Journal: International journal of molecular medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis.

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Figure 1. Effect of XBJ on the mRNA expression of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 in lung tissue. Groups of mice were challenged with CLP and treated with XBJ 24 h later. The expression of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 in lung tissue was determined by RT-PCR. Representative RT-PCR shows the level of Tollip, IRAK1, TLR4, NF-κB65, and TRAF6 expression in the four rat groups. M, marker; A, normal control group; B, sham operation group; C, control group; D, treatment group.

Article Snippet: Rabbit anti-mouse Tollip, TLR4, TRAF6, p-IRAK1, VEGF-α, HO-1 and NF-κB polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Control

Journal: International journal of molecular medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis.

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Figure 2. Administration of XBJ led to increased expression levels of Tollip mRNA, and inhibition of TLR4, NF-κB65 and TRAF6 mRNA expression in lung tissue in CLP-ALI mice. The expression of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 in lung tissue was determined by RT-PCR. Statistical summary of the densitometric analysis of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 expression in the four rat groups. Data are presented as mean ± stan dard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate; ﹡P<0.05 and ﹡﹡P<0.01 vs. normal control group and sham operation group. ﹟P<0.05 and ﹟﹟P<0.01 vs. control group.

Article Snippet: Rabbit anti-mouse Tollip, TLR4, TRAF6, p-IRAK1, VEGF-α, HO-1 and NF-κB polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Expressing, Inhibition, Reverse Transcription Polymerase Chain Reaction, Control

Journal: International journal of molecular medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis.

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Figure 4. Administration of XBJ enhanced the expression of Tollip protein protein, and inhibition TLR4, NF-κB65, p-IRAK1 and TRAF6 protein expression in lung tissue in CLP-ALI mice. Groups of mice were challenged with LPS and treated with salidroside 24 h later. Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 were assayed by western blot analysis. Statistical summary of the densitometric analysis of Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 protein expression in the four rat groups. Data are presented as mean ± standard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate; ﹡﹡P<0.01 vs. the normal control group and sham operation group. ﹟P<0.05, ﹟﹟P<0.01 vs. the control group.

Article Snippet: Rabbit anti-mouse Tollip, TLR4, TRAF6, p-IRAK1, VEGF-α, HO-1 and NF-κB polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Expressing, Inhibition, Western Blot, Standard Deviation, Control

Journal: International journal of molecular medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis.

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Figure 3. Administration of XBJ enhanced the expression of Tollip protein, and inhibition of TLR4, NF-κB65, p-IRAK1 and TRAF6 protein expression in lung tissue in CLP-ALI mice. Groups of mice were challenged with LPS and treated with salidroside 24 h later. Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 were assayed by western blot analysis. Representative western blots show the level of Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 protein expression in the four rat groups. A, normal control group; B, sham operation group; C, control group; D, treatment group.

Article Snippet: Rabbit anti-mouse Tollip, TLR4, TRAF6, p-IRAK1, VEGF-α, HO-1 and NF-κB polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Expressing, Inhibition, Western Blot, Control

Journal: International journal of molecular medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis.

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Figure 5. Administration of XBJ upregulated Tollip positive protein in lung tissue in CLP-ALI mice. Groups of mice were challenged with CLP and treated with XBJ 24 h later. Tollip positive protein levels of lung tissue were determined using immunohistochemistry and the average proportion of positive expres sion in each field was counted using the true color multi-functional cell image analysis management system. Values are expressed as mean ± SD; ﹡﹡P<0.01 vs. normal control group and sham operation group. ﹟P<0.05 and ﹟﹟P<0.01 vs. control group.

Article Snippet: Rabbit anti-mouse Tollip, TLR4, TRAF6, p-IRAK1, VEGF-α, HO-1 and NF-κB polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Immunohistochemistry, Functional Assay, Control

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation.

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: Figure 1: Overexpression profiles of TUBB3 and FOXO3a in a panel of cancer cells with ABCB1-associated acquired drug resistance. (A) Characterization of indicated parental or drug-resistant phenotype cell lines for TUBB3 expression at both mRNA (upper panel) and protein (lower panel) levels. (B) Characterization of indicated parental or drug-resistant phenotype cell lines for FOXO3a expression at both mRNA (upper panel) and protein (lower panel) levels. (C) Identification of ABCB1-association expression in a panel of indicated parental or drug resistant cancer cell lines at the mRNA level. (D) Confirmation of P-gp protein overexpression in drug resistant cancer cell lines. (E) Intracellular distribution and localization of P-gp expression in both wild-type and drug-resistant phenotype cell lines. Cells were stained with human P-gp antibody and DAPI and analyzed through confocal microscopy. Images shown were magnified at ×200.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [80].

Techniques: Over Expression, Expressing, Staining, Confocal Microscopy

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation.

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: Figure 2: Occurrence of cross-resistance in PTX-resistant cancer cells is highly associated with ATP-dependent P-gp/ ABCB1 efflux activity. (A and B) Fraction of drug-intolerant A549, A549-PacR (A) and HEK293, HEK293/ABCB1 (B) cells. Cells were treated with PTX, 5-FU, DCT, or CIS for 24 hr, and the cell viability was determined by MTT assay. Data are represented as means ± SEM. (C) Representative drug intolerant cell colonies (right) and quantified colony numbers of A549-PacR cells (left). Cells were exposed to 5-FU-conditioned media (CM) and continuously grown for > 7 days and formed colonies of established PTX-resistant cells were stained with sapphire 700. Data are represented as means ± SEM. (D and E) ATP-dependent P-gp efflux activity. Cells were treated with 60 µM 5-FU for 24 hr and assayed for Rho-123 incorporation. Flow cytometry was used to quantify Rho-123 fluorescence (D). Drug-resistant cells were treated with DMSO, 60 µM 5-FU, or 50 µM DCT for 24 hr and was assayed for ABCB1 ATPase activity (E). Data are represented as means ± SEM. (F and G) Association of P-gp expression with occurrence of cross-resistance. Drug-resistant cells were treated with 5-FU, DCT, or CIS for 24 hr (F) and time-dependently treated with 5-FU for (G). Cells were treated with 20 µM 5-FU then assayed for qRT-PCR using ABCB1-specific primer. (H) P-gp-specific ATPase activity. Cells were treated with increasing 5-FU concentration for 24 hr and cells were subjected to P-gp luminescent ATPase assay. Data are represented as means ± SEM.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [80].

Techniques: Activity Assay, MTT Assay, Staining, Flow Cytometry, Fluorescence, Expressing, Quantitative RT-PCR, Concentration Assay, ATPase Assay

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation.

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: Figure 3: High acquired cross-resistance correlates to regulated TUBB3 and FOXO3a expressions with distinct hyperactive ABCB1 transcription in paclitaxel-resistant cancer cells. (A) Schematic diagram of general strategy used for the generation of transient cross-resistance to 5-FU, DCT, or CIS in PacR phenotype cancer cells or ABCB1-GFP transfected HEK293 cells. (B) Growth rate response of indicated cells (lower panel) to 5-FU treatment in a dose-dependent manner. Schematic schedule of treatment is also displayed (upper panel). Cell viability was determined using MTT assay. Data are represented as means ± SEM. (C) Characterization for maintained ABCB1 and ABCC1 mRNA expressions in developed A549-PacR/5-FU and PC-3-PacR/5-FU cells after indicated subsequent cell cultures. Passages of cells were maintained with 1 µM 5-FU final concentration. (D–F) Characterization for TUBB3 and FOXO3a mRNA expressions in indicated developed transient cross-resistance in PacR phenotype derived from A549 (D), PC-3 (E), and in developed transient 5-FU cross-resistance derived from HEK293 (F) cells. (G and H) Western blot analysis of A549, PC-3-PacR cells (G) and HEK293 cells transfected with either empty vector or ABCB1-GFP (H) cells, all with developed transient cross-resistance to indicated drugs. Cells were assessed for expressions of indicated proteins after 24 hr cell culture. (I) Flow cytometric determination of verapamil-induced apoptosis in indicated cells. Cells were treated with or without 100 µM verapamil for 24 hr. Data are shown as bar graph represented as means ± SEM. (J) Western blot analysis of indicated cells for expressions of apoptotic markers Bax, Bcl-2, and p53. Cells were treated with or without 100 µM verapamil for 24 hr. (K) Intracellular ATP level assessment in indicated cells. Cells were treated with or without 100 µM verapamil for 24 hr and ATP levels were determined in 104 fraction of cells. RLU, relative luciferase units. Data are represented as means ± SEM. (L) Determination of verapamil-induced inhibition of P-gp/ABCB1 in indicated cells assessed through qRT-PCR (left), Western blotting (center) and confocal microscopy (right). Cells were treated with 60 µM verapamil for 24 hr. Confocal images shown were magnified to 80 µm.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [80].

Techniques: Transfection, MTT Assay, Concentration Assay, Derivative Assay, Western Blot, Plasmid Preparation, Cell Culture, Luciferase, Inhibition, Quantitative RT-PCR, Confocal Microscopy

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation.

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: Figure 4: FOXO3a activity involves ABCB1 regulation to control TUBB3 response in paclitaxel-resistant cancer cells with transient 5-FU cross-resistance. (A) Characterization of ABCB1 and TUBB3 expressions at both protein (upper panel) and mRNA (lower panel) levels after transient transfections with indicated vectors or siRNA in A549-PacR and PC-3-PacR both with developed 5-FU transient cross-resistance. Cells were transfected with empty vector or FOXO3a-GFP and scrambled siRNA or ABCB1 siRNA for 48 hr. Data are represented as means ± SEM. (B) Protein expressions (upper panel) of ABCB1 and TUBB3 and mRNA levels of TUBB3 (lower panel) in cells same as in A after transient transfection with either scrambled siRNA or FOXO3a siRNA for 48 hr. Data are represented as means ± SEM. (C) Effect of proteasome inhibitor, MG132 (MG), on βIII-tubulin distribution. Cells treated with or without 50 µM MG for 24 h were subjected to immunocytochemistry. Magnified images were zoomed at ×80. The cells were stained with TUBB3 antibody and DAPI. (D) Effect of MG on TUBB3 protein expressions in same cells as in A. Cells were transfected with either empty vector or FOXO3a- GFP for 48 hr and treated with or without 50 µM MG132 for 24 hr. Whole-cell lysates were assayed by Western blotting. (E) Detection of ubiquitinated TUBB3 protein in A549-PacR/5-FU cells. Cells were transfected with either scramble siRNA or FOXO3a-siRNA for 48 hr and cells were further transfected with an empty vector or myc-tagged ubiquitin-encoding vector for 24 hr. Immunoprecipitation was performed by TUBB3 antibody, then ubiquitinated proteins were detected by myc antibody assayed by Western blotting. (F) Methylation- specific PCR of ABCB1 -50GC and -110GC boxes in A549-PacR/5-FU cells. Primer sets are designed to amplify methylated (M) and unmethylated (U) alleles. A primer set encoding the whole GC region (T) was used as loading control. Cells were transiently transfected with either empty vector or FOXO3a-GFP for 48 hr and cells were treated with 10 µM PTX for 12 hr (left lane) or 24 hr (right lane). (G) Combined bisulphite restriction analysis of the Inr ABCB1 promoter region in A549-PacR/5-FU cells treated with or without 10 µM PTX, or 40 µM 5-FU for 24 hr or in combination for 24 or 48 hr as indicated. Figures represent the methylation percentages observed in the indicated drug-treated cells obtained from two separate independent experiments. (H) Effect of doxorubicin (Dox) on FOXO3a-induced regulation of Akt-related signals. Cells were transiently transfected with either empty vector or FOXO3a-GFP for 48 hr and cells were treated with or without 2 µM Dox for 8 hr. Protein expression levels were analyzed by Western blotting. (I) Methylated FOXO3a status in

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [80].

Techniques: Activity Assay, Control, Transfection, Plasmid Preparation, Immunocytochemistry, Staining, Western Blot, Ubiquitin Proteomics, Immunoprecipitation, Methylation, Expressing

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation.

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: Figure 5: Drug-induced secretome factors influence MDR promotion of FOXO3a-regulated P-gp activity in PTX- resistant A549 cells with multiple cross-resistance. (A) Schematic diagram of the drug conditioned media (CM)-exposure procedure used in various assays (B–J). (B and C) Characterization of FOXO3a-mediated multiple cross-drug resistance in A549-PacR cells (B). Cells were preincubated with indicated 1:1 combination ratio of CM from DMSO-, DCT-, 5-FU-treated cells. After 24 hr, cells were transiently transfected with indicated vectors or siRNA for 48 hr and assessed for colony formation (C). Established drug resistant colonies were stained and visualized by crystal violet or sapphire 700. (D and E) TUBB3 knockdown or FOXO3a overexpression (D) and overexpression of ABCB1 and FOXO3a effects on growth rate of A549-PacR/5-FU cells (E). A549-PacR/DCT cells were treated with indicated 5-FU concentrations for 24 h then CM was collected. Adherent A549 cells were exposed to the collected CM for 24 h followed by transient transfection with indicated vectors or siRNA for 48 hr. Cell viability was assessed by MTT assay. Data are represented as means ± SEM. (F) Confocal microscopic analysis of A549-PacR/5-FU cells. Cells were transiently transfected with indicated vectors or siRNA for 48 hr and stained with P-gp antibody and DAPI. Images shown were magnified to 80 µm. (G) Western blot analysis (left) and agarose gel electrophoresis of qPCR products of A549-PacR/5-FU cells. Cells were transiently transfected with indicated vectors or siRNA for 48 hr and assessed for expression studies. (H) ABCB1 drug efflux activity of A549-PacR/5-FU cells. Cells were transiently transfected with either empty vector or FOXO3a-GFP for 48 hr and cells were treated with or without 100 µM 5-FU or 120 µM DCT for 24 hr. Drug-treated cells were assayed for Rhodamine-123 (Rho-123) uptake (left) and untreated cells were assayed for ABCB1 ATPase activity (right). Data are represented as means ± SEM. (I) ABCB1 drug efflux activity of A549-PacR/5-FU cells. Cells were transiently transfected with either scrambled siRNA or TUBB3 siRNA for 48 hr followed by drug treatment and assayed as in H. Data are represented as means ± SEM. (J) Multidrug resistant colonies. Cells were transiently transfected with either scramble siRNA or TUBB3 siRNA for 48 hr and cells were treated with 30 µM 5-FU for 7 days. Resistant colonies were visualized by sapphire 700. (K) Schematic diagram of drug CM exposure and respective TUBB3 feedback after induced gene expression changes via transient gene transfection or siRNA gene silencing. Indicated gene or siRNA transfection schemes were used in L and M for P-gp functional assays. (L) Growth rate of A549-PacR/5-FU cells. Cells were pre-incubated with indicated drug-CM and cells were transiently transfected with indicated genes or siRNA for 48 hr. Cell viability was assessed by MTT assay. Data are represented as means ± SEM. (M) ABCB1 drug efflux activity A549-PacR/5-FU cells. Cells were pre- incubated with indicated drug-CM and cells were transiently transfected with indicated genes or siRNA for 48 hr. Cells were then treated with indicated drugs and concentration for 36 hr. Data are represented as means ± SEM.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [80].

Techniques: Activity Assay, Transfection, Staining, Knockdown, Over Expression, MTT Assay, Western Blot, Agarose Gel Electrophoresis, Expressing, Plasmid Preparation, Gene Expression, Functional Assay, Incubation, Concentration Assay

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation.

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: Figure 7: TUBB3 feedback inactivation reverses impaired microtubule stability in A549-PacR cells with developed transient multiple cross-resistance. (A) Microtubule stability in parental, PacR, and developed PacR phenotype cells with multiple transient cross-resistance. Cells were grown in the presence of indicated PTX concentrations for 18 hr. Following cell lysis, pellet (P) and the supernatant (S) protein fractions were separated by centrifugation and resolved on adjacent lanes by electrophoresis. Transferred filters were probed with tubulin antibody. (B) Acetylated tubulin status of parental, PacR, and developed PacR phenotype cells with multiple transient cross-resistance. Cells were grown in the absence or presence 20 nM PTX for 18 hr, and the amount of acetylated tubulin was measured by immunoblotting using antibody specific to acetylated tubulin. (C) Mixing tubulin experiment. Indicated cells were harvested by adding hypotonic buffer with 50 µg/mL PTX for 15 min. Whole cell lysate from PacR phenotype was added to that of developed PacR/5- FU subline at different ratios as indicated, and incubated for an additional 10 min. The polymerized (Pol) and soluble (Sol) protein fractions were processed as described in Materials and Methods. (D) Schematic diagram of transient gene transfection and/or siRNA silencing with respective TUBB3 feedback results used in E and F in A549-PacR/5-FU cells. (E) Confirmation of TUBB3 feedback gene expressions after transient transfection and/or siRNA silencing of indicated genes with C as control (respective empty vector and/or scramble siRNA) and TS as the transfection scheme shown in D. (F) Microtubule stability of A549-PacR/5-FU cells through tubulin polymerization after indicated transient gene and/or siRNA transfections. Cells were grown in the presence of 60 nM PTX for 18 hr. The polymerized (Pol) and soluble (Sol) protein fractions were processed as in C. (G) Illustration of proposed mechanism behind the feedback response of TUBB3 to silencing of FOXO3a and ABCB1 in regulating microtubule stability in A549-PacR/5-FU cells.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [80].

Techniques: Lysis, Centrifugation, Electrophoresis, Western Blot, Incubation, Transfection, Control, Plasmid Preparation

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation.

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: Figure 8: Model representation of the feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation inducing multiplicity in acquired cross-drug resistance. Multiplicity of acquired cross-resistance to structurally different drugs in cancer cells selected for taxane resistance is associated with feedback control of TUBB3 through FOXO3a-mediated ABCB1 regulation inducing hyperfunctional P-gp-associated drug efflux and escape from potential inhibition.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [80].

Techniques: Control, Inhibition

Journal: Frontiers in cellular neuroscience

Article Title: Neurovascular Interaction Promotes the Morphological and Functional Maturation of Cortical Neurons.

doi: 10.3389/fncel.2017.00290

Figure Lengend Snippet: FIGURE 5 | SU1498 and SB203580 inhibit the increase of mEPSC frequency induced by B-CM. (A) Representative blots for vascular endothelial growth factor (VEGF) and GAPDH in neurons cultured alone (N) or treated with B-CM for 2 days as well as VEGF in cell-free B-CM. The relative quantity of VEGF protein was calculated after normalization to GAPDH (data from three independent experiments. ∗p < 0.05). (B,C) Cumulative probability plots of interevent interval and the quantification of mEPSC frequency are shown (N: n = 7; B-CM: n = 10; B-CM+SU1498: n = 8; SU1498: n = 10. ∗∗p < 0.01). (D) Representative blots for p-p38, p38 and GAPDH in neurons cultured alone or treated with B-CM for 2 days. The relative quantity of p-p38 protein was calculated after normalization to GAPDH (data from seven independent experiments. ∗∗p < 0.01). (E) Representative blots for p-p38, p38 and GAPDH in neurons cultured alone or treated with SU1498, B-CM or both. The relative quantity of p-p38 protein was calculated after normalization to GAPDH (data from four independent experiments. ∗p < 0.05; ∗∗p < 0.01). (F,G) Cumulative probability plot of interevent interval and the quantification of mEPSC frequency are shown (N: n = 10; B-CM: n = 10; B-CM+SB203580: n = 8; SB203580: n = 7. ∗∗∗p < 0.001). Details see Supplementary Figure S1.

Article Snippet: Proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes, and incubated with primary antibodies against VEGF (1:500; catalog sc-152; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p38 (1:1000; catalog 9212; Cell Signaling Technology, Beverly, MA, USA) and phosphorylated p38 (p-p38, 1:1000; catalog 4511; Cell Signaling Technology), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; catalog sc-2004; Santa Cruz Biotechnology).

Techniques: Cell Culture

Journal: Frontiers in cellular neuroscience

Article Title: Neurovascular Interaction Promotes the Morphological and Functional Maturation of Cortical Neurons.

doi: 10.3389/fncel.2017.00290

Figure Lengend Snippet: FIGURE 6 | SU1498 and SB203580 inhibit the increase of VGlut-1 and PSD95 cluster density induced by B-CM. (A) Representative staining in clusters for VGlut-1 and PSD95 in cortical neurons cultured alone or treated with SU1498 (an inhibitor of Flk-1), SB203580 (an inhibitor of p38 MAPK), B-CM, B-CM+SU1498 or B-CM+SB203580. (B) Summary graphs showing the cluster density for VGlut-1, PSD95 and PSD95/VGlut-1 co-localization in the different treated neurons with values normalized to neuron alone group (n = 11 per condition. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).

Article Snippet: Proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes, and incubated with primary antibodies against VEGF (1:500; catalog sc-152; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p38 (1:1000; catalog 9212; Cell Signaling Technology, Beverly, MA, USA) and phosphorylated p38 (p-p38, 1:1000; catalog 4511; Cell Signaling Technology), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; catalog sc-2004; Santa Cruz Biotechnology).

Techniques: Staining, Cell Culture

Journal: Frontiers in cellular neuroscience

Article Title: Neurovascular Interaction Promotes the Morphological and Functional Maturation of Cortical Neurons.

doi: 10.3389/fncel.2017.00290

Figure Lengend Snippet: FIGURE 7 | Working model. (A) Neurovascular interaction promotes neurite outgrowth in cortical neurons in the early stage of in vitro development. (B) Neurovascular interaction promotes functional maturation of neurons, as evidenced by facilitating APs production and increasing mEPSC frequency. Signaling pathways: neuronal Flk-1 engagement by VEGF results in the activation of p38 MAPK which induces presynaptic VGlut-1 and postsynaptic PSD95 and increases the excitatory synapse formation and transmission.

Article Snippet: Proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes, and incubated with primary antibodies against VEGF (1:500; catalog sc-152; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p38 (1:1000; catalog 9212; Cell Signaling Technology, Beverly, MA, USA) and phosphorylated p38 (p-p38, 1:1000; catalog 4511; Cell Signaling Technology), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; catalog sc-2004; Santa Cruz Biotechnology).

Techniques: In Vitro, Functional Assay, Protein-Protein interactions, Activation Assay, Transmission Assay

Journal: Cellular and Molecular Life Sciences

Article Title: New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome

doi: 10.1007/s00018-022-04301-6

Figure Lengend Snippet: GZD824 inhibits downstream signaling of ROR1. a Schematic representation of GZD824 binding to ROR1 PK domain, which induces inhibition of its downstream PI3K/AKT, STAT3, and NF-κB signaling. b Immunoblot analysis of the BaF3-ROR1 cells left untreated of pre-treated with Src inhibitor dasatinib or GZD824 (1 µM) for 2 h before the addition of Wnt5a (100 ng/ml) as indicated. Downstream ROR1 signaling levels or pERK/ERK, pAKT/AKT, and pSrc/Src are shown. β-tubulin was used as a loading control. c Venn diagram showing the number of shared and specific interactors of ROR1 before and after GZD284 treatment as identified by AP-MS, BioID, or both. For each set of interactors, the protein-coding genes involved in MAPK signaling (red), Rho GTPases signaling (yellow), or actin/cytoskeleton signaling (blue) are indicated. d , e Bar graphs of the top non-redundant enriched ontology clusters from multiple functional annotation databases in ROR1 interactomes identified with both AP-MS and BioID when before ( d ) and after ( e ) GZD824 treatment. The color scale represents statistical significance

Article Snippet: For Western blotting, the following antibodies were used: pAKT (S473, #6942), AKT (#9272), Bcl-xL (#2764), pERK1/2 (#9101), MEK1/2 (#4694), pMEK1/2 (#9121), ERK1/2 (#4696), NF-κB p65 (#6956), pNF-κB p65 (#3033), PARP (#9532), PI3K p85α (#13,666), pPI3K p85/p55 (#4228), PTK7 (#25618), Rac-1 (#4651), RhoA (#2117), ROR1 (#16,540), ROR2 (#88,639), Src (#2109), STAT3 (#9139), pSTAT3 (#9145) from Cell Signaling Technology (CST, Danvers, MA, USA); anti-pTYR 4G10 (#05–321) from Merck Millipore (Burlington, MA, USA); β-tubulin (#sc-166729) from Santa Cruz Biotechnology (Dallas, TX, USA); HA (#901,513) from BioLegend (San Diego, CA, USA).

Techniques: Binding Assay, Inhibition, Western Blot, Control, Protein-Protein interactions, Functional Assay

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 1. A schematic drawing illustrates the four signaling pathways utilized by TGFb to affect cellular function. This diagram was prepared based on reviews [18, 19, 21]. Original articles that describe the discovery of the key molecules depicted in these pathways can be found in these reviews. MEKKs, MAP/ERK kinase kinases, which include MEKK1, MEKK2, MEKK3, and others; JNK, c-Jun NH2-terminal kinase also known as Jun kinase or stress-activated protein kinase, SAPK; JNKK, c-Jun NH2-terminal kinase kinase; MKK3, MAP kinase kinase 3; MEK1/2, MAP/ERK kinase 1 and MAP/ERK kinase 2; ERK1/2, extracellular signal-regulated kinase 1 and extracellular signal-regulated kinase 2.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Protein-Protein interactions, Cell Function Assay

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 4. Relative expression of the TGFb- upstream signal transducers in Sertoli and germ cells isolated from 20-day-old rat testes. A) Semiquantitative RT-PCR was performed to assess the steady-state mRNA levels of Smad2, Cdc42, Rac2, MEKK2, and N-Ras in Sertoli and germ cells. Im- munoblots were performed in parallel ex- periments to assess the relative protein levels of these signal transducers in Sertoli and germ cells using the corresponding specific antibodies (see Materials and Methods). (C). B) This figure shows the corresponding densitometrically scanned results using autoradiograms or immuno- blots such as those shown in A and C. Re- sults are expressed as mean 6 SD using three batches of cells from three different experiments normalized against S16. Each experiment had triplicate cultures. These analyses revealed that results of immuno- blots are consistent with the RT-PCR data. Statistical analysis was performed by Stu- dent t-test comparing germ cells with the corresponding Sertoli cells, which was ar- bitrarily set at one. *, Significantly different from Sertoli cells by Student t-test, P , 0.05; **, significantly different from Sertoli cells by Student t-test, P , 0.01.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

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

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 5. Developmental regulation of the steady-state mRNA levels of the TGFb up- stream signal transducers in Sertoli (A, B) and germ (C, D) cells. RT-PCR was per- formed to assess the steady-state mRNA levels of Smad2, Cdc42, Rac2, MEKK2, and N-Ras in Sertoli cells (A) and germ cells (C) during maturation. B, D) The cor- responding densitometrically scanned re- sults using autoradiograms such as those shown in A and C. Results are expressed as mean 6 SD using two batches of cells normalized against S16 from two different experiments. Each experiment had tripli- cate cultures. ns, Not significantly different from cultures isolated from rats at 20 days of age in B and 5 or 10 days of age in D, which was arbitrarily set at one, by Stu- dent t-test; *, significantly different by Stu- dent t-test, P , 0.05; **, significantly dif- ferent by Student t-test, P , 0.01; nd, not detectable.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 6. Changes in the steady-state mRNA levels of TGFb upstream signal transducers in the testis during develop- ment. A) Semiquantitative RT-PCR was performed to assess the steady-state mRNA levels of Smad2, Cdc42, Rac2, MEKK2, and N-Ras in testes during maturation. B) This panel shows the corresponding densi- tometrically scanned results using autora- diograms such as those shown in A. Re- sults are expressed as mean 6 SD using testes from three different rats normalized against S16. ns, Not significantly different from rats at 5 days of age, which was arbi- trarily set at one, by Student t-test; *, signif- icantly different by Student t-test, P , 0.05; **, significantly different by Student t-test, P , 0.01.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 8. Change in the steady-state mRNA and protein levels of the TGFb upstream signal transducers when the Sertoli cell TJ barrier was assembled in vitro in the ab- sence (control) and presence (test) of TGFb3. Sertoli cells (0.5 3 106 cells/cm2) cultured on Matrigel-coated dishes in the absence (A, C, E) or presence (B, D, F) of TGFb3 (3 ng/ml) were terminated by RNA STAT-60 or lysed in SDS sample buffer at specified time points. Cell lysates (;200 mg protein) from each time point were re- solved by SDS-PAGE under reducing con- ditions using 10% T SDS-polyacrylamide gels. Immunoblotting was performed to as- sess changes in the levels of Smad2, Cdc42, Rac2, N-Ras, and MEKK2 in the absence (A, E) and presence (B, F) of re- combinant TGFb3 (3 ng/ml). RT-PCR was performed to assess changes in the MEKK2 steady-state mRNA level in the absence (C) or presence (D) of recombinant TGFb3 (3 ng/ml). G, H) Corresponding densito- metrically scanned results using autoradio- grams and immunoblots shown in C–F. Results are expressed as mean 6 SD from three separate experiments using different batches of cells and normalized against S16. Each time point had duplicate cul- tures. ns, Not significantly different by AN- OVA, in which each sample at a given time point was compared with samples of all other time points within the same ex- perimental group; *, significantly different by ANOVA, P , 0.01; D, days.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: In Vitro, Control, Cell Culture, SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction, Recombinant

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of STAT5 induces G1 cell cycle arrest and reduces tumor cell invasion in human colorectal cancer cells.

doi: 10.1038/labinvest.2009.11

Figure Lengend Snippet: Figure 1 RNAi treatment decreases STAT5 signaling in CRC cells. (a) Western blot analysis demonstrated that both STAT5 and pSTAT5 were depleted in SW1116 cells after transfection with Dharmacon STAT5 siRNA. The lowest levels of STAT5 expression were detected on day 3, and the proteins were recovered at day 4 post-transfection. (b) At 72 h post- transfection, western blot analysis showed that STAT5 siRNA (Santa Cruz) induced a downregulation of STAT5 and pSTAT5 in CRC cells. In addition, STAT5 siRNA-induced alterations in several, but not all downstream targets of STATs in CRC cells at 72 h after treatment. Bcl-2 protein was downregulated simultaneous to an upregulation of p16ink4a, p21waf1/cip1

Article Snippet: All antibodies in this study were purchased from Cell Signaling Technology Inc. (USA), except the following antibodies: phospho-STAT5 (pSTAT5Tyr694/Tyr699) (Santa Cruz, CA, USA), Bcl-2 (R&D, USA) and GAPDH (Kangchen, China).

Techniques: Western Blot, Transfection, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of STAT5 induces G1 cell cycle arrest and reduces tumor cell invasion in human colorectal cancer cells.

doi: 10.1038/labinvest.2009.11

Figure Lengend Snippet: Figure 2 The functional role of STAT5 signaling on CRC cell growth and cell cycle progression. (a) Cell viability was determined by the CCK-8 assay following treatment of CRC cells with STAT5 siRNAs. The percentage of viable cells was determined as described in the Materials and Methods section. STAT5 siRNA inhibited CRC cell growth. This suppression persisted for 72 h, after which the cell recovered at 96 h post-transfection. The results represent mean±s.d. of three experiments. (b) Cell cycle analysis was performed after treating CRC cells with STAT5 siRNA (Santa Cruz). STAT5 knockdown led to a block in the cell cycle at the G1 phase at 72 h after transfection.

Article Snippet: All antibodies in this study were purchased from Cell Signaling Technology Inc. (USA), except the following antibodies: phospho-STAT5 (pSTAT5Tyr694/Tyr699) (Santa Cruz, CA, USA), Bcl-2 (R&D, USA) and GAPDH (Kangchen, China).

Techniques: Functional Assay, CCK-8 Assay, Transfection, Cell Cycle Assay, Knockdown, Blocking Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of STAT5 induces G1 cell cycle arrest and reduces tumor cell invasion in human colorectal cancer cells.

doi: 10.1038/labinvest.2009.11

Figure Lengend Snippet: Figure 3 The functional role of STAT5 signaling on the invasive ability of CRC cells. (a) CRC cells transfected with STAT5 siRNA were examined for their invasive capability. At 48 h post-transfection, the number of migrated cells significantly decreased compared with untreated cells (*Po0.05). (b) Western blot analysis showed that STAT5 siRNA (Santa Cruz) induced an upregulation of E-cadherin, simultaneous with a downregulation of FAK in CRC cells at 72 h post-transfection of the cells. (c) Effects of STAT5 siRNA on the secretion of VEGF, MMP2 and MMP9. At 48 h post-transfection, the concentrations of VEGF and MMP2 were decreased compared with that of untreated cells (*Po0.05). The experiment was performed three times with consistent findings.

Article Snippet: All antibodies in this study were purchased from Cell Signaling Technology Inc. (USA), except the following antibodies: phospho-STAT5 (pSTAT5Tyr694/Tyr699) (Santa Cruz, CA, USA), Bcl-2 (R&D, USA) and GAPDH (Kangchen, China).

Techniques: Functional Assay, Transfection, Western Blot

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of STAT5 induces G1 cell cycle arrest and reduces tumor cell invasion in human colorectal cancer cells.

doi: 10.1038/labinvest.2009.11

Figure Lengend Snippet: Figure 4 Immunohistochemical staining of the tissue microarray. Predominantly cytoplasmic staining of STAT5 was detected in normal colonic epithelium, adenomas and primary colon adenocarcinomas (as indicated by red arrows). pSTAT5 localized to the cytoplasm of adenoma cells and colon adenocarcinoma cells, although predominantly found in the nucleus of normal epithelium cells (Black arrows indicate pSTAT5 protein is shuttled from nucleus to cytoplasm in the oncogenesis of CRC; magnification ¼ 400).

Article Snippet: All antibodies in this study were purchased from Cell Signaling Technology Inc. (USA), except the following antibodies: phospho-STAT5 (pSTAT5Tyr694/Tyr699) (Santa Cruz, CA, USA), Bcl-2 (R&D, USA) and GAPDH (Kangchen, China).

Techniques: Immunohistochemical staining, Staining, Microarray

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of STAT5 induces G1 cell cycle arrest and reduces tumor cell invasion in human colorectal cancer cells.

doi: 10.1038/labinvest.2009.11

Figure Lengend Snippet: Figure 5 Interaction of STAT5 with MAPK, PI3K and AKT in CRC cells. (a) Protein–protein interactions were determined by co-immunoprecipitation analysis. Co-immunoprecipitation analysis revealed that STAT5 formed a complex with p44/42 MAPK and SAPK/JNK in SW1116 cells. However, no significant interactions of STAT5 with PI3K, p38, AKT were detected in our studies. (b) Reciprocal immunoprecipitations were performed with antibodies directed against p44/42 or SAPK/JNK or a non-relevant antibody (purified human IgG, negative control), then detected STAT5 by western blot. These results also validate the interactions of STAT5 with p44/42 MAPK or SAPK/JNK in CRC cells.

Article Snippet: All antibodies in this study were purchased from Cell Signaling Technology Inc. (USA), except the following antibodies: phospho-STAT5 (pSTAT5Tyr694/Tyr699) (Santa Cruz, CA, USA), Bcl-2 (R&D, USA) and GAPDH (Kangchen, China).

Techniques: Protein-Protein interactions, Immunoprecipitation, Purification, Negative Control, Western Blot

Journal: Proteomics

Article Title: Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

doi: 10.1002/pmic.201500501

Figure Lengend Snippet: Identification of proteins with the most differential change at 40 and 80% cell density in response to Nutlin‐3 treatment. (A) Identification of proteins perturbed by Nutlin‐3 using SWATH MS. HCT116 cells were incubated for 4 h with Nutlin‐3 under conditions in which MDM2 is just beginning to be stabilized (Fig. B). This ensures that we capture changes in the cellular proteome just when MDM2 is starting to be perturbed in cells by Nutlin‐3. The cell pellets were processed using MS SWATH to identify differentially perturbed proteins (Supporting Information Tables 1 and 2), some of which are highlighted (in green) as a function of 40 or 80% cell density. (B) A scatter plot of the total protein changes as a function of cell density and fold change (log 2 ) with mitochondrial proteins highlighted in red.

Article Snippet: The cells were fixed onto slides with 4% paraformaldehyde in PBS for 20 min at RT, permeabilized for 10 min in 0.25% Triton x‐100 in PBS and blocked with 3% BSA in PBS for 30 min. Antibodies from different species were then incubated on the slides, with combinations of MDM2 mouse mAb (4B2) with either rabbit pAb DLD (sc‐135027; Santa Cruz) or p53 (CM‐1), at a 1:250 dilution for 1 h at RT.

Techniques: Data-independent acquisition, Incubation

Journal: Proteomics

Article Title: Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

doi: 10.1002/pmic.201500501

Figure Lengend Snippet: The allosteric effect of Nutlin‐3 on MDM2 functions. (A) MDM2 has multiple functional domains including an N‐terminal peptide‐binding groove that is the binding site of Nutlin‐3 ; a central domain that responds allosterically to N‐terminal MDM2‐binding ligands (like Nutlin‐3) to increase p53 binding and ubiquitination (arrows) , and a RING domain that is required for E2 docking and allosteric control of ubiquitination by the E2, UBCH5 . N‐terminal domain Nutlin‐3 binding by MDM2 can stimulate p53 ubiquitination , stimulate ubiquitin‐dependent protein activation of Notch , and direct binding to alter target protein oligomerization of NPM . Thus, Nutlin‐3 can dissociate or induce various protein–protein interactions due to the allosteric effects of ligands on MDM2 function. These data suggest a complex effect of MDM2 ligands on changes in the steady‐state cellular proteome. (B–D) HCT116 cells (p53+ and p53‐null, as indicated) where grown to 40 and 80% density as defined in the methods. Cells were treated with Nutlin‐3 (20 μM final concentration) or DMSO control and after 4 h cells were harvested for lysis without proteasome inhibitor treatment (that would artificially elevate levels of target protein). Lysates were blotted with the indicated antibodies (MDM2, p53, and tubulin as a loading control). The data demonstrate that p53 protein is induced equivalently by Nutlin‐3 at either cell density, but MDM2 protein exhibits higher steady‐state levels at the lower density after Nutlin‐3 treatment, as reported previously .

Article Snippet: The cells were fixed onto slides with 4% paraformaldehyde in PBS for 20 min at RT, permeabilized for 10 min in 0.25% Triton x‐100 in PBS and blocked with 3% BSA in PBS for 30 min. Antibodies from different species were then incubated on the slides, with combinations of MDM2 mouse mAb (4B2) with either rabbit pAb DLD (sc‐135027; Santa Cruz) or p53 (CM‐1), at a 1:250 dilution for 1 h at RT.

Techniques: Functional Assay, Binding Assay, Ubiquitin Proteomics, Control, Activation Assay, Protein-Protein interactions, Concentration Assay, Lysis

Journal: Proteomics

Article Title: Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

doi: 10.1002/pmic.201500501

Figure Lengend Snippet: Immunochemical analysis of dihydrolipoamide dehydrogenase protein levels after MDM2 perturbation. (A and B) HCT116 cells (p53+) where grown to 40 or 80% density. Cells were treated with Nutlin‐3 (20 μM final concentration) or DMSO control and after 4 h, cells were harvested for lysis without proteasome inhibitor treatment that would artificially elevate levels of target protein. Lysates were blotted with the indicated antibodies to dihydrolipoamide dehydrogenase and tubulin as a loading control. (C–F). HCT116 cells grown at 80% density and then were treated with control siRNA (C) or siRNA to deplete MDM2 (M) for 24 h, followed by treatment with DMSO or Nutlin‐3. The lysates were then immunoblotted for p53, MDM2, and dihydrolipoamide dehydrogenase, and the loading control.

Article Snippet: The cells were fixed onto slides with 4% paraformaldehyde in PBS for 20 min at RT, permeabilized for 10 min in 0.25% Triton x‐100 in PBS and blocked with 3% BSA in PBS for 30 min. Antibodies from different species were then incubated on the slides, with combinations of MDM2 mouse mAb (4B2) with either rabbit pAb DLD (sc‐135027; Santa Cruz) or p53 (CM‐1), at a 1:250 dilution for 1 h at RT.

Techniques: Concentration Assay, Control, Lysis

Journal: Proteomics

Article Title: Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

doi: 10.1002/pmic.201500501

Figure Lengend Snippet: Nutlin‐3 dissociates dihydrolipoamide dehydrogenase and dihydrolipoamide acetyltransferase protein–protein interactions within the pyruvate dehydrogenase holoenzyme complex. (A–F) HCT116 cells were treated with DMSO or Nutlin‐3 (20 μM) for 4 h. Cells were fixed and processed for proximity ligation as recorded in the Methods using antibodies to dihydrolipoamide dehydrogenase (mouse) and dihydrolipoamide acetyltransferase (rabbit). Cells were imaged using FITC as a readout of proximity ligation (representative images in A, B, and C), DAPI plus merged FITC (D, E, and F). (A, D) DMSO control; (B, E) Nutlin‐3 effects; (C, F) DMSO with BSA controls without primary antibodies (G and H). MDM2:p53 complexes were evaluated with DMSO control (G) or with Nutlin‐3 (H; representative images are a merge of FITC proximity ligation and DAPI to localize the nucleus). The quantitation of the average dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase foci in cells is summarized in I (using ImageJ software).

Article Snippet: The cells were fixed onto slides with 4% paraformaldehyde in PBS for 20 min at RT, permeabilized for 10 min in 0.25% Triton x‐100 in PBS and blocked with 3% BSA in PBS for 30 min. Antibodies from different species were then incubated on the slides, with combinations of MDM2 mouse mAb (4B2) with either rabbit pAb DLD (sc‐135027; Santa Cruz) or p53 (CM‐1), at a 1:250 dilution for 1 h at RT.

Techniques: Protein-Protein interactions, Ligation, Control, Quantitation Assay, Software

Journal: Proteomics

Article Title: Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

doi: 10.1002/pmic.201500501

Figure Lengend Snippet: The effects of Nutlin‐3 on dihydrolipoamide dehydrogenase and MDM2 complexes protein in vitro and in vivo. (A and B) The effects of Nutlin‐3 on MDM2 protein–protein interactions. An ELISA was used to measure the binding of MDM2 to A, dihydrolipoamide dehydrogenase or B, p53 . The purity of the indicated proteins measure by Coomassie blue is in Supporting Information Fig. 2. Target protein p53 or dihydrolipoamide dehydrogenase was coated onto the solid phase as indicated in the Methods. Ligand‐free MDM2 (DMSO control) or Nutlin‐3 (20 μM in DMSO) bound MDM2 was titrated into reactions followed by immunochemical quantitation of the amounts of MDM2 bound. MDM2 activity is depicted in relative light units as a function of increasing MDM2 protein levels (in nanograms). (C–H) In vivo binding of MDM2 and dihydrolipoamide dehydrogenase. Proximity ligation assays were used according to the Methods to measure MDM2 and dihydrolipoamide dehydrogenase binding in situ. (C and D) FITC and DAPI merged images, respectively, in proliferating cells treated with DMSO control. (E and F) FITC and DAPI merged images, respectively, in cells treated with Nutlin‐3 (20 μM) for 4 h. (G and H) FITC and DAPI merged images, respectively, in proliferating cells treated with DMSO control using BSA in place of primary antibodies as a negative control. (I and J) Total cellular distribution of MDM2 and dihydrolipoamide dehydrogenase. Immunofluorescence was used with specific antibodies in fixed cells according to the Methods to measure the total distribution of MDM2 and dihydrolipoamide dehydrogenase pools in the absence or presence of Nutlin‐3 (20 μM). (K) Quantitation of MDM2 and dihydrolipoamide dehydrogenase protein–protein interaction foci in the absence and presence of Nutlin‐3 using proximity ligation .

Article Snippet: The cells were fixed onto slides with 4% paraformaldehyde in PBS for 20 min at RT, permeabilized for 10 min in 0.25% Triton x‐100 in PBS and blocked with 3% BSA in PBS for 30 min. Antibodies from different species were then incubated on the slides, with combinations of MDM2 mouse mAb (4B2) with either rabbit pAb DLD (sc‐135027; Santa Cruz) or p53 (CM‐1), at a 1:250 dilution for 1 h at RT.

Techniques: In Vitro, In Vivo, Protein-Protein interactions, Enzyme-linked Immunosorbent Assay, Binding Assay, Control, Quantitation Assay, Activity Assay, Ligation, In Situ, Negative Control, Immunofluorescence

Journal: Proteomics

Article Title: Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

doi: 10.1002/pmic.201500501

Figure Lengend Snippet: The effects of Leptomycin B on dihydrolipoamide dehydrogenase and MDM2 complexes in cells using proximity ligation assays. (A and B) The effects of Leptomycin B after four hours of treatment on MDM2 and dihydrolipoamide dehydrogenase protein–protein interactions with images depicting FITC proximity ligation [24] (A) and DAPI nuclear stain (in blue) and MitoTracker Red CMXros (in red) as a merged image (B). (C and D) The effects of Nutlin‐3 on MDM2 and dihydrolipoamide dehydrogenase protein–protein interactions with images depicting FITC proximity ligation (C) and DAPI nuclear stain (in blue) and MitoTracker Red CMXros (in red) as a merged image (D). (E and F) The effects of Leptomycin B and Nutlin‐3 combined on MDM2 and dihydrolipoamide dehydrogenase protein–protein interactions with images depicting FITC proximity ligation (E) and DAPI nuclear stain (in blue) and MitoTracker Red CMXros (in red) as a merged image (F). (G) Quantitation of protein–protein interaction foci with the indicated treatment with DMSO, Leptomycin B, Nutlin‐3, or Nutlin‐3, and Leptomycin B combined.

Article Snippet: The cells were fixed onto slides with 4% paraformaldehyde in PBS for 20 min at RT, permeabilized for 10 min in 0.25% Triton x‐100 in PBS and blocked with 3% BSA in PBS for 30 min. Antibodies from different species were then incubated on the slides, with combinations of MDM2 mouse mAb (4B2) with either rabbit pAb DLD (sc‐135027; Santa Cruz) or p53 (CM‐1), at a 1:250 dilution for 1 h at RT.

Techniques: Ligation, Protein-Protein interactions, Staining, Quantitation Assay

Journal: Proteomics

Article Title: Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

doi: 10.1002/pmic.201500501

Figure Lengend Snippet: Model summarizing the effects of Nutlin‐3 on dihydrolipoamide dehydrogenase and MDM2 localizations. (Left panel) In proliferating cells, there are at least two pools of dihydrolipoamide dehydrogenase. One pool interacts predominantly with components of the pyruvate dehydrogenase complex and the second pool with MDM2, predominantly in the cytosol. (Right panel) Following Nutlin‐3 treatment, perturbation of the mitochondrial proteome results in dissociation of the dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase, suggesting a disruption of pyruvate dehydrogenase holoenzyme complex. In addition, although minimal dihydrolipoamide dehydrogenase is observed in the nucleus in untreated cells, the Nutlin‐3‐dependent import of MDM2 into the nucleus coincides with increased dihydrolipoamide dehydrogenase: MDM2 complexes in the nucleus. It remains to be determined if the binding of dihydrolipoamide dehydrogenase to MDM2 in the nucleus has a direct effect on p53 protein activation.

Article Snippet: The cells were fixed onto slides with 4% paraformaldehyde in PBS for 20 min at RT, permeabilized for 10 min in 0.25% Triton x‐100 in PBS and blocked with 3% BSA in PBS for 30 min. Antibodies from different species were then incubated on the slides, with combinations of MDM2 mouse mAb (4B2) with either rabbit pAb DLD (sc‐135027; Santa Cruz) or p53 (CM‐1), at a 1:250 dilution for 1 h at RT.

Techniques: Disruption, Binding Assay, Activation Assay