Journal: Cell Reports Medicine

Article Title: Arachidonic acid released by PIK3CA mutant tumor cells triggers malignant transformation of colonic epithelium by inducing chromatin remodeling

doi: 10.1016/j.xcrm.2024.101510

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal antibody anti-PDK1 , Cell Signaling Technology , Cat# 5662; RRID: AB_10839264.

Techniques: Microarray, Recombinant, Cloning, Mutagenesis, Immunohistochemistry, Protein Purification, In Vitro, Sequencing, Methylation

Journal: Journal of Cell Science

Article Title: Nuclear factor-κB signaling and ezrin are essential for L1-mediated metastasis of colon cancer cells

doi: 10.1242/jcs.069542

Figure Lengend Snippet: L1 induces NF-κB signaling and the phosphorylation of IκB in colon cancer cells. (A) The NF-κB-responsive reporter plasmid 3xκB.luc was transfected together with pSV β-galactosidase control vector (for transfection efficiency normalization) into human Ls174T CRC cells that do not express L1, and into two individually selected clones stably transfected to express L1 (L1 Cl1 and Cl2) and a control clone (pcDNA3). Fold NF-κB activation was determined after dividing luciferase activity by the values obtained with an empty reporter plasmid. (B) 293T cells were transfected as in A with either an empty pcDNA3 plasmid or with the same plasmid containing L1. Luciferase activity was then determined. (C) Human SW620 CRC cells that express endogenous L1, two individually selected clones stably transfected with shRNA targeting L1 (shRNA L1 Cl1 and Cl2) and a control clone (control) were transfected as in A, and luciferase activity was determined. (D) The cells described in C were analyzed for L1 RNA and protein levels by PCR and western blotting (bottom and top blots, respectively. (E) Ls174T cells stably transfected with the empty pcDNA3 vector or vector expressing L1 were either left untreated (lanes 1,3) or were treated with the proteasome inhibitor MG132 for 3 hours before harvesting (lanes 2,4). Levels of IκB total (IκB) and phosphorylated IκB (IκB-P) were determined by western blotting. Tubulin served as loading control. (F) Ls174T cells stably transfected with either pcDNA3 or vector expressing L1 were immunostained with anti-p65-P and anti-L1 antibodies (red and green staining, respectively). Nuclei were stained with DAPI (blue). Scale bar: 20 μm. pIκB is IκB-P and pp65 is p65-P.

Article Snippet: For L1 and p65- P detection, polyclonal rabbit anti-L1 antibody (described above; diluted 1:1000), rabbit antiserum against p65- P (no. 3037, Cell Signaling, Frankfurt, Germany; diluted 1:50), and mouse anti-ezrin antibody (described above) were used.

Techniques: Plasmid Preparation, Transfection, Clone Assay, Stable Transfection, Activation Assay, Luciferase, Activity Assay, shRNA, Western Blot, Expressing, Staining

Journal: Journal of Cell Science

Article Title: Nuclear factor-κB signaling and ezrin are essential for L1-mediated metastasis of colon cancer cells

doi: 10.1242/jcs.069542

Figure Lengend Snippet: The juxtamembrane L1 cytodomain contains a tyrosine residue at position 1151 that is necessary for L1-mediated cancer progression. (A) Diagram of the L1 cytoplasmic domain in wt and mutant molecules. (B) The NF-κB-responsive reporter plasmid 3xκB.luc was transfected into 293T cells together with an empty pcDNA3 plasmid (control), or with the same plasmid expressing wt L1 or mutant L1 in the cytodomain. Constructs L1-1176 and L1-1180 express truncated versions of L1, whereas constructs L1-4A and L1-Y1151A express L1 point mutants. (C) Cells stably transfected with either pcDNA3 or L1-Y1151A were immunostained with anti-L1 antibody, and their levels of expression were determined by western blotting with antibodies against L1 and IκB (D). (E) Lysates of 293T cells transfected to express L1 or L1-Y1151A together with IκB-SR were immunoprecipitated (IP) with antibodies against L1 and analyzed by western blotting with antibodies against L1 and IκB. (F) Ls174T cells stably transfected with empty pcDNA3 vector, or plasmids expressing L1, L1-Y1151A or the p65 NF-κB subunit were either left untreated (lanes 1,3,5,7) or were treated with the proteasome inhibitor MG132 for 3 hours before cell harvesting (lanes 2,4,6,8), and levels of phosphorylated IκB (IκB-P) were determined. (G) Proliferation of CRC cell clones described in C in the presence of 10% or 0% serum (H) was determined during 7 days in quadruplicate. (I) Cell clones described in C were injected into the flanks of nude mice – on one side L1-expressing cells and on the other side of the same mouse L1-Y1151A-expressing cells or cells expressing the empty pcDNA3 vector (control). Tumor size was determined 14 days after injection. (J) The metastatic capacity of the CRC cell clones described in C was determined as described for Fig. 2G. IκB is IκB-P.

Article Snippet: For L1 and p65- P detection, polyclonal rabbit anti-L1 antibody (described above; diluted 1:1000), rabbit antiserum against p65- P (no. 3037, Cell Signaling, Frankfurt, Germany; diluted 1:50), and mouse anti-ezrin antibody (described above) were used.

Techniques: Mutagenesis, Plasmid Preparation, Transfection, Expressing, Construct, Stable Transfection, Western Blot, Immunoprecipitation, Cell Harvesting, Clone Assay, Injection

Journal: Journal of Cell Science

Article Title: Nuclear factor-κB signaling and ezrin are essential for L1-mediated metastasis of colon cancer cells

doi: 10.1242/jcs.069542

Figure Lengend Snippet: Overexpression of the NF-κB p65 subunit promotes cell proliferation and motility, and confers metastasis in human CRC cells. (A) Two cell clones that stably expressed p65 (p65 Cl1 and Cl2) or pcDNA3 (western blot, left panel) and also L1-transferred cells (Fig. 2A, lane 3) were transiently transfected with 3xκB.luc and β-galactosidase together. Activation of NF-κB was determined (bar graph, right panel) as described for Fig. 1A. (B) Proliferation (in 0.5% of serum) of pcDNA3-transfected cells and the two p65-transfected cell clones was compared. (C) The capacity of a p65-transfected cell clone to close an artificial wound was compared with that of control Ls174T cells after 24 hours. The same areas were photographed immediately after wounding (0 hours) and 24 hours later. (D) The motility of two p65 overexpressing cell clones was compared with that of control Ls174T cells. Wound closure was determined simultaneously in four wounds for each cell line. (E) The metastatic capacity of Ls174T cells stably transfected with pcDNA3, L1 or p65 was determined as described for Fig. 2G. (F) Sections of livers stained with hematoxylin and eosin show tumor metastases in mice that had been injected with either L1- or p65-expressing CRC cells. Scale bar: 250 μm.

Article Snippet: For L1 and p65- P detection, polyclonal rabbit anti-L1 antibody (described above; diluted 1:1000), rabbit antiserum against p65- P (no. 3037, Cell Signaling, Frankfurt, Germany; diluted 1:50), and mouse anti-ezrin antibody (described above) were used.

Techniques: Over Expression, Clone Assay, Stable Transfection, Western Blot, Transfection, Activation Assay, Staining, Injection, Expressing

Journal: Journal of Cell Science

Article Title: Nuclear factor-κB signaling and ezrin are essential for L1-mediated metastasis of colon cancer cells

doi: 10.1242/jcs.069542

Figure Lengend Snippet: Ezrin is required for L1-mediated metastasis, and L1, IκB and ezrin form a complex in CRC cells. (A) Lysates of 293T cells transfected with different combinations of ezrin, L1 and L1-Y1151A, were immunoprecipitated (IP) with antibodies against ezrin. Samples of cell lysates (Total) and immunoprecipitated proteins (IP:Ezrin) were analyzed by western blotting with antibodies against L1 and ezrin. (B) L1-expressing Ls174T cell clones were transfected with shRNA targeting ezrin or a scrambled siRNA sequence (L1+siRNA-Sc). In two independently isolated cell clones (lanes 4,5) the expression of ezrin was suppressed. (C) The proliferation of CRC cell clones (described in B) in the presence of 0.5% serum was determined during 5 days. (D,E) An artificial wound was introduced into confluent monolayers of the cell clones described in B, and the extent of wound closure in four different wounds for each cell clone was determined after 24 hours (an image of one representative wound is shown in E). (F) The metastatic capacity of the CRC cell clones described in B was determined as described for Fig. 2G. (G) Cells stably transfected with either the empty pcDNA3 vector, the construct expressing L1 alone or together with shRNA targeting ezrin, or with the construct expressing the p65 subunit of NF-κB were either left untreated (−) or were treated with the proteasome inhibitor MG132 (+) for 3 hours before cell harvesting. Protein levels of L1 and IκB-P were determined. Ponceau staining shows the protein gel loading. (H) 293T cells were transfected with different combinations of L1, L1-Y1151A, ezrin and IκB-SR, and cell lysates were immunoprecipitated (IP) with antibodies against ezrin. Immunoprecipitated proteins were analyzed by western blotting with antibodies against L1, IκB and ezrin. Ezrin co-precipitates with IκB and wt L1 (lanes 1,3), but to a much lesser extent with L1-Y1151A and IκB. pIκB is IκB-P.

Article Snippet: For L1 and p65- P detection, polyclonal rabbit anti-L1 antibody (described above; diluted 1:1000), rabbit antiserum against p65- P (no. 3037, Cell Signaling, Frankfurt, Germany; diluted 1:50), and mouse anti-ezrin antibody (described above) were used.

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Clone Assay, shRNA, Sequencing, Isolation, Stable Transfection, Plasmid Preparation, Construct, Cell Harvesting, Staining

Journal: Journal of Cell Science

Article Title: Nuclear factor-κB signaling and ezrin are essential for L1-mediated metastasis of colon cancer cells

doi: 10.1242/jcs.069542

Figure Lengend Snippet: NF-κB, L1 and ezrin are preferentially co-expressed at the invading edge of human CRC tissue. Immunohistochemical staining for L1 (red; A-C,G-I), p65-P (D-F) and ezrin (J-L) was conducted in sequential paraffin sections of human colorectal adenocarcinoma tissue with differentiated (diff) central regions indicated by tubular structures and undifferentiated invading tumor cells (inv). Boxed areas in the first column of images indicate respective areas at higher magnifications that are shown in the middle and right column. Note that L1-expressing invading tumor cells (inv) contain nuclear p65-P (B,E); these invading tumor cells also coexpress ezrin (H,K). By contrast, tumor cells in central tumor regions lack L1 (diff), and p65-P is only detected in some mitotic cells (diff, arrows) (D,F). Ezrin staining in more central regions of the tumor is weak (J,L). (M-O) Staining of normal colon tissue. Scale bar: 400 μm (A,D,G,J), 75 μm (all other panels). In the magnification boxes seen in panels (H,I,K,L) the bar represent 40 μm. pp65 is p65-P.

Article Snippet: For L1 and p65- P detection, polyclonal rabbit anti-L1 antibody (described above; diluted 1:1000), rabbit antiserum against p65- P (no. 3037, Cell Signaling, Frankfurt, Germany; diluted 1:50), and mouse anti-ezrin antibody (described above) were used.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Experimental and therapeutic medicine

Article Title: PDK1 inhibition reduces autophagy and cell senescence through the PI3K/AKT signalling pathway in a cigarette smoke mouse emphysema model.

doi: 10.3892/etm.2023.11922

Figure Lengend Snippet: Figure 1. Evaluation of the mouse emphysema model. H&E staining of lung tissues from the (A) control, (B) emphysema, (C) PI3K inhibitor and (D) PDK1 inhibitor groups. Magnification, x400. (E) IL‑6 protein levels and (F) total cell count in BALF. Numbers of (G) neutrophils and (H) macrophages in BALF. (I) MLI and (J) DI were measured show the extent of airway remodelling in lung tissues. aP<0.05 vs. control. bP<0.05 vs. emphysema. BALF, bronchoalveolar lavage fluid; MLI, mean linear intercept; DI, destructive index; CS + CSE, emphysema group; CS3, PI3K inhibitor group; CS1, PDK1 inhibitor group; PI3K, phosphatidylinositol‑3‑kinase; PDK1, phosphoinositide dependent protein kinase 1.

Article Snippet: After the PVDF membrane was blocked with 5% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, it was incubated overnight at 4 ̊C with the following primary anti‐ bodies: PI3K (1:5,000; cat. no. 60225‐1‐1g); PDK1 (1:500; cat. no. 18262‐1‐AP); AKT (1:5,000; cat. no. 60203‐1‐1g); LC3B (1:500; cat. no. 18725‐1‐AP) (ProteinTech Group, Inc.) and p16 (1:2,000; cat. no. R22878; Zen BioScience Co., Ltd.).

Techniques: Staining, Control, Cell Counting

Journal: Experimental and therapeutic medicine

Article Title: PDK1 inhibition reduces autophagy and cell senescence through the PI3K/AKT signalling pathway in a cigarette smoke mouse emphysema model.

doi: 10.3892/etm.2023.11922

Figure Lengend Snippet: Figure 3. Expression of LC3B protein in airway epithelial tissues. (A) Immunofluorescence staining for LC3BII in airway epithelial tissue. Magnification, x400. (B) Comparison of LC3B protein expression among the experimental groups. aP<0.05 vs. control. bP<0.05 vs. emphysema. CS + CSE, emphysema group; CS3, PI3K inhibitor group; CS1, PDK1 inhibitor group; PDK1, phosphoinositide dependent protein kinase 1; MFI, mean fluorescence intensity.

Article Snippet: After the PVDF membrane was blocked with 5% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, it was incubated overnight at 4 ̊C with the following primary anti‐ bodies: PI3K (1:5,000; cat. no. 60225‐1‐1g); PDK1 (1:500; cat. no. 18262‐1‐AP); AKT (1:5,000; cat. no. 60203‐1‐1g); LC3B (1:500; cat. no. 18725‐1‐AP) (ProteinTech Group, Inc.) and p16 (1:2,000; cat. no. R22878; Zen BioScience Co., Ltd.).

Techniques: Expressing, Immunofluorescence, Staining, Comparison, Control, Fluorescence

Journal: Experimental and therapeutic medicine

Article Title: PDK1 inhibition reduces autophagy and cell senescence through the PI3K/AKT signalling pathway in a cigarette smoke mouse emphysema model.

doi: 10.3892/etm.2023.11922

Figure Lengend Snippet: Figure 2. Expression of PI3K, PDK1 and AKT proteins in the airway epithelial tissue. Immunohistochemical staining for (A) PI3K (B) PDK1 (C) AKT expression in the airway epithelial tissues. Magnification, x400. Quantification of (D) PI3K (E) PDK1 (F) AKT protein expression in the airway epithelial tissues in each group. aP<0.05 vs. control. bP<0.05 vs. emphysema. CS + CSE, emphysema group; CS3, PI3K inhibitor group; CS1, PDK1 inhibitor group; PDK1, phosphoinositide dependent protein kinase 1; AOD, average optical density.

Article Snippet: After the PVDF membrane was blocked with 5% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, it was incubated overnight at 4 ̊C with the following primary anti‐ bodies: PI3K (1:5,000; cat. no. 60225‐1‐1g); PDK1 (1:500; cat. no. 18262‐1‐AP); AKT (1:5,000; cat. no. 60203‐1‐1g); LC3B (1:500; cat. no. 18725‐1‐AP) (ProteinTech Group, Inc.) and p16 (1:2,000; cat. no. R22878; Zen BioScience Co., Ltd.).

Techniques: Expressing, Immunohistochemical staining, Staining, Control

Journal: Experimental and therapeutic medicine

Article Title: PDK1 inhibition reduces autophagy and cell senescence through the PI3K/AKT signalling pathway in a cigarette smoke mouse emphysema model.

doi: 10.3892/etm.2023.11922

Figure Lengend Snippet: Figure 4. Expression of p16 protein in the airway epithelial tissues. (A) Immunofluorescence staining for the analysis of p16 protein expression in airway epithelial tissues. Magnification, x400. (B) Comparison of p16 protein expression among the experimental groups. aP<0.05 vs. control. bP<0.05 vs. emphysema. CS + CSE, emphysema group; CS3, PI3K inhibitor group; CS1, PDK1 inhibitor group; PDK1, phosphoinositide dependent protein kinase 1; p16, cyclin‑dependent kinase inhibitor 2A.

Article Snippet: After the PVDF membrane was blocked with 5% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, it was incubated overnight at 4 ̊C with the following primary anti‐ bodies: PI3K (1:5,000; cat. no. 60225‐1‐1g); PDK1 (1:500; cat. no. 18262‐1‐AP); AKT (1:5,000; cat. no. 60203‐1‐1g); LC3B (1:500; cat. no. 18725‐1‐AP) (ProteinTech Group, Inc.) and p16 (1:2,000; cat. no. R22878; Zen BioScience Co., Ltd.).

Techniques: Expressing, Immunofluorescence, Staining, Comparison, Control

Journal: Experimental and therapeutic medicine

Article Title: PDK1 inhibition reduces autophagy and cell senescence through the PI3K/AKT signalling pathway in a cigarette smoke mouse emphysema model.

doi: 10.3892/etm.2023.11922

Figure Lengend Snippet: Figure 5. PI3K, PDK1, AKT, LC3B II and p16 protein expression levels. Western blotting for (A) PI3K, (B) PDK1, (C) AKT, (D) LC3B and (E) p16 expression in the airway epithelial tissues. GAPDH as an internal control. Semi‑quantification of PI3K, PDK1, AKT, LC3B and p16 protein expression in airway epithelial tissues in each group. aP<0.05 vs. control. bP<0.05 vs. emphysema. The protein expression levels were expressed as the ratio of band intensity for the target protein relative to that for the internal control GAPDH. Values are expressed as the mean ± standard deviation. CS + CSE, emphysema group; CS3, PI3K inhibitor group; CS1, PDK1 inhibitor group; PDK1, phosphoinositide dependent protein kinase 1; p16, cyclin‑dependent kinase inhibitor 2A.

Article Snippet: After the PVDF membrane was blocked with 5% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, it was incubated overnight at 4 ̊C with the following primary anti‐ bodies: PI3K (1:5,000; cat. no. 60225‐1‐1g); PDK1 (1:500; cat. no. 18262‐1‐AP); AKT (1:5,000; cat. no. 60203‐1‐1g); LC3B (1:500; cat. no. 18725‐1‐AP) (ProteinTech Group, Inc.) and p16 (1:2,000; cat. no. R22878; Zen BioScience Co., Ltd.).

Techniques: Expressing, Western Blot, Control, Standard Deviation

Journal: Cancer Science

Article Title: WDR54 exerts oncogenic roles in T‐cell acute lymphoblastic leukemia

doi: 10.1111/cas.15872

Figure Lengend Snippet: WDR54 modulates PDPK1, AKT, ERK, cleaved caspase‐3, cleaved caspase‐9, Bcl‐2, and Bcl‐xL signal pathways in T‐cell acute lymphoblastic leukemia (T‐ALL) cell lines. Cell lysates from shC or shWDR54 cells at 3 days post‐infection were analyzed. (A) The protein expression of p‐AKT, AKT, p‐ERK, and ERK in Molt4 (left) and Jurkat (right) cells was detected by immunoblots, respectively. (B) Analysis of cleaved caspase‐3, cleaved caspase‐9, Bcl‐2, and Bcl‐xL was performed by immunoblots. (C) Heatmap analysis of RNAseq expression data showing differentially expressed genes in Jurkat cells ( p < 0.05). (D) The mRNA level of PDPK1 was detected by real‐time PCR in Molt4 cells (left) and Jurkat cells (right). (E) The protein expression of PDPK1 in Molt4 cells (left) and Jurkat cells (right). *** p < 0.001.

Article Snippet: Primary antibodies used in the experiments include β‐actin (1:50000, AC026, ABclonal), WDR54 (1:1000, HPA043257, Sigma), AKT (1:1000, 9272, Cell Signaling Technology), p‐AKT (1:1000, 4060, Cell Signaling Technology), ERK (1:1000, 4370, Cell Signaling Technology), p‐ERK (1:1000, 4695, Cell Signaling Technology), Bcl‐2 (1:1000, 3498, Cell Signaling Technology), Bcl‐xL (1:1000, ab98143, Abcam), cleaved Caspase‐3 (1:1000, 9664, Cell Signaling Technology), Caspase‐9 (1:1000, 9502, Cell Signaling Technology), GAPDH (1:5000, 60,004‐1‐Ig, Proteintech), and PDPK1 (1:1000, 17,086‐1‐AP, Proteintech).

Techniques: Infection, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: BMC Cardiovascular Disorders

Article Title: Metformin improves lipid metabolism and reverses the Warburg effect in a canine model of chronic atrial fibrillation

doi: 10.1186/s12872-020-01359-7

Figure Lengend Snippet: Metformin reverses the Warburg effect in chronic AF. a Quantitative analysis of key fators of the Warburg effect including HIF-1α, GLUT-1, PDK-1, PDH, HK, and LDH. b Analysis of the production of atrial lactate. c Representative images of the protein expression. SR, sinus rhythm; AF: atrial fibrillation; MET, metformin; HIF-1α, hypoxia-inducible factor 1α; GLUT-1, glucose transporter-1; PDK-1, pyruvate dehydrogenase kinase 1; PDH, pyruvate dehydrogenase; HK, hexokinase; LDH, lactate dehydrogenase. * P < 0.05 versus SR group; # P < 0.05 versus AF group; n = 6 per group

Article Snippet: Then, the membranes were incubated overnight at 4 °C with the following primary antibodies: anti-FAT/CD36 antibody (diluted 1:500, Bioss); anti-CPT-1 antibody (diluted 1:1000, Abcam); anti-VLCAD antibody (diluted 1:1000, Abcam); anti-GLUT1 antibody (diluted 1:500, Biorad); anti-HK antibody (diluted 1:500, Abcam); anti-PDK1 antibody (diluted 1:500, Biorbyt); anti-PDH antibody (diluted 1:500, Bioss); anti-LDH antibody (diluted 1:1000, Abcam); anti-AMPKα1 antibody (diluted 1:1000, Abcam); anti-p-AMPK (diluted 1:1000, Abcam); anti-HIF-1α antibody (diluted 1:1000, Thermofisher); anti-PGC-1α antibody (diluted 1:500, Abcam); anti-PPARα antibody (diluted 1:1500, Abcam); anti-β-actin antibody (diluted 1:10000, TDYbio); Secondary antibodies were goat anti-rabbit or anti-mouse, or rabbit anti-goat HRP-conjugated antibody (diluted 1:10000, ASPEN) for 30 min at room temperature.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: DRP1 Promotes BRAF V600E -Driven Tumor Progression and Metabolic Reprogramming in Colorectal Cancer

doi: 10.3389/fonc.2020.592130

Figure Lengend Snippet: Pyruvate dehydrogenase kinase 1 (PDK 1) regulates DRP1 mediated mitochondrial fission and glycolytic phenotype in BRAF V600E cells. (A, B) Western blot and representative confocal micrographs (2.5x zoom, 60x original magnification) of si-PDK1 silenced BRAF V600E CRC cells, showing a reduction in both pDRP1 S616 levels as well as mitochondrial fission after PDK1 knockdown, 30-40 cells were quantified (n=3, mean± SEM), red= mitochondria, CMX Ros stained and blue= Nuclei, DAPI stained, statistical analysis was done by two way ANOVA, followed by Bonferroni post-tests comparing replicate means by column with corresponding p values shown. (C, D) Genetic and pharmacologic knockdown of PDK1 in BRAF V600E CRC cells leads to a reduction in cell proliferation (n= 4, mean± SEM) and clonogenic rates in soft agar (n=3, mean± SEM) respectively; statistical analysis for proliferation was done by 2-way ANOVA followed by Bonferroni post-tests comparing replicate means by row with corresponding p values shown, and statistical analysis for soft agar clonogenic assay was done by unpaired t-test followed by Welch’s correction assuming unequal variance in means, corresponding p values shown.

Article Snippet: The primary antibodies used were against DRP1 (SC-271583, Santa Cruz), pDRP1 S616 (PA5-64821, Thermo Scientific), Mfn1/2 (NBP1-51841/STJ-94105, R & D/St Johns Laboratory), β- actin (NB600-501, Novus Biologicals), E cadherin (NBP2-19051, Novus Biologicals), N cadherin (NBP1-48309, Novus Biologicals), Vimentin (STJ140133, St Johns Laboratory), PDK1 (STJ24945, St Johns Laboratory), p-MEK 1/2 (sc-81503), MEK 1/2 (sc-81504) and the corresponding secondary antibodies used were either goat anti-rabbit IgG-HRP (NB7160, Novus Biologicals) or goat anti-mouse IgG-HRP (NB7539, Novus Biologicals).

Techniques: Western Blot, Knockdown, Staining, Clonogenic Assay

Journal: Frontiers in Oncology

Article Title: DRP1 Promotes BRAF V600E -Driven Tumor Progression and Metabolic Reprogramming in Colorectal Cancer

doi: 10.3389/fonc.2020.592130

Figure Lengend Snippet: Mitochondrial fission regulates migration and invasion in BRAF V600E CRC cells through glucose metabolic reprograming. (A) Relative cell invasion post vemurafenib treatment in BRAF V600E cells (n=3, mean ± SEM); statistical analysis was done by unpaired t-test followed by Welch’s correction assuming unequal variance in means, corresponding p values shown. (B) Western blot showing a reduction in EMT markers upon vemurafenib treatment in BRAF V600E cells. Relative cellular invasion post (C) DRP1 silencing, (D) PDK1 silencing, and (E) 2-deoxyglucose treatment in Colo205 and HT29 cells (n=3, mean± SEM); statistical analysis was done by unpaired t-test followed by Welch’s correction assuming unequal variance in means, corresponding p values shown. (F) Western blots showing a reduction in epithelial mesenchymal transition (EMT) markers upon DRP1 silencing, PDK1 knockdown, and 2-DG treatment in Colo205 and HT29 cells.

Article Snippet: The primary antibodies used were against DRP1 (SC-271583, Santa Cruz), pDRP1 S616 (PA5-64821, Thermo Scientific), Mfn1/2 (NBP1-51841/STJ-94105, R & D/St Johns Laboratory), β- actin (NB600-501, Novus Biologicals), E cadherin (NBP2-19051, Novus Biologicals), N cadherin (NBP1-48309, Novus Biologicals), Vimentin (STJ140133, St Johns Laboratory), PDK1 (STJ24945, St Johns Laboratory), p-MEK 1/2 (sc-81503), MEK 1/2 (sc-81504) and the corresponding secondary antibodies used were either goat anti-rabbit IgG-HRP (NB7160, Novus Biologicals) or goat anti-mouse IgG-HRP (NB7539, Novus Biologicals).

Techniques: Migration, Western Blot, Knockdown