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

Journal: Molecular and Cellular Biology

Article Title: Regulation of Insulin Signaling by the Phosphatidylinositol 3,4,5-Triphosphate Phosphatase SKIP through the Scaffolding Function of Pak1

doi: 10.1128/MCB.00636-12

Figure Lengend Snippet: Insulin-dependent formation of a SKIP-Akt2 complex. (A) Induction of protein complex formation between SKIP, insulin receptor β, and Nck by insulin stimulation. C2C12 cells were transfected with the indicated constructs and stimulated with insulin for 0 or 30 min. Lysates from these cells were immunoprecipitated (I.P.) with anti-FLAG antibody. (B) Colocalization of endogenous SKIP and insulin receptor β in insulin-stimulated C2C12 cells. Cells were visualized by confocal microscopy. F-actin was visualized with Alexa Fluor 647-labeled phalloidin. Yellow indicates regions of colocalization of SKIP and insulin receptor β; white shows regions of colocalization of SKIP, insulin receptor β, and actin. Scale bar, 20 μm. (C) Insulin-dependent formation of a SKIP-Akt2 complex. C2C12 cells were transfected with 3× FLAG-tagged SKIP WT and then stimulated with insulin for the indicated times. Lysates were immunoprecipitated with anti-FLAG antibodies. (D) Pak1 mediates complex formation between endogenous SKIP and Akt2. C2C12 cells were transfected with control or Pak1 siRNA. Relative expression of Pak1 in these cells is shown (upper panels). These cells were stimulated with insulin for 0 or 30 min. Lysates were immunoprecipitated with anti-SKIP antibody, and the immunoprecipitates were detected with anti-Akt2, -PDK1, or -Pak1 antibodies. The amounts of Akt2 and PDK1 immunoprecipitated with SKIP are shown (lower panels). Results are presented as the means ± SEMs of 5 independent experiments. *, P < 0.05; **, P < 0.01. (E) Pak1 predominantly associated with Akt2 in C2C12 cells. C2C12 cells were serum deprived for 24 h and then stimulated with insulin for the indicated times. Lysates were immunoprecipitated with anti-rabbit IgG and anti-Pak1 antibody. Immunoprecipitates were subjected to Western blot analysis.

Article Snippet: Anti-insulin receptor β, Rac1, and PDK1 antibodies were purchased from BD Biosciences (Franklin Lakes, NJ).

Techniques: Transfection, Construct, Immunoprecipitation, Confocal Microscopy, Labeling, Expressing, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Regulation of Insulin Signaling by the Phosphatidylinositol 3,4,5-Triphosphate Phosphatase SKIP through the Scaffolding Function of Pak1

doi: 10.1128/MCB.00636-12

Figure Lengend Snippet: Dissociation of Akt2 from the Pak1 complex, as mediated by the PIP3 phosphatase activity of SKIP. (A) Decreased Rac1 activity in insulin-stimulated C2C12 cells caused by expression of SKIP WT but not the D310G mutant. Results are presented as the means ± SDs of 3 independent experiments. **, P < 0.01. (B) Suppression of Pak1 activity caused by expression of SKIP WT but not the D310G mutant. Results are presented as the means ± SDs of 3 independent experiments. *, P < 0.05. (C) Increased Rac1 activity caused by the silencing of SKIP. Results are presented as the means ± SDs of 3 independent experiments. *, P < 0.05. (D) Increased Pak1 activity, measured as insulin-induced phosphorylation of Bad at Ser-112, caused by SKIP attenuation in C2C12 cells. Results are presented as the means ± SDs of 3 independent experiments. **, P < 0.01. (E) Decreased binding of Akt2 and PDK1 to Pak1 caused by the PIP3 phosphatase activity of SKIP. (F) Suppression of Akt2 activity caused by expression of SKIP WT but not the D310G mutant. Results are presented as the means ± SDs of 3 independent experiments. *, P < 0.05.

Article Snippet: Anti-insulin receptor β, Rac1, and PDK1 antibodies were purchased from BD Biosciences (Franklin Lakes, NJ).

Techniques: Activity Assay, Expressing, Mutagenesis, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: Regulation of Insulin Signaling by the Phosphatidylinositol 3,4,5-Triphosphate Phosphatase SKIP through the Scaffolding Function of Pak1

doi: 10.1128/MCB.00636-12

Figure Lengend Snippet: Working model of SKIP regulation of insulin signaling. (A) Under resting conditions, SKIP is localized to the ER. (B) Insulin-dependent PIP3 generation and Rac1-dependent Pak1 activation. Pak1 activation triggers its recruitment to the insulin receptor complex and subsequent complex formation with Akt2 and PDK1. Activated signaling molecules are highlighted in red. (C) Insulin induces translocation of SKIP to the plasma membrane, where SKIP binds to active Pak1 (the open form). Formation of a complex including SKIP, Pak1, Akt2, and Rac1 is induced at the membrane ruffles. (D) SKIP hydrolyzes PIP3 bound to Akt2. Localization of SKIP in proximity to Akt2 facilitates its inactivation. Signaling molecules inactivated by SKIP are indicated in blue. (E) SKIP negatively regulates Rac1 and Pak1 activity, leading to the inactive conformation of Pak1. Dissociation of Akt2 and PDK1 from inactive Pak1 terminates insulin signaling.

Article Snippet: Anti-insulin receptor β, Rac1, and PDK1 antibodies were purchased from BD Biosciences (Franklin Lakes, NJ).

Techniques: Activation Assay, Translocation Assay, Activity Assay

Journal: Aging (Albany NY)

Article Title: ROR2 knockdown suppresses breast cancer growth through PI3K/ATK signaling

doi: 10.18632/aging.103400

Figure Lengend Snippet: ROR2 induces PI3K/AKT signaling in BC cells. ( A ) qRT-PCR of PI3K, AKT, PDK1, p21, and cyclin D1 in MDA-MB-231 and MCF-7 cells after siROR2 and pLenti-ROR2 transfection. ( B , C ) Western blotting of PI3K, AKT, pAKT, PDK1, p21, and cyclin D1 in MDA-MB-231 and MCF-7 cells after siROR2 and pLenti-ROR2 transfection. Results are shown as means ± SD; n=3; *p<0.05, **p<0.01.

Article Snippet: Subsequently, the membranes were incubated with anti-ROR2 (Biovision, Cat. 6702-100), anti-Bax (EnoGene, Cat. E11-0132C), anti-Bak (EnoGene, Cat. E11-0131C), anti-Bcl-2 (EnoGene, Cat. E10-30077), anti-Bcl-xl (EnoGene, Cat. E90209), anti-mTOR (EnoGene, Cat. E11-7156B), anti-survivin 1 (Biorbyt, Cat. orb394299), anti-PI3K (Biorbyt Cat. orb137259), anti-AKT (bioss, Cat. bs-0115R-1), anti-pAKT (bioss, Cat. bs-12458R-1), anti-PDK1 (EnoGene, Cat. E10-30154), anti-p21 (Abcam, Cat. ab215971-p21), anti-cyclin D1 (Abcam, Cat. ab185241 - Cyclin D1), and control anti-GAPDH (EnoGene, Cat. E12-052) antibodies at 4 °C overnight.

Techniques: Quantitative RT-PCR, Transfection, Western Blot

Journal: Aging (Albany NY)

Article Title: ROR2 knockdown suppresses breast cancer growth through PI3K/ATK signaling

doi: 10.18632/aging.103400

Figure Lengend Snippet: ROR2 induces PI3K/AKT signaling in vivo . ( A ) qRT-PCR of Bax, Bak, Bcl-2, Bcl-xl, mTOR, and survivin 1 in MDA-MB-231 xenografts with ROR2 knockdown, and in ROR2-overexpressing MCF-7 xenografts. ( B ) qRT-PCR of PI3K, AKT, pAKT, PDK1, p21, and cyclin D1 in the above tumors. Results are shown as means ± SD; n=3; *p<0.05, **p<0.01.

Article Snippet: Subsequently, the membranes were incubated with anti-ROR2 (Biovision, Cat. 6702-100), anti-Bax (EnoGene, Cat. E11-0132C), anti-Bak (EnoGene, Cat. E11-0131C), anti-Bcl-2 (EnoGene, Cat. E10-30077), anti-Bcl-xl (EnoGene, Cat. E90209), anti-mTOR (EnoGene, Cat. E11-7156B), anti-survivin 1 (Biorbyt, Cat. orb394299), anti-PI3K (Biorbyt Cat. orb137259), anti-AKT (bioss, Cat. bs-0115R-1), anti-pAKT (bioss, Cat. bs-12458R-1), anti-PDK1 (EnoGene, Cat. E10-30154), anti-p21 (Abcam, Cat. ab215971-p21), anti-cyclin D1 (Abcam, Cat. ab185241 - Cyclin D1), and control anti-GAPDH (EnoGene, Cat. E12-052) antibodies at 4 °C overnight.

Techniques: In Vivo, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: ECM-Dependent HIF Induction Directs Trophoblast Stem Cell Fate via LIMK1-Mediated Cytoskeletal Rearrangement

doi: 10.1371/journal.pone.0056949

Figure Lengend Snippet: Immunofluorescence microscopy of terminally differentiated control TGCs (A) and Hif-1/2 α −/− SynTs (B) with a β -Catenin (red) and LIMK1 (green) antibody (arrows = perinunclear LIMK1 staining). Immunofluorescence microscopy of terminally differentiated control TGCs (C) and Hif-1/2 α −/− SynTs (D) with a β -Catenin (green) and p-Cofilin (red) antibody (arrows = perinunclear p-Cof staining, arrowheads = cofilin rods). (E, F) Two representative images of TGC formation (arrows) following transient myc-LIMK1 expression in Hif-1/2 −/− TSCs while untransfected cells primarily form SynTs (arrowheads) (red = myc-LIMK1, green = β -catenin). (G) Quantification of the percentage of LIMK1 transfected HIF-null TSCs differentiated into TGCs vs. SynTs. (H) Immunoblot analysis of LIMK1 levels in differentiated wild-type (+/+) TSCs without and with U0126 (U0). Integrated densitometry confirmed the decreased expression of LIMK1, relative to total Cofilin, in control TSCs differentiated in the presence of U0126.

Article Snippet: The following antibodies were used for immunoblotting, EMSA, immunoprecipitaion and immunofluoresence staining: CDX2 (Biogenex), EOMES (Orbigen), anti human/mouse HIF-1α (R&D Systems, Minneapolis, MN), HIF-1α c-terminal (Cayman Chemical, Ann Arbor, MI), HIF-2α NB 100–122 (Novus Biologicals), ARNT 2B10 (Abcam, Cambridge, MA), pMAPK3/1 (pERK; Cell Signaling, Danvers, MA), MAPK1(ERK2; Epitomics, Burlingame, CA), α-Tubulin (NeoMarkers, Fremont, CA), Ac- α-Tubulin (Sigma-Aldrich), LIMK1 (BD Biosciences), LIMK2 (Proteintech), Cofilin (BD Biosciences), p-Cofilin (CellSignal), PDK1 (StressGen), BNIP3, VHL M-20, HOPX1 (Santa Cruz Biotechnology, Santa Cruz, CA), β -catenin (Cell Signaling), c-MYC (SCBT), HA (Zymed, South San Francisco, CA), GFP (Aves), E-cadherin (BD Transduction Pharmingen), and HDAC2 (Zymed).

Techniques: Immunofluorescence, Microscopy, Control, Staining, Expressing, Transfection, Western Blot

Journal: PLoS ONE

Article Title: ECM-Dependent HIF Induction Directs Trophoblast Stem Cell Fate via LIMK1-Mediated Cytoskeletal Rearrangement

doi: 10.1371/journal.pone.0056949

Figure Lengend Snippet: (A) Immunoblot analysis of LIMK1 protein levels in Hif-1/2 α −/− TSCs stably reconstituted with full length HIF-1α or -2α, as well as versions lacking their basic domains. (B) Immunoblot analysis of LIMK1 and LIMK2 expression in control (+), Hif-1/2 α −/− ( Hif −/− ), and HIF-2α and HIF-2αΔb reconstituted Hif-1/2 α −/− TSCs. Integrated densitometric analysis confirmed that both HIF-2α, as well as HIF-2αΔb, restored LIMK1 expression to control levels in Hif-1/2α −/− TSCs. (C) Immunoprecipitation with an anti-HA antibody of HA-tagged HIF-2αΔb followed by immunoblot with anti-HA, c-MYC, β -Catenin, α-Tubulin and GFP antibodies. (D) Schematic representation of E-box element identified within the Limk1 promoter. Chromatin immunoprecipitation (ChIP) analysis indicated specific binding of c-MYC and HA-tagged HIF-2αΔb to this element. (E) Immunoblot analysis of LIMK1 protein levels in HIF-2αΔb expressing Hif-1/2 α −/− TSCs without (-) or with (+) c-MYC inhibitor. Integrated densitometric analysis confirmed reduced expression of LIMK1 relative to α-Tubulin in drug treated cells. (F) Schematic representation of HIF-2α interacting with MYC:MAX heterodimers at the Limk1 promoter.

Article Snippet: The following antibodies were used for immunoblotting, EMSA, immunoprecipitaion and immunofluoresence staining: CDX2 (Biogenex), EOMES (Orbigen), anti human/mouse HIF-1α (R&D Systems, Minneapolis, MN), HIF-1α c-terminal (Cayman Chemical, Ann Arbor, MI), HIF-2α NB 100–122 (Novus Biologicals), ARNT 2B10 (Abcam, Cambridge, MA), pMAPK3/1 (pERK; Cell Signaling, Danvers, MA), MAPK1(ERK2; Epitomics, Burlingame, CA), α-Tubulin (NeoMarkers, Fremont, CA), Ac- α-Tubulin (Sigma-Aldrich), LIMK1 (BD Biosciences), LIMK2 (Proteintech), Cofilin (BD Biosciences), p-Cofilin (CellSignal), PDK1 (StressGen), BNIP3, VHL M-20, HOPX1 (Santa Cruz Biotechnology, Santa Cruz, CA), β -catenin (Cell Signaling), c-MYC (SCBT), HA (Zymed, South San Francisco, CA), GFP (Aves), E-cadherin (BD Transduction Pharmingen), and HDAC2 (Zymed).

Techniques: Western Blot, Stable Transfection, Expressing, Control, Immunoprecipitation, Chromatin Immunoprecipitation, Binding Assay

Journal: PLoS ONE

Article Title: ECM-Dependent HIF Induction Directs Trophoblast Stem Cell Fate via LIMK1-Mediated Cytoskeletal Rearrangement

doi: 10.1371/journal.pone.0056949

Figure Lengend Snippet: ECM composition regulates HIF stabilization likely downstream of cell surface integrin ligation via MAP2K1/2 activation. Inside-out integrin signaling mechanisms may also be operative. Oxygen sensing and signaling pathways intersect with this signaling cascade to stabilize HIF, when ECM-dependent cues are absent. Stabilized HIF can act via canonical and non-canonical target genes. Non-canonical HIF-2, by interacting with MYC:MAX heterodimers, bind the Limk1 promoter to activate its expression. LIMK1 promotes microtubule and actin stability, critical for TGC formation, and thereby prevents SynT formation. HIF, therefore, can integrate divergent environmental inputs from within the placenta to regulate cell fate via non-canonical gene expression.

Article Snippet: The following antibodies were used for immunoblotting, EMSA, immunoprecipitaion and immunofluoresence staining: CDX2 (Biogenex), EOMES (Orbigen), anti human/mouse HIF-1α (R&D Systems, Minneapolis, MN), HIF-1α c-terminal (Cayman Chemical, Ann Arbor, MI), HIF-2α NB 100–122 (Novus Biologicals), ARNT 2B10 (Abcam, Cambridge, MA), pMAPK3/1 (pERK; Cell Signaling, Danvers, MA), MAPK1(ERK2; Epitomics, Burlingame, CA), α-Tubulin (NeoMarkers, Fremont, CA), Ac- α-Tubulin (Sigma-Aldrich), LIMK1 (BD Biosciences), LIMK2 (Proteintech), Cofilin (BD Biosciences), p-Cofilin (CellSignal), PDK1 (StressGen), BNIP3, VHL M-20, HOPX1 (Santa Cruz Biotechnology, Santa Cruz, CA), β -catenin (Cell Signaling), c-MYC (SCBT), HA (Zymed, South San Francisco, CA), GFP (Aves), E-cadherin (BD Transduction Pharmingen), and HDAC2 (Zymed).

Techniques: Ligation, Activation Assay, Protein-Protein interactions, Expressing, Gene Expression