, Expressing, Real-time Polymerase Chain Reaction

A panel of NSCLC cell lines exhibits differential dependencies on FOXA1. (A) Knockdown of FOXA1 in a panel of cell lines was verified by Q-RTPCR in all cell lines. RQ = relative quantification, internally normalized to GAPDH, calculated from three technical replicates. (B) Proliferation rates after knockdown of FOXA1 as measured by MTS assay, calculated from at least three technical replicates. RP = relative proliferation, normalized to non-targeting control shRNA (shNTC). (C) Representative colony formation assays in a panel of NSCLC cell lines with knockdown of FOXA1. (D) Quantitation of clonogenicity after knockdown of FOXA1. RC = relative clonogenicity, normalized to shNTC, calculated from at least three technical replicates. (E) Relative clonogenicity compared to FOXA1 copy number and FOXA1 Illumina array-based mRNA expression levels.
Figure Legend Snippet: A panel of NSCLC cell lines exhibits differential dependencies on FOXA1. (A) Knockdown of FOXA1 in a panel of cell lines was verified by Q-RTPCR in all cell lines. RQ = relative quantification, internally normalized to GAPDH, calculated from three technical replicates. (B) Proliferation rates after knockdown of FOXA1 as measured by MTS assay, calculated from at least three technical replicates. RP = relative proliferation, normalized to non-targeting control shRNA (shNTC). (C) Representative colony formation assays in a panel of NSCLC cell lines with knockdown of FOXA1. (D) Quantitation of clonogenicity after knockdown of FOXA1. RC = relative clonogenicity, normalized to shNTC, calculated from at least three technical replicates. (E) Relative clonogenicity compared to FOXA1 copy number and FOXA1 Illumina array-based mRNA expression levels.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, MTS Assay, shRNA, Quantitation Assay, Expressing

FOXA1 is amplified and overexpressed in NSCLCs. (A) FOXA1 is frequently co-amplified with NKX2-1 in NSCLC tumors. Segmented copy number profiles of the genomic region that includes NKX2-1 and FOXA1 , from SNP array datasets on primary lung adenocarcinomas from TCGA (left), and on NSCLC cell lines (right). Samples with ≥4 copies of NKX2-1 are shown in rank order of NKX2-1 amplification from left to right, with corresponding chromosome band regions indicated on the left. (B) Scatter plots comparing FOXA1 copy number with FOXA1 mRNA expression level in cell lines (top), and tumors (bottom). (C) Average FOXA1 expression levels in cell lines compared to HBEC and HSAEC cell lines (top), and in primary tumors compared to normal tissues (bottom). (D) FOXA1 mRNA levels measured by Q-RTPCR in a panel of NSCLC cell lines. RQ = relative quantification, normalized to GAPDH and calculated from three technical replicates. Error bars indicate the upper and lower limits of the RQ value. Copy number for NKX2-1 and FOXA1 are indicated on the bottom. Inset: Scatter plot comparing copy number levels in NSCLC cell lines with mRNA levels. (E) FOXA1 protein expression levels in a panel of cell lines, with copy number indicated on the bottom. (F) Representative images of lung adenocarcinomas from the PROSPECT tumor array harboring 14q-ampflication and high FOXA1 expression (top), or no amplification and low expression (bottom). (G) Scatter plot comparing FOXA1 copy number (aCGH) with quantified IHC staining for FOXA1 on PROSPECT tumor array specimens.
Figure Legend Snippet: FOXA1 is amplified and overexpressed in NSCLCs. (A) FOXA1 is frequently co-amplified with NKX2-1 in NSCLC tumors. Segmented copy number profiles of the genomic region that includes NKX2-1 and FOXA1 , from SNP array datasets on primary lung adenocarcinomas from TCGA (left), and on NSCLC cell lines (right). Samples with ≥4 copies of NKX2-1 are shown in rank order of NKX2-1 amplification from left to right, with corresponding chromosome band regions indicated on the left. (B) Scatter plots comparing FOXA1 copy number with FOXA1 mRNA expression level in cell lines (top), and tumors (bottom). (C) Average FOXA1 expression levels in cell lines compared to HBEC and HSAEC cell lines (top), and in primary tumors compared to normal tissues (bottom). (D) FOXA1 mRNA levels measured by Q-RTPCR in a panel of NSCLC cell lines. RQ = relative quantification, normalized to GAPDH and calculated from three technical replicates. Error bars indicate the upper and lower limits of the RQ value. Copy number for NKX2-1 and FOXA1 are indicated on the bottom. Inset: Scatter plot comparing copy number levels in NSCLC cell lines with mRNA levels. (E) FOXA1 protein expression levels in a panel of cell lines, with copy number indicated on the bottom. (F) Representative images of lung adenocarcinomas from the PROSPECT tumor array harboring 14q-ampflication and high FOXA1 expression (top), or no amplification and low expression (bottom). (G) Scatter plot comparing FOXA1 copy number (aCGH) with quantified IHC staining for FOXA1 on PROSPECT tumor array specimens.

Techniques Used: Amplification, Expressing, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Staining

Knockdown of FOXA1 reduces clonogenicity and xenograft growth in H1819 but not in H1299. (A) QPCR for FOXA1 mRNA with shFOXA1. RQ = relative quantification, normalized to GAPDH and calculated from three technical replicates. Error bars indicate upper and lower limits of the RQ value. (B) Proliferation over 5 days as measured by MTS. RP = relative proliferation, calculated from at least three technical replicates. Error bars indicate standard deviations. Statistical significance was determined by unpaired t -test ( p
Figure Legend Snippet: Knockdown of FOXA1 reduces clonogenicity and xenograft growth in H1819 but not in H1299. (A) QPCR for FOXA1 mRNA with shFOXA1. RQ = relative quantification, normalized to GAPDH and calculated from three technical replicates. Error bars indicate upper and lower limits of the RQ value. (B) Proliferation over 5 days as measured by MTS. RP = relative proliferation, calculated from at least three technical replicates. Error bars indicate standard deviations. Statistical significance was determined by unpaired t -test ( p

Techniques Used: Real-time Polymerase Chain Reaction

11) Product Images from "TBK1 Mutation Spectrum in an Extended European Patient Cohort with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis"

Article Title: TBK1 Mutation Spectrum in an Extended European Patient Cohort with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis

Journal: Human Mutation

doi: 10.1002/humu.23161

Transcript and protein analysis of TBK1 LoF and single amino acid deletion mutations. A : gDNA and cDNA sequence traces around the c.288delT (p. Val97Phefs * 2) mutation, showing reduced expression of the mutant transcript on cDNA extracted from blood. B : gDNA and cDNA sequence traces around the c.379C > T (p.Arg127 * ) mutation, showing the absence of the mutant transcript on cDNA extracted from blood. C : Sizing of cDNA fragments generated with primers in TBK1 exon 10 and exon 13 of the c.1340+1G > A (p.Ala417 * ) carrier on cDNA extracted from blood. Sequence traces from the low‐expressed aberrant transcript demonstrates skipping of exon 11. D : Transcript and protein analysis on brain frontal cortex from the c.235_237delACA (p.Thr79del) carrier and four age‐matched control brains. The graph on the left shows the relative expression in the patient sample (blue) compared with the control samples (black) measured by quantitative real‐time PCR (qRT‐PCR). In the middle, Western blot analysis is shown of protein extracts from the patient carrier compared with control individuals. The upper band represents TBK1 (84 kDa) and the lower band represents the housekeeping protein GAPDH (37 kDa). The graph on the right shows the quantification in the patient sample (blue) and control samples (black) of the TBK1 signal normalized to the signal of GAPDH. Error bars represent the SD. E : Western blot analysis of phosphorylated TBK1 (Ser172, p‐TBK1) (upper band, 84 kDa) in HEK293T cells overexpressing the in‐frame single amino acid deletions (p.Thr79del, p.Asp167del, and p.Glu643del) compared with wild type, relative to GAPDH (lower band, 37 kDa). Mock and kinase dead (p.Ser172Ala, KD) were used as negative control. cDNA numbering according to reference sequence NM_013254.3, in addition, for intronic variants, the genomic reference sequence NC_000012.12 was used. Nucleotide positions refer to cDNA sequence and nucleotide numbering uses +1 as the A of the ATG translation initiation codon in the reference sequence, with the initiation codon as codon 1. Protein numbering according to reference sequence NP_037386.1.
Figure Legend Snippet: Transcript and protein analysis of TBK1 LoF and single amino acid deletion mutations. A : gDNA and cDNA sequence traces around the c.288delT (p. Val97Phefs * 2) mutation, showing reduced expression of the mutant transcript on cDNA extracted from blood. B : gDNA and cDNA sequence traces around the c.379C > T (p.Arg127 * ) mutation, showing the absence of the mutant transcript on cDNA extracted from blood. C : Sizing of cDNA fragments generated with primers in TBK1 exon 10 and exon 13 of the c.1340+1G > A (p.Ala417 * ) carrier on cDNA extracted from blood. Sequence traces from the low‐expressed aberrant transcript demonstrates skipping of exon 11. D : Transcript and protein analysis on brain frontal cortex from the c.235_237delACA (p.Thr79del) carrier and four age‐matched control brains. The graph on the left shows the relative expression in the patient sample (blue) compared with the control samples (black) measured by quantitative real‐time PCR (qRT‐PCR). In the middle, Western blot analysis is shown of protein extracts from the patient carrier compared with control individuals. The upper band represents TBK1 (84 kDa) and the lower band represents the housekeeping protein GAPDH (37 kDa). The graph on the right shows the quantification in the patient sample (blue) and control samples (black) of the TBK1 signal normalized to the signal of GAPDH. Error bars represent the SD. E : Western blot analysis of phosphorylated TBK1 (Ser172, p‐TBK1) (upper band, 84 kDa) in HEK293T cells overexpressing the in‐frame single amino acid deletions (p.Thr79del, p.Asp167del, and p.Glu643del) compared with wild type, relative to GAPDH (lower band, 37 kDa). Mock and kinase dead (p.Ser172Ala, KD) were used as negative control. cDNA numbering according to reference sequence NM_013254.3, in addition, for intronic variants, the genomic reference sequence NC_000012.12 was used. Nucleotide positions refer to cDNA sequence and nucleotide numbering uses +1 as the A of the ATG translation initiation codon in the reference sequence, with the initiation codon as codon 1. Protein numbering according to reference sequence NP_037386.1.

Techniques Used: Sequencing, Mutagenesis, Expressing, Generated, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Negative Control

12) Product Images from "DNA damage-induced regulatory interplay between DAXX, p53, ATM kinase and Wip1 phosphatase"

Article Title: DNA damage-induced regulatory interplay between DAXX, p53, ATM kinase and Wip1 phosphatase

Journal: Cell Cycle

doi: 10.4161/15384101.2014.988019

DAXX depletion or S564A mutation does not affect Mdm2/p53 stability or p53‑mediated gene expression. ( A ) BJ fibroblasts stably transduced with empty lentiviral pCDH vector or either pCDH-DAXX WT or pCDH-DAXX S564A were treated with 40 μM VP16 for 0, 2, 4 or 6 hours and RNA expression of the indicated p53-dependent genes was analyzed by quantitative RT‑PCR. Expression values were normalized to the average of 3 reference genes (β-actin, SDH and ALAS). ( B ) Transduced BJ fibroblast as in ( A ) were exposed to 40 μM VP16 for the indicated times and subjected to protein gel blotting analysis using add p53 - antibodies against DAXX, phospho-p53 (S15), p53 or p21. ( C ) U2OS cells transfected with pXJ41 Hdm2 (human Mdm2) together with empty FLAG-CMV, FLAG-DAXX WT or FLAG-DAXX S564A were treated with 50 μl/ml CHX alone or together with 10 μM VP16 for the specified time points. Cell were harvested and lysates separated by SDS–PAGE and probed with indicated antibodies. ( D ) BJ fibroblasts were depleted by control siRNA (siLuc) or siRNA against Wip1 and 3 d after transfection treated with 4 nM NCS. Cells were lysed at the indicated time points after DNA damage and analyzed by western blotting using labeled antibodies. GAPDH was used as a loading control.
Figure Legend Snippet: DAXX depletion or S564A mutation does not affect Mdm2/p53 stability or p53‑mediated gene expression. ( A ) BJ fibroblasts stably transduced with empty lentiviral pCDH vector or either pCDH-DAXX WT or pCDH-DAXX S564A were treated with 40 μM VP16 for 0, 2, 4 or 6 hours and RNA expression of the indicated p53-dependent genes was analyzed by quantitative RT‑PCR. Expression values were normalized to the average of 3 reference genes (β-actin, SDH and ALAS). ( B ) Transduced BJ fibroblast as in ( A ) were exposed to 40 μM VP16 for the indicated times and subjected to protein gel blotting analysis using add p53 - antibodies against DAXX, phospho-p53 (S15), p53 or p21. ( C ) U2OS cells transfected with pXJ41 Hdm2 (human Mdm2) together with empty FLAG-CMV, FLAG-DAXX WT or FLAG-DAXX S564A were treated with 50 μl/ml CHX alone or together with 10 μM VP16 for the specified time points. Cell were harvested and lysates separated by SDS–PAGE and probed with indicated antibodies. ( D ) BJ fibroblasts were depleted by control siRNA (siLuc) or siRNA against Wip1 and 3 d after transfection treated with 4 nM NCS. Cells were lysed at the indicated time points after DNA damage and analyzed by western blotting using labeled antibodies. GAPDH was used as a loading control.

Techniques Used: Mutagenesis, Expressing, Stable Transfection, Transduction, Plasmid Preparation, RNA Expression, Quantitative RT-PCR, Transfection, SDS Page, Western Blot, Labeling

(See previous page) DAXX is phosphorylated on S564 rapidly after DNA damage. ( A ) HEK 293T cells were transfected with a vector expressing FLAG-DAXX WT and treated or not, as indicated, with 8 nM NCS or10 μM VP16 for 1 hour. Cells were lysed and FLAG-tagged proteins immunoprecipitated using anti-FLAG M2 beads, prior to analysis by SDS-PAGE and western blotting with antibodies against phospho-(S/T) ATM/ATR substrate, FLAG M2, phospho-Chk2 (T68) and DAXX. ( B ) HEK 293T cells were transfected with expression constructs encoding FLAG-tagged wild-type DAXX, single (DAXX S564A ), triple or quadruple mutant as indicated and treated with 10 μM VP16 for 1 hour. Cells were lysed, immunoprecipitated using anti-FLAG M2 beads and analyzed by protein gel blotting with indicated antibodies. ( C ) BJ fibroblasts were exposed to 10 Gy of IR, collected at the indicated time points and subjected to western blotting analysis using specific phospho-DAXX (S564), DAXX, phospho-p53 (S15) and phospho-Chk2 (T68) antibodies. GAPDH was used as a loading control. ( D ) BJ fibroblasts were transfected with control (siCTRL) or DAXX siRNA and 3 d later treated or not as indicated with 10 μM VP16 for 1 hour. Immunofluorescence analysis using α-PML (red) and α-P-DAXX (S564) (green) showed that upon DNA damage DAXX is preferentially phosphorylated at PML nuclear bodies.
Figure Legend Snippet: (See previous page) DAXX is phosphorylated on S564 rapidly after DNA damage. ( A ) HEK 293T cells were transfected with a vector expressing FLAG-DAXX WT and treated or not, as indicated, with 8 nM NCS or10 μM VP16 for 1 hour. Cells were lysed and FLAG-tagged proteins immunoprecipitated using anti-FLAG M2 beads, prior to analysis by SDS-PAGE and western blotting with antibodies against phospho-(S/T) ATM/ATR substrate, FLAG M2, phospho-Chk2 (T68) and DAXX. ( B ) HEK 293T cells were transfected with expression constructs encoding FLAG-tagged wild-type DAXX, single (DAXX S564A ), triple or quadruple mutant as indicated and treated with 10 μM VP16 for 1 hour. Cells were lysed, immunoprecipitated using anti-FLAG M2 beads and analyzed by protein gel blotting with indicated antibodies. ( C ) BJ fibroblasts were exposed to 10 Gy of IR, collected at the indicated time points and subjected to western blotting analysis using specific phospho-DAXX (S564), DAXX, phospho-p53 (S15) and phospho-Chk2 (T68) antibodies. GAPDH was used as a loading control. ( D ) BJ fibroblasts were transfected with control (siCTRL) or DAXX siRNA and 3 d later treated or not as indicated with 10 μM VP16 for 1 hour. Immunofluorescence analysis using α-PML (red) and α-P-DAXX (S564) (green) showed that upon DNA damage DAXX is preferentially phosphorylated at PML nuclear bodies.

Techniques Used: Polyacrylamide Gel Electrophoresis, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, SDS Page, Western Blot, Construct, Mutagenesis, Immunofluorescence

DAXX deletion does not affect Mdm2/p53 stability or p53-mediated gene expression. ( A ) Mdm2 and p53 protein stability was examined in control U2OS cells, 2 independent DAXX +/+ clones (0–4, 0–18), 3 independent DAXX −/- clones (17-7, 17-18, 17–42) and in 17-7 DAXX −/- clone stably transduced with pCDH empty vector (EV) or pCDH-DAXX WT . Cells were collected after the treatment with 50 μl/ml CHX at the indicated time and subjected to protein gel blotting analysis using labeled antibodies. THIIF was used as a loading control. ( B ) DAXX +/+ (clone 0–18) and DAXX −/- (clones 17-18) cells were exposed to 4 nM NCS for the specified time points. Lysed cells were then separated by SDS–PAGE and immunoblotted with indicated antibodies. GAPDH was used as a loading control. ( C ) RNA expression of p53-dependent genes 8 hours after the treatment with 10 μM VP16 (expressed as fold change after VP16) in U2OS clones. RNA was analyzed by quantitative RT‑PCR and the expression values were normalized to the average of 3 reference genes (β-actin, SDH and ALAS). ( D ) U2OS cells transfected with control non-targeting siRNA and siRNA against p53 were treated with 10 μM VP16 for 8 hours and RNA expression of p53 and indicated p53-dependent genes was analyzed by quantitative RT‑PCR. The expression values were normalized to the average of 3 reference genes (β-actin, SDH and ALAS).
Figure Legend Snippet: DAXX deletion does not affect Mdm2/p53 stability or p53-mediated gene expression. ( A ) Mdm2 and p53 protein stability was examined in control U2OS cells, 2 independent DAXX +/+ clones (0–4, 0–18), 3 independent DAXX −/- clones (17-7, 17-18, 17–42) and in 17-7 DAXX −/- clone stably transduced with pCDH empty vector (EV) or pCDH-DAXX WT . Cells were collected after the treatment with 50 μl/ml CHX at the indicated time and subjected to protein gel blotting analysis using labeled antibodies. THIIF was used as a loading control. ( B ) DAXX +/+ (clone 0–18) and DAXX −/- (clones 17-18) cells were exposed to 4 nM NCS for the specified time points. Lysed cells were then separated by SDS–PAGE and immunoblotted with indicated antibodies. GAPDH was used as a loading control. ( C ) RNA expression of p53-dependent genes 8 hours after the treatment with 10 μM VP16 (expressed as fold change after VP16) in U2OS clones. RNA was analyzed by quantitative RT‑PCR and the expression values were normalized to the average of 3 reference genes (β-actin, SDH and ALAS). ( D ) U2OS cells transfected with control non-targeting siRNA and siRNA against p53 were treated with 10 μM VP16 for 8 hours and RNA expression of p53 and indicated p53-dependent genes was analyzed by quantitative RT‑PCR. The expression values were normalized to the average of 3 reference genes (β-actin, SDH and ALAS).

Techniques Used: Expressing, Clone Assay, Stable Transfection, Transduction, Plasmid Preparation, Labeling, SDS Page, RNA Expression, Quantitative RT-PCR, Transfection

DAXX is a substrate of Wip1 phosphatase. ( A ) BJ fibroblasts were exposed to 10 Gy or 2 Gy of IR and lysed at the indicated time points after DNA damage. Western blotting analysis using antibodies against phospho-DAXX (S564), DAXX, Wip1 or GAPDH showed that the DAXX S564 dephosphorylation coincides with increased expression of Wip1. ( B ) In vitro phosphatase assay was performed with recombinant wild-type Wip1 or phosphatase-dead Wip1-D314A mutant on FLAG-DAXX WT immunopurified from transfected U2OS cells exposed to DNA damage. Samples were separated by SDS–PAGE and probed with indicated antibodies. As control, phosphatase buffer without Mg 2+ was used or treatment with lambda protein phosphatase (λPP). ( C ) Wip1 was depleted by siRNA in U2OS cells stably expressing FLAG-DAXX WT . Western blotting analysis using indicated antibodies showed that after DNA damage more phosphorylated DAXX is present in Wip1 siRNA treated cells compared to control GAPDH siRNA transfected cells.
Figure Legend Snippet: DAXX is a substrate of Wip1 phosphatase. ( A ) BJ fibroblasts were exposed to 10 Gy or 2 Gy of IR and lysed at the indicated time points after DNA damage. Western blotting analysis using antibodies against phospho-DAXX (S564), DAXX, Wip1 or GAPDH showed that the DAXX S564 dephosphorylation coincides with increased expression of Wip1. ( B ) In vitro phosphatase assay was performed with recombinant wild-type Wip1 or phosphatase-dead Wip1-D314A mutant on FLAG-DAXX WT immunopurified from transfected U2OS cells exposed to DNA damage. Samples were separated by SDS–PAGE and probed with indicated antibodies. As control, phosphatase buffer without Mg 2+ was used or treatment with lambda protein phosphatase (λPP). ( C ) Wip1 was depleted by siRNA in U2OS cells stably expressing FLAG-DAXX WT . Western blotting analysis using indicated antibodies showed that after DNA damage more phosphorylated DAXX is present in Wip1 siRNA treated cells compared to control GAPDH siRNA transfected cells.

Techniques Used: Western Blot, De-Phosphorylation Assay, Expressing, In Vitro, Phosphatase Assay, Recombinant, Mutagenesis, Transfection, SDS Page, Stable Transfection

13) Product Images from "Conditional Deletion of the L-Type Calcium Channel Cav1.2 in NG2-Positive Cells Impairs Remyelination in Mice"

Article Title: Conditional Deletion of the L-Type Calcium Channel Cav1.2 in NG2-Positive Cells Impairs Remyelination in Mice

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1787-17.2017

Black Gold II staining for myelin in the Cav1.2 KO brain. A , Black Gold II staining in representative coronal sections of brain tissue collected from untreated animals (untreated), mice treated with CPZ for 7 weeks (CPZ), and control and Cav1.2 KO mice at 4 weeks of recovery (4W Rec). Scale bar, 360 μm. Control and Cav1.2 KO mice were injected following Protocol ( a ). B , Black Gold II integrated staining intensity was quantified in the lateral area of the corpus callosum (CC), cingulate cortex (CX), and striatum (ST) at 1 and 4 weeks of recovery (1, 4W Rec). C , Total proteins were collected from the whole brain to assess the expression of CNP, MOG, and MBP by Western blot. GAPDH was used as the internal standard and data from four independent experiments are summarized based on the relative spot intensities and plotted as percentage of the control. Comparisons between experimental groups were made by the unpaired t test. Data represent pooled results from at least four brains per experimental group and values are expressed as mean ± SEM. ## p
Figure Legend Snippet: Black Gold II staining for myelin in the Cav1.2 KO brain. A , Black Gold II staining in representative coronal sections of brain tissue collected from untreated animals (untreated), mice treated with CPZ for 7 weeks (CPZ), and control and Cav1.2 KO mice at 4 weeks of recovery (4W Rec). Scale bar, 360 μm. Control and Cav1.2 KO mice were injected following Protocol ( a ). B , Black Gold II integrated staining intensity was quantified in the lateral area of the corpus callosum (CC), cingulate cortex (CX), and striatum (ST) at 1 and 4 weeks of recovery (1, 4W Rec). C , Total proteins were collected from the whole brain to assess the expression of CNP, MOG, and MBP by Western blot. GAPDH was used as the internal standard and data from four independent experiments are summarized based on the relative spot intensities and plotted as percentage of the control. Comparisons between experimental groups were made by the unpaired t test. Data represent pooled results from at least four brains per experimental group and values are expressed as mean ± SEM. ## p

Techniques Used: Staining, Mouse Assay, Injection, Expressing, Western Blot

14) Product Images from "Berberine Derivatives Suppress Cellular Proliferation and Tumorigenesis In Vitro in Human Non-Small-Cell Lung Cancer Cells"

Article Title: Berberine Derivatives Suppress Cellular Proliferation and Tumorigenesis In Vitro in Human Non-Small-Cell Lung Cancer Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms21124218

Cell cycle profile for in non-small-cell lung cancer cells after incubation with B6 and B7. ( A ) A549 and ( B ) H1435 cells were incubated with control medium or B6 and B7 for 24 h, and the cell cycle was examined with FACS flow cytometry. Additionally, the total cell lysate was isolated and the expressions of CDK2, CDK4, and p21 were verified with western blotting. DMSO was used as a negative control. GAPDH was used as a loading control. Three independent experiments were conducted.
Figure Legend Snippet: Cell cycle profile for in non-small-cell lung cancer cells after incubation with B6 and B7. ( A ) A549 and ( B ) H1435 cells were incubated with control medium or B6 and B7 for 24 h, and the cell cycle was examined with FACS flow cytometry. Additionally, the total cell lysate was isolated and the expressions of CDK2, CDK4, and p21 were verified with western blotting. DMSO was used as a negative control. GAPDH was used as a loading control. Three independent experiments were conducted.

Techniques Used: Incubation, FACS, Flow Cytometry, Isolation, Western Blot, Negative Control

15) Product Images from "EEF1A1 deacetylation enables transcriptional activation of remyelination"

Article Title: EEF1A1 deacetylation enables transcriptional activation of remyelination

Journal: Nature Communications

doi: 10.1038/s41467-020-17243-z

Theophylline promotes CNS remyelination in young and old adults. a Co-immunofluorescence of Ac-eEF1A and CC1 (marker of mature OLs) and DAPI labeling in white matter of adult mouse spinal cord 24 h after treatment with mocetinostat or its vehicle. b , c Co-immunofluorescence of Ac-eEF1A ( b ) or Sox10 ( c ) with CC1 and DAPI labeling in differentiated primary OLs treated with mocetinostat or its vehicle for 24 h. d – f Co-immunoprecipitation (IP) of Ac-eEF1A with Sox10 ( d ) or Sox10 Western blot ( e ) and quantification normalized to GAPDH, or co-immunofluorescence of HDAC2 or Sox10 with CC1 and DAPI labeling ( f ) in spinal cords of PLPCreERT2 ; Hdac1fl/fl ; Hdac2fl/fl (dKO) mice or control littermate (Ctrl) at 14 days post tamoxifen injections. g Co-immunofluorescence of HDAC2, Sox10 and MBP and DAPI labeling in the spinal cord lesion site of adult mice (3–4-month old) at 14dpdl after a 4-day treatment with theophylline (Theo) or its vehicle. Arrows indicate OLs. h – j Electron micrographs of spinal cord ultrathin sections in the lesion site ( h , i ) or unlesioned site ( i , j ) at 14 and/or 30dpdl in young adult (4-month old) ( h , j ) or old (19-month old) ( i ) mice, and % of remyelinated axons in the lesion site (delineated by axons with thin myelin, g ratio ≥ 0.835) or g ratio in unlesioned site. Data are presented as mean values ± SEM. Unpaired ( h , i , j ) or paired ( d , e ) one-tailed (gray asterisk) or two-tailed (black asterisks) Student’s t -tests, p values: 0.047387 ( d ), 0.036845 ( e ), 0.043182 ( h , 14dpl), 0.018162 ( h , 30dpl), 0.127958 ( i , unlesioned), 0.03517 ( i , lesion), 0.328856 ( j , 14dpl), 0.447458 ( j , 30dpl). d n = 5 animals per group, ( e ) n = 3 animals per group, ( h – j ) n = 3 animals per group per time-point. Representative images of 3 animals per group ( a , f , g ) or of 3 independent experiments ( b , c ) are shown. Scale bars: white = 10 µm, black = 5 µM. Source data are provided as a Source Data file.
Figure Legend Snippet: Theophylline promotes CNS remyelination in young and old adults. a Co-immunofluorescence of Ac-eEF1A and CC1 (marker of mature OLs) and DAPI labeling in white matter of adult mouse spinal cord 24 h after treatment with mocetinostat or its vehicle. b , c Co-immunofluorescence of Ac-eEF1A ( b ) or Sox10 ( c ) with CC1 and DAPI labeling in differentiated primary OLs treated with mocetinostat or its vehicle for 24 h. d – f Co-immunoprecipitation (IP) of Ac-eEF1A with Sox10 ( d ) or Sox10 Western blot ( e ) and quantification normalized to GAPDH, or co-immunofluorescence of HDAC2 or Sox10 with CC1 and DAPI labeling ( f ) in spinal cords of PLPCreERT2 ; Hdac1fl/fl ; Hdac2fl/fl (dKO) mice or control littermate (Ctrl) at 14 days post tamoxifen injections. g Co-immunofluorescence of HDAC2, Sox10 and MBP and DAPI labeling in the spinal cord lesion site of adult mice (3–4-month old) at 14dpdl after a 4-day treatment with theophylline (Theo) or its vehicle. Arrows indicate OLs. h – j Electron micrographs of spinal cord ultrathin sections in the lesion site ( h , i ) or unlesioned site ( i , j ) at 14 and/or 30dpdl in young adult (4-month old) ( h , j ) or old (19-month old) ( i ) mice, and % of remyelinated axons in the lesion site (delineated by axons with thin myelin, g ratio ≥ 0.835) or g ratio in unlesioned site. Data are presented as mean values ± SEM. Unpaired ( h , i , j ) or paired ( d , e ) one-tailed (gray asterisk) or two-tailed (black asterisks) Student’s t -tests, p values: 0.047387 ( d ), 0.036845 ( e ), 0.043182 ( h , 14dpl), 0.018162 ( h , 30dpl), 0.127958 ( i , unlesioned), 0.03517 ( i , lesion), 0.328856 ( j , 14dpl), 0.447458 ( j , 30dpl). d n = 5 animals per group, ( e ) n = 3 animals per group, ( h – j ) n = 3 animals per group per time-point. Representative images of 3 animals per group ( a , f , g ) or of 3 independent experiments ( b , c ) are shown. Scale bars: white = 10 µm, black = 5 µM. Source data are provided as a Source Data file.

Techniques Used: Immunofluorescence, Marker, Labeling, Immunoprecipitation, Western Blot, Mouse Assay, One-tailed Test, Two Tailed Test

Tip60 acetylates eEF1A1 in a Stat3-dependent manner. a , e eEF1A1 immunoprecipitation (IP) in lysates of SCs induced to de-differentiate for 1 day and treated with TH1864 (Tip60 inhibitor, Tip60i) or vehicle, and Tip60, Stat3 ( a ) or Ac-eEF1A ( e ) Western blot and quantification (normalized to eEF1A1) of Tip60 co-immunoprecipitated with eEF1A1 ( a ) or of Ac-eEF1A1 ( e ) in Tip60 inhibitor- compared to vehicle-treated SCs. b IP Stat3 or Flag (Ctrl) in lysates of SCs induced to de-differentiate for 1 day, Tip60 Western blot and Stat3 inputs. c Tip60, GAPDH (cytoplasmic marker) and Lamin A/C (nuclear marker) Western blots in cytoplasmic and nuclear fractions of SCs induced to de-differentiate for 1 day. Dashed lines: samples run on the same gel, but not on consecutive lanes. d Tip60 and GAPDH Western blots in lysates of crushed (Cr) and contralateral (Co) mouse sciatic nerves at 1-3-5-12dpl, and Tip60 quantification normalized to GAPDH in Cr compared to Co. f Sox10 and GAPDH Western blots in lysates of SCs induced to de-differentiate and treated with Tip60i or vehicle for 3 days and Sox10 quantification normalized to GAPDH. g Stat3 and Tip60 Western blots in SCs transduced with Stat3-specific shRNA (Stat3 sh) or non-targeting control shRNA (Ctrl sh) lentiviruses and quantification of sumoylated (higher molecular weight) and non-sumoylated (lower molecular weight) Tip60 isoforms normalized to GAPDH. h eEF1A1 IP and Tip60 Western blot in SCs transduced with Stat3 sh or Ctrl sh lentiviruses. i Stat3 and Ac-eEF1A co-immunofluorescence and DAPI labeling in primary SCs transduced with Stat3 sh or Ctrl sh lentiviruses and induced to de-differentiate for 2 days. Scale bar: 20 µm. Data presented as mean values ± SEM. Unpaired ( f ) or paired ( a , d , e , g ) one-tailed (gray asterisk) or two-tailed (black asterisks) Student’s t -tests. P values = 0.029197 ( a ), 0.139771 ( d , 1dpl), 0.047935 ( d , 3dpl), 0.030298 ( d , 5dpl), 0.061845 ( d , 12dpl), 0.001796 ( e ), 0.033551 ( f ), 0.047979 ( g , sumoylated), 0.300207 ( g , non-sumoylated). N = 3 independent experiments or 3 animals per time-point. For each panel, representative images of 3 independent experiments are shown. Source data are provided as a Source Data file.
Figure Legend Snippet: Tip60 acetylates eEF1A1 in a Stat3-dependent manner. a , e eEF1A1 immunoprecipitation (IP) in lysates of SCs induced to de-differentiate for 1 day and treated with TH1864 (Tip60 inhibitor, Tip60i) or vehicle, and Tip60, Stat3 ( a ) or Ac-eEF1A ( e ) Western blot and quantification (normalized to eEF1A1) of Tip60 co-immunoprecipitated with eEF1A1 ( a ) or of Ac-eEF1A1 ( e ) in Tip60 inhibitor- compared to vehicle-treated SCs. b IP Stat3 or Flag (Ctrl) in lysates of SCs induced to de-differentiate for 1 day, Tip60 Western blot and Stat3 inputs. c Tip60, GAPDH (cytoplasmic marker) and Lamin A/C (nuclear marker) Western blots in cytoplasmic and nuclear fractions of SCs induced to de-differentiate for 1 day. Dashed lines: samples run on the same gel, but not on consecutive lanes. d Tip60 and GAPDH Western blots in lysates of crushed (Cr) and contralateral (Co) mouse sciatic nerves at 1-3-5-12dpl, and Tip60 quantification normalized to GAPDH in Cr compared to Co. f Sox10 and GAPDH Western blots in lysates of SCs induced to de-differentiate and treated with Tip60i or vehicle for 3 days and Sox10 quantification normalized to GAPDH. g Stat3 and Tip60 Western blots in SCs transduced with Stat3-specific shRNA (Stat3 sh) or non-targeting control shRNA (Ctrl sh) lentiviruses and quantification of sumoylated (higher molecular weight) and non-sumoylated (lower molecular weight) Tip60 isoforms normalized to GAPDH. h eEF1A1 IP and Tip60 Western blot in SCs transduced with Stat3 sh or Ctrl sh lentiviruses. i Stat3 and Ac-eEF1A co-immunofluorescence and DAPI labeling in primary SCs transduced with Stat3 sh or Ctrl sh lentiviruses and induced to de-differentiate for 2 days. Scale bar: 20 µm. Data presented as mean values ± SEM. Unpaired ( f ) or paired ( a , d , e , g ) one-tailed (gray asterisk) or two-tailed (black asterisks) Student’s t -tests. P values = 0.029197 ( a ), 0.139771 ( d , 1dpl), 0.047935 ( d , 3dpl), 0.030298 ( d , 5dpl), 0.061845 ( d , 12dpl), 0.001796 ( e ), 0.033551 ( f ), 0.047979 ( g , sumoylated), 0.300207 ( g , non-sumoylated). N = 3 independent experiments or 3 animals per time-point. For each panel, representative images of 3 independent experiments are shown. Source data are provided as a Source Data file.

Techniques Used: Immunoprecipitation, Western Blot, Marker, Transduction, shRNA, Molecular Weight, Immunofluorescence, Labeling, One-tailed Test, Two Tailed Test

Theophylline increases Sox10, Krox20, and P0 expression and eEF1A1 deacetylation. a HDAC2 (12dpl: n = 3, p = 0.025883, 14dpl: n = 5, p = 0.011697), Sox10 (12dpl: n = 3, p = 0.031422, 14dpl: n = 3–4, p = 0.032671), Krox20 (12dpl: n = 3, p = 0.030486, 14dpl and 30dpl: n = 7, p = 0.033493 and 0.022041) and P0 (12dpl: n = 3, p = 0.022239, 14dpl: n = 4-5, p = 0.019084, 30dpl: n = 6, p = 0.0456) Western blots at 12, 14 and 30dpl in lysates of crushed (Cr) or contralateral (Co) sciatic nerves of mice treated at 10dpl with theophylline (Theo) or vehicle (Veh.) for 2 days (12dpl) or 4 days (14 and 30dpl), and quantification normalized to GAPDH and Co. b , c EEF1A1 IP and Ac-eEF1A Western blot ( b ) and Ac-eEF1A and S100 (SC marker) co-immunofluorescence and DAPI labeling ( c ) at 12dpl in Cr ( c ) or in Cr and Co ( b ) 20 h after one theophylline or vehicle injection, and ( b ) Ac-eEF1A1 quantification normalized to GAPDH input and Co in theophylline- compared to vehicle-treated groups. N = 3 animals per group ( b , c ), p = 0.000597 ( b ). Representative images are shown. d – f GFP ( d ) and/or Sox10 ( d – f ) chromatin immunoprecipitation at 12dpl on Krox20 MSE or P0 intron 1 in ( d ) Cr and fold enrichment normalized to Sox10 input compared to GFP ( n = 3 animals, Sox10 and GFP IPs on the same nerve lysate, p Krox20 -MSE = 0.032616, p P0 -intron-1 = 0.027702) or in Cr ( e ) collected 20 h after theophylline (Theo) or vehicle (Veh.) treatment or ( f ) after mocetinostat (Mocet.) or Veh. treatment for 2 days. e N = 5–6 animals per group, p Krox20 -MSE = 0.030466, p P0 -intron-1 = 0.04003. f N = 3 animals per group, p Krox20 -MSE = 0.037396, p P0 -intron-1 = 0.023672. g Sox10, P0, HDAC2, and GAPDH Western blots in lysates of P4 DhhCre ; Hdac1 fl/fl; Hdac2 fl/fl dKO and control littermate sciatic nerves treated at P1 with Theo or Veh. for 3 days, and quantification normalized to GAPDH in Theo compared to Veh. N = 6 animals per group, pSox10 = 0.04567, pP0 = 0.033358, pHDAC2 = 0.02891. Dashed lines: samples loaded on the same gel but not on consecutive lanes. Data are presented as mean values ± SEM. Paired ( a : HDAC2 and Sox10-12dpl, Krox20-14dpl-30dpl; b , d , e , and f : P 0-intron-1; g ) or unpaired one-tailed (gray asterisks) or two-tailed (black asterisks) Student’s t -tests. Scale bar: 20 µm ( c ). Source data are provided as a Source Data file.
Figure Legend Snippet: Theophylline increases Sox10, Krox20, and P0 expression and eEF1A1 deacetylation. a HDAC2 (12dpl: n = 3, p = 0.025883, 14dpl: n = 5, p = 0.011697), Sox10 (12dpl: n = 3, p = 0.031422, 14dpl: n = 3–4, p = 0.032671), Krox20 (12dpl: n = 3, p = 0.030486, 14dpl and 30dpl: n = 7, p = 0.033493 and 0.022041) and P0 (12dpl: n = 3, p = 0.022239, 14dpl: n = 4-5, p = 0.019084, 30dpl: n = 6, p = 0.0456) Western blots at 12, 14 and 30dpl in lysates of crushed (Cr) or contralateral (Co) sciatic nerves of mice treated at 10dpl with theophylline (Theo) or vehicle (Veh.) for 2 days (12dpl) or 4 days (14 and 30dpl), and quantification normalized to GAPDH and Co. b , c EEF1A1 IP and Ac-eEF1A Western blot ( b ) and Ac-eEF1A and S100 (SC marker) co-immunofluorescence and DAPI labeling ( c ) at 12dpl in Cr ( c ) or in Cr and Co ( b ) 20 h after one theophylline or vehicle injection, and ( b ) Ac-eEF1A1 quantification normalized to GAPDH input and Co in theophylline- compared to vehicle-treated groups. N = 3 animals per group ( b , c ), p = 0.000597 ( b ). Representative images are shown. d – f GFP ( d ) and/or Sox10 ( d – f ) chromatin immunoprecipitation at 12dpl on Krox20 MSE or P0 intron 1 in ( d ) Cr and fold enrichment normalized to Sox10 input compared to GFP ( n = 3 animals, Sox10 and GFP IPs on the same nerve lysate, p Krox20 -MSE = 0.032616, p P0 -intron-1 = 0.027702) or in Cr ( e ) collected 20 h after theophylline (Theo) or vehicle (Veh.) treatment or ( f ) after mocetinostat (Mocet.) or Veh. treatment for 2 days. e N = 5–6 animals per group, p Krox20 -MSE = 0.030466, p P0 -intron-1 = 0.04003. f N = 3 animals per group, p Krox20 -MSE = 0.037396, p P0 -intron-1 = 0.023672. g Sox10, P0, HDAC2, and GAPDH Western blots in lysates of P4 DhhCre ; Hdac1 fl/fl; Hdac2 fl/fl dKO and control littermate sciatic nerves treated at P1 with Theo or Veh. for 3 days, and quantification normalized to GAPDH in Theo compared to Veh. N = 6 animals per group, pSox10 = 0.04567, pP0 = 0.033358, pHDAC2 = 0.02891. Dashed lines: samples loaded on the same gel but not on consecutive lanes. Data are presented as mean values ± SEM. Paired ( a : HDAC2 and Sox10-12dpl, Krox20-14dpl-30dpl; b , d , e , and f : P 0-intron-1; g ) or unpaired one-tailed (gray asterisks) or two-tailed (black asterisks) Student’s t -tests. Scale bar: 20 µm ( c ). Source data are provided as a Source Data file.

Techniques Used: Expressing, Western Blot, Mouse Assay, Marker, Immunofluorescence, Labeling, Injection, Chromatin Immunoprecipitation, One-tailed Test, Two Tailed Test

EEF1A1 is deacetylated by HDAC1/2 in SCs. a Sox10 Western blot and quantification normalized to GAPDH showing low Sox10 levels at 1dpl in crushed as compared to contralateral sciatic nerves of adult mice. Paired two-tailed Student’s t -tests, p value = 0.008938, n = 8 animals per group. b , c Co-immunofluorescence of Ac-eEF1A and S100 (SC marker) and DAPI (nuclei) labeling ( b ) and immunoprecipitation (IP) of eEF1A1 and Western blot of Ac-eEF1A ( c ) in sciatic nerves of P4 control (Ctrl) and DhhCre;Hdac1;Hdac2 knockout (dKO) mice showing increased levels of Ac-eEF1A in the absence of HDAC1/2 in SCs. n.s. = non-specific. d Western blot of total eEF1A1 and GAPDH (loading control) in sciatic nerves of P4 Ctrl and dKO mice and quantification of eEF1A1 levels normalized to GAPDH levels showing no significant difference between the two groups. Paired one-tailed Student’s t -tests, p value = 0.297774, n = 4 animals per group. e IP eEF1A1 followed by Western blot of Ac-eEF1A in nuclear and cytoplasmic fractions of primary SCs cultured under de-differentiating conditions and treated with the HDAC1/2 inhibitor mocetinostat (Mocet.) or its vehicle (Veh.) for 3 days. Lamin A/C (nuclear marker), GAPDH (cytoplasmic marker) and eEF1A1 Western blots on lysates used for IP show the inputs. f Immunofluorescence of Ac-eEF1A and DAPI labeling in primary SCs cultured under de-differentiation conditions and treated with mocetinostat or its vehicle for 3 days. Arrowheads ( b ) and arrows ( f ) show SC nuclei. Z-series projections ( b ) or single optical sections ( f ) are shown. b , f Representative images of 3 different animals per group or of 3 independent experiments are shown. b , d Data are presented as mean values ± SEM. Scale bars: 10 µm. Source data are provided as a Source Data file.
Figure Legend Snippet: EEF1A1 is deacetylated by HDAC1/2 in SCs. a Sox10 Western blot and quantification normalized to GAPDH showing low Sox10 levels at 1dpl in crushed as compared to contralateral sciatic nerves of adult mice. Paired two-tailed Student’s t -tests, p value = 0.008938, n = 8 animals per group. b , c Co-immunofluorescence of Ac-eEF1A and S100 (SC marker) and DAPI (nuclei) labeling ( b ) and immunoprecipitation (IP) of eEF1A1 and Western blot of Ac-eEF1A ( c ) in sciatic nerves of P4 control (Ctrl) and DhhCre;Hdac1;Hdac2 knockout (dKO) mice showing increased levels of Ac-eEF1A in the absence of HDAC1/2 in SCs. n.s. = non-specific. d Western blot of total eEF1A1 and GAPDH (loading control) in sciatic nerves of P4 Ctrl and dKO mice and quantification of eEF1A1 levels normalized to GAPDH levels showing no significant difference between the two groups. Paired one-tailed Student’s t -tests, p value = 0.297774, n = 4 animals per group. e IP eEF1A1 followed by Western blot of Ac-eEF1A in nuclear and cytoplasmic fractions of primary SCs cultured under de-differentiating conditions and treated with the HDAC1/2 inhibitor mocetinostat (Mocet.) or its vehicle (Veh.) for 3 days. Lamin A/C (nuclear marker), GAPDH (cytoplasmic marker) and eEF1A1 Western blots on lysates used for IP show the inputs. f Immunofluorescence of Ac-eEF1A and DAPI labeling in primary SCs cultured under de-differentiation conditions and treated with mocetinostat or its vehicle for 3 days. Arrowheads ( b ) and arrows ( f ) show SC nuclei. Z-series projections ( b ) or single optical sections ( f ) are shown. b , f Representative images of 3 different animals per group or of 3 independent experiments are shown. b , d Data are presented as mean values ± SEM. Scale bars: 10 µm. Source data are provided as a Source Data file.

Techniques Used: Western Blot, Mouse Assay, Two Tailed Test, Immunofluorescence, Marker, Labeling, Immunoprecipitation, Knock-Out, One-tailed Test, Cell Culture

Sox10 re-localizes to the cytoplasm with Ac-eEF1A1 in de-differentiated SCs. Immunofluorescence of Ac-eEF1A ( a ) and Sox10 ( c ), and DAPI (nuclei) labeling in differentiated and de-differentiated SCs. b Immunoprecipitation (IP) of eEF1A1 or GFP (control) carried out on the same pool of two adult mouse crushed or unlesioned contralateral sciatic nerves at 3dpl and Western blot of Ac-eEF1A followed by HDAC2. EEF1A1 and GAPDH Western blots on lysates show the input, n = 3 (6 animals). The graph shows the quantification of Ac-eEF1A levels (measured on a longer exposure to obtain a value for the contra IP eEF1A1) normalized to eEF1A1 input. Data are presented as mean values ± SEM. Paired two-tailed Student’s t -tests, p value = 0.03376. IP Sox10 or GFP ( d ) or IP eEF1A1 ( e ) followed by Ac-eEF1A ( d ) or HDAC2 ( e ) Western blot in nuclear and cytoplasmic fractions of primary SCs cultured under de-differentiating conditions and treated with the HDAC1/2 inhibitor mocetinostat or its vehicle for 3 days. Lamin A/C (nuclear marker), GAPDH (cytoplasmic marker), Sox10 and eEF1A1 Western blots on lysates used for IP show the inputs. Dashed lines indicate that samples were run on the same gel but not on consecutive lanes. Arrows point to SC nuclei ( a , c ) and arrowheads ( c ) to SC cytoplasm. a – e Representative images of 3 independent experiments are shown. Scale bars: 10 µm. Source data are provided as a Source Data file.
Figure Legend Snippet: Sox10 re-localizes to the cytoplasm with Ac-eEF1A1 in de-differentiated SCs. Immunofluorescence of Ac-eEF1A ( a ) and Sox10 ( c ), and DAPI (nuclei) labeling in differentiated and de-differentiated SCs. b Immunoprecipitation (IP) of eEF1A1 or GFP (control) carried out on the same pool of two adult mouse crushed or unlesioned contralateral sciatic nerves at 3dpl and Western blot of Ac-eEF1A followed by HDAC2. EEF1A1 and GAPDH Western blots on lysates show the input, n = 3 (6 animals). The graph shows the quantification of Ac-eEF1A levels (measured on a longer exposure to obtain a value for the contra IP eEF1A1) normalized to eEF1A1 input. Data are presented as mean values ± SEM. Paired two-tailed Student’s t -tests, p value = 0.03376. IP Sox10 or GFP ( d ) or IP eEF1A1 ( e ) followed by Ac-eEF1A ( d ) or HDAC2 ( e ) Western blot in nuclear and cytoplasmic fractions of primary SCs cultured under de-differentiating conditions and treated with the HDAC1/2 inhibitor mocetinostat or its vehicle for 3 days. Lamin A/C (nuclear marker), GAPDH (cytoplasmic marker), Sox10 and eEF1A1 Western blots on lysates used for IP show the inputs. Dashed lines indicate that samples were run on the same gel but not on consecutive lanes. Arrows point to SC nuclei ( a , c ) and arrowheads ( c ) to SC cytoplasm. a – e Representative images of 3 independent experiments are shown. Scale bars: 10 µm. Source data are provided as a Source Data file.

Techniques Used: Immunofluorescence, Labeling, Immunoprecipitation, Western Blot, Two Tailed Test, Cell Culture, Marker

EEF1A1 acetylation increases eEF1A1 nuclear localization and decreases Sox10 levels. a GFP Western blot in nuclear (Nucl.) and cytoplasmic (Cyt.) fractions of primary SCs cultured 1 day under de-differentiating conditions and transfected with eEF1A1-GFP, K41Q-GFP, K179Q-GFP or K273Q-GFP and % of protein localized in nucleus or cytoplasm normalized to Lamin A/C and GAPDH ( n = 3 independent experiments per group). b – d Sox10 immunofluorescence with GFP fluorescence and DAPI (nuclei) labeling in SCs overexpressing eEF1A1-GFP, K41R-GFP, K179R-GFP or K273R-GFP and cultured under de-differentiating conditions for 3 days, and % of cells with eEF1A1 or mutants localized in cytoplasm only (Cyt. only) or in nucleus and cytoplasm (Nucl. + Cyt., b ) or % of low Sox10-expressing cells ( c , d ). N = 3 independent experiments per group, 18-84 cells counted per group per n . e , f Sox10 immunofluorescence with GFP fluorescence and DAPI (nuclei) labeling in SCs overexpressing eEF1A1-GFP, K41Q-GFP, K179Q-GFP or K273Q-GFP and cultured under proliferating conditions for 2 days, and % of cells with eEF1A1 or mutants localized in cytoplasm only or in nucleus and cytoplasm of SCs ( e ) or % of low Sox10-expressing cells ( f ). The lower images are magnifications of the dashed white boxes on the upper images. N = 3 independent experiments per group, 17–71 cells counted per group per n. Arrows show transfected SCs. Orange arrows indicate SCs where eEF1A1 or the mutants are present in the nucleus of SCs. Scale bars: 10 µm ( b ), 20 µm ( e ). Data are presented as mean values ± SEM. Unpaired one-tailed (gray asterisks) or two-tailed (black asterisks) Student’s t -tests, p values = 0.029415 ( a , K41Q), 0.04884 ( a , K179Q), 0.0329 ( a , K273Q), 0.023698 ( b , K41R), 0.008772 ( b , K179R), 0.00946 ( b , K273R), 0.189827 ( c , K41R), 0.027448 ( c , K179R), 0.018314 ( c , K273R), 0.405588 ( d , K41R), 0.03836 ( d , K179R), 0.406686 ( d , K273R), 0.011923 ( e , K41Q), 0.019373 ( e , K179Q), 0.021749 ( e , K273Q), 0.009059 ( f , K41Q), 0.009399 ( f , K179Q), 0.02223 ( f , K273Q). Source data are provided as a Source Data file.
Figure Legend Snippet: EEF1A1 acetylation increases eEF1A1 nuclear localization and decreases Sox10 levels. a GFP Western blot in nuclear (Nucl.) and cytoplasmic (Cyt.) fractions of primary SCs cultured 1 day under de-differentiating conditions and transfected with eEF1A1-GFP, K41Q-GFP, K179Q-GFP or K273Q-GFP and % of protein localized in nucleus or cytoplasm normalized to Lamin A/C and GAPDH ( n = 3 independent experiments per group). b – d Sox10 immunofluorescence with GFP fluorescence and DAPI (nuclei) labeling in SCs overexpressing eEF1A1-GFP, K41R-GFP, K179R-GFP or K273R-GFP and cultured under de-differentiating conditions for 3 days, and % of cells with eEF1A1 or mutants localized in cytoplasm only (Cyt. only) or in nucleus and cytoplasm (Nucl. + Cyt., b ) or % of low Sox10-expressing cells ( c , d ). N = 3 independent experiments per group, 18-84 cells counted per group per n . e , f Sox10 immunofluorescence with GFP fluorescence and DAPI (nuclei) labeling in SCs overexpressing eEF1A1-GFP, K41Q-GFP, K179Q-GFP or K273Q-GFP and cultured under proliferating conditions for 2 days, and % of cells with eEF1A1 or mutants localized in cytoplasm only or in nucleus and cytoplasm of SCs ( e ) or % of low Sox10-expressing cells ( f ). The lower images are magnifications of the dashed white boxes on the upper images. N = 3 independent experiments per group, 17–71 cells counted per group per n. Arrows show transfected SCs. Orange arrows indicate SCs where eEF1A1 or the mutants are present in the nucleus of SCs. Scale bars: 10 µm ( b ), 20 µm ( e ). Data are presented as mean values ± SEM. Unpaired one-tailed (gray asterisks) or two-tailed (black asterisks) Student’s t -tests, p values = 0.029415 ( a , K41Q), 0.04884 ( a , K179Q), 0.0329 ( a , K273Q), 0.023698 ( b , K41R), 0.008772 ( b , K179R), 0.00946 ( b , K273R), 0.189827 ( c , K41R), 0.027448 ( c , K179R), 0.018314 ( c , K273R), 0.405588 ( d , K41R), 0.03836 ( d , K179R), 0.406686 ( d , K273R), 0.011923 ( e , K41Q), 0.019373 ( e , K179Q), 0.021749 ( e , K273Q), 0.009059 ( f , K41Q), 0.009399 ( f , K179Q), 0.02223 ( f , K273Q). Source data are provided as a Source Data file.

Techniques Used: Western Blot, Cell Culture, Transfection, Immunofluorescence, Fluorescence, Labeling, Expressing, One-tailed Test, Two Tailed Test

EEF1A1 re-localizes Sox10 to the cytoplasm and reduces its expression. a Sox10 and 20 S co-immunofluorescence in SCs cultured under de-differentiation conditions. Representative images (z-series projections) are shown. The right image is a magnified 3D view of the region highlighted by a dashed box on the left images. Arrows point to proteasome structures containing Sox10. b Sox10 and GAPDH Western blot in lysates of SCs induced to de-differentiate for 1 day and treated with the proteasome inhibitor MG132 (proteasome inhib.) or vehicle (Veh.) for 12 h, and Sox10 quantification normalized to GAPDH ( n = 3 independent experiments). c GFP immunoprecipitation (IP) and Sox10 Western blot in nuclear and cytoplasmic fractions of SCs transfected with eEF1A1-GFP or GFP and induced to de-differentiate for 1 day, and input GFP, LaminA/C (nuclear fraction) and GAPDH (cytoplasmic fraction) on lysates used for IPs. d Sox10 and GFP Western blots in nuclear and cytoplasmic fractions of SCs transfected with a construct expressing eEF1A1-GFP or control GFP and induced to de-differentiate for 1 day, and Sox10 quantification (normalized to GAPDH or Lamin A/C) in each fraction. n.s. = non-specific. e Sox10 immunofluorescence with GFP fluorescence and DAPI (nuclei) labeling in SCs expressing eEF1A1-GFP or control GFP and cultured under de-differentiating conditions for 3 days, and % of low Sox10-expressing cells among cells expressing GFP or eEF1A1-GFP in the cytoplasm only (Cyt. only) or in both nucleus and cytoplasm (Nucl. + Cyt.). Arrows point to transfected SCs ( e ). Orange arrows indicate transfected SCs with low Sox10 levels and/or with eEF1A1 localized in the nucleus ( e ). Dashed lines indicate that samples were run on the same gel but not on consecutive lanes. Data are presented as mean values ± SEM. Unpaired ( b , e ) or paired ( d ) two-tailed Student’s t -tests. P values: 0.001725 ( b ), 0.045156 ( d , cytoplasmic), 0.024327 ( d , nuclear), 0.02473 ( e ). N = 3 independent experiments ( b , d , e ), 15–25 (GFP) and 48–58 (eEF1A1-GFP) transfected cells counted per n ( e ). a – e Representative images of 3 independent experiments are shown. Scale bars: 10 µm. Source data are provided as a Source Data file.
Figure Legend Snippet: EEF1A1 re-localizes Sox10 to the cytoplasm and reduces its expression. a Sox10 and 20 S co-immunofluorescence in SCs cultured under de-differentiation conditions. Representative images (z-series projections) are shown. The right image is a magnified 3D view of the region highlighted by a dashed box on the left images. Arrows point to proteasome structures containing Sox10. b Sox10 and GAPDH Western blot in lysates of SCs induced to de-differentiate for 1 day and treated with the proteasome inhibitor MG132 (proteasome inhib.) or vehicle (Veh.) for 12 h, and Sox10 quantification normalized to GAPDH ( n = 3 independent experiments). c GFP immunoprecipitation (IP) and Sox10 Western blot in nuclear and cytoplasmic fractions of SCs transfected with eEF1A1-GFP or GFP and induced to de-differentiate for 1 day, and input GFP, LaminA/C (nuclear fraction) and GAPDH (cytoplasmic fraction) on lysates used for IPs. d Sox10 and GFP Western blots in nuclear and cytoplasmic fractions of SCs transfected with a construct expressing eEF1A1-GFP or control GFP and induced to de-differentiate for 1 day, and Sox10 quantification (normalized to GAPDH or Lamin A/C) in each fraction. n.s. = non-specific. e Sox10 immunofluorescence with GFP fluorescence and DAPI (nuclei) labeling in SCs expressing eEF1A1-GFP or control GFP and cultured under de-differentiating conditions for 3 days, and % of low Sox10-expressing cells among cells expressing GFP or eEF1A1-GFP in the cytoplasm only (Cyt. only) or in both nucleus and cytoplasm (Nucl. + Cyt.). Arrows point to transfected SCs ( e ). Orange arrows indicate transfected SCs with low Sox10 levels and/or with eEF1A1 localized in the nucleus ( e ). Dashed lines indicate that samples were run on the same gel but not on consecutive lanes. Data are presented as mean values ± SEM. Unpaired ( b , e ) or paired ( d ) two-tailed Student’s t -tests. P values: 0.001725 ( b ), 0.045156 ( d , cytoplasmic), 0.024327 ( d , nuclear), 0.02473 ( e ). N = 3 independent experiments ( b , d , e ), 15–25 (GFP) and 48–58 (eEF1A1-GFP) transfected cells counted per n ( e ). a – e Representative images of 3 independent experiments are shown. Scale bars: 10 µm. Source data are provided as a Source Data file.

Techniques Used: Expressing, Immunofluorescence, Cell Culture, Western Blot, Inhibition, Immunoprecipitation, Transfection, Construct, Fluorescence, Labeling, Two Tailed Test

16) Product Images from "Autophagy Pathway Is Required for IL-6 Induced Neuroendocrine Differentiation and Chemoresistance of Prostate Cancer LNCaP Cells"

Article Title: Autophagy Pathway Is Required for IL-6 Induced Neuroendocrine Differentiation and Chemoresistance of Prostate Cancer LNCaP Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0088556

IL-6 induces NED in LNCaP cells and this is concomitant with increased autophagy. (A) LNCaP cells were treated with 2.5% CDT or 2.5% CDT plus 100 ng/ml IL-6 for 48 hours. The induced neurite elongation was assessed using brightfield microscopy images (40× magnification). (B) The neurite elongation was quantified using the average from 3–5 microscopic fields; bars , SD. (C) LNCaP cells were treated as described in (A). Total cell lysates (TCLs) were prepared and then immunoblotted to detect tubulin III, androgen receptor (AR) and LC3. GAPDH was used as the loading control.
Figure Legend Snippet: IL-6 induces NED in LNCaP cells and this is concomitant with increased autophagy. (A) LNCaP cells were treated with 2.5% CDT or 2.5% CDT plus 100 ng/ml IL-6 for 48 hours. The induced neurite elongation was assessed using brightfield microscopy images (40× magnification). (B) The neurite elongation was quantified using the average from 3–5 microscopic fields; bars , SD. (C) LNCaP cells were treated as described in (A). Total cell lysates (TCLs) were prepared and then immunoblotted to detect tubulin III, androgen receptor (AR) and LC3. GAPDH was used as the loading control.

Techniques Used: Microscopy

Regulation of NED by REST in LNCaP cells. (A) The level of REST protein declines during IL-6 treatment. LNCaP cells were treated with 100 ng/ml IL-6 for 48 and 96 hours. The expression level of REST was analyzed by immunoblotting using anti-REST antibody. GAPDH was used as the loading control. (B) LNCaP-TR-shREST cells were treated with or without Dox for 48 hours. TCLs were analyzed by immunoblotting using anti-REST antibody. (C) LNCaP-TR-shREST cells were treated with Dox for 6 days. The promotion of neurite outgrowth by REST knockdown was assessed using brightfield microscopy images (40× magnification). (D) The neurite elongation was quantified using the average from 3–5 microscopic fields; bars , SD. (E) LNCaP-TR-shREST cells were treated as described in (C). TCLs were prepared and analyzed by immunoblotting using the antibodies as indicated. (F) LNCaP-TR-REST cells were treated with 1 µg/ml Dox in the absence (control) or presence of 100 ng/ml IL-6 for 4 days. Inhibition of IL-6-induced neurite outgrowth by REST overexpression was assessed using brightfield microscopy images (40× magnification). (G) TCLs were obtained from LNCaP-TR-REST cells treated as described in (F); these were then analyzed by immunoblotting using the indicated antibodies. (H) RT-qPCR analysis of total RNA isolated from LNCaP-TR-shREST cells treated as described in (C). The relative mRNA levels of REST, Atg5, beclin1 and LC3 were normalized against GAPDH. Values from three independent data points are reported as mean±S.D.
Figure Legend Snippet: Regulation of NED by REST in LNCaP cells. (A) The level of REST protein declines during IL-6 treatment. LNCaP cells were treated with 100 ng/ml IL-6 for 48 and 96 hours. The expression level of REST was analyzed by immunoblotting using anti-REST antibody. GAPDH was used as the loading control. (B) LNCaP-TR-shREST cells were treated with or without Dox for 48 hours. TCLs were analyzed by immunoblotting using anti-REST antibody. (C) LNCaP-TR-shREST cells were treated with Dox for 6 days. The promotion of neurite outgrowth by REST knockdown was assessed using brightfield microscopy images (40× magnification). (D) The neurite elongation was quantified using the average from 3–5 microscopic fields; bars , SD. (E) LNCaP-TR-shREST cells were treated as described in (C). TCLs were prepared and analyzed by immunoblotting using the antibodies as indicated. (F) LNCaP-TR-REST cells were treated with 1 µg/ml Dox in the absence (control) or presence of 100 ng/ml IL-6 for 4 days. Inhibition of IL-6-induced neurite outgrowth by REST overexpression was assessed using brightfield microscopy images (40× magnification). (G) TCLs were obtained from LNCaP-TR-REST cells treated as described in (F); these were then analyzed by immunoblotting using the indicated antibodies. (H) RT-qPCR analysis of total RNA isolated from LNCaP-TR-shREST cells treated as described in (C). The relative mRNA levels of REST, Atg5, beclin1 and LC3 were normalized against GAPDH. Values from three independent data points are reported as mean±S.D.

Techniques Used: Expressing, Microscopy, Inhibition, Over Expression, Quantitative RT-PCR, Isolation

17) Product Images from "Autophagy gene haploinsufficiency drives chromosome instability, increases migration, and promotes early ovarian tumors"

Article Title: Autophagy gene haploinsufficiency drives chromosome instability, increases migration, and promotes early ovarian tumors

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1008558

Titrated knockdown of BECN1 or LC3B reduces autophagosome accumulation markers. A, Commonly used ovarian cancer cell lines were tested for acidic vacuole accumulation following chloroquine treatment (10μM 24h) by flow cytometric measurement of acridine orange (AO). Shown are the averages of 4 flow cytometry assays. The x-axis depicts a pathway network calculation from HAPTRIG defining the levels of autophagy pathway haploinsufficiency within each cell line due to monoallelic losses of KEGG autophagy pathway genes. B, SKOV3 cells, which do not have losses in BECN1 nor LC3B , were stably knocked-down by shRNA lentivirus. A western blot was performed for the targets LC3B and BECN1, along with GAPDH loading control. C, IGROV1 cells were similarly assayed.
Figure Legend Snippet: Titrated knockdown of BECN1 or LC3B reduces autophagosome accumulation markers. A, Commonly used ovarian cancer cell lines were tested for acidic vacuole accumulation following chloroquine treatment (10μM 24h) by flow cytometric measurement of acridine orange (AO). Shown are the averages of 4 flow cytometry assays. The x-axis depicts a pathway network calculation from HAPTRIG defining the levels of autophagy pathway haploinsufficiency within each cell line due to monoallelic losses of KEGG autophagy pathway genes. B, SKOV3 cells, which do not have losses in BECN1 nor LC3B , were stably knocked-down by shRNA lentivirus. A western blot was performed for the targets LC3B and BECN1, along with GAPDH loading control. C, IGROV1 cells were similarly assayed.

Techniques Used: Flow Cytometry, Cytometry, Stable Transfection, shRNA, Western Blot

18) Product Images from "Nrf2 is the key to chemotherapy resistance in MCF7 breast cancer cells under hypoxia"

Article Title: Nrf2 is the key to chemotherapy resistance in MCF7 breast cancer cells under hypoxia

Journal: Oncotarget

doi: 10.18632/oncotarget.7406

Nrf2 contributes to the increase of drug resistance MCF7 cells were transfected with vector control (vector) and Nrf2 plasmid (Nrf2). A. The protein levels of Nrf2, NQO1, GCLC, GCLM and GAPDH were detected by western blotting. B. MCF7 cells were treated with 1 μg/ml CDDP for 24 hours, and cell viability was determined with MTT. N=3, *, P
Figure Legend Snippet: Nrf2 contributes to the increase of drug resistance MCF7 cells were transfected with vector control (vector) and Nrf2 plasmid (Nrf2). A. The protein levels of Nrf2, NQO1, GCLC, GCLM and GAPDH were detected by western blotting. B. MCF7 cells were treated with 1 μg/ml CDDP for 24 hours, and cell viability was determined with MTT. N=3, *, P

Techniques Used: Transfection, Plasmid Preparation, Western Blot, MTT Assay

Regulation of antioxidant enzyme activities leads to the alteration of drug resistance under hypoxia A-D. MCF7 cells were exposed to hypoxia for 0, 4, 8, 24 hours. (A) The intracellular ROS levels were detected by flow cytometry with DCFHDA staining. (B) The total proteins were extracted from the cells and the Hif-1α, NQO1, GCLC, GCLM, GAPDH levels were detected by western blot. (C) The intracellular total GSH (tGSH) activities were measured by glutathione assay. N=3, *, P
Figure Legend Snippet: Regulation of antioxidant enzyme activities leads to the alteration of drug resistance under hypoxia A-D. MCF7 cells were exposed to hypoxia for 0, 4, 8, 24 hours. (A) The intracellular ROS levels were detected by flow cytometry with DCFHDA staining. (B) The total proteins were extracted from the cells and the Hif-1α, NQO1, GCLC, GCLM, GAPDH levels were detected by western blot. (C) The intracellular total GSH (tGSH) activities were measured by glutathione assay. N=3, *, P

Techniques Used: Flow Cytometry, Cytometry, Staining, Western Blot, Glutathione Assay

Nuclear translocation of Nrf2 leads to drug resistance under hypoxia A-C. MCF7 cells were exposed to hypoxia for 0, 4, 8, and 24 hours. (A) Nrf2 localization was determined by immunocytochemistry (ICC) with an anti-Nrf2 antibody (green fluorescence). Nuclear location was determined by DAPI staining (blue fluorescence). (B, C) Total proteins and cytosolic (C)/nuclear (N) proteins were extracted. The protein levels of Hif-1α, Nrf2, GAPDH, and Histone H3 were detected by western blotting. GAPDH was the loading control for the cytosolic fraction, and Histone H3 was the loading control for the nuclear fraction. D. MCF7 cells were transfected with pARE-CMV and pRL-CMV plasmids. After transfection, the cells were exposed to hypoxia for 0, 4, 8, and 24 hours, and the firefly and Renilla luminescence activities were detected by Dual-Glo luciferase assay. The luciferase activity was represented by firefly luminescence normalized with Renilla luminescence. N=3, *, P
Figure Legend Snippet: Nuclear translocation of Nrf2 leads to drug resistance under hypoxia A-C. MCF7 cells were exposed to hypoxia for 0, 4, 8, and 24 hours. (A) Nrf2 localization was determined by immunocytochemistry (ICC) with an anti-Nrf2 antibody (green fluorescence). Nuclear location was determined by DAPI staining (blue fluorescence). (B, C) Total proteins and cytosolic (C)/nuclear (N) proteins were extracted. The protein levels of Hif-1α, Nrf2, GAPDH, and Histone H3 were detected by western blotting. GAPDH was the loading control for the cytosolic fraction, and Histone H3 was the loading control for the nuclear fraction. D. MCF7 cells were transfected with pARE-CMV and pRL-CMV plasmids. After transfection, the cells were exposed to hypoxia for 0, 4, 8, and 24 hours, and the firefly and Renilla luminescence activities were detected by Dual-Glo luciferase assay. The luciferase activity was represented by firefly luminescence normalized with Renilla luminescence. N=3, *, P

Techniques Used: Translocation Assay, Immunocytochemistry, Fluorescence, Staining, Western Blot, Transfection, Luciferase, Activity Assay

19) Product Images from "Ethyl Acetate Extract of Scindapsus cf. hederaceus Exerts the Inhibitory Bioactivity on Human Non-Small Cell Lung Cancer Cells through Modulating ER Stress"

Article Title: Ethyl Acetate Extract of Scindapsus cf. hederaceus Exerts the Inhibitory Bioactivity on Human Non-Small Cell Lung Cancer Cells through Modulating ER Stress

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19071832

SH-EAE reduces the EGFR and VEGF signaling in NSCLC cells. Expression of phospho-EGFR (Tyr845), EGFR, and VEGF were analyzed by western blot in NSCLC cell line H1299. GAPDH was used as the loading control. Data are representative of three independent experiments.
Figure Legend Snippet: SH-EAE reduces the EGFR and VEGF signaling in NSCLC cells. Expression of phospho-EGFR (Tyr845), EGFR, and VEGF were analyzed by western blot in NSCLC cell line H1299. GAPDH was used as the loading control. Data are representative of three independent experiments.

Techniques Used: Expressing, Western Blot

20) Product Images from "Houshiheisan promotes angiogenesis via HIF-1α/VEGF and SDF-1/CXCR4 pathways: in vivo and in vitro"

Article Title: Houshiheisan promotes angiogenesis via HIF-1α/VEGF and SDF-1/CXCR4 pathways: in vivo and in vitro

Journal: Bioscience Reports

doi: 10.1042/BSR20191006

Western blotting results for HIF-1α, VEGFA, Ang-1, Ang-2, and GAPDH Quantitative results of Western blotting for HIF-1α, VEGFA, Ang-1, Ang-2 relative to GAPDH. n =3–5 (3 for Ang-1 and 5 for others). Data are presented as mean ± SD. * P
Figure Legend Snippet: Western blotting results for HIF-1α, VEGFA, Ang-1, Ang-2, and GAPDH Quantitative results of Western blotting for HIF-1α, VEGFA, Ang-1, Ang-2 relative to GAPDH. n =3–5 (3 for Ang-1 and 5 for others). Data are presented as mean ± SD. * P

Techniques Used: Western Blot

21) Product Images from "Zfp423 Binds Autoregulatory Sites in P19 Cell Culture Model"

Article Title: Zfp423 Binds Autoregulatory Sites in P19 Cell Culture Model

Journal: PLoS ONE

doi: 10.1371/journal.pone.0066514

Cell culture models express Zfp423 and its binding partners. ( A ) Inverted gel images from semi-quantitative RT-PCR show expression of ZNF423 and EBF family members in IMR32 (neuroblastoma), as well as D238 (medulloblastoma) cell lines. ZNF423 was not detected from either U87 or U251 (glioblastoma) lines. Cycle numbers are indicated to the left. ( B ) Among four neuroblastoma cell lines, RT-qPCR shows high relative expression of ZNF423 in IMR32 and SK-N-SH cells. Graph shows average and standard deviation for technical replicates of a single experiment. ( C ) Graph shows RT-qPCR expression values for Zfp423 and Ebf RNAs in P19 cells, normalized to the geometric mean of Gapdh , Pitpna , and Ppig as reference genes. Error bars indicate range in technical replicates only. Analysis of an independent second culture showed similar results. ( D ) RT-qPCR expression values from postnatal day 3 mouse cerebellum as a primer control, normalized and scaled as in ( C ). Note significantly higher expression levels in tissue compared to P19. ( E ) Western blots show detection of Zfp423 in 25 µg total cellular protein from IMR32 or P19 cells, detected with a goat polyclonal antibody (E20) and visualized by infrared imaging. The doublet appearance is sometimes observed due to incomplete denaturation in the gel. Blot was re-probed with antibodies to β-actin and GAPDH as loading controls. Relative amounts of total Zfp423 reactivity compared to the loading controls are indicated.
Figure Legend Snippet: Cell culture models express Zfp423 and its binding partners. ( A ) Inverted gel images from semi-quantitative RT-PCR show expression of ZNF423 and EBF family members in IMR32 (neuroblastoma), as well as D238 (medulloblastoma) cell lines. ZNF423 was not detected from either U87 or U251 (glioblastoma) lines. Cycle numbers are indicated to the left. ( B ) Among four neuroblastoma cell lines, RT-qPCR shows high relative expression of ZNF423 in IMR32 and SK-N-SH cells. Graph shows average and standard deviation for technical replicates of a single experiment. ( C ) Graph shows RT-qPCR expression values for Zfp423 and Ebf RNAs in P19 cells, normalized to the geometric mean of Gapdh , Pitpna , and Ppig as reference genes. Error bars indicate range in technical replicates only. Analysis of an independent second culture showed similar results. ( D ) RT-qPCR expression values from postnatal day 3 mouse cerebellum as a primer control, normalized and scaled as in ( C ). Note significantly higher expression levels in tissue compared to P19. ( E ) Western blots show detection of Zfp423 in 25 µg total cellular protein from IMR32 or P19 cells, detected with a goat polyclonal antibody (E20) and visualized by infrared imaging. The doublet appearance is sometimes observed due to incomplete denaturation in the gel. Blot was re-probed with antibodies to β-actin and GAPDH as loading controls. Relative amounts of total Zfp423 reactivity compared to the loading controls are indicated.

Techniques Used: Cell Culture, Binding Assay, Quantitative RT-PCR, Expressing, Standard Deviation, Western Blot, Imaging

Zfp423 binds consensus sites in introns 3 and 5. ( A ) Semi-quantitative ChIP-PCR assays for the ZNF423 intron 5 site in IMR32 cells, with commercial antibodies against the indicated factors compared with normal serum from same host species and titrated input chromatin. Cycle numbers are indicated to the left. ( B ) Frequency of observed enrichment for predicted sites tested in replicate experiments in IMR32 cells. Schematic indicates predicted binding sites for Zfp423 (oval), Ebf (circle) and SMAD (diamond). ( C – F ) Fold enrichment at the orthologous sites in mouse P19 cells, before or after 4 hour treatment with 200 ng/ml BMP2, measured by ChIP-qPCR. Data from Zfp423 antibody E20 are shown. ( C ) Zfp423 intron 3, ( D ) Zfp423 intron 5, ( E ) Ebf1 , ( F ) Ebf3 . ( G–H ) ChIP-qPCR using a custom, affinity-purified antibody against ZNF423 fusion protein shows higher fold discrimination at Zfp423 sites in P19 cells. Experiments in A–B, C–F and G–H were performed independently by different investigators among the authors. * p≥0.05, ** p≥0.01, *** p≥0.001, t-test for comparison to IgG control for same condition and primer pair. ( I ) Alignment of the predicted binding site in intron 5 and syntenic sites from the indicated species shows strong sequence constraint that fits the overlapping Zfp423 (ROAZ) and EBF (OLF1) consensus motifs. ( J ) Western blot of mouse forebrain (Brain) and cerebellum (Cbm) extracts from wild-type littermate (+/+) or Zfp423 null mutant ( nur12 ) mice. The same blot was stripped and re-probed, using species-specific secondary antibodies coupled to alternate infrared fluors. Reactivity for β-actin and Gapdh are shown as internal loading controls. ( K ) Western blot of nuclear extracts from P19 cells treated with the indicated shRNA shows high degree of specificity for each Zfp423 antibody. Nxf1 is used as a loading control. Normalized shZfp423 signal
Figure Legend Snippet: Zfp423 binds consensus sites in introns 3 and 5. ( A ) Semi-quantitative ChIP-PCR assays for the ZNF423 intron 5 site in IMR32 cells, with commercial antibodies against the indicated factors compared with normal serum from same host species and titrated input chromatin. Cycle numbers are indicated to the left. ( B ) Frequency of observed enrichment for predicted sites tested in replicate experiments in IMR32 cells. Schematic indicates predicted binding sites for Zfp423 (oval), Ebf (circle) and SMAD (diamond). ( C – F ) Fold enrichment at the orthologous sites in mouse P19 cells, before or after 4 hour treatment with 200 ng/ml BMP2, measured by ChIP-qPCR. Data from Zfp423 antibody E20 are shown. ( C ) Zfp423 intron 3, ( D ) Zfp423 intron 5, ( E ) Ebf1 , ( F ) Ebf3 . ( G–H ) ChIP-qPCR using a custom, affinity-purified antibody against ZNF423 fusion protein shows higher fold discrimination at Zfp423 sites in P19 cells. Experiments in A–B, C–F and G–H were performed independently by different investigators among the authors. * p≥0.05, ** p≥0.01, *** p≥0.001, t-test for comparison to IgG control for same condition and primer pair. ( I ) Alignment of the predicted binding site in intron 5 and syntenic sites from the indicated species shows strong sequence constraint that fits the overlapping Zfp423 (ROAZ) and EBF (OLF1) consensus motifs. ( J ) Western blot of mouse forebrain (Brain) and cerebellum (Cbm) extracts from wild-type littermate (+/+) or Zfp423 null mutant ( nur12 ) mice. The same blot was stripped and re-probed, using species-specific secondary antibodies coupled to alternate infrared fluors. Reactivity for β-actin and Gapdh are shown as internal loading controls. ( K ) Western blot of nuclear extracts from P19 cells treated with the indicated shRNA shows high degree of specificity for each Zfp423 antibody. Nxf1 is used as a loading control. Normalized shZfp423 signal

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Real-time Polymerase Chain Reaction, Affinity Purification, Sequencing, Western Blot, Mutagenesis, Mouse Assay, shRNA

22) Product Images from "Expression profiling of cell-intrinsic regulators in the process of differentiation of human iPSCs into retinal lineages"

Article Title: Expression profiling of cell-intrinsic regulators in the process of differentiation of human iPSCs into retinal lineages

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-018-0848-7

Effect of knockdown of EBF1 on differentiation of RGCs. a Western blot analysis demonstrating expression levels of EBF1 and indicated RGC markers in RGCs differentiated from control and EBF1 knockdown (EBF KD1 and EBF KD2) hiPSCs. GAPDH used as loading control. b Immunofluorescent microscopy showing expression of EBF1, BRN3A and Tuj1 in control and EBF1 knockdown RGCs (EBF1 KD2). c Bright-field microscopy demonstrating morphology of OVs (top) and RGCs (bottom) derived from control and EBF1 knockdown (EBF1 KD2) hiPSCs. Arrows indicate neural retina precursor layer of brighter color. DAPI 4′,6-diamidino-2-phenylindole, OV optic vesicle, RGC retinal ganglion cell
Figure Legend Snippet: Effect of knockdown of EBF1 on differentiation of RGCs. a Western blot analysis demonstrating expression levels of EBF1 and indicated RGC markers in RGCs differentiated from control and EBF1 knockdown (EBF KD1 and EBF KD2) hiPSCs. GAPDH used as loading control. b Immunofluorescent microscopy showing expression of EBF1, BRN3A and Tuj1 in control and EBF1 knockdown RGCs (EBF1 KD2). c Bright-field microscopy demonstrating morphology of OVs (top) and RGCs (bottom) derived from control and EBF1 knockdown (EBF1 KD2) hiPSCs. Arrows indicate neural retina precursor layer of brighter color. DAPI 4′,6-diamidino-2-phenylindole, OV optic vesicle, RGC retinal ganglion cell

Techniques Used: Western Blot, Expressing, Microscopy, Derivative Assay

23) Product Images from "Genotypic and Phenotypic Diversity of Herpes Simplex Virus 2 within the Infected Neonatal Population"

Article Title: Genotypic and Phenotypic Diversity of Herpes Simplex Virus 2 within the Infected Neonatal Population

Journal: mSphere

doi: 10.1128/mSphere.00590-18

Increased plaque size in culture is not determined by viral entry, DNA replication, protein expression, or infectious virus production. Viral growth characteristics were compared for representative neonatal isolates, including large-plaque formers (green) and small-plaque formers (black). (A and B) Viral entry kinetics. (A) Viral isolates were applied to Vero cell monolayers at 4°C for 1 h to allow virus binding and were then moved to 37°C to allow virus entry. Extracellular virus was inactivated by a low-pH buffer at the times indicated (orange arrowheads). Cell monolayers were washed and overlaid with methylcellulose. Plaques were scored after 100 h of incubation. PBS, phosphate-buffered saline. (B) Viral entry was quantified as the fraction of plaques formed following citrate buffer application, where 100% represents the number of plaques formed on a monolayer not treated with citrate buffer (control). These data represent results from three independent experiments. Two-way ANOVA followed by Tukey’s multiple-comparison test was applied. (C to E) Single-cycle viral replication kinetics. Vero cell monolayers were infected at MOI = 5 and incubated in the presence of 0.1% human serum. Cell monolayers were harvested at the time points indicated. (C) The quantity of viral genomes present was evaluated by qPCR for UL27. (D and E) Infectious virion production (titer) was evaluated by plaque formation on U2OS (D) or Vero (E) cells. These data represent results from three independent experiments. Two-way ANOVA followed by Tukey’s multiple-comparison test was applied. (F and G) Protein production. Vero cell monolayers were infected at MOI = 5 for 6 h (F) or 24 h (G). Whole-cell lysates were subjected to immunoblot analysis with the following antibodies: gC (UL44), gD (US6), gE (US8), gH (UL22), four virion glycoproteins; VP5 (UL19), capsid protein; ICP8 (UL29), viral single-strand DNA-binding protein; HSV, viral antibody against whole HSV-1; GAPDH, cellular glyceraldehyde-3 phosphate dehydrogenase as a loading control.
Figure Legend Snippet: Increased plaque size in culture is not determined by viral entry, DNA replication, protein expression, or infectious virus production. Viral growth characteristics were compared for representative neonatal isolates, including large-plaque formers (green) and small-plaque formers (black). (A and B) Viral entry kinetics. (A) Viral isolates were applied to Vero cell monolayers at 4°C for 1 h to allow virus binding and were then moved to 37°C to allow virus entry. Extracellular virus was inactivated by a low-pH buffer at the times indicated (orange arrowheads). Cell monolayers were washed and overlaid with methylcellulose. Plaques were scored after 100 h of incubation. PBS, phosphate-buffered saline. (B) Viral entry was quantified as the fraction of plaques formed following citrate buffer application, where 100% represents the number of plaques formed on a monolayer not treated with citrate buffer (control). These data represent results from three independent experiments. Two-way ANOVA followed by Tukey’s multiple-comparison test was applied. (C to E) Single-cycle viral replication kinetics. Vero cell monolayers were infected at MOI = 5 and incubated in the presence of 0.1% human serum. Cell monolayers were harvested at the time points indicated. (C) The quantity of viral genomes present was evaluated by qPCR for UL27. (D and E) Infectious virion production (titer) was evaluated by plaque formation on U2OS (D) or Vero (E) cells. These data represent results from three independent experiments. Two-way ANOVA followed by Tukey’s multiple-comparison test was applied. (F and G) Protein production. Vero cell monolayers were infected at MOI = 5 for 6 h (F) or 24 h (G). Whole-cell lysates were subjected to immunoblot analysis with the following antibodies: gC (UL44), gD (US6), gE (US8), gH (UL22), four virion glycoproteins; VP5 (UL19), capsid protein; ICP8 (UL29), viral single-strand DNA-binding protein; HSV, viral antibody against whole HSV-1; GAPDH, cellular glyceraldehyde-3 phosphate dehydrogenase as a loading control.

Techniques Used: Expressing, Binding Assay, Incubation, Infection, Real-time Polymerase Chain Reaction

24) Product Images from "BNIP3L/NIX and FUNDC1-mediated mitophagy is required for mitochondrial network remodeling during cardiac progenitor cell differentiation"

Article Title: BNIP3L/NIX and FUNDC1-mediated mitophagy is required for mitochondrial network remodeling during cardiac progenitor cell differentiation

Journal: Autophagy

doi: 10.1080/15548627.2019.1580095

Mitophagy of depolarized mitochondria is functional in POLG CPCs. Cells were infected with β-Gal or mCherry-PRKN prior to treatment with 25 μM FCCP for 24 h. (a) Representative western blots of LC3-II and GAPDH in WT and POLG CPCs. (b) Quantification of LC3-II:GAPDH in WT (n = 4) and POLG CPCs (n = 3). (c) Representative western blots of the mitochondrial protein TIMM23 and GAPDH in WT and POLG CPCs. (d) Quantitation of TIMM23:GAPDH in WT (n = 4) and POLG CPCs (n = 3). Data are mean ± SEM. *p
Figure Legend Snippet: Mitophagy of depolarized mitochondria is functional in POLG CPCs. Cells were infected with β-Gal or mCherry-PRKN prior to treatment with 25 μM FCCP for 24 h. (a) Representative western blots of LC3-II and GAPDH in WT and POLG CPCs. (b) Quantification of LC3-II:GAPDH in WT (n = 4) and POLG CPCs (n = 3). (c) Representative western blots of the mitochondrial protein TIMM23 and GAPDH in WT and POLG CPCs. (d) Quantitation of TIMM23:GAPDH in WT (n = 4) and POLG CPCs (n = 3). Data are mean ± SEM. *p

Techniques Used: Functional Assay, Infection, Western Blot, Quantitation Assay

PRKN is not required for mitophagy in CPCs. (a) Representative western blots of PRKN and GAPDH in mouse CPCs and adult hearts. (b) Real-time PCR analysis of Prkn transcript levels in CPCs and heart tissue (n = 3). (c) Representative western blots of TIMM23 and ACTA1 in WT and prkn −/- CPCs after treatment with 25 μM FCCP for 24 h. (d) Quantification of TIMM23:ACTA1 in WT and prkn −/- CPCs (n = 3). (e) The number and percentage of cells with mRNA detected by single-cell RNA sequencing for Prkn and mitophagy genes in mouse CPCs at passage 0 (fresh) or passage 5 (cultured). Violin plots display gene expression of mitophagy genes in mouse CPCs. (f) The number and percentage of cells with mRNA detected by single-cell RNA sequencing for PRKN and mitophagy receptors in human CPCs at passage 5 (cultured). Violin plots display gene expression of mitophagy genes in human CPCs. Data are mean ± SEM. ***p
Figure Legend Snippet: PRKN is not required for mitophagy in CPCs. (a) Representative western blots of PRKN and GAPDH in mouse CPCs and adult hearts. (b) Real-time PCR analysis of Prkn transcript levels in CPCs and heart tissue (n = 3). (c) Representative western blots of TIMM23 and ACTA1 in WT and prkn −/- CPCs after treatment with 25 μM FCCP for 24 h. (d) Quantification of TIMM23:ACTA1 in WT and prkn −/- CPCs (n = 3). (e) The number and percentage of cells with mRNA detected by single-cell RNA sequencing for Prkn and mitophagy genes in mouse CPCs at passage 0 (fresh) or passage 5 (cultured). Violin plots display gene expression of mitophagy genes in mouse CPCs. (f) The number and percentage of cells with mRNA detected by single-cell RNA sequencing for PRKN and mitophagy receptors in human CPCs at passage 5 (cultured). Violin plots display gene expression of mitophagy genes in human CPCs. Data are mean ± SEM. ***p

Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Cell Culture, Expressing

25) Product Images from "DUSP2 regulates extracellular vesicle-VEGF-C secretion and pancreatic cancer early dissemination"

Article Title: DUSP2 regulates extracellular vesicle-VEGF-C secretion and pancreatic cancer early dissemination

Journal: Journal of Extracellular Vesicles

doi: 10.1080/20013078.2020.1746529

VEGF-C is associated with extracellular vesicles . (a) Representative immunohistochemical staining images (serial section) show expression of Lyve-1 and VEGF-C in the pancreas of Lox-Stop-Lox (LSL)- Trp53 R172 H (WT) and in the tumour of LSL-Kras G12D ; LSL-Trp53 R172 H ; Pdx1-cre (KPC) transgenic mouse. (b) Serum-free conditioned medium from MIA PaCa-2 cells was collected and fractions were isolated based on size exclusion chromatography according to the manufacturer’s protocol. Expression of VEGF-C, CD63 and HSP70 was detected in vesicle-associated fractions by Western blotting (upper). Three fractions (as indicated) were sent for NTA analysis (bottom left). Protein concentration in each fraction was measured (bottom right). (c) VEGF-C is highly expressed in EV fraction. Conditioned medium from MIA PaCa-2 cells was collected and ultracentrifugation was performed to isolate microvesicles (MV), exosomes (Ex) and supernatant (Sup). Western blotting was performed to detect the expression of VEGF-C, CD63 and ALBUMIN (ALB) in whole cell lysate (WCL), MV, Ex and Sup with equal amount of protein. (d) EV was isolated by ExoQuick-TC from CM of MIA PaCa-2 cells. Equal protein amount was loaded to compare VEGF-C expression in WCL, EV and CM. CD63, TSG101 and HSP70 were used as EV markers. ALB was detected to demonstrate the purity of EV (left). EV isolated by ExoQuick-TC was sent for NTA analysis (right). (e) Representative transmission electron microscopic images show that VEGF-C is associated with EV. VEGF-C (dark particles) was stained with gold particle-labelled anti-VEGF-C antibody. (f) VEGF-C is associated with surface of EV. Isolated EV (by ExoQuick-TC) was treated with proteinase K, triton X, proteinase K plus triton X and trypsin. Western blotting was performed to detect VEGF-C and GAPDH. (g) Expression of VEGF-C in AsPC1 cells. VEGF-C cDNA was cloned into pCDH lentivirus vector under the control of CMV promoter (upper panel). Expression of secreted VEGF-C is decreased by treatment with GW4869 (40 μM). (h) VEGF-C is enriched in the small EV fraction. Conditioned medium from AsPC-VEGF-C cells was collected and the EV fraction (MV and Ex) was purified by ultracentrifugation. CD63 was detected as a marker for EV. (i) NTA analysis (left) and Western blotting (right) show particle numbers and VEGF-C expression in AsPC-Ctrl and AsPC-VEGF-C cells. EV was isolated by ExoQuick-TC. (j) LECs uptake EV from MIA PaCa-2 cells. Mia PaCa-2 cells were labelled with PKH67 and serum-free conditioned medium from PKH67 labelled Mia PaCa-2 cells were collected and isolated by ExoQuick-TC. LECs were treated EVs for 6 h and fixed for image taken. (k) Quantification of LECs proliferation treated with EV from AsPC-control and AsPC-VEGF-C cells. Ki67 staining was performed as the indicator of the proliferation of LECs. ** P
Figure Legend Snippet: VEGF-C is associated with extracellular vesicles . (a) Representative immunohistochemical staining images (serial section) show expression of Lyve-1 and VEGF-C in the pancreas of Lox-Stop-Lox (LSL)- Trp53 R172 H (WT) and in the tumour of LSL-Kras G12D ; LSL-Trp53 R172 H ; Pdx1-cre (KPC) transgenic mouse. (b) Serum-free conditioned medium from MIA PaCa-2 cells was collected and fractions were isolated based on size exclusion chromatography according to the manufacturer’s protocol. Expression of VEGF-C, CD63 and HSP70 was detected in vesicle-associated fractions by Western blotting (upper). Three fractions (as indicated) were sent for NTA analysis (bottom left). Protein concentration in each fraction was measured (bottom right). (c) VEGF-C is highly expressed in EV fraction. Conditioned medium from MIA PaCa-2 cells was collected and ultracentrifugation was performed to isolate microvesicles (MV), exosomes (Ex) and supernatant (Sup). Western blotting was performed to detect the expression of VEGF-C, CD63 and ALBUMIN (ALB) in whole cell lysate (WCL), MV, Ex and Sup with equal amount of protein. (d) EV was isolated by ExoQuick-TC from CM of MIA PaCa-2 cells. Equal protein amount was loaded to compare VEGF-C expression in WCL, EV and CM. CD63, TSG101 and HSP70 were used as EV markers. ALB was detected to demonstrate the purity of EV (left). EV isolated by ExoQuick-TC was sent for NTA analysis (right). (e) Representative transmission electron microscopic images show that VEGF-C is associated with EV. VEGF-C (dark particles) was stained with gold particle-labelled anti-VEGF-C antibody. (f) VEGF-C is associated with surface of EV. Isolated EV (by ExoQuick-TC) was treated with proteinase K, triton X, proteinase K plus triton X and trypsin. Western blotting was performed to detect VEGF-C and GAPDH. (g) Expression of VEGF-C in AsPC1 cells. VEGF-C cDNA was cloned into pCDH lentivirus vector under the control of CMV promoter (upper panel). Expression of secreted VEGF-C is decreased by treatment with GW4869 (40 μM). (h) VEGF-C is enriched in the small EV fraction. Conditioned medium from AsPC-VEGF-C cells was collected and the EV fraction (MV and Ex) was purified by ultracentrifugation. CD63 was detected as a marker for EV. (i) NTA analysis (left) and Western blotting (right) show particle numbers and VEGF-C expression in AsPC-Ctrl and AsPC-VEGF-C cells. EV was isolated by ExoQuick-TC. (j) LECs uptake EV from MIA PaCa-2 cells. Mia PaCa-2 cells were labelled with PKH67 and serum-free conditioned medium from PKH67 labelled Mia PaCa-2 cells were collected and isolated by ExoQuick-TC. LECs were treated EVs for 6 h and fixed for image taken. (k) Quantification of LECs proliferation treated with EV from AsPC-control and AsPC-VEGF-C cells. Ki67 staining was performed as the indicator of the proliferation of LECs. ** P

Techniques Used: Immunohistochemistry, Staining, Expressing, Transgenic Assay, Isolation, Size-exclusion Chromatography, Western Blot, Protein Concentration, Transmission Assay, Clone Assay, Plasmid Preparation, Purification, Marker

26) Product Images from "Enhanced oligodendrocyte maturation and myelination in a mouse model of Timothy syndrome"

Article Title: Enhanced oligodendrocyte maturation and myelination in a mouse model of Timothy syndrome

Journal: Glia

doi: 10.1002/glia.23468

Evaluation of myelin proteins expression in the postnatal TS2 mouse ( A ) MBP and MOG immunostaining in the brains of control and TS2 mice at P60. Representative brain coronal sections are shown. Scale bar = 180μm. ( B ) Myelin was quantified by analyzing fluorescence intensity of MBP and MOG in the central area of the corpus callosum ( CC ), in the cingulate cortex ( CX ) and in the striatum ( ST ) at P60. ( C ) Total proteins were collected from the corpus callosum and cerebellum ( CB ) at P60 to assess the expression of myelin proteins by western blot. Representative western blots are shown. GAPDH was used as the internal standard and data from five independent experiments are summarized based on the relative spot intensities and plotted as percent of controls. Five brains per experimental condition were analyzed and values are expressed as mean ± SEM. *p
Figure Legend Snippet: Evaluation of myelin proteins expression in the postnatal TS2 mouse ( A ) MBP and MOG immunostaining in the brains of control and TS2 mice at P60. Representative brain coronal sections are shown. Scale bar = 180μm. ( B ) Myelin was quantified by analyzing fluorescence intensity of MBP and MOG in the central area of the corpus callosum ( CC ), in the cingulate cortex ( CX ) and in the striatum ( ST ) at P60. ( C ) Total proteins were collected from the corpus callosum and cerebellum ( CB ) at P60 to assess the expression of myelin proteins by western blot. Representative western blots are shown. GAPDH was used as the internal standard and data from five independent experiments are summarized based on the relative spot intensities and plotted as percent of controls. Five brains per experimental condition were analyzed and values are expressed as mean ± SEM. *p

Techniques Used: Expressing, Immunostaining, Mouse Assay, Fluorescence, Western Blot

27) Product Images from "Developmental Changes in Hepatic Organic Cation Transporter OCT1 Protein Expression from Neonates to Children"

Article Title: Developmental Changes in Hepatic Organic Cation Transporter OCT1 Protein Expression from Neonates to Children

Journal: Drug Metabolism and Disposition

doi: 10.1124/dmd.116.072256

. OCT1 levels were calculated from membrane GAPDH– (A and C) and membrane protein–normalized (B and D) OCT1 signal density from a minimum of three immunoblots per sample. Final OCT1 values were normalized to the mean protein level for subjects aged 8 to 12 years and are expressed in AU. (A and B) Relative OCT1 protein levels for individual samples in each age group. Each symbol represents individual data and bars represent the group mean value ± S.D. (C and D) Statistical analysis of differential OCT1 protein expression by age group. Statistical differences in OCT1 expression were evaluated based on the nonparametric comparison of each pair by means of the Wilcoxon method using JMP statistical software (version 10; SAS Institute Inc.). Significant differences are reported as P values; where none are reported, associated groups were not significantly different in OCT1 protein concentration.
Figure Legend Snippet: . OCT1 levels were calculated from membrane GAPDH– (A and C) and membrane protein–normalized (B and D) OCT1 signal density from a minimum of three immunoblots per sample. Final OCT1 values were normalized to the mean protein level for subjects aged 8 to 12 years and are expressed in AU. (A and B) Relative OCT1 protein levels for individual samples in each age group. Each symbol represents individual data and bars represent the group mean value ± S.D. (C and D) Statistical analysis of differential OCT1 protein expression by age group. Statistical differences in OCT1 expression were evaluated based on the nonparametric comparison of each pair by means of the Wilcoxon method using JMP statistical software (version 10; SAS Institute Inc.). Significant differences are reported as P values; where none are reported, associated groups were not significantly different in OCT1 protein concentration.

Techniques Used: Western Blot, Expressing, Software, Protein Concentration

Example immunoblot of OCT1. Isolated crude membrane fractions (20 µ . In the lower blot, the immunoblot signal for internal standard protein GAPDH appeared as a distinct band of approximately 42 kDa and did not vary by tissue donor age.
Figure Legend Snippet: Example immunoblot of OCT1. Isolated crude membrane fractions (20 µ . In the lower blot, the immunoblot signal for internal standard protein GAPDH appeared as a distinct band of approximately 42 kDa and did not vary by tissue donor age.

Techniques Used: Isolation

Relative levels of hepatic membrane OCT1 protein levels by assumed phenotype groups. Pediatric donors were classified into three assumed phenotype groups: (A) wild type (having two *1 genotypes, n = 19), (B) heterozygotes (having one *1 genotype, n = 10), and (C) homozygotes ( n = 3). Relative OCT1 protein levels, which were calculated from membrane GAPDH-normalized OCT1 signal density, are presented for individual samples at each age group. Each symbol represents individual data. Asterisks indicate individual donors with the OCT1-Arg61Cys variant (rs12208357).
Figure Legend Snippet: Relative levels of hepatic membrane OCT1 protein levels by assumed phenotype groups. Pediatric donors were classified into three assumed phenotype groups: (A) wild type (having two *1 genotypes, n = 19), (B) heterozygotes (having one *1 genotype, n = 10), and (C) homozygotes ( n = 3). Relative OCT1 protein levels, which were calculated from membrane GAPDH-normalized OCT1 signal density, are presented for individual samples at each age group. Each symbol represents individual data. Asterisks indicate individual donors with the OCT1-Arg61Cys variant (rs12208357).

Techniques Used: Variant Assay

28) Product Images from "REST reduction is essential for hypoxia-induced neuroendocrine differentiation of prostate cancer cells by activating autophagy signaling"

Article Title: REST reduction is essential for hypoxia-induced neuroendocrine differentiation of prostate cancer cells by activating autophagy signaling

Journal: Oncotarget

doi: 10.18632/oncotarget.8433

Inhibition of neurite elongation by REST overexpression ( A ) LNCaP-TR-REST cells were treated with 0.001 μg/ml Dox under normoxia or hypoxia (2% O 2 ) conditions for 4 days. LNCaP-TR-REST cells without Dox treatment under normoxia was used as control. Representative photos of control and hypoxia-treated cells with or without REST overexpression were stained with Hoechst. ( B ) The neurite length was assessed using brightfield microscopy images (40× magnification) and quantified by the average from 10 microscopic fields; bars, SD. ( C ) The expression of REST and NSE under hypoxia treatment as described in (A) was confirmed using anti-REST and anti-NSE specific antibodies. GAPDH was used as the loading control.
Figure Legend Snippet: Inhibition of neurite elongation by REST overexpression ( A ) LNCaP-TR-REST cells were treated with 0.001 μg/ml Dox under normoxia or hypoxia (2% O 2 ) conditions for 4 days. LNCaP-TR-REST cells without Dox treatment under normoxia was used as control. Representative photos of control and hypoxia-treated cells with or without REST overexpression were stained with Hoechst. ( B ) The neurite length was assessed using brightfield microscopy images (40× magnification) and quantified by the average from 10 microscopic fields; bars, SD. ( C ) The expression of REST and NSE under hypoxia treatment as described in (A) was confirmed using anti-REST and anti-NSE specific antibodies. GAPDH was used as the loading control.

Techniques Used: Inhibition, Over Expression, Staining, Microscopy, Expressing

AMPK/mTOR pathway is activated by hypoxia treatment and REST knockdown ( A ) LNCaP cells were treated with normoxia (N) or hypoxia (H) (2% O 2 ) for 3 days (left panel). LNCaP-TR-shREST cells were treated with 1 μg/ml Dox for 3 days to knockdown REST (right panel). Total cell lysates (TLCs) were analyzed by immunoblotting using anti-REST, anti-AR, anti-p-AMPK, and anti-p-mTOR antibodies. Total AMPK and mTOR are used as controls. GAPDH was used as loading control. ( B ) The expression of each protein in three independent experiments was quantified; bars, SD.
Figure Legend Snippet: AMPK/mTOR pathway is activated by hypoxia treatment and REST knockdown ( A ) LNCaP cells were treated with normoxia (N) or hypoxia (H) (2% O 2 ) for 3 days (left panel). LNCaP-TR-shREST cells were treated with 1 μg/ml Dox for 3 days to knockdown REST (right panel). Total cell lysates (TLCs) were analyzed by immunoblotting using anti-REST, anti-AR, anti-p-AMPK, and anti-p-mTOR antibodies. Total AMPK and mTOR are used as controls. GAPDH was used as loading control. ( B ) The expression of each protein in three independent experiments was quantified; bars, SD.

Techniques Used: Expressing

Hypoxia induces NED of LNCaP cells concomitant with down-regulation REST protein levels but not REST mRNA ( A ) LNCaP cells were treated with hypoxia (2% O 2 ) for 3 days. Representative photos of control and hypoxia-treated cells were stained with Hoechst. ( B ) The induced neurite length was assessed using brightfield microscopy images (40× magnification) and quantified by the average from 10 microscopic fields; bars, SD. ( C ) Total cell lysates (TCLs) were prepared from LNCaP cells treated as described in (A) for 1, 2 and 3 days and then immunoblotted to detect REST, AR, β-tubulin III, NSE, and β-TrCP. GAPDH was used as the loading control. ( D ) RT-qPCR analysis of total RNA from LNCaP cells treated as described in (A). The relative mRNA level of REST was normalized with B2M. Values from 3 independent experiments are reported as mean ± SD. ( E ) LNCaP cells were treated with hypoxia for 3 days in the presence or absence of 0.09 μM MG-132. The expression of REST was detected by immunoblotting using anti-REST antibody. GAPDH was used as the loading control.
Figure Legend Snippet: Hypoxia induces NED of LNCaP cells concomitant with down-regulation REST protein levels but not REST mRNA ( A ) LNCaP cells were treated with hypoxia (2% O 2 ) for 3 days. Representative photos of control and hypoxia-treated cells were stained with Hoechst. ( B ) The induced neurite length was assessed using brightfield microscopy images (40× magnification) and quantified by the average from 10 microscopic fields; bars, SD. ( C ) Total cell lysates (TCLs) were prepared from LNCaP cells treated as described in (A) for 1, 2 and 3 days and then immunoblotted to detect REST, AR, β-tubulin III, NSE, and β-TrCP. GAPDH was used as the loading control. ( D ) RT-qPCR analysis of total RNA from LNCaP cells treated as described in (A). The relative mRNA level of REST was normalized with B2M. Values from 3 independent experiments are reported as mean ± SD. ( E ) LNCaP cells were treated with hypoxia for 3 days in the presence or absence of 0.09 μM MG-132. The expression of REST was detected by immunoblotting using anti-REST antibody. GAPDH was used as the loading control.

Techniques Used: Staining, Microscopy, Quantitative RT-PCR, Expressing

29) Product Images from "Conditional Deletion of the L-Type Calcium Channel Cav1.2 in Oligodendrocyte Progenitor Cells Affects Postnatal Myelination in Mice"

Article Title: Conditional Deletion of the L-Type Calcium Channel Cav1.2 in Oligodendrocyte Progenitor Cells Affects Postnatal Myelination in Mice

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1770-16.2016

Delayed in vitro maturation of Cav1.2 KO OPCs. A , After 3 d of 4-OH-tamoxifen treatment, semiquantitative RT-PCR and Western blot analysis of Cav1.2 expression in OPCs was performed using GAPDH and β-actin, respectively, as internal standards.
Figure Legend Snippet: Delayed in vitro maturation of Cav1.2 KO OPCs. A , After 3 d of 4-OH-tamoxifen treatment, semiquantitative RT-PCR and Western blot analysis of Cav1.2 expression in OPCs was performed using GAPDH and β-actin, respectively, as internal standards.

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

30) Product Images from "TNF-α-induced miR-450a mediates TMEM182 expression to promote oral squamous cell carcinoma motility"

Article Title: TNF-α-induced miR-450a mediates TMEM182 expression to promote oral squamous cell carcinoma motility

Journal: PLoS ONE

doi: 10.1371/journal.pone.0213463

TNFα upregulates miR-450a versus ERK and NFκB pathways to inhibit TMEM182 stabilized OSCC cells mobility. (A) OSCC cells were subjected into negative control DMSO (-), ERK inhibitor (ERKi), NF-κB inhibitor (NF-κBi), or p38 inhibitor (p38i) as described above. After 6 hrs, TNF-α and ddH 2 O (negative control) were added as indicated treatments. Twenty-four hrs later, miR-450a levels were evaluated with qPCR. Levels of miR-450a in described conditions were standardized to negative controls (DMSO + ddH 2 O one). TNF-α only was used as reference. (B) RT-PCR analyses revealed that TNF-α suppressed TMEM182 expression in human OSCC cells was restored by ERKi. ddH 2 O and DMSO (-) were used as negative controls. GAPDH was used as the loading control. (C) Cell adhesion analyses revealed that ERKi or NF-κBi pretreated SAS cells successfully abolished TNF-α reduced cell adhesion ability. (D) TNF-α-induced miR-450a repressed TMEM182 expression to promote tumor malignancy through both intrinsic ERK and NF-κB pathways. Results were represented as mean±SEM;** P
Figure Legend Snippet: TNFα upregulates miR-450a versus ERK and NFκB pathways to inhibit TMEM182 stabilized OSCC cells mobility. (A) OSCC cells were subjected into negative control DMSO (-), ERK inhibitor (ERKi), NF-κB inhibitor (NF-κBi), or p38 inhibitor (p38i) as described above. After 6 hrs, TNF-α and ddH 2 O (negative control) were added as indicated treatments. Twenty-four hrs later, miR-450a levels were evaluated with qPCR. Levels of miR-450a in described conditions were standardized to negative controls (DMSO + ddH 2 O one). TNF-α only was used as reference. (B) RT-PCR analyses revealed that TNF-α suppressed TMEM182 expression in human OSCC cells was restored by ERKi. ddH 2 O and DMSO (-) were used as negative controls. GAPDH was used as the loading control. (C) Cell adhesion analyses revealed that ERKi or NF-κBi pretreated SAS cells successfully abolished TNF-α reduced cell adhesion ability. (D) TNF-α-induced miR-450a repressed TMEM182 expression to promote tumor malignancy through both intrinsic ERK and NF-κB pathways. Results were represented as mean±SEM;** P

Techniques Used: Negative Control, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing

Overexpression of TMEM182 renders human OSCC cells resistance to miR-450a-decreased cell adhesion. (A) Changes of TMEM182 levels in OSCC cells transfected with control miRNA (scramble)/miR-450a and pCDH-CMV-GFP puro+ (vehicle)/TMEM182 (TMEM182-flag) were assessed by RT-PCR as described in panel. GAPDH was used as a loading control. (B) Cell adhesion assays of OSCC cells transfected with scramble/miR-450a and vehicle/TMEM182-flag were as described above. (C) Transwell invasion assays were used to measure the effect of scramble/miR-450a and vehicle/TMEM182-flag in DOK and SAS cells after 48 hrs transfection. (D) TMEM182 overexpression in SAS cells was confirmed with RT-PCR after transfection of GFP-linked TMEM182 (TMEM182-GFP) and empty vector pEGFPN1 (vehicle). (E) Representative epifluorescence images of SAS cells transiently transfected with TMEM182-GFP/ Vehicle and co-immunolabeled endogenous E-cadherin. Vehicle (Green) expression was scattered inside of cells (D-i, D-ii) . Co-stained E-cadherin (Red), as a plasma membrane marker, was localized at the lateral membrane and intracellular junctional area. Nuclei were labeled with DAPI (Blue).TMEM182-driven GFP(green) was co-localized with E-cadherin (red) at the sites of cell-cell contact on the plasma membrane (D-iii, D-iv) . Original magnification, x400. Data was represented as mean±SEM; * P
Figure Legend Snippet: Overexpression of TMEM182 renders human OSCC cells resistance to miR-450a-decreased cell adhesion. (A) Changes of TMEM182 levels in OSCC cells transfected with control miRNA (scramble)/miR-450a and pCDH-CMV-GFP puro+ (vehicle)/TMEM182 (TMEM182-flag) were assessed by RT-PCR as described in panel. GAPDH was used as a loading control. (B) Cell adhesion assays of OSCC cells transfected with scramble/miR-450a and vehicle/TMEM182-flag were as described above. (C) Transwell invasion assays were used to measure the effect of scramble/miR-450a and vehicle/TMEM182-flag in DOK and SAS cells after 48 hrs transfection. (D) TMEM182 overexpression in SAS cells was confirmed with RT-PCR after transfection of GFP-linked TMEM182 (TMEM182-GFP) and empty vector pEGFPN1 (vehicle). (E) Representative epifluorescence images of SAS cells transiently transfected with TMEM182-GFP/ Vehicle and co-immunolabeled endogenous E-cadherin. Vehicle (Green) expression was scattered inside of cells (D-i, D-ii) . Co-stained E-cadherin (Red), as a plasma membrane marker, was localized at the lateral membrane and intracellular junctional area. Nuclei were labeled with DAPI (Blue).TMEM182-driven GFP(green) was co-localized with E-cadherin (red) at the sites of cell-cell contact on the plasma membrane (D-iii, D-iv) . Original magnification, x400. Data was represented as mean±SEM; * P

Techniques Used: Over Expression, Transfection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Immunolabeling, Expressing, Staining, Marker, Labeling

TMEM182 decreases OSCC cell motility. (A) RT-PCR analyses were performed to detect mRNA expression level of TMEM182 in DOK and SAS cells transfected withTMEM182 knockdown clones-shRNA clone 1 (sh182#1), clone 2 (sh182#2), or empty vector (shCTRL). GAPDH was used as a loading control. (B) Suppression of cell adhesive ability was found in TMEM182-knockdown cells towards to fibronectin and matrigel. (C) RT-PCR and Western blot analyses measured the levels of TMEM182 in vehicle or TMEM182-flag transfected cells. GAPDH and α-tubulin were used as loading controls. (D-E) Cell adhesion and invasion analyses of TMEM182 overexpression in DOK and SAS were measured. Data was represented as mean±SEM; * P
Figure Legend Snippet: TMEM182 decreases OSCC cell motility. (A) RT-PCR analyses were performed to detect mRNA expression level of TMEM182 in DOK and SAS cells transfected withTMEM182 knockdown clones-shRNA clone 1 (sh182#1), clone 2 (sh182#2), or empty vector (shCTRL). GAPDH was used as a loading control. (B) Suppression of cell adhesive ability was found in TMEM182-knockdown cells towards to fibronectin and matrigel. (C) RT-PCR and Western blot analyses measured the levels of TMEM182 in vehicle or TMEM182-flag transfected cells. GAPDH and α-tubulin were used as loading controls. (D-E) Cell adhesion and invasion analyses of TMEM182 overexpression in DOK and SAS were measured. Data was represented as mean±SEM; * P

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Clone Assay, shRNA, Plasmid Preparation, Western Blot, Over Expression

miR-450a decreases TMEM182 by directly targeting 3'-UTR. (A) Flowchart for in silico analysis of miR-450a-regulated genes from OSCC cell lines (DOK and SAS cells) and our previous OSCC clinical samples data (n = 40)(accession number GSE37991). (B) Twelve of miR-450a-targeted candidates were evaluated on the basis of down-regulated rates (fold change) and Pearson r correlation against miR-450a expression in previous OSCC clinical samples (n = 40) data. TMEM182 (black circle) presented the best negative correlation with miR-450a. (C) Levels of TMEM182 changes in DOK and SAS cells were assessed with RT-PCR and western blot after miR-450a mimics/scramble transfection for 48 hrs. Numerical values for band intensities are shown below the gels. The values were quantitated by densitometry and normalized to GAPDH or α-tubulin. (D) Schematic representation of predicted miR-450a binding sequence in the 3'-UTR of TMEM182 with wild-type form (3'UTR-WT), and with miR-450a binding site deleted form (3'UTR-DEL). (E) miR-450a regulated TMEM182 3'-UTRluciferase activities of 3'-UTR-WTor 3'-UTR-DEL in DOK and SAS cells after 48 hrs transfection as described in panel. The relative luciferase activities are the ratios of Renilla luciferase normalized to scramble. (F) Levels of TMEM182 in OSCC human samples (n = 35) was assessed with qPCR. (Student’s t test, p
Figure Legend Snippet: miR-450a decreases TMEM182 by directly targeting 3'-UTR. (A) Flowchart for in silico analysis of miR-450a-regulated genes from OSCC cell lines (DOK and SAS cells) and our previous OSCC clinical samples data (n = 40)(accession number GSE37991). (B) Twelve of miR-450a-targeted candidates were evaluated on the basis of down-regulated rates (fold change) and Pearson r correlation against miR-450a expression in previous OSCC clinical samples (n = 40) data. TMEM182 (black circle) presented the best negative correlation with miR-450a. (C) Levels of TMEM182 changes in DOK and SAS cells were assessed with RT-PCR and western blot after miR-450a mimics/scramble transfection for 48 hrs. Numerical values for band intensities are shown below the gels. The values were quantitated by densitometry and normalized to GAPDH or α-tubulin. (D) Schematic representation of predicted miR-450a binding sequence in the 3'-UTR of TMEM182 with wild-type form (3'UTR-WT), and with miR-450a binding site deleted form (3'UTR-DEL). (E) miR-450a regulated TMEM182 3'-UTRluciferase activities of 3'-UTR-WTor 3'-UTR-DEL in DOK and SAS cells after 48 hrs transfection as described in panel. The relative luciferase activities are the ratios of Renilla luciferase normalized to scramble. (F) Levels of TMEM182 in OSCC human samples (n = 35) was assessed with qPCR. (Student’s t test, p

Techniques Used: In Silico, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Binding Assay, Sequencing, Luciferase, Real-time Polymerase Chain Reaction

TMEM182 is down-regulated by miR-450a in response to TNF-α in OSCC cells. (A) Levels of TMEM182 in DOK and SAS cells treated with either ddH 2 O (CTRL) or TNF-αwere assessed with RT-PCR. GAPDH was used as a control. (B) miR-450a changes in DOK and SAS cells treated with or without TNF-α were measured with qPCR and normalized to RNU44. (C) Luciferase activity measured that TNF-α regulated TMEM182 through miR-450a binding site at 3'-UTR. Cells were transfected with TMEM182 3'-UTRs constructed either with wild-type (3'-UTR-WT) or miR-450a binding site truncated (3'-UTR-DEL), before TNF-α was added for 24 hrs as described in panel. Relative luciferase activities were the ratios of Renilla luciferase normalized to the wild-type control. (D-E) Cells were transfected with either empty vector pCDH-CMV-GFP puro+ (Vehicle) or TMEM182 (TMEM182-flag) followed by the stimulation with TNF-α for 24 hrs, and then, corresponding adhesion ability and invasion ability were measured. Data are represented as mean±SEM; ** P
Figure Legend Snippet: TMEM182 is down-regulated by miR-450a in response to TNF-α in OSCC cells. (A) Levels of TMEM182 in DOK and SAS cells treated with either ddH 2 O (CTRL) or TNF-αwere assessed with RT-PCR. GAPDH was used as a control. (B) miR-450a changes in DOK and SAS cells treated with or without TNF-α were measured with qPCR and normalized to RNU44. (C) Luciferase activity measured that TNF-α regulated TMEM182 through miR-450a binding site at 3'-UTR. Cells were transfected with TMEM182 3'-UTRs constructed either with wild-type (3'-UTR-WT) or miR-450a binding site truncated (3'-UTR-DEL), before TNF-α was added for 24 hrs as described in panel. Relative luciferase activities were the ratios of Renilla luciferase normalized to the wild-type control. (D-E) Cells were transfected with either empty vector pCDH-CMV-GFP puro+ (Vehicle) or TMEM182 (TMEM182-flag) followed by the stimulation with TNF-α for 24 hrs, and then, corresponding adhesion ability and invasion ability were measured. Data are represented as mean±SEM; ** P

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Binding Assay, Transfection, Construct, Plasmid Preparation

31) Product Images from "Transcription Factor Elf3 Modulates Vasopressin-Induced Aquaporin-2 Gene Expression in Kidney Collecting Duct Cells"

Article Title: Transcription Factor Elf3 Modulates Vasopressin-Induced Aquaporin-2 Gene Expression in Kidney Collecting Duct Cells

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2019.01308

Vasopressin did not induce Elf3 nuclear translocation in the mpkCCD cells. (A) confocal immunofluorescence micrographs of mpkCCD cells transfected with the V5-tagged Elf3 isoform 1 expression vector and stimulated with dDAVP for 30 min. (B) Nucleus (Nc) vs. cytosol (Cyt) fractionation followed by immunoblotting for V5-tagged Elf3 in the mpkCCD cells under vehicle or dDAVP-stimulated conditions. GAPDH and histone H2A were stained as a cytosol and a nucleus marker, respectively. In, input i.e., cell lysate.
Figure Legend Snippet: Vasopressin did not induce Elf3 nuclear translocation in the mpkCCD cells. (A) confocal immunofluorescence micrographs of mpkCCD cells transfected with the V5-tagged Elf3 isoform 1 expression vector and stimulated with dDAVP for 30 min. (B) Nucleus (Nc) vs. cytosol (Cyt) fractionation followed by immunoblotting for V5-tagged Elf3 in the mpkCCD cells under vehicle or dDAVP-stimulated conditions. GAPDH and histone H2A were stained as a cytosol and a nucleus marker, respectively. In, input i.e., cell lysate.

Techniques Used: Translocation Assay, Immunofluorescence, Transfection, Expressing, Plasmid Preparation, Fractionation, Staining, Marker

32) Product Images from "MiR-30a-5p Inhibits Epithelial-to-Mesenchymal Transition and Upregulates Expression of Tight Junction Protein Claudin-5 in Human Upper Tract Urothelial Carcinoma Cells"

Article Title: MiR-30a-5p Inhibits Epithelial-to-Mesenchymal Transition and Upregulates Expression of Tight Junction Protein Claudin-5 in Human Upper Tract Urothelial Carcinoma Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18081826

miR-30a-5p overexpression inhibits epithelial-to-mesenchymal transition marker expression in UTUC cells. Western blot analysis of expression levels of epithelial marker E-cadherin ( A ), and mesenchymal markers, including vimentin ( B ), fibronectin ( C ), and α-SMA ( D ) in cultured BFTC-909 cells transfected with miR-30a-5p or miR-NC. GAPDH was used as the loading control. Data are expressed as means ± SEM ( n = 3). * indicates that p
Figure Legend Snippet: miR-30a-5p overexpression inhibits epithelial-to-mesenchymal transition marker expression in UTUC cells. Western blot analysis of expression levels of epithelial marker E-cadherin ( A ), and mesenchymal markers, including vimentin ( B ), fibronectin ( C ), and α-SMA ( D ) in cultured BFTC-909 cells transfected with miR-30a-5p or miR-NC. GAPDH was used as the loading control. Data are expressed as means ± SEM ( n = 3). * indicates that p

Techniques Used: Over Expression, Marker, Expressing, Western Blot, Cell Culture, Transfection

Claudin-5 expression is enhanced by the miR-30a-5p. The qPCR ( A ) and Western blot ( B ) analysis of CLDN-5 expression levels in cultured BFTC-909 cells transfected with miR-30a-5p or miR-NC. GAPDH was used as the loading control. Data are expressed as means ± SEM ( n = 3). * indicates that p
Figure Legend Snippet: Claudin-5 expression is enhanced by the miR-30a-5p. The qPCR ( A ) and Western blot ( B ) analysis of CLDN-5 expression levels in cultured BFTC-909 cells transfected with miR-30a-5p or miR-NC. GAPDH was used as the loading control. Data are expressed as means ± SEM ( n = 3). * indicates that p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, Transfection

33) Product Images from "ETV7-Mediated DNAJC15 Repression Leads to Doxorubicin Resistance in Breast Cancer Cells"

Article Title: ETV7-Mediated DNAJC15 Repression Leads to Doxorubicin Resistance in Breast Cancer Cells

Journal: Neoplasia (New York, N.Y.)

doi: 10.1016/j.neo.2018.06.008

ETV7 can repress DNAJC15 expression at the transcriptional level and DNAJC15 over-expression can rescue Doxorubicin sensitivity. A) RT-qPCR analysis of ETV7 and DNAJC15 expression in MCF7 cells transfected with pCMV6-Entry-Empty or pCMV6-Entry-ETV7 plasmids. B) Gene reporter assay of MCF7 cells transiently over-expressing pCMV6-Entry-Empty or pCMV6-Entry-ETV7 along with pGL4.26-DNAJC15 reporter plasmid or the pGL4.26-DNAJC15-BS1 or -BS2 plasmids mutated in the putative ETV7 binding sites. Data are normalized using pRL-SV40 and are shown as fold of induction relative to the empty control. C) ChIP-PCR of DNAJC15 and GAPDH (control) promoter regions in MCF7 transfected with pCMV6-ETV7. Shown is the percentage of enrichment of ETV7 or control (IgG) bound to DNAJC15 promoter region in respect to INPUT DNA. For panels A-C, bars represent averages and standard deviations of at least three biological replicates. D-E) MTT Assay of ETV7-over-expressing MCF7 (D) and MDA-MB-231 (E) cells transiently transfected with pCMV6-Entry-Empty or pCMV6-Entry-DNAJC15 plasmids and treated with Doxorubicin 1.5 μM or 3 μM for 72 hours. Experiments are done in quadruplicate. * = P -value
Figure Legend Snippet: ETV7 can repress DNAJC15 expression at the transcriptional level and DNAJC15 over-expression can rescue Doxorubicin sensitivity. A) RT-qPCR analysis of ETV7 and DNAJC15 expression in MCF7 cells transfected with pCMV6-Entry-Empty or pCMV6-Entry-ETV7 plasmids. B) Gene reporter assay of MCF7 cells transiently over-expressing pCMV6-Entry-Empty or pCMV6-Entry-ETV7 along with pGL4.26-DNAJC15 reporter plasmid or the pGL4.26-DNAJC15-BS1 or -BS2 plasmids mutated in the putative ETV7 binding sites. Data are normalized using pRL-SV40 and are shown as fold of induction relative to the empty control. C) ChIP-PCR of DNAJC15 and GAPDH (control) promoter regions in MCF7 transfected with pCMV6-ETV7. Shown is the percentage of enrichment of ETV7 or control (IgG) bound to DNAJC15 promoter region in respect to INPUT DNA. For panels A-C, bars represent averages and standard deviations of at least three biological replicates. D-E) MTT Assay of ETV7-over-expressing MCF7 (D) and MDA-MB-231 (E) cells transiently transfected with pCMV6-Entry-Empty or pCMV6-Entry-DNAJC15 plasmids and treated with Doxorubicin 1.5 μM or 3 μM for 72 hours. Experiments are done in quadruplicate. * = P -value

Techniques Used: Expressing, Over Expression, Quantitative RT-PCR, Transfection, Reporter Assay, Plasmid Preparation, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, MTT Assay, Multiple Displacement Amplification

34) Product Images from "Thermal cycling protects SH-SY5Y cells against hydrogen peroxide and β-amyloid-induced cell injury through stress response mechanisms involving Akt pathway"

Article Title: Thermal cycling protects SH-SY5Y cells against hydrogen peroxide and β-amyloid-induced cell injury through stress response mechanisms involving Akt pathway

Journal: bioRxiv

doi: 10.1101/2019.12.19.877373

Effect of TC-HT on Akt/Nrf2 and Akt/CREB signalling pathways and related protein expressions. Western blot analysis of p-Akt, Nrf2, p-CREB, and HO-1 proteins. Quantification of p-Akt, Nrf2, p-CREB, HO-1 expressions after H 2 O 2 , TC+H 2 O 2 , or HT+H 2 O 2 treatment. The expression levels were normalized to GAPDH. Data represent the mean ± standard deviation (n=3). ***P
Figure Legend Snippet: Effect of TC-HT on Akt/Nrf2 and Akt/CREB signalling pathways and related protein expressions. Western blot analysis of p-Akt, Nrf2, p-CREB, and HO-1 proteins. Quantification of p-Akt, Nrf2, p-CREB, HO-1 expressions after H 2 O 2 , TC+H 2 O 2 , or HT+H 2 O 2 treatment. The expression levels were normalized to GAPDH. Data represent the mean ± standard deviation (n=3). ***P

Techniques Used: Western Blot, Expressing, Standard Deviation

35) Product Images from "Effect of high frequency and low intensity pulsed electric field on protecting SH-SY5Y cells against hydrogen peroxide and β-amyloid-induced cell injury via ERK pathway"

Article Title: Effect of high frequency and low intensity pulsed electric field on protecting SH-SY5Y cells against hydrogen peroxide and β-amyloid-induced cell injury via ERK pathway

Journal: bioRxiv

doi: 10.1101/2019.12.30.891499

Effect of H-LIPEF on ERK/Nrf2 and ERK/CREB signalling pathways and related protein expressions. Western blot analysis of p-ERK (A), Nrf2 (B), p-CREB (C), Bcl-2 and Bax (D) protein expressions. The expression levels were normalized to GAPDH and each relative expression level was compared with control. Data represent the mean ± standard deviation (n=3). *** P
Figure Legend Snippet: Effect of H-LIPEF on ERK/Nrf2 and ERK/CREB signalling pathways and related protein expressions. Western blot analysis of p-ERK (A), Nrf2 (B), p-CREB (C), Bcl-2 and Bax (D) protein expressions. The expression levels were normalized to GAPDH and each relative expression level was compared with control. Data represent the mean ± standard deviation (n=3). *** P

Techniques Used: Western Blot, Expressing, Standard Deviation

36) Product Images from "Ethyl Acetate Extract of Scindapsus cf. hederaceus Exerts the Inhibitory Bioactivity on Human Non-Small Cell Lung Cancer Cells through Modulating ER Stress"

Article Title: Ethyl Acetate Extract of Scindapsus cf. hederaceus Exerts the Inhibitory Bioactivity on Human Non-Small Cell Lung Cancer Cells through Modulating ER Stress

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19071832

SH-EAE reduces the EGFR and VEGF signaling in NSCLC cells. Expression of phospho-EGFR (Tyr845), EGFR, and VEGF were analyzed by western blot in NSCLC cell line H1299. GAPDH was used as the loading control. Data are representative of three independent experiments.
Figure Legend Snippet: SH-EAE reduces the EGFR and VEGF signaling in NSCLC cells. Expression of phospho-EGFR (Tyr845), EGFR, and VEGF were analyzed by western blot in NSCLC cell line H1299. GAPDH was used as the loading control. Data are representative of three independent experiments.

Techniques Used: Expressing, Western Blot

37) Product Images from "TBK1 Mutation Spectrum in an Extended European Patient Cohort with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis"

Article Title: TBK1 Mutation Spectrum in an Extended European Patient Cohort with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis

Journal: Human Mutation

doi: 10.1002/humu.23161

Transcript and protein analysis of TBK1 LoF and single amino acid deletion mutations. A : gDNA and cDNA sequence traces around the c.288delT (p. Val97Phefs * 2) mutation, showing reduced expression of the mutant transcript on cDNA extracted from blood. B : gDNA and cDNA sequence traces around the c.379C > T (p.Arg127 * ) mutation, showing the absence of the mutant transcript on cDNA extracted from blood. C : Sizing of cDNA fragments generated with primers in TBK1 exon 10 and exon 13 of the c.1340+1G > A (p.Ala417 * ) carrier on cDNA extracted from blood. Sequence traces from the low‐expressed aberrant transcript demonstrates skipping of exon 11. D : Transcript and protein analysis on brain frontal cortex from the c.235_237delACA (p.Thr79del) carrier and four age‐matched control brains. The graph on the left shows the relative expression in the patient sample (blue) compared with the control samples (black) measured by quantitative real‐time PCR (qRT‐PCR). In the middle, Western blot analysis is shown of protein extracts from the patient carrier compared with control individuals. The upper band represents TBK1 (84 kDa) and the lower band represents the housekeeping protein GAPDH (37 kDa). The graph on the right shows the quantification in the patient sample (blue) and control samples (black) of the TBK1 signal normalized to the signal of GAPDH. Error bars represent the SD. E : Western blot analysis of phosphorylated TBK1 (Ser172, p‐TBK1) (upper band, 84 kDa) in HEK293T cells overexpressing the in‐frame single amino acid deletions (p.Thr79del, p.Asp167del, and p.Glu643del) compared with wild type, relative to GAPDH (lower band, 37 kDa). Mock and kinase dead (p.Ser172Ala, KD) were used as negative control. cDNA numbering according to reference sequence NM_013254.3, in addition, for intronic variants, the genomic reference sequence NC_000012.12 was used. Nucleotide positions refer to cDNA sequence and nucleotide numbering uses +1 as the A of the ATG translation initiation codon in the reference sequence, with the initiation codon as codon 1. Protein numbering according to reference sequence NP_037386.1.
Figure Legend Snippet: Transcript and protein analysis of TBK1 LoF and single amino acid deletion mutations. A : gDNA and cDNA sequence traces around the c.288delT (p. Val97Phefs * 2) mutation, showing reduced expression of the mutant transcript on cDNA extracted from blood. B : gDNA and cDNA sequence traces around the c.379C > T (p.Arg127 * ) mutation, showing the absence of the mutant transcript on cDNA extracted from blood. C : Sizing of cDNA fragments generated with primers in TBK1 exon 10 and exon 13 of the c.1340+1G > A (p.Ala417 * ) carrier on cDNA extracted from blood. Sequence traces from the low‐expressed aberrant transcript demonstrates skipping of exon 11. D : Transcript and protein analysis on brain frontal cortex from the c.235_237delACA (p.Thr79del) carrier and four age‐matched control brains. The graph on the left shows the relative expression in the patient sample (blue) compared with the control samples (black) measured by quantitative real‐time PCR (qRT‐PCR). In the middle, Western blot analysis is shown of protein extracts from the patient carrier compared with control individuals. The upper band represents TBK1 (84 kDa) and the lower band represents the housekeeping protein GAPDH (37 kDa). The graph on the right shows the quantification in the patient sample (blue) and control samples (black) of the TBK1 signal normalized to the signal of GAPDH. Error bars represent the SD. E : Western blot analysis of phosphorylated TBK1 (Ser172, p‐TBK1) (upper band, 84 kDa) in HEK293T cells overexpressing the in‐frame single amino acid deletions (p.Thr79del, p.Asp167del, and p.Glu643del) compared with wild type, relative to GAPDH (lower band, 37 kDa). Mock and kinase dead (p.Ser172Ala, KD) were used as negative control. cDNA numbering according to reference sequence NM_013254.3, in addition, for intronic variants, the genomic reference sequence NC_000012.12 was used. Nucleotide positions refer to cDNA sequence and nucleotide numbering uses +1 as the A of the ATG translation initiation codon in the reference sequence, with the initiation codon as codon 1. Protein numbering according to reference sequence NP_037386.1.

Techniques Used: Sequencing, Mutagenesis, Expressing, Generated, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Negative Control

38) Product Images from "p63α protein up-regulates heat shock protein 70 expression via E2F1 transcription factor 1, promoting Wasf3/Wave3/MMP9 signaling and bladder cancer invasion"

Article Title: p63α protein up-regulates heat shock protein 70 expression via E2F1 transcription factor 1, promoting Wasf3/Wave3/MMP9 signaling and bladder cancer invasion

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M117.792010

Wave3 was an Hsp70 downstream mediator responsible for p63α promotion of BC invasion. A , T24T(Vector), T24T(p63α/Nonsense), T24T(p63α/shHsp70-1), and T24T(p63α/shHsp70–2) cells were extracted upon the density reaching 80–90%, and the cell extracts were subjected to Western blotting with the specific antibodies as indicated. GAPDH was used as a protein-loading control. B and C , the invasion abilities of T24T(p63α/Nonsense), T24T(p63α/shHsp70-1), and T24T(p63α/shHsp70–2) cells were subjected to a transwell invasion assay. B , the invasion rate was normalized with the insert control according to the manufacturer's instruction, and the results are presented as relative invasion cells. C , the asterisk (*) indicates a significant difference of invasion abilities between T24T(p63α/Nonsense) and T24T(p63α/shHsp70) cells ( p
Figure Legend Snippet: Wave3 was an Hsp70 downstream mediator responsible for p63α promotion of BC invasion. A , T24T(Vector), T24T(p63α/Nonsense), T24T(p63α/shHsp70-1), and T24T(p63α/shHsp70–2) cells were extracted upon the density reaching 80–90%, and the cell extracts were subjected to Western blotting with the specific antibodies as indicated. GAPDH was used as a protein-loading control. B and C , the invasion abilities of T24T(p63α/Nonsense), T24T(p63α/shHsp70-1), and T24T(p63α/shHsp70–2) cells were subjected to a transwell invasion assay. B , the invasion rate was normalized with the insert control according to the manufacturer's instruction, and the results are presented as relative invasion cells. C , the asterisk (*) indicates a significant difference of invasion abilities between T24T(p63α/Nonsense) and T24T(p63α/shHsp70) cells ( p

Techniques Used: Western Blot, Transwell Invasion Assay

Hsp70 and Wave3 were consistently up-regulated in p63α ectopic expressed human BC cells. A–C , the indicated cells were seeded into 6-well plates. The cells were extracted upon the cell density reaching 80–90%, and the cell extracts were subjected to Western blot for determination of protein expression as indicated. GAPDH was used as a protein loading control.
Figure Legend Snippet: Hsp70 and Wave3 were consistently up-regulated in p63α ectopic expressed human BC cells. A–C , the indicated cells were seeded into 6-well plates. The cells were extracted upon the cell density reaching 80–90%, and the cell extracts were subjected to Western blot for determination of protein expression as indicated. GAPDH was used as a protein loading control.

Techniques Used: Western Blot, Expressing

E2F1 was the transcription factor mediating p63α promotion of Hsp70 transcription and cell invasion in human BC cells. A and C , knockdown efficiency of Sp1 or E2F1 ectopic expression in T24T cells was evaluated by Western blotting. GAPDH was used as a protein loading control. B and D , T24T(Nonsense) versus T24T(shSp1) cells or T24T(Vector) versus T24T(E2F1) cells were extracted for total RNA with TRIzol reagent. RT-PCR was used to determine hsp70 mRNA expression, whereas GAPDH was used as an internal control. E , T24T(Vector) and T24T(E2F1) cells were transfected with an hsp70 promoter-driven luciferase reporter together with pRL-TK. The transfectants were used to determine hsp70 promoter activity by measuring luciferase activity. pRL-TK was used as an internal control to normalize the transfection efficiency. Each bar indicates the mean ± S.D. from three replicate assays. F , ChIP assay was carried out as described under “Experimental Procedures,” anti-E2F1 specific antibody was used to pull down its E2F1-bound DNA fragments, and primers for E2F1-binding site and negative control Sp1-binding site were used to carry out PCR. IP , immunoprecipitation. G and H , the invasion abilities of T24T(Vector) and T24T(E2F1) cells were determined using a transwell invasion assay. The results are presented as the number of invasive T24T(E2F1) cells relative to invasive T24T(Vector) transfectants ( H ). I , the knockdown efficiency of E2F1 in T24T(p63α) cells was determined by Western blotting. GAPDH was used as a protein loading control. J and K , the invasion abilities of T24T(p63α/Nonsense), T24T(p63α/shE2F1–1), and T24T(p63α/shE2F1–2) cells were determined using a transwell invasion assay. L and M , IHC-P was carried out to evaluate E2F1 protein expression in mouse BC tissues and normal bladder tissues. The optical density was analyzed as described under “Experimental Procedures.” The asterisk (*) indicates a significant increase in p63α protein expression in comparison with that observed in normal tissues ( p
Figure Legend Snippet: E2F1 was the transcription factor mediating p63α promotion of Hsp70 transcription and cell invasion in human BC cells. A and C , knockdown efficiency of Sp1 or E2F1 ectopic expression in T24T cells was evaluated by Western blotting. GAPDH was used as a protein loading control. B and D , T24T(Nonsense) versus T24T(shSp1) cells or T24T(Vector) versus T24T(E2F1) cells were extracted for total RNA with TRIzol reagent. RT-PCR was used to determine hsp70 mRNA expression, whereas GAPDH was used as an internal control. E , T24T(Vector) and T24T(E2F1) cells were transfected with an hsp70 promoter-driven luciferase reporter together with pRL-TK. The transfectants were used to determine hsp70 promoter activity by measuring luciferase activity. pRL-TK was used as an internal control to normalize the transfection efficiency. Each bar indicates the mean ± S.D. from three replicate assays. F , ChIP assay was carried out as described under “Experimental Procedures,” anti-E2F1 specific antibody was used to pull down its E2F1-bound DNA fragments, and primers for E2F1-binding site and negative control Sp1-binding site were used to carry out PCR. IP , immunoprecipitation. G and H , the invasion abilities of T24T(Vector) and T24T(E2F1) cells were determined using a transwell invasion assay. The results are presented as the number of invasive T24T(E2F1) cells relative to invasive T24T(Vector) transfectants ( H ). I , the knockdown efficiency of E2F1 in T24T(p63α) cells was determined by Western blotting. GAPDH was used as a protein loading control. J and K , the invasion abilities of T24T(p63α/Nonsense), T24T(p63α/shE2F1–1), and T24T(p63α/shE2F1–2) cells were determined using a transwell invasion assay. L and M , IHC-P was carried out to evaluate E2F1 protein expression in mouse BC tissues and normal bladder tissues. The optical density was analyzed as described under “Experimental Procedures.” The asterisk (*) indicates a significant increase in p63α protein expression in comparison with that observed in normal tissues ( p

Techniques Used: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Binding Assay, Negative Control, Polymerase Chain Reaction, Immunoprecipitation, Transwell Invasion Assay, Immunohistochemistry

p63α promoted E2F1 transcription. A , the indicated stable transfectants were extracted with TRIzol reagent for total RNA isolation. e2f1 mRNA was determined with RT-PCR using the specific primers. GAPDH was used as an internal control. B , the T24T(Vector) and T24T(p63α) cells were treated with actinomycin D ( Act D ) for the indicated time points, then total RNA was isolated and subjected to RT-PCR for analysis of e2f1 mRNA degradation. GAPDH was used as a loading control. C and D , T24T(Vector) versus T24T(p63α) cells ( C ) and T24T(Vector) versus T24T(E2F1) cells ( D ) were transfected with an E2F1 promoter-driven luciferase reporter together with pRL-TK. The transfectants were seeded into 96-well plates to determine E2F1 promoter transcriptional activity. pRL-TK was used as an internal control to normalize the transfection efficiency. Each bar indicates the mean ± S.D. from three replicate assays. The asterisk indicates significant increases in E2F1 promoter-driven reporter activity in p63α-overexpressed cells ( C ) or in E2F1-overexpressed cells ( D ) in comparison with Vector transfectants ( p
Figure Legend Snippet: p63α promoted E2F1 transcription. A , the indicated stable transfectants were extracted with TRIzol reagent for total RNA isolation. e2f1 mRNA was determined with RT-PCR using the specific primers. GAPDH was used as an internal control. B , the T24T(Vector) and T24T(p63α) cells were treated with actinomycin D ( Act D ) for the indicated time points, then total RNA was isolated and subjected to RT-PCR for analysis of e2f1 mRNA degradation. GAPDH was used as a loading control. C and D , T24T(Vector) versus T24T(p63α) cells ( C ) and T24T(Vector) versus T24T(E2F1) cells ( D ) were transfected with an E2F1 promoter-driven luciferase reporter together with pRL-TK. The transfectants were seeded into 96-well plates to determine E2F1 promoter transcriptional activity. pRL-TK was used as an internal control to normalize the transfection efficiency. Each bar indicates the mean ± S.D. from three replicate assays. The asterisk indicates significant increases in E2F1 promoter-driven reporter activity in p63α-overexpressed cells ( C ) or in E2F1-overexpressed cells ( D ) in comparison with Vector transfectants ( p

Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Activated Clotting Time Assay, Transfection, Luciferase, Activity Assay, Plasmid Preparation

Hsp70 was up-regulated at transcriptional level by p63α in bladder cancer cells. A , the indicated cells were extracted with TRIzol reagent to isolate total RNA upon the density reaching 80–90%. hsp70 mRNA levels were determined with RT-PCR by using the specific primers. GAPDH was used as an internal control. B , T24T(Vector) and T24T(p63α) cells were seeded into 6-well plates. After synchronization, T24T(Vector) and T24T(p63α) cells were treated with actinomycin D ( Act D ) for the indicated time points, then total RNA was isolated and subjected to RT-PCR analysis to evaluate mRNA levels of hsp70 and GAPDH. C , the indicated cells were transfected with Hsp70 promoter-driven luciferase reporter together with pRL-TK. The transfectants were seeded into 96-well plates and then subjected to determine Hsp70 promoter activity by measuring luciferase activity. pRL-TK was used as an internal control to normalize the transfection efficiency. Each bar indicates the mean ± S.D. from three replicate assays. The asterisk (*) indicates a significant increase in promoter-driven promoter activity in p63-overexpressed cells in comparison with Vector transfectants ( p
Figure Legend Snippet: Hsp70 was up-regulated at transcriptional level by p63α in bladder cancer cells. A , the indicated cells were extracted with TRIzol reagent to isolate total RNA upon the density reaching 80–90%. hsp70 mRNA levels were determined with RT-PCR by using the specific primers. GAPDH was used as an internal control. B , T24T(Vector) and T24T(p63α) cells were seeded into 6-well plates. After synchronization, T24T(Vector) and T24T(p63α) cells were treated with actinomycin D ( Act D ) for the indicated time points, then total RNA was isolated and subjected to RT-PCR analysis to evaluate mRNA levels of hsp70 and GAPDH. C , the indicated cells were transfected with Hsp70 promoter-driven luciferase reporter together with pRL-TK. The transfectants were seeded into 96-well plates and then subjected to determine Hsp70 promoter activity by measuring luciferase activity. pRL-TK was used as an internal control to normalize the transfection efficiency. Each bar indicates the mean ± S.D. from three replicate assays. The asterisk (*) indicates a significant increase in promoter-driven promoter activity in p63-overexpressed cells in comparison with Vector transfectants ( p

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Activated Clotting Time Assay, Isolation, Transfection, Luciferase, Activity Assay, Plasmid Preparation

39) Product Images from "Caspase-2 and oxidative stress underlie the immunogenic potential of high hydrostatic pressure-induced cancer cell death"

Article Title: Caspase-2 and oxidative stress underlie the immunogenic potential of high hydrostatic pressure-induced cancer cell death

Journal: Oncoimmunology

doi: 10.1080/2162402X.2016.1258505

Caspase-8 knockdown reduces CALR exposure induced by HHP. (A) The kinetics of pro-caspase-8, pro-caspase-3 and pro-caspase-2 cleavage in OV-90 control and OV-90 shcaspase-8 cells 1, 2, and 6 h after UV-B, HHP, or idarubicin treatment was analyzed by western blotting. Caspase-8 knockdown was verified by western blotting and equal protein loading was demonstrated by using GAPDH as a loading control. Experiments were performed in triplicate. (B) CALR surface exposure was measured in OV-90 control and OV-90 shcaspase-8 cells 1, 2, and 6 h after UV-B or HHP treatment by flow cytometry. Data are presented as the mean ± SD for three independent experiments. Significant differences are shown (* p
Figure Legend Snippet: Caspase-8 knockdown reduces CALR exposure induced by HHP. (A) The kinetics of pro-caspase-8, pro-caspase-3 and pro-caspase-2 cleavage in OV-90 control and OV-90 shcaspase-8 cells 1, 2, and 6 h after UV-B, HHP, or idarubicin treatment was analyzed by western blotting. Caspase-8 knockdown was verified by western blotting and equal protein loading was demonstrated by using GAPDH as a loading control. Experiments were performed in triplicate. (B) CALR surface exposure was measured in OV-90 control and OV-90 shcaspase-8 cells 1, 2, and 6 h after UV-B or HHP treatment by flow cytometry. Data are presented as the mean ± SD for three independent experiments. Significant differences are shown (* p

Techniques Used: Western Blot, Flow Cytometry, Cytometry

HHP induces ER stress (PERK and eIF2α phosphorylation) involved in CALR surface exposure. (A) Kinetics of the PERK and eIF2α phosphorylation and CHOP activation in CT26 cells 1, 2, and 6 h after HHP treatment. Thapsigargin was used as a positive control. Equal protein loading was demonstrated by GAPDH reprobing. (B) Western blot analysis of eIF2α phosphorylation kinetics in OV-90 control and OV-90 shPERK cells 1, 2, and 6 h after UV-B, HHP, and idarubicin treatment. The activity of eIF2α was determined by a phospho-specific antibody, and then the membranes were reprobed with antibody against total eIF2α. Equal protein loading was demonstrated by using GAPDH as a loading control. Experiments were performed in triplicate. (C) Western blot analysis of pro-caspase-2 cleavage in non-transfected OV-90 cells, SCR, and shPERK cells. PERK knockdown was verified by western blotting. Equal protein loading was demonstrated by using GAPDH as loading control. (D) Western blot analysis of eIF2α phosphorylation and pro-caspase-2 cleavage in non-transfected OV-90 cells, SCR and shcaspase-2 cells 2 h after HHP treatment. Caspase-2 knockdown was verified by western blotting. Equal protein loading was demonstrated by using GAPDH as loading control. (E) OV-90 cells were transfected with siRNA against caspase-2 and PERK. Knockout was verified by western blotting. The activity of eIF2α was determined by a phospho-specific antibody and the membranes were reprobed with antibody against total eIF2α. Equal protein loading was demonstrated by using GAPDH as a loading control. (F) CALR surface exposure was measured in untreated, UV-B and HHP treated OV-90 shPERK and OV-90 control cells after 1, 2, and 6 h by flow cytometry. The data show the compiled results (mean ± SD) of three independent experiments. Significant differences are shown (* p
Figure Legend Snippet: HHP induces ER stress (PERK and eIF2α phosphorylation) involved in CALR surface exposure. (A) Kinetics of the PERK and eIF2α phosphorylation and CHOP activation in CT26 cells 1, 2, and 6 h after HHP treatment. Thapsigargin was used as a positive control. Equal protein loading was demonstrated by GAPDH reprobing. (B) Western blot analysis of eIF2α phosphorylation kinetics in OV-90 control and OV-90 shPERK cells 1, 2, and 6 h after UV-B, HHP, and idarubicin treatment. The activity of eIF2α was determined by a phospho-specific antibody, and then the membranes were reprobed with antibody against total eIF2α. Equal protein loading was demonstrated by using GAPDH as a loading control. Experiments were performed in triplicate. (C) Western blot analysis of pro-caspase-2 cleavage in non-transfected OV-90 cells, SCR, and shPERK cells. PERK knockdown was verified by western blotting. Equal protein loading was demonstrated by using GAPDH as loading control. (D) Western blot analysis of eIF2α phosphorylation and pro-caspase-2 cleavage in non-transfected OV-90 cells, SCR and shcaspase-2 cells 2 h after HHP treatment. Caspase-2 knockdown was verified by western blotting. Equal protein loading was demonstrated by using GAPDH as loading control. (E) OV-90 cells were transfected with siRNA against caspase-2 and PERK. Knockout was verified by western blotting. The activity of eIF2α was determined by a phospho-specific antibody and the membranes were reprobed with antibody against total eIF2α. Equal protein loading was demonstrated by using GAPDH as a loading control. (F) CALR surface exposure was measured in untreated, UV-B and HHP treated OV-90 shPERK and OV-90 control cells after 1, 2, and 6 h by flow cytometry. The data show the compiled results (mean ± SD) of three independent experiments. Significant differences are shown (* p

Techniques Used: Activation Assay, Positive Control, Western Blot, Activity Assay, Transfection, Knock-Out, Flow Cytometry, Cytometry

Caspase-2 is involved in the CALR cell surface exposure induced by HHP. (A) The kinetics of pro-caspase-8, pro-caspase-3, and pro-caspase-2 cleavage upon UV-B, HHP, or idarubicin treatment in OV-90 control and OV-90 shcaspase-2 cells at the indicated time points (1, 2, and 6 h). Caspase-2 knockdown was verified by western blotting and equal protein loading was demonstrated by using GAPDH as a loading control. Experiments were performed in triplicate. (B) CALR surface exposure was measured in OV-90 control and OV-90 shcaspase-2 cells 1, 2, and 6 h after UV-B or HHP treatment by flow cytometry. Data are presented as the mean ± SD for three independent experiments. Significant differences are shown (* p
Figure Legend Snippet: Caspase-2 is involved in the CALR cell surface exposure induced by HHP. (A) The kinetics of pro-caspase-8, pro-caspase-3, and pro-caspase-2 cleavage upon UV-B, HHP, or idarubicin treatment in OV-90 control and OV-90 shcaspase-2 cells at the indicated time points (1, 2, and 6 h). Caspase-2 knockdown was verified by western blotting and equal protein loading was demonstrated by using GAPDH as a loading control. Experiments were performed in triplicate. (B) CALR surface exposure was measured in OV-90 control and OV-90 shcaspase-2 cells 1, 2, and 6 h after UV-B or HHP treatment by flow cytometry. Data are presented as the mean ± SD for three independent experiments. Significant differences are shown (* p

Techniques Used: Western Blot, Flow Cytometry, Cytometry

40) Product Images from "The oncogenic role of MST3 in human gastric cancer"

Article Title: The oncogenic role of MST3 in human gastric cancer

Journal: American Journal of Cancer Research

doi:

MST3 shRNA attenuated the cell growth of gastric cancer cell lines. A. Protein expression level of the MST3 in gastric cancer cell lines (MKN45 and NCI-N87) by western blotting. B. shRNA against MST3 downregulates MST3 expression and induces p21 upregulation in MKN45. MKN45 and NCI-N87 were transfected with MST3 shRNA or control Lucifrease shRNA and the stable clones were selected. The cell lysates were analyzed with western blotting probed by anti-MST3 Ab and anti-p21 Ab. GAPDH was used as control. C. The cell growth rates of parental MKN45 and NCI-N87, control transfectants, and MST3 shRNA transfectants were determined with WST-1 Cell Proliferation assays at different time intervals ranging from 0 to 96 h. Mean ± SEM of absorbance from each group (n = 3) is shown. All P values were obtained by two-way ANOVA.
Figure Legend Snippet: MST3 shRNA attenuated the cell growth of gastric cancer cell lines. A. Protein expression level of the MST3 in gastric cancer cell lines (MKN45 and NCI-N87) by western blotting. B. shRNA against MST3 downregulates MST3 expression and induces p21 upregulation in MKN45. MKN45 and NCI-N87 were transfected with MST3 shRNA or control Lucifrease shRNA and the stable clones were selected. The cell lysates were analyzed with western blotting probed by anti-MST3 Ab and anti-p21 Ab. GAPDH was used as control. C. The cell growth rates of parental MKN45 and NCI-N87, control transfectants, and MST3 shRNA transfectants were determined with WST-1 Cell Proliferation assays at different time intervals ranging from 0 to 96 h. Mean ± SEM of absorbance from each group (n = 3) is shown. All P values were obtained by two-way ANOVA.

Techniques Used: shRNA, Expressing, Western Blot, Transfection, Clone Assay

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Incubation:

Article Title: MicroRNA let-7g inhibits angiotensin II-induced endothelial senescence via the LOX-1-independent mechanism
Article Snippet: .. The membranes were incubated with primary antibodies at 4°C for 16 h, including IGF1 (cat. no. ab9572; 0.1 μ g/ml; Abcam, Cambridge, UK) and GAPDH (cat. no. GTX10011; 1:5,000 dilution; GeneTex, Inc., Irvine, CA, USA). .. Following three washes in PBST, the membranes were incubated with the secondary rabbit immunoglobulin G antibody conjugated to horseradish peroxidase (cat. no. GTX213110-01; 1:10,000 dilution; GeneTex, Inc.) at room temperature for 1 h. The enhanced chemiluminescence non-radioactive detection system was used to detect the antibody-protein complexes with the LAS-3000 imaging system (Fujifilm, Tokyo, Japan).

Immunofluorescence:

Article Title: Proteomic Analysis of the Mammalian Katanin Family of Microtubule-severing Enzymes Defines Katanin p80 subunit B-like 1 (KATNBL1) as a Regulator of Mammalian Katanin Microtubule-severing *
Article Snippet: .. Antibodies Immunofluorescence, immunoblotting, and immunoprecipitations were carried out using the following antibodies: GFP (Abcam, Cambridge, UK), Gapdh (GeneTex, Irvine, CA), α-tubulin (Serotec, Raleigh, NC), HA (Cell Signaling, Danvers, MA). .. Secondary antibodies conjugated to FITC, Cy3 and Cy5 were from Jackson Immuno Research (West Grove, PA).

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    GeneTex gapdh
    Diaph1 deletion improved calcium dynamics after I/R. (a) <t>SERCA2a/GAPDH</t> protein levels were increased by Diaph1 deletion in both mouse hearts after LAD/reperfusion (n = 10/group; *p
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    Diaph1 deletion improved calcium dynamics after I/R. (a) SERCA2a/GAPDH protein levels were increased by Diaph1 deletion in both mouse hearts after LAD/reperfusion (n = 10/group; *p

    Journal: EBioMedicine

    Article Title: The Formin, DIAPH1, is a Key Modulator of Myocardial Ischemia/Reperfusion Injury

    doi: 10.1016/j.ebiom.2017.11.012

    Figure Lengend Snippet: Diaph1 deletion improved calcium dynamics after I/R. (a) SERCA2a/GAPDH protein levels were increased by Diaph1 deletion in both mouse hearts after LAD/reperfusion (n = 10/group; *p

    Article Snippet: We used primary antibodies (1:1000) as follows: mDia1 (DIAPH1), β-actin, ROCK2, nucleoporin p62 (BD Biosciences, San Jose, CA), pan-actin (Cytoskeleton, Denver, CO), GAPDH (Genetex, Irvine, CA), EGR1, SERCA2a (Santa Cruz, Dallas, TX), DIAPH1, myocardin, NCX1, calsequestrin, DIAPH2, histone 3, GAPDH (AbCam, Cambridge, UK), phosphorylated (ser16) and total phospholamban (EMD Millipore, Billerica, MA), GAPDH (Genetex, Irvine, CA), phosphorylated and total Rac1, RhoA, phosphorylated (ser9) and total GSK3β (Cell Signaling, Danvers, MA).

    Techniques:

    Effect of acute F − intoxication on myocardial protein expression of Hsf1 and Hsps in rats. Western blotting and its densitometric analysis of myocardial Hsf1 and Hsps (Hsp27, Hsp32, Hsp40, Hsp60, Hsp70, and Hsp90) in control and F − -treated rats. GAPDH was used as loading control. The values represent mean ± SD ( n = 3). *p

    Journal: Cell Stress & Chaperones

    Article Title: Differential expression of myocardial heat shock proteins in rats acutely exposed to fluoride

    doi: 10.1007/s12192-017-0801-1

    Figure Lengend Snippet: Effect of acute F − intoxication on myocardial protein expression of Hsf1 and Hsps in rats. Western blotting and its densitometric analysis of myocardial Hsf1 and Hsps (Hsp27, Hsp32, Hsp40, Hsp60, Hsp70, and Hsp90) in control and F − -treated rats. GAPDH was used as loading control. The values represent mean ± SD ( n = 3). *p

    Article Snippet: Sodium fluoride (Loba Chemie, Mumbai, India), antibodies of Hsp40, Hsp70, Hsp90, and Hsf1 (Cell Signaling Technology, Inc., USA), Hsp60 (Santa Cruz Biotechnology, Inc., USA), Hsp27, Hsp32 (Enzo Life Science, Inc., USA), GAPDH (GeneTex, Inc., USA), and secondary antibodies conjugated with horseradish peroxidase (Merck, Darmstadt, Germany) were used in this study.

    Techniques: Expressing, Western Blot

    Effect of acute F − intoxication on myocardial mRNA expression of Hsf1 and Hsps in rats. mRNA expression and its densitometric analysis of myocardial Hsf1 and Hsps (Hsp27, Hsp32, Hsp40, Hsp60, Hsp70, and Hsp90) in control and F − -treated rats. GAPDH was used as loading control. The values represent mean ± SD ( n = 3). *p

    Journal: Cell Stress & Chaperones

    Article Title: Differential expression of myocardial heat shock proteins in rats acutely exposed to fluoride

    doi: 10.1007/s12192-017-0801-1

    Figure Lengend Snippet: Effect of acute F − intoxication on myocardial mRNA expression of Hsf1 and Hsps in rats. mRNA expression and its densitometric analysis of myocardial Hsf1 and Hsps (Hsp27, Hsp32, Hsp40, Hsp60, Hsp70, and Hsp90) in control and F − -treated rats. GAPDH was used as loading control. The values represent mean ± SD ( n = 3). *p

    Article Snippet: Sodium fluoride (Loba Chemie, Mumbai, India), antibodies of Hsp40, Hsp70, Hsp90, and Hsf1 (Cell Signaling Technology, Inc., USA), Hsp60 (Santa Cruz Biotechnology, Inc., USA), Hsp27, Hsp32 (Enzo Life Science, Inc., USA), GAPDH (GeneTex, Inc., USA), and secondary antibodies conjugated with horseradish peroxidase (Merck, Darmstadt, Germany) were used in this study.

    Techniques: Expressing

    Reduction of COX-2 expression and catalytic activity by shRNA or NS398 reduce DENV replication. COX-2 shRNA reduced DENV replication in ( A ) DENV replicon cells and ( B to D ) a DENV infection system. Huh-7-D2-FLuc-SGR-Neo DENV replicon cells were transfected with GFP or COX-2 shRNA at the indicated concentrations for 3 days and the cell lysates were subjected to a luciferase activity assay and western blotting. Huh-7 cells were transfected with COX-2 shRNA at the indicated concentrations, and the transfected cells were infected with DENV-2 at an MOI of 1. After 3 days of treatment, the cell lysate, cellular RNA and supernatants were analyzed by western blotting, RT-qPCR or plaque assay, respectively. NS398 reduced DENV replication in ( E ) DENV replicon cells and ( F to H ) a DENV infection system. Huh-7-D2-FLuc-SGR-Neo DENV replicon cells were treated with NS398 at different concentrations (0, 5, 10, 20, and 40 μM) for 3 days, and the cell lysates were subjected to a luciferase activity assay and western blotting. Huh-7 cells were infected with DENV-2 at an MOI of 1 and then treated with NS398 at different concentrations (0, 5, 10, 20, and 40 μM) for 3 days. Western blotting was performed with anti-COX-2, anti-NS2B, and anti-GAPDH antibodies. The relative RNA level of DENV-2 was determined by RT-qPCR following normalization to the cellular gapdh mRNA level. All data are indicative of at least three independent experiments, with each measurement performed in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Journal: Scientific Reports

    Article Title: Cyclooxygenase‐2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents

    doi: 10.1038/srep44701

    Figure Lengend Snippet: Reduction of COX-2 expression and catalytic activity by shRNA or NS398 reduce DENV replication. COX-2 shRNA reduced DENV replication in ( A ) DENV replicon cells and ( B to D ) a DENV infection system. Huh-7-D2-FLuc-SGR-Neo DENV replicon cells were transfected with GFP or COX-2 shRNA at the indicated concentrations for 3 days and the cell lysates were subjected to a luciferase activity assay and western blotting. Huh-7 cells were transfected with COX-2 shRNA at the indicated concentrations, and the transfected cells were infected with DENV-2 at an MOI of 1. After 3 days of treatment, the cell lysate, cellular RNA and supernatants were analyzed by western blotting, RT-qPCR or plaque assay, respectively. NS398 reduced DENV replication in ( E ) DENV replicon cells and ( F to H ) a DENV infection system. Huh-7-D2-FLuc-SGR-Neo DENV replicon cells were treated with NS398 at different concentrations (0, 5, 10, 20, and 40 μM) for 3 days, and the cell lysates were subjected to a luciferase activity assay and western blotting. Huh-7 cells were infected with DENV-2 at an MOI of 1 and then treated with NS398 at different concentrations (0, 5, 10, 20, and 40 μM) for 3 days. Western blotting was performed with anti-COX-2, anti-NS2B, and anti-GAPDH antibodies. The relative RNA level of DENV-2 was determined by RT-qPCR following normalization to the cellular gapdh mRNA level. All data are indicative of at least three independent experiments, with each measurement performed in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Article Snippet: The antibodies used in the present study included anti-DENV NS2B (1:3000; GeneTex, Irvine, CA, USA), anti-GAPDH (1:3000; GeneTex, Irvine, CA, USA), anti-COX-2 (1:1000, Cayman, ML, USA), and anti-Myc (1:2000; Abcam, Cambridge, MA, USA).

    Techniques: Expressing, Activity Assay, shRNA, Infection, Transfection, Luciferase, Western Blot, Quantitative RT-PCR, Plaque Assay

    COX-2 overexpression and PGE 2 treatment increase DENV-2 replication. COX-2 overexpression induced DENV-2 replication in ( A ) DENV-2 replicon cells and ( B and C ) the DENV infection system. Huh-7-D2-FLuc-SGR-Neo DENV replicon reporter cells were transfected with pcDNA4/Myc or pcDNA4-COX-2-Myc at the indicated concentrations. After 3 days of incubation, the cell lysates were subjected to a luciferase activity assay. Huh-7 cells were transfected with pcDNA4/Myc or pcDNA4-COX-2-Myc at the indicated concentrations, and the transfected cells were infected with DENV-2 at an MOI of 1. After 3 days of incubation, the cell lysates and cellular RNA were subjected to western blotting and RT-qPCR. ( D ) COX-2 overexpression increased DENV-2 propagation. The transfected Huh-7 cells were infected by DENV-2 at a MOI of 1 for 3 days. Supernatants were collected and subjected to a viral plaque assay. PGE 2 treatment induced DENV-2 replication in ( E ) viral replicon cells and ( F and G ) the DENV infection system. Huh-7-D2-FLuc-SGR-Neo DENV replicon reporter cells were treated with PGE 2 at the indicated concentrations for 3 days and the cell lysates were subjected to a luciferase activity assay. Huh-7 cells were infected with DENV-2 at an MOI of 1, and the infected cells were treated with PGE 2 at the indicated concentrations for 3 days. Western blotting was performed with anti-NS2B, anti-Myc, and anti-GAPDH antibodies. Relative RNA levels of DENV-2 was determined by RT-qPCR following the normalization of cellular gapdh mRNA levels. ( H ) PGE 2 treatment induced DENV-2 propagation. Huh-7 cells were infected with DENV-2 at an MOI of 1 and then treated with PGE 2 . Supernatants were collected and subjected to a viral plaque assay. ( I ) PGE 2 treatment induced DENV-2 NS5 polymerase activity. Huh-7 cells were cotransfected with p(+)RLuc-(−)DV-UTRΔC-Fluc reporter template (0.5 μg) and pcDNA-NS5-Myc expression plasmid (0.5 μg), and the transfected cells were treated with PGE 2 at the indicated concentrations for 3 days. The cell lysates were subjected to a Dual-Glo Luciferase Assay. All data were indicative of at least three independent experiments, with each measurement carried out in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Journal: Scientific Reports

    Article Title: Cyclooxygenase‐2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents

    doi: 10.1038/srep44701

    Figure Lengend Snippet: COX-2 overexpression and PGE 2 treatment increase DENV-2 replication. COX-2 overexpression induced DENV-2 replication in ( A ) DENV-2 replicon cells and ( B and C ) the DENV infection system. Huh-7-D2-FLuc-SGR-Neo DENV replicon reporter cells were transfected with pcDNA4/Myc or pcDNA4-COX-2-Myc at the indicated concentrations. After 3 days of incubation, the cell lysates were subjected to a luciferase activity assay. Huh-7 cells were transfected with pcDNA4/Myc or pcDNA4-COX-2-Myc at the indicated concentrations, and the transfected cells were infected with DENV-2 at an MOI of 1. After 3 days of incubation, the cell lysates and cellular RNA were subjected to western blotting and RT-qPCR. ( D ) COX-2 overexpression increased DENV-2 propagation. The transfected Huh-7 cells were infected by DENV-2 at a MOI of 1 for 3 days. Supernatants were collected and subjected to a viral plaque assay. PGE 2 treatment induced DENV-2 replication in ( E ) viral replicon cells and ( F and G ) the DENV infection system. Huh-7-D2-FLuc-SGR-Neo DENV replicon reporter cells were treated with PGE 2 at the indicated concentrations for 3 days and the cell lysates were subjected to a luciferase activity assay. Huh-7 cells were infected with DENV-2 at an MOI of 1, and the infected cells were treated with PGE 2 at the indicated concentrations for 3 days. Western blotting was performed with anti-NS2B, anti-Myc, and anti-GAPDH antibodies. Relative RNA levels of DENV-2 was determined by RT-qPCR following the normalization of cellular gapdh mRNA levels. ( H ) PGE 2 treatment induced DENV-2 propagation. Huh-7 cells were infected with DENV-2 at an MOI of 1 and then treated with PGE 2 . Supernatants were collected and subjected to a viral plaque assay. ( I ) PGE 2 treatment induced DENV-2 NS5 polymerase activity. Huh-7 cells were cotransfected with p(+)RLuc-(−)DV-UTRΔC-Fluc reporter template (0.5 μg) and pcDNA-NS5-Myc expression plasmid (0.5 μg), and the transfected cells were treated with PGE 2 at the indicated concentrations for 3 days. The cell lysates were subjected to a Dual-Glo Luciferase Assay. All data were indicative of at least three independent experiments, with each measurement carried out in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Article Snippet: The antibodies used in the present study included anti-DENV NS2B (1:3000; GeneTex, Irvine, CA, USA), anti-GAPDH (1:3000; GeneTex, Irvine, CA, USA), anti-COX-2 (1:1000, Cayman, ML, USA), and anti-Myc (1:2000; Abcam, Cambridge, MA, USA).

    Techniques: Over Expression, Infection, Transfection, Incubation, Luciferase, Activity Assay, Western Blot, Quantitative RT-PCR, Viral Plaque Assay, Expressing, Plasmid Preparation

    COX-2 expression is required for viral replication. ( A to C ) Exogenous COX-2 expression restored DENV-2 protein synthesis, RNA replication and viral propagation in COX-2 shRNA-transfected cells. Huh-7 cells were cotransfected with COX-2 shRNA (1.0 μg) and pCMV-COX-2-Myc (0.25, 0.5, and 1.0 μg), followed by DENV-2 infection at an MOI of 1. ( D to F ) Exogenous COX-2 expression restored DENV-2 protein synthesis, RNA replication and viral propagation in NS398-treated cells. Huh-7 cells were transfected with pcDNA4/Myc (0.5 μg) or pCMV-COX-2-Myc (0.25, 0.5, and 1.0 μg), followed by DENV-2 infection at an MOI of 1. The cells were treated with DMSO or 40 μM NS398 for 3 days. Western blotting was performed with anti-NS2B, anti-Myc, and anti-GAPDH antibodies. The relative RNA level of DENV-2 was determined by RT-qPCR following normalization to the cellular gapdh mRNA level. All data are indicative of at least three independent experiments, with each measurement performed in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Journal: Scientific Reports

    Article Title: Cyclooxygenase‐2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents

    doi: 10.1038/srep44701

    Figure Lengend Snippet: COX-2 expression is required for viral replication. ( A to C ) Exogenous COX-2 expression restored DENV-2 protein synthesis, RNA replication and viral propagation in COX-2 shRNA-transfected cells. Huh-7 cells were cotransfected with COX-2 shRNA (1.0 μg) and pCMV-COX-2-Myc (0.25, 0.5, and 1.0 μg), followed by DENV-2 infection at an MOI of 1. ( D to F ) Exogenous COX-2 expression restored DENV-2 protein synthesis, RNA replication and viral propagation in NS398-treated cells. Huh-7 cells were transfected with pcDNA4/Myc (0.5 μg) or pCMV-COX-2-Myc (0.25, 0.5, and 1.0 μg), followed by DENV-2 infection at an MOI of 1. The cells were treated with DMSO or 40 μM NS398 for 3 days. Western blotting was performed with anti-NS2B, anti-Myc, and anti-GAPDH antibodies. The relative RNA level of DENV-2 was determined by RT-qPCR following normalization to the cellular gapdh mRNA level. All data are indicative of at least three independent experiments, with each measurement performed in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Article Snippet: The antibodies used in the present study included anti-DENV NS2B (1:3000; GeneTex, Irvine, CA, USA), anti-GAPDH (1:3000; GeneTex, Irvine, CA, USA), anti-COX-2 (1:1000, Cayman, ML, USA), and anti-Myc (1:2000; Abcam, Cambridge, MA, USA).

    Techniques: Expressing, shRNA, Transfection, Infection, Western Blot, Quantitative RT-PCR

    DENV induces COX-2 expression and PGE 2 production in DF patients, DENV-infected mice, and human hepatoma cells. ( A and B ) Elevated COX-2 expression and PGE 2 levels in the blood of dengue fever patients. COX-2 mRNA and PGE 2 levels in blood samples from 13 clinical DF patients and 6 healthy donors were determined by RT-qPCR or ELISA, respectively. ( C ) The induced COX-2 expression in DENV-2-infected ICR suckling mice. Six-day-old suckling mice were injected with 2.5 × 10 5 pfu of DENV-2 or heat-inactivated DENV-2 (iDENV) by intracerebral injection. Each group comprised six suckling mice (n = 6). Six days after inoculation, COX-2 mRNA levels of mouse brain tissues were determined by RT-qPCR. DENV-2 time-dependently induced ( D ) COX-2 protein expression, ( E ) COX-2 RNA replication, and ( F ) PGE 2 production. Huh-7 cells were infected with DENV-2 at an MOI of 0.1, and the cell lysate and cellular RNA were extracted at the indicated time points (24, 48, and 72 hpi). Western blotting was performed with anti-COX-2, anti-NS2B, and anti-GAPDH antibodies. Relative RNA levels of DENV-2 and COX-2 were determined by RT-qPCR following the normalization of cellular gapdh mRNA levels. Supernatants were collected at the indicated time points and subjected to a PGE 2 ELISA assay. All data from cell-based experiments are indicative of at least three independent experiments, with each measurement carried out in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Journal: Scientific Reports

    Article Title: Cyclooxygenase‐2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents

    doi: 10.1038/srep44701

    Figure Lengend Snippet: DENV induces COX-2 expression and PGE 2 production in DF patients, DENV-infected mice, and human hepatoma cells. ( A and B ) Elevated COX-2 expression and PGE 2 levels in the blood of dengue fever patients. COX-2 mRNA and PGE 2 levels in blood samples from 13 clinical DF patients and 6 healthy donors were determined by RT-qPCR or ELISA, respectively. ( C ) The induced COX-2 expression in DENV-2-infected ICR suckling mice. Six-day-old suckling mice were injected with 2.5 × 10 5 pfu of DENV-2 or heat-inactivated DENV-2 (iDENV) by intracerebral injection. Each group comprised six suckling mice (n = 6). Six days after inoculation, COX-2 mRNA levels of mouse brain tissues were determined by RT-qPCR. DENV-2 time-dependently induced ( D ) COX-2 protein expression, ( E ) COX-2 RNA replication, and ( F ) PGE 2 production. Huh-7 cells were infected with DENV-2 at an MOI of 0.1, and the cell lysate and cellular RNA were extracted at the indicated time points (24, 48, and 72 hpi). Western blotting was performed with anti-COX-2, anti-NS2B, and anti-GAPDH antibodies. Relative RNA levels of DENV-2 and COX-2 were determined by RT-qPCR following the normalization of cellular gapdh mRNA levels. Supernatants were collected at the indicated time points and subjected to a PGE 2 ELISA assay. All data from cell-based experiments are indicative of at least three independent experiments, with each measurement carried out in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Article Snippet: The antibodies used in the present study included anti-DENV NS2B (1:3000; GeneTex, Irvine, CA, USA), anti-GAPDH (1:3000; GeneTex, Irvine, CA, USA), anti-COX-2 (1:1000, Cayman, ML, USA), and anti-Myc (1:2000; Abcam, Cambridge, MA, USA).

    Techniques: Expressing, Infection, Mouse Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Injection, Western Blot

    NF-κB and MAPK/JNK-mediated C/EBP are responsible for DENV-2-induced COX-2 expression and viral replication. ( A ) DENV-2 induced activation of the NF-κB signaling pathway. Huh-7 cells were infected by DENV-2 at an MOI of 1 and the cell lysates were extracted at the indicated time points. Western blotting was performed and the relative blot intensities were quantified by densitometry scanning. ( B ) CAPE significantly suppressed DENV-2-induced COX-2 expression. Huh-7 cells were pretreated with DMSO or CAPE (20 μM) for 2 h and then infected with DENV-2 at an MOI of 1. The cell lysates were analyzed at 2 dpi by western blotting. ( C and D ) CAPE dose-dependently suppressed DENV-2 protein synthesis and RNA replication. Huh-7 cells were infected by DENV-2 and treated with CAPE at different concentrations (0, 5, 10, and 20 μM). After 3 days of treatment, the cell lysates and cellular RNA were analyzed by western blotting or RT-qPCR, respectively. ( E ) DENV-2 induced the activation of the MAPK/JNK pathway. Huh-7 cells were infected by DENV-2, and the cell lysates were extracted at the indicated time points. Western blotting was performed and the relative blot intensities were quantified by densitometry scanning. ( F ) SP600125 significantly suppressed DENV-2-induced COX-2 expression. Huh-7 cells were pretreated with DMSO or SP600125 (20 μM) for 2 h, and then, the cells were infected by DENV-2. After 2 days of treatment, cell lysates were subjected to western blotting. ( G and H ) SP600125 dose-dependently suppressed DENV-2 protein synthesis and RNA replication. Huh-7 cells were infected by DENV-2, and the cells were treated with DMSO or SP600125 at different concentrations (0, 5, 10, and 20 μM). After 3 days of treatment, the cell lysates and cellular RNA were analyzed by western blotting or RT-qPCR, respectively. GAPDH served as a loading control in western blotting. The relative RNA level of DENV-2 was determine by RT-qPCR following normalization to the cellular gapdh mRNA level. All data are indicative of at least three independent experiments, with each measurement carried out in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Journal: Scientific Reports

    Article Title: Cyclooxygenase‐2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents

    doi: 10.1038/srep44701

    Figure Lengend Snippet: NF-κB and MAPK/JNK-mediated C/EBP are responsible for DENV-2-induced COX-2 expression and viral replication. ( A ) DENV-2 induced activation of the NF-κB signaling pathway. Huh-7 cells were infected by DENV-2 at an MOI of 1 and the cell lysates were extracted at the indicated time points. Western blotting was performed and the relative blot intensities were quantified by densitometry scanning. ( B ) CAPE significantly suppressed DENV-2-induced COX-2 expression. Huh-7 cells were pretreated with DMSO or CAPE (20 μM) for 2 h and then infected with DENV-2 at an MOI of 1. The cell lysates were analyzed at 2 dpi by western blotting. ( C and D ) CAPE dose-dependently suppressed DENV-2 protein synthesis and RNA replication. Huh-7 cells were infected by DENV-2 and treated with CAPE at different concentrations (0, 5, 10, and 20 μM). After 3 days of treatment, the cell lysates and cellular RNA were analyzed by western blotting or RT-qPCR, respectively. ( E ) DENV-2 induced the activation of the MAPK/JNK pathway. Huh-7 cells were infected by DENV-2, and the cell lysates were extracted at the indicated time points. Western blotting was performed and the relative blot intensities were quantified by densitometry scanning. ( F ) SP600125 significantly suppressed DENV-2-induced COX-2 expression. Huh-7 cells were pretreated with DMSO or SP600125 (20 μM) for 2 h, and then, the cells were infected by DENV-2. After 2 days of treatment, cell lysates were subjected to western blotting. ( G and H ) SP600125 dose-dependently suppressed DENV-2 protein synthesis and RNA replication. Huh-7 cells were infected by DENV-2, and the cells were treated with DMSO or SP600125 at different concentrations (0, 5, 10, and 20 μM). After 3 days of treatment, the cell lysates and cellular RNA were analyzed by western blotting or RT-qPCR, respectively. GAPDH served as a loading control in western blotting. The relative RNA level of DENV-2 was determine by RT-qPCR following normalization to the cellular gapdh mRNA level. All data are indicative of at least three independent experiments, with each measurement carried out in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Article Snippet: The antibodies used in the present study included anti-DENV NS2B (1:3000; GeneTex, Irvine, CA, USA), anti-GAPDH (1:3000; GeneTex, Irvine, CA, USA), anti-COX-2 (1:1000, Cayman, ML, USA), and anti-Myc (1:2000; Abcam, Cambridge, MA, USA).

    Techniques: Expressing, Activation Assay, Infection, Western Blot, Quantitative RT-PCR

    Effects of azacytidine (AZA) and decitabine (DAC) on the cell cycle and p53 expression ( A ) HCT116 cells were treated the different doses of AZA or DAC for 24 and 48 h, and the cell cycle was analyzed by flow cytometry as described in “Materials and Methods”. ( B ) HCT116 cells were treated the different doses of AZA or DAC for 24 h, and whole-cell lysates were subjected to a Western blot analysis using antibodies against p53, p53R2, γH2AX, or GAPDH.

    Journal: Oncotarget

    Article Title: Systematic discovery of drug action mechanisms by an integrated chemical genomics approach: identification of functional disparities between azacytidine and decitabine

    doi: 10.18632/oncotarget.8455

    Figure Lengend Snippet: Effects of azacytidine (AZA) and decitabine (DAC) on the cell cycle and p53 expression ( A ) HCT116 cells were treated the different doses of AZA or DAC for 24 and 48 h, and the cell cycle was analyzed by flow cytometry as described in “Materials and Methods”. ( B ) HCT116 cells were treated the different doses of AZA or DAC for 24 h, and whole-cell lysates were subjected to a Western blot analysis using antibodies against p53, p53R2, γH2AX, or GAPDH.

    Article Snippet: DNMT1, c-MYC, p53, p53R2, γH2AX, LC3B, and GAPDH antibodies were purchased form GeneTex (Hsinchu, Taiwan).

    Techniques: Expressing, Flow Cytometry, Cytometry, Western Blot

    The relationship between apoptosis and autophagy induced by azacytidine (AZA) ( A ) HCT116 cells were treated with different doses of doxorubicin for 72 h, and the cell viability was analyzed by an MTT assay. ( B ) HCT116 cells were treated with different doses of AZA or decitabine (DAC), or 0.5 μM doxorubicin for 24 and 48 h, and whole-cell lysates were subjected to a Western blot analysis using antibodies against PARP1, LC3B, or GAPDH. ( C ) HCT116 cells were pretreated with 5 mM 3-MA or 50 μM Z-DEVD-FMK for 1 h and then exposed to 50 μM AZA for 24 h. Whole-cell lysates were subjected to a Western blot analysis using antibodies against PARP1, LC3B, or GAPDH. ( D ) ATG7-WT and ATG7-KO DLD-1 cells were treated with different doses of AZA for 72 h, and cell viability was analyzed by an MTT assay.

    Journal: Oncotarget

    Article Title: Systematic discovery of drug action mechanisms by an integrated chemical genomics approach: identification of functional disparities between azacytidine and decitabine

    doi: 10.18632/oncotarget.8455

    Figure Lengend Snippet: The relationship between apoptosis and autophagy induced by azacytidine (AZA) ( A ) HCT116 cells were treated with different doses of doxorubicin for 72 h, and the cell viability was analyzed by an MTT assay. ( B ) HCT116 cells were treated with different doses of AZA or decitabine (DAC), or 0.5 μM doxorubicin for 24 and 48 h, and whole-cell lysates were subjected to a Western blot analysis using antibodies against PARP1, LC3B, or GAPDH. ( C ) HCT116 cells were pretreated with 5 mM 3-MA or 50 μM Z-DEVD-FMK for 1 h and then exposed to 50 μM AZA for 24 h. Whole-cell lysates were subjected to a Western blot analysis using antibodies against PARP1, LC3B, or GAPDH. ( D ) ATG7-WT and ATG7-KO DLD-1 cells were treated with different doses of AZA for 72 h, and cell viability was analyzed by an MTT assay.

    Article Snippet: DNMT1, c-MYC, p53, p53R2, γH2AX, LC3B, and GAPDH antibodies were purchased form GeneTex (Hsinchu, Taiwan).

    Techniques: MTT Assay, Western Blot

    Different effects of azacytidine (AZA) and decitabine (DAC) on the cell viability of human colorectal cancer cells ( A ) Chemical structures of cytidine, AZA, and DAC, and the metabolic pathways of AZA (5-Aza-CR) and DAC (5-Aza-CdR). MP, DP, and TP, mono-, di-, and triphosphate, respectively; PPase, phosphatase; UrdK/CydK, uridine/cytidine kinase; dCydk, deoxycytidine kinase. ( B ) HCT116 cells were treated with different doses of AZA or DAC for 24 and 72 h. The cell viability at 72 h was analyzed by an MTT assay (upper part). Whole-cell lysates at 24 h were subjected to a Western blot analysis using antibodies against DNMT1 or GAPDH (lower part). ( C ) RKO, LoVo, HCT-15, DLD-1, and HT-29 cells were treated with different doses of AZA or DAC for 72 h, and the cell viability was analyzed by an MTT assay.

    Journal: Oncotarget

    Article Title: Systematic discovery of drug action mechanisms by an integrated chemical genomics approach: identification of functional disparities between azacytidine and decitabine

    doi: 10.18632/oncotarget.8455

    Figure Lengend Snippet: Different effects of azacytidine (AZA) and decitabine (DAC) on the cell viability of human colorectal cancer cells ( A ) Chemical structures of cytidine, AZA, and DAC, and the metabolic pathways of AZA (5-Aza-CR) and DAC (5-Aza-CdR). MP, DP, and TP, mono-, di-, and triphosphate, respectively; PPase, phosphatase; UrdK/CydK, uridine/cytidine kinase; dCydk, deoxycytidine kinase. ( B ) HCT116 cells were treated with different doses of AZA or DAC for 24 and 72 h. The cell viability at 72 h was analyzed by an MTT assay (upper part). Whole-cell lysates at 24 h were subjected to a Western blot analysis using antibodies against DNMT1 or GAPDH (lower part). ( C ) RKO, LoVo, HCT-15, DLD-1, and HT-29 cells were treated with different doses of AZA or DAC for 72 h, and the cell viability was analyzed by an MTT assay.

    Article Snippet: DNMT1, c-MYC, p53, p53R2, γH2AX, LC3B, and GAPDH antibodies were purchased form GeneTex (Hsinchu, Taiwan).

    Techniques: MTT Assay, Western Blot

    Effects of azacytidine (AZA) and decitabine (DAC) on protein synthesis and stability ( A ) HCT116 cells were treated with 5 μg/mL cycloheximide (CHX), or different doses of AZA or DAC for 6 and 24 h, and protein synthesis was examined by a puromycin-incorporation assay as described in “Materials and Methods”. Upper part: ponceau S staining of nitrocellulose membranes. Lower part: Western blots of puromycin. ( B ) HCT116 and RKO cells were treated with 5 μg/mL CHX, 50 μM AZA, or 50 μM DAC for the indicated time intervals, and whole-cell lysates were subjected to a Western blot analysis using antibodies against c-MYC or GAPDH.

    Journal: Oncotarget

    Article Title: Systematic discovery of drug action mechanisms by an integrated chemical genomics approach: identification of functional disparities between azacytidine and decitabine

    doi: 10.18632/oncotarget.8455

    Figure Lengend Snippet: Effects of azacytidine (AZA) and decitabine (DAC) on protein synthesis and stability ( A ) HCT116 cells were treated with 5 μg/mL cycloheximide (CHX), or different doses of AZA or DAC for 6 and 24 h, and protein synthesis was examined by a puromycin-incorporation assay as described in “Materials and Methods”. Upper part: ponceau S staining of nitrocellulose membranes. Lower part: Western blots of puromycin. ( B ) HCT116 and RKO cells were treated with 5 μg/mL CHX, 50 μM AZA, or 50 μM DAC for the indicated time intervals, and whole-cell lysates were subjected to a Western blot analysis using antibodies against c-MYC or GAPDH.

    Article Snippet: DNMT1, c-MYC, p53, p53R2, γH2AX, LC3B, and GAPDH antibodies were purchased form GeneTex (Hsinchu, Taiwan).

    Techniques: Staining, Western Blot