Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a ) TCF-4 potentiates the binding between NF-κB p65 and the MMP-15 promoter. The LLC cells were transfected with si-NC (20 nmol/ml), si-TCF4-1 (20 nmol/ml) or si-TCF4-2 (20 nmol/ml) for 36 hours, and then harvested for ChIP assay. The cell lysates were immunoprecipitated by p65 antibody or rabbit IgG as control. ( b ) TCF-4 interacts with NF-κB p65 which binds to the promoter of MMP-15. The LLC cells were transfected with si-NC (20 nmol/ml), si-TCF4-1 (20 nmol/ml) or si-TCF4-2 (20 nmol/ml) for 36 hours, and then harvested for ChIP assay. The cell lysates were immunoprecipitated by TCF-4 antibody or rabbit IgG as control. ( c ) Protein-protein interaction between TCF-4 and NF-κB p65 in LLC cells. The LLC cells were transfected with si-NC (20 nmol/ml), si-TCF4-1 (20 nmol/ml) or si-TCF4-2 (20 nmol/ml) for 36 hours, and then harvested for immunoprecipitation (IP) and Western blotting assay of p65 protein. The cell lysates were immunoprecipitated by TCF-4 antibody or rabbit IgG as control. ( d ) Protein-protein interaction between TCF-4 and NF-κB p65 in SAEC cells. The SAEC cells were transfected with PCMV (0.4 μg/ml) or PCMV-TCF4 (0.4 μg/ml) for 36 hours, and then collected for IP and Western blotting assay of p65. The cell lysates were immunoprecipitated by TCF-4 antibody or rabbit IgG as control. Experiment from ( a – d ) was repeated for 3 times, and the representative results were displayed.

Article Snippet: Mouse endogenous p65 was knocked down by using the two mixed commercial siRNAs (sc-29411 and sc-44213, Santa Cruz).

Techniques: Binding Assay, Transfection, Immunoprecipitation, Control, Western Blot

Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a ) The mRNA levels of MMP-15, TCF-4 and p65 in the LLC cells transfected with PCMV (0.4 μg/ml) or PCMV-TCF-4 (0.4 μg/ml) plus si-NC (20 nmol/ml) or a siRNA for mouse NF-κB p65 (si-p65, 20 nmol/ml) for 36 hours (n = 5, **P < 0.01). ( b ) The mRNA levels of MMP-15, TCF-4 and p65 in the LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus pCDNA3.1 (0.4 μg/ml) or pCDNA-p65 (0.4 μg/ml) for 36 hours (n = 5, **P < 0.01). ( c ) Immunoblotting assay of TCF-4 and p65 in the cytosol or nucleus of LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) for 36 hours. ( d ) Relative luciferase activity of LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus a reporter construct (0.4 μg/ml) containing the promoter of CCL20 for 36 hours (n = 4, **P < 0.01). The tests from ( a – d ) were repeated for 3 times, and the representative results were displayed.

Article Snippet: Mouse endogenous p65 was knocked down by using the two mixed commercial siRNAs (sc-29411 and sc-44213, Santa Cruz).

Techniques: Transfection, Western Blot, Luciferase, Activity Assay, Construct

Journal: PLoS Pathogens

Article Title: Orientia tsutsugamushi uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation

doi: 10.1371/journal.ppat.1007023

Figure Lengend Snippet: HeLa cells (A and B) or BMDMs (C and D) were infected with O . tsutsugamushi at an MOI of 1. At 2, 4, 8, 12, or 24 h, the cells were fixed and screened with antibodies against O . tsutsugamushi TSA56 ( Ot ) and p65 prior to examination by confocal microscopy. (A and C) Representative fluorescence images of cells viewed for Ot , p65, and merged images plus DAPI, which stains the nucleus, are presented. White arrows denote representative individual O . tsutsugamushi bacteria. (B and D) The mean percentage + SD of cells exhibiting p65 in the nucleus was determined at each time point. Triplicate samples of 100 cells each were counted per time point. Results are representative of three separate experiments.

Article Snippet: Samples were then incubated at room temperature with antibody targeting endogenous p65 at a 1:250 (Santa Cruz, Dallas, TX) or 1:1000 (Invitrogen) dilution, rabbit anti- O . tsutsugamushi TSA56 at a 1:1,000 dilution [ ], and/or mouse anti-Flag epitope (Sigma-Aldrich) at a 1:1,000 dilution in PBS containing 5% (vol/vol) BSA for 1 h. The coverslips were washed three times in PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) at a 1:1,000 dilution in 5% BSA for 1 h at room temperature.

Techniques: Infection, Confocal Microscopy, Fluorescence, Bacteria

Journal: PLoS Pathogens

Article Title: Orientia tsutsugamushi uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation

doi: 10.1371/journal.ppat.1007023

Figure Lengend Snippet: HeLa cells (A and B) or BMDMs (C and D) were infected with O . tsutsugamushi at an MOI of 50 or 10, respectively, or mock infected. At 24, 48, or 72 h, the cells were treated with TNFα or vehicle control (Ctrl) for 30 min after which they were fixed, screened with antibodies against O . tsutsugamushi TSA56 ( Ot ) and p65, and visualized by confocal microscopy. (A and C) Representative fluorescence images of cells viewed for Ot , p65, and merged images plus DAPI, which stains the nucleus, are presented. (B and D) The mean percentage + SD of cells exhibiting p65 in the nucleus were determined at each time point. Triplicate samples of 100 cells each were counted per time point. Statistically significant (* P < 0.05; ** P <0.01; *** P < 0.001; **** P < 0.0001) values are indicated. n.s., not significant. Data are the mean + SD of three independent experiments performed in triplicate.

Article Snippet: Samples were then incubated at room temperature with antibody targeting endogenous p65 at a 1:250 (Santa Cruz, Dallas, TX) or 1:1000 (Invitrogen) dilution, rabbit anti- O . tsutsugamushi TSA56 at a 1:1,000 dilution [ ], and/or mouse anti-Flag epitope (Sigma-Aldrich) at a 1:1,000 dilution in PBS containing 5% (vol/vol) BSA for 1 h. The coverslips were washed three times in PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) at a 1:1,000 dilution in 5% BSA for 1 h at room temperature.

Techniques: Infection, Control, Confocal Microscopy, Fluorescence

Journal: PLoS Pathogens

Article Title: Orientia tsutsugamushi uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation

doi: 10.1371/journal.ppat.1007023

Figure Lengend Snippet: RAW264.7 (A and B) or HeLa cells (C and D) were infected with O . tsutsugamushi at an MOI of 25 or 10, respectively, or mock infected. (A) Whole cell lysates were analyzed by Western blotting with antibodies to O . tsutsugamushi TSA56 and p65. The blots were stripped and reprobed with antibody against GAPDH or β-actin as a loading control. (B and D) Mean normalized ratios + SD of p65:GAPDH (B) and p65:β-actin (D) from three separate experiments were calculated using densitometry. Statistically significant ( *P < 0.05) values are indicated. n.s., not significant. The arrowhead and asterisk in A and B denote the expected size for TSA56 and a non-specific host cell-derived band, respectively.

Article Snippet: Samples were then incubated at room temperature with antibody targeting endogenous p65 at a 1:250 (Santa Cruz, Dallas, TX) or 1:1000 (Invitrogen) dilution, rabbit anti- O . tsutsugamushi TSA56 at a 1:1,000 dilution [ ], and/or mouse anti-Flag epitope (Sigma-Aldrich) at a 1:1,000 dilution in PBS containing 5% (vol/vol) BSA for 1 h. The coverslips were washed three times in PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) at a 1:1,000 dilution in 5% BSA for 1 h at room temperature.

Techniques: Infection, Western Blot, Control, Derivative Assay

Journal: PLoS Pathogens

Article Title: Orientia tsutsugamushi uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation

doi: 10.1371/journal.ppat.1007023

Figure Lengend Snippet: HeLa cells were infected with O . tsutsugamushi at an MOI of 10 (A and B) or 25 (C and D) or incubated with mock control. At 4 or 72 h, the cells were treated with TNFα or vehicle control for 30 min followed by homogenization. Whole cell lysates (A) or nuclear and cytosolic fractions (C) were analyzed by Western blotting with antibodies to IκBα, O . tsutsugamushi TSA56, and β-actin (loading control) (C) or p65, lamin B1, TSA56, and GADPH (D). Mean normalized ratios + SD of IκBα:β-actin (B) and p65:lamin B1 (D) were calculated from three separate experiments performed in (A) and (C), respectively, using densitometry. Statistically significant ( *P < 0.05) values are indicated. n.s., not significant.

Article Snippet: Samples were then incubated at room temperature with antibody targeting endogenous p65 at a 1:250 (Santa Cruz, Dallas, TX) or 1:1000 (Invitrogen) dilution, rabbit anti- O . tsutsugamushi TSA56 at a 1:1,000 dilution [ ], and/or mouse anti-Flag epitope (Sigma-Aldrich) at a 1:1,000 dilution in PBS containing 5% (vol/vol) BSA for 1 h. The coverslips were washed three times in PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) at a 1:1,000 dilution in 5% BSA for 1 h at room temperature.

Techniques: Infection, Incubation, Control, Homogenization, Western Blot

Journal: PLoS Pathogens

Article Title: Orientia tsutsugamushi uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation

doi: 10.1371/journal.ppat.1007023

Figure Lengend Snippet: (A) HeLa cells were transfected to express Flag-tagged BAP, Ank1, Ank6, or IκBα SR. At 16 h, the cells were exposed to TNFα or vehicle control for 30 min, after which they were fixed, screened with antibodies specific for the Flag epitope and p65, and examined by confocal microscopy. (B) The mean percentage + SD of transfected cells exhibiting p65 in the nucleus was determined. Triplicate samples of 100 cells each were counted per time point. Statistically significant (**** P < 0.0001) values indicated. Results are representative of three independent experiments.

Article Snippet: Samples were then incubated at room temperature with antibody targeting endogenous p65 at a 1:250 (Santa Cruz, Dallas, TX) or 1:1000 (Invitrogen) dilution, rabbit anti- O . tsutsugamushi TSA56 at a 1:1,000 dilution [ ], and/or mouse anti-Flag epitope (Sigma-Aldrich) at a 1:1,000 dilution in PBS containing 5% (vol/vol) BSA for 1 h. The coverslips were washed three times in PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) at a 1:1,000 dilution in 5% BSA for 1 h at room temperature.

Techniques: Transfection, Control, FLAG-tag, Confocal Microscopy

Journal: PLoS Pathogens

Article Title: Orientia tsutsugamushi uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation

doi: 10.1371/journal.ppat.1007023

Figure Lengend Snippet: (A) HeLa cells were transfected to express Flag-tagged BAP, Ank1, Ank6, or IκBα SR. At 16 h, they were treated with TNFα or vehicle control (Ctrl) for 30 min. Western blotted nuclear or cytosolic fractions were screened with antibodies against p65, lamin B1, GAPDH, or the Flag epitope. (B) Mean normalized ratios + SD of p65:lamin B1 densitomery signals were calculated from three separate experiments performed in (A). Statistically significant values (* P < 0.05; *** P < 0.001) are indicated. Data presented are representative of three separate experiments.

Article Snippet: Samples were then incubated at room temperature with antibody targeting endogenous p65 at a 1:250 (Santa Cruz, Dallas, TX) or 1:1000 (Invitrogen) dilution, rabbit anti- O . tsutsugamushi TSA56 at a 1:1,000 dilution [ ], and/or mouse anti-Flag epitope (Sigma-Aldrich) at a 1:1,000 dilution in PBS containing 5% (vol/vol) BSA for 1 h. The coverslips were washed three times in PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) at a 1:1,000 dilution in 5% BSA for 1 h at room temperature.

Techniques: Transfection, Control, Western Blot, FLAG-tag

Journal: PLoS Pathogens

Article Title: Orientia tsutsugamushi uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation

doi: 10.1371/journal.ppat.1007023

Figure Lengend Snippet: HeLa cells ectopically expressing Flag-tagged BAP, Ank1, or Ank6 and were treated with TNFα or vehicle for 30 min. Whole cell lysates were incubated with Flag antibody-conjugated agarose beads to immunoprecipitate (IP) Flag-tagged proteins and their interacting proteins. Resulting Western blots were probed with p65 antibody to determine if any Flag-tagged protein co-immunoprecipitated endogenous p65. As a control to ensure that the conditions allowed for p65 and IκBα to interact, a parallel experiment was performed in which lysates of TNFα or vehicle control stimulated Flag-p65 expressing HeLa cells were incubated with the Flag antibody coated beads followed by Western blot analysis of the resulting eluate with antibody against endogenous IκBα. Expression of each Flag-tagged protein of interest was confirmed by subjecting input lysate to Western blotting using Flag antibody. Data presented are representative of three independent experiments.

Article Snippet: Samples were then incubated at room temperature with antibody targeting endogenous p65 at a 1:250 (Santa Cruz, Dallas, TX) or 1:1000 (Invitrogen) dilution, rabbit anti- O . tsutsugamushi TSA56 at a 1:1,000 dilution [ ], and/or mouse anti-Flag epitope (Sigma-Aldrich) at a 1:1,000 dilution in PBS containing 5% (vol/vol) BSA for 1 h. The coverslips were washed three times in PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) at a 1:1,000 dilution in 5% BSA for 1 h at room temperature.

Techniques: Expressing, Incubation, Western Blot, Immunoprecipitation, Control

Journal: PLoS Pathogens

Article Title: Orientia tsutsugamushi uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation

doi: 10.1371/journal.ppat.1007023

Figure Lengend Snippet: Whole cell lysates of HeLa cells expressing Flag-tagged BAP, Ank1, Ank6, or Ank7 were incubated with Flag antibody-conjugated agarose beads to immunoprecipitate Flag-tagged proteins and their interacting proteins. Resulting Western blots were probed with antibodies specific for importin β1, exportin 1, and p65. Expression of each Flag-tagged protein of interest was confirmed by subjecting input lysate to Western blotting using Flag antibody. Data presented are representative of three independent experiments.

Article Snippet: Samples were then incubated at room temperature with antibody targeting endogenous p65 at a 1:250 (Santa Cruz, Dallas, TX) or 1:1000 (Invitrogen) dilution, rabbit anti- O . tsutsugamushi TSA56 at a 1:1,000 dilution [ ], and/or mouse anti-Flag epitope (Sigma-Aldrich) at a 1:1,000 dilution in PBS containing 5% (vol/vol) BSA for 1 h. The coverslips were washed three times in PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) at a 1:1,000 dilution in 5% BSA for 1 h at room temperature.

Techniques: Expressing, Incubation, Western Blot

Journal: PLoS Pathogens

Article Title: Orientia tsutsugamushi uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation

doi: 10.1371/journal.ppat.1007023

Figure Lengend Snippet: HeLa cells were transfected to express Flag-tagged BAP, Ank1, Ank6, or IκBα SR. At 16 h, the cells were treated with LMB or vehicle control for 1 h. The media was replaced with media containing TNFα or vehicle for 30 min. The cells were then fixed, screened with antibodies specific for the Flag epitope and p65, and examined by confocal microscopy. Representative fluorescence images are presented in . The mean percentage + SD of cells exhibiting p65 in the nucleus was determined. Quadruplicate samples of 100 cells each were counted per time point. Statistically significant (* P < 0.05; *** P < 0.001) values are indicated. n.s., not significant. Results are representative of three independent experiments.

Article Snippet: Samples were then incubated at room temperature with antibody targeting endogenous p65 at a 1:250 (Santa Cruz, Dallas, TX) or 1:1000 (Invitrogen) dilution, rabbit anti- O . tsutsugamushi TSA56 at a 1:1,000 dilution [ ], and/or mouse anti-Flag epitope (Sigma-Aldrich) at a 1:1,000 dilution in PBS containing 5% (vol/vol) BSA for 1 h. The coverslips were washed three times in PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) at a 1:1,000 dilution in 5% BSA for 1 h at room temperature.

Techniques: Transfection, Control, FLAG-tag, Confocal Microscopy, Fluorescence

Journal: PLoS Pathogens

Article Title: Orientia tsutsugamushi uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation

doi: 10.1371/journal.ppat.1007023

Figure Lengend Snippet: (A and B) O . tsutsugamushi mediates p65 nuclear export in an LMB-sensitive manner. HeLa cells were infected with O . tsutsugamushi at an MOI of 10 for 24 h or 72 h. Next, the cells were treated with LMB or vehicle control for 1 h after which they were fixed, screened with antibodies against O . tsutsugamushi TSA56 ( Ot ) and p65, and examined by confocal microscopy. (A) Representative fluorescence images of cells viewed for Ot , p65, and merged images plus DAPI for infected cells treated with LMB or vehicle control (Ctrl) are presented. (B) The mean percentage + SD of cells exhibiting p65 in the nucleus was determined for each condition. Triplicate samples of 100 cells each were counted per time point. n.s., not significant. Results are representative of three separate experiments. (C and D) O . tsutsugamushi inhibits NF-κB-dependent transcriptional activation in an LMB-insensitive manner. Stably transfected HeLa cells bearing four copies of the NF-κB response element upstream of the Luc gene were infected with O . tsutsugamushi for 24 h at an MOI of 10 after which they were incubated with LMB or vehicle control in the presence or absence of TNFα for 8 h. (C) Representative fluorescence images of LMB-treated infected cells viewed for TSA56 ( Ot ), p65, and merged images plus DAPI. (D) The cells were lysed followed by mixing with Luc substrate. Data are presented as the mean percentage + SD of Luc activity normalized to the Luc activity of uninfected vehicle control treated cells that were exposed to TNFα from triplicate samples. Statistically significant (*** P < 0.001) values are indicated. n.s., not significant. Results are representative of three independent experiments.

Article Snippet: Samples were then incubated at room temperature with antibody targeting endogenous p65 at a 1:250 (Santa Cruz, Dallas, TX) or 1:1000 (Invitrogen) dilution, rabbit anti- O . tsutsugamushi TSA56 at a 1:1,000 dilution [ ], and/or mouse anti-Flag epitope (Sigma-Aldrich) at a 1:1,000 dilution in PBS containing 5% (vol/vol) BSA for 1 h. The coverslips were washed three times in PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) at a 1:1,000 dilution in 5% BSA for 1 h at room temperature.

Techniques: Infection, Control, Confocal Microscopy, Fluorescence, Activation Assay, Stable Transfection, Transfection, Incubation, Activity Assay

Journal: PLoS Pathogens

Article Title: Orientia tsutsugamushi uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation

doi: 10.1371/journal.ppat.1007023

Figure Lengend Snippet: Whole cell lysates of HeLa cells expressing the indicated Flag-tagged proteins were incubated with Flag antibody-conjugated agarose beads to immunoprecipitate Flag-tagged proteins and their interacting proteins. (A) Western blots of input lysates and immunoprecipitates were probed with p65, exportin 1 (Exp 1), and importin β1 (Imp β1) antibodies. Expression of each Flag-tagged protein of interest was confirmed by subjecting input lysate to Western blotting using Flag antibody. GAPDH immunoblotting of input lysates served as a loading control. (B) Mean + SD of co-immunoprecipitated p65, exportin 1, or importin β1. Values are presented as the percent change in densitometric signal relative that for each respective protein of interest that was coimmunoprecipitated by Flag-tagged Ank1 or Ank6. Data presented were calculated from three separate experiments formed in (A). Statistically significant (* P < 0.05; ** P < 0.01) values are indicated.

Article Snippet: Samples were then incubated at room temperature with antibody targeting endogenous p65 at a 1:250 (Santa Cruz, Dallas, TX) or 1:1000 (Invitrogen) dilution, rabbit anti- O . tsutsugamushi TSA56 at a 1:1,000 dilution [ ], and/or mouse anti-Flag epitope (Sigma-Aldrich) at a 1:1,000 dilution in PBS containing 5% (vol/vol) BSA for 1 h. The coverslips were washed three times in PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) at a 1:1,000 dilution in 5% BSA for 1 h at room temperature.

Techniques: Expressing, Incubation, Western Blot, Control, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Orientia tsutsugamushi uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation

doi: 10.1371/journal.ppat.1007023

Figure Lengend Snippet: HeLa cells were transfected to express Flag-tagged BAP, Ank1, Ank1△ISR, Ank1△F-box, Ank6, Ank6△ISR, or Ank6△F-box. At 16 h, the cells were exposed to TNFα for 30 min, after which they either were fixed, screened with antibodies specific for the Flag epitope and p65, and examined by confocal microscopy (A and B) or resolved into nuclear and cytosolic fractions that were subjected to Western blot and densitometry analyses (C and D). (A) Representative fluorescence images of cells viewed for Flag-tagged protein, p65, and merged images plus DAPI. (B) The mean percentage + SD of transfected cells exhibiting p65 in the nucleus was determined. Triplicate samples of 100 cells each were counted per time point. (C) Western blotted nuclear and/or cytosolic fractions were screened with antibodies against the Flag epitope, GAPDH, p65, or lamin C. (D) Mean normalized ratios + SD of p65:lamin C densitometry signals were calculated from three separate experiments performed in (C). Results in each panel are representative of three independent experiments. Statistically significant (* P < 0.05; ** P < 0.01; **** P <0.0001) values are indicated. n.s., not significant. Indicators of statistical significance relative to values obtained for Flag-BAP, Flag-Ank1, and Flag-Ank6 are colored black, red, and blue, respectively.

Article Snippet: Samples were then incubated at room temperature with antibody targeting endogenous p65 at a 1:250 (Santa Cruz, Dallas, TX) or 1:1000 (Invitrogen) dilution, rabbit anti- O . tsutsugamushi TSA56 at a 1:1,000 dilution [ ], and/or mouse anti-Flag epitope (Sigma-Aldrich) at a 1:1,000 dilution in PBS containing 5% (vol/vol) BSA for 1 h. The coverslips were washed three times in PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen) at a 1:1,000 dilution in 5% BSA for 1 h at room temperature.

Techniques: Transfection, FLAG-tag, Confocal Microscopy, Western Blot, Fluorescence

Journal: Cell Death Discovery

Article Title: METTL3 overexpression aggravates LPS-induced cellular inflammation in mouse intestinal epithelial cells and DSS-induced IBD in mice

doi: 10.1038/s41420-022-00849-1

Figure Lengend Snippet: A , B METTL3 knockdown was achieved in mouse intestinal epithelial cell line MODE-K by transfecting lentivirus containing specific short hairpin RNA (#1 sh-METTL3 or #2 sh-METTL3) and confirmed using qRT-PCR and immunoblotting. C MODE-K cells were transduced with #1 sh-METTL3 or #2 sh-METTL3 and examined for cell viability by CCK-8 assay; #1 sh-METTL3 was selected for further experiments. D MODE-K cells were transfected with sh-METTL3 and examined for cell apoptosis by flow cytometry. E The protein levels of cleaved-caspase3 and cleaved-caspase9 by immunoblotting. F The mRNA expression of IL-1β, TNF-α, IL-6, IL-18, COX-2, and iNOS by qRT-PCR. G The protein levels of IL-1β, TNF-α, IL-6, IL-18, COX-2, and iNOS by immunoblotting. H MODE-K cells were transfected with lentivirus-overexpressing METTL3 (Lv-METTL3) and examined for the protein levels of METTL3 using immunoblotting. I MODE-K cells were transfected with Lv-METTL3 or sh-METTL3, stimulated with LPS, and examined for the protein levels of p-p65 and p65 using immunoblotting. J MODE-K cells were transfected with Lv-METTL3, stimulated with LPS or co-stimulated with LPS and JSH-23 (NF-κB inhibitor), and examined for the protein levels of p-p65 and p65 using immunoblotting. N = 3, ** p < 0.01, compared with sh-NC group.

Article Snippet: The primary antibodies were against METTL3 (15073-AP, Proteintech, USA), TNF-α (60291-1-Ig, Proteintech), iNOS (18985-1-AP, Proteintech), IL-6 (CSB-PA06757A0RB, CUSABIO, China), cleaved-caspase3 (ab34287, Abcam, USA), cleaved-caspase9 (10380-1-AP, Proteintech), IL-1β (12242, Cell signaling technology, USA), IL-18 (60070-1-Ig, Preoteintech), COX-2 (12375-1-AP, Proteintech), p-p65 (ab76302, Abcam), p65 (10745-1-AP, Proteintech) and GAPDH (endogenous control, 60004-1-Ig, Proteintech).

Techniques: Knockdown, shRNA, Quantitative RT-PCR, Western Blot, Transduction, CCK-8 Assay, Transfection, Flow Cytometry, Expressing

Journal: Cell Death Discovery

Article Title: METTL3 overexpression aggravates LPS-induced cellular inflammation in mouse intestinal epithelial cells and DSS-induced IBD in mice

doi: 10.1038/s41420-022-00849-1

Figure Lengend Snippet: A Mice were divided into four groups: control, DSS-induced IBD model, DSS-induced mice received sh-NC injection, and DSS-induced mice received sh-METTL3 injection. DSS-induced IBD model and sh-NC/sh-METTL3 administration were conducted as described in the Materials and methods section. Colon length of mice in each group was determined. B The protein levels of METTL3 in colon tissues were examined using Immunoblotting. C – D Basal body weight and the disease activity index (DAI) score were examined on day 0, to 7. E At the end of the modeling, mice were sacrificed and the histopathological features of mice colon were examined using H&E staining. F Nitric oxide (NO), malondialdehyde (MDA), and myeloperoxidase activity (MPO) in colon tissues were examined. G The mRNA expression of IL-1β, TNF-α, iNOS, IL-6, IL-18, and COX2 were examined in colon tissues using qRT-PCR. H The protein levels of METTL3, IL-1β, TNF-α, iNOS, IL-6, IL-18, and COX2 in colon tissues were examined using Immunoblotting. I The protein levels of p-p65 and p65 were examined in colon tissues using Immunoblotting. N = 6, ** p < 0.01, compared with control group; && p < 0.01, compared with DSS group; ## p < 0.01, compared with DSS+sh-NC group.

Article Snippet: The primary antibodies were against METTL3 (15073-AP, Proteintech, USA), TNF-α (60291-1-Ig, Proteintech), iNOS (18985-1-AP, Proteintech), IL-6 (CSB-PA06757A0RB, CUSABIO, China), cleaved-caspase3 (ab34287, Abcam, USA), cleaved-caspase9 (10380-1-AP, Proteintech), IL-1β (12242, Cell signaling technology, USA), IL-18 (60070-1-Ig, Preoteintech), COX-2 (12375-1-AP, Proteintech), p-p65 (ab76302, Abcam), p65 (10745-1-AP, Proteintech) and GAPDH (endogenous control, 60004-1-Ig, Proteintech).

Techniques: Control, Injection, Western Blot, Activity Assay, Staining, Expressing, Quantitative RT-PCR

Journal: Cell Death Discovery

Article Title: METTL3 overexpression aggravates LPS-induced cellular inflammation in mouse intestinal epithelial cells and DSS-induced IBD in mice

doi: 10.1038/s41420-022-00849-1

Figure Lengend Snippet: A Mice were divided into four groups: control, DSS-induced IBD model, DSS-induced mice received vehicle control, and DSS-induced mice received STM2457 administration. DSS-induced IBD model and vehicle/STM2457 administration were conducted as described in the Materials and methods section. The colon length of mice in each group was determined. B , C Basal body weight and the DAI score were examined on day 0 to 7. D At the end of the modeling, mice were sacrificed and the histopathological features of mice colon were examined using H&E staining. E NO, MDA and MPO levels in colon tissues were examined. F The mRNA expression of IL-1β, TNF-α, iNOS, IL-6, IL-18, and COX2 were examined in colon tissues using qRT-PCR. G The protein levels of IL-1β, TNF-α, iNOS, IL-6, IL-18, and COX2 in colon tissues were examined using Immunoblotting. H The protein levels of p-p65 and p65 were examined in colon tissues using Immunoblotting. N = 6, ** p < 0.01, compared with control group; && p < 0.01, compared with DSS group; ## p < 0.01, compared with DSS + vehicle group.

Article Snippet: The primary antibodies were against METTL3 (15073-AP, Proteintech, USA), TNF-α (60291-1-Ig, Proteintech), iNOS (18985-1-AP, Proteintech), IL-6 (CSB-PA06757A0RB, CUSABIO, China), cleaved-caspase3 (ab34287, Abcam, USA), cleaved-caspase9 (10380-1-AP, Proteintech), IL-1β (12242, Cell signaling technology, USA), IL-18 (60070-1-Ig, Preoteintech), COX-2 (12375-1-AP, Proteintech), p-p65 (ab76302, Abcam), p65 (10745-1-AP, Proteintech) and GAPDH (endogenous control, 60004-1-Ig, Proteintech).

Techniques: Control, Staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Oncology

Article Title: The lncRNA CASC2 Modulates Hepatocellular Carcinoma Cell Sensitivity and Resistance to TRAIL Through Apoptotic and Non-Apoptotic Signaling

doi: 10.3389/fonc.2021.726622

Figure Lengend Snippet: Effects of lncRNA CASC2 on caspase-cascades and the NF-κB pathway. (A) A schematic diagram showing two different modes of TRAIL acting on TRAIL-sensitive and TRAIL-resistant cell lines. (B) Huh-7 and HCCLM3 cells were exposed to 1, 10, 100, and 1,000 ng/ml rhTRAIL protein and examined for the cell viability by MTT assay. The IC50 values were calculated and shown. Regular Huh-7 and HCCLM3 cells were then divided into TRAIL-sensitive Huh-7 (S) and HCCLM3 (S) and TRAIL-resistant Huh-7 (R) and HCCLM3 (R). (C) Huh-7 (S), HCCLM3 (S), Huh-7 (R), and HCCLM3 (R) cells were treated with 0 or 150 ng/ml rhTRAIL and examined for the protein levels of RIPK1, caspase-8, caspase-3, IKKβ, p-IκBα, and p-p65 using Immunoblotting. (D) CASC2 expression was determined in Huh-7 (S), HCCLM3 (S), Huh-7 (R), and HCCLM3 (R) cells using qRT-PCR. (E) Huh-7 (S) and HCCLM3 (S) cells were transfected with CASC2 or sh-CASC2 and examined for the protein levels of caspase-8 and caspase-3 using Immunoblotting. (F) Huh-7 (R) and HCCLM3 (R) cells were transfected with CASC2 or sh-CASC2 and examined for the protein levels of IKKβ, p-IκBα, and p-p65 using Immunoblotting. **p < 0.01, compared with sensitive group or vector-NC group, ## p < 0.01, compared with sh-NC group.

Article Snippet: The protein levels of cyclin D1, ki67, caspase-8, caspase-3, cleaved-caspase-8, cleaved-caspase-3, RIPK1, IKKβ, p-IκBα, and p-p65 were examined by immunoblotting following the methods described before ( ) with antibodies against cyclin D1 (60186-1-Ig; Proteintech, Wuhan, China), ki67 (27309-1-AP, Proteintech), caspase3 (19677-1-AP, Proteintech), cleaved-caspase 3 (ab2302, Abcam, Cambridge, MA, USA), caspase-8 (ab32397, Abcam), cleaved-caspase-8 (# 9496S; Cell Signaling; Danvers, MA, USA), IKKβ (07-1008; Sigma-Aldrich, St. Louis, MO, USA), p-IκBα (#9246; CST, Danvers, MA, USA), p-p65 (ab194726, Abcam), and β-actin (60008-1-Ig, Proteintech). β-actin was taken as an endogenous control.

Techniques: MTT Assay, Western Blot, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: The lncRNA CASC2 Modulates Hepatocellular Carcinoma Cell Sensitivity and Resistance to TRAIL Through Apoptotic and Non-Apoptotic Signaling

doi: 10.3389/fonc.2021.726622

Figure Lengend Snippet: Dynamic effects of the CASC2/miR-18a/RIPK1 axis on TRAIL-resistant HCC cell proliferation. Huh-7 (R) and HCCLM3 (R) cells were divided into six groups: NC, sh-CASC2, sh-RIPK1, miR-18a inhibitor, sh-CASC2+ miR-18a inhibitor, and sh-RIPK1+ miR-18a inhibitor; cells in different groups were transfected accordingly and examined for cell viability using CCK-8 assay (A) ; DNA synthesusing EdU assay (B) ; the protein levels of IKKβ, p-IκBα, and p-p65 using Immunoblotting (C) . *p < 0.05 compared with NC group.

Article Snippet: The protein levels of cyclin D1, ki67, caspase-8, caspase-3, cleaved-caspase-8, cleaved-caspase-3, RIPK1, IKKβ, p-IκBα, and p-p65 were examined by immunoblotting following the methods described before ( ) with antibodies against cyclin D1 (60186-1-Ig; Proteintech, Wuhan, China), ki67 (27309-1-AP, Proteintech), caspase3 (19677-1-AP, Proteintech), cleaved-caspase 3 (ab2302, Abcam, Cambridge, MA, USA), caspase-8 (ab32397, Abcam), cleaved-caspase-8 (# 9496S; Cell Signaling; Danvers, MA, USA), IKKβ (07-1008; Sigma-Aldrich, St. Louis, MO, USA), p-IκBα (#9246; CST, Danvers, MA, USA), p-p65 (ab194726, Abcam), and β-actin (60008-1-Ig, Proteintech). β-actin was taken as an endogenous control.

Techniques: Transfection, CCK-8 Assay, EdU Assay, Western Blot

Journal: Frontiers in Oncology

Article Title: The lncRNA CASC2 Modulates Hepatocellular Carcinoma Cell Sensitivity and Resistance to TRAIL Through Apoptotic and Non-Apoptotic Signaling

doi: 10.3389/fonc.2021.726622

Figure Lengend Snippet: The transcription factor NF-κB inhibits lncRNA CASC2 transcription. (A) TCGA hepatocellular carcinoma data (TCGA_LIHC) were analyzed using Spearman’s correlation analysis for the correlation between lncRNA CASC2 and RELA. (B) NF-κB overexpression or knockdown was achieved in Huh-7 (R) and HCCLM3 (R) cells by transfecting NF-κB-overexpressing plasmid (NF-κB) or short hairpin RNA targeting NF-κB (sh-NF-κB); the transfection efficiency was confirmed using qRT-PCR. (C, D) Wild- and mutant-type CASC2 promoter luciferase reporter plasmids were co-transfected with NF-κB or sh-NF-κB, and the luciferase activity was determined. (E) ChIP assay was performed using anti-IgG or anti-NF-κB, and the levels of CASC2 promoter in immunoprecipitate by anti-IgG or anti-NF-κB were determined using qRT-PCR. (F) Huh-7 (R) and HCCLM3 (R) cells were transfected with NF-κB or sh-NF-κB and examined for CASC2 expression using qRT-PCR. **p < 0.01, compared with vector or IgG group, ## p < 0.01, compared with shRNA-NC group.

Article Snippet: The protein levels of cyclin D1, ki67, caspase-8, caspase-3, cleaved-caspase-8, cleaved-caspase-3, RIPK1, IKKβ, p-IκBα, and p-p65 were examined by immunoblotting following the methods described before ( ) with antibodies against cyclin D1 (60186-1-Ig; Proteintech, Wuhan, China), ki67 (27309-1-AP, Proteintech), caspase3 (19677-1-AP, Proteintech), cleaved-caspase 3 (ab2302, Abcam, Cambridge, MA, USA), caspase-8 (ab32397, Abcam), cleaved-caspase-8 (# 9496S; Cell Signaling; Danvers, MA, USA), IKKβ (07-1008; Sigma-Aldrich, St. Louis, MO, USA), p-IκBα (#9246; CST, Danvers, MA, USA), p-p65 (ab194726, Abcam), and β-actin (60008-1-Ig, Proteintech). β-actin was taken as an endogenous control.

Techniques: Over Expression, Knockdown, Plasmid Preparation, shRNA, Transfection, Quantitative RT-PCR, Mutagenesis, Luciferase, Activity Assay, Expressing