Journal: Oncogene

Article Title: Upregulation of PIP3-Dependent Rac Exchanger 1 (P-Rex1) Promotes Prostate Cancer Metastasis

doi: 10.1038/onc.2009.30

Figure Lengend Snippet: P-Rex1 promotes prostate cancer cell migration and invasion via Rac activation. (a) CWR22Rv1 cells (left panel) or PC3-LN4 cells (right panel) were transfected with control GFP, GFP-tagged wild-type P-Rex1 or P-Rex1(-DH) mutant for 48 h, and then subjected to Transwell migration assay for 12 h (CWR22Rv1 cells) or 5 h (PC3-LN4) toward NIH3T3 CM. Transfected PC3-LN4 cells were selected by flow cytometry cell sorting based on GFP fluorescence prior to migration assays due to a low transfection efficiency (~25%). Bars show mean ± S.E. with *p<0.05 compared to control set at 100% (n=3). Inset: Expression of GFP-tagged P-Rex1 and P-Rex1(-DH) protein detected by western blot analysis using anti-GFP antibody. (b) CWR22Rv1 cells stably expressing wild-type P-Rex1, P-Rex1 “GEF-dead” mutant or control vector were subjected to migration and invasion assays for 12 h. Cells stably expressing wild-type P-Rex1 were pretreated without (none) or with 50 μM NSC for 15 min. Bars show mean ± S.E. with *p<0.01 compared to control (n=3). Inset: Western blot analysis of expression levels of P-Rex1 and its mutant. (c) P-Rex1 activates Rac1 in prostate cancer cells. The amount of activated GTP-bound Rac1 was measured by a Rac activation assay. The image shown is representative of 3 separate experiments. (d) P-Rex1 increases lamellipodia formation. Serum-starved CWR22Rv1 cells or PC-3 cells (positive control) were incubated with NIH3T3 CM for 5 h and then stained with rhodamine-conjugated phalloidin (red) for detection of F-actin.

Article Snippet: Additional reagents were NSC23766, Y27632 and anti-GFP antibody (EMD Biosciences); cell permeable C3 transferase and F-actin visualization kit (Cytoskeleton); Rac activation assay kit, Rho assay reagent, anti-Rac1 antibody (clone 23A8), and Immobilon-FL (Millipore); mouse anti-RhoA (26C4), and rabbit anti-HA and anti-β-actin antibodies (Santa Cruz); FBS (Hyclone); Lipofectamine TM 2000, AlexaF488-labeled secondary antibody, RPMI-1640 and DMEM (Invitrogen); EGF, Matrigel and fibronectin (BD Biosciences); CXCL12 (R&D Systems); and IRDye-labeled secondary antibodies (LI-COR Biosciences).

Techniques: Migration, Activation Assay, Transfection, Control, Mutagenesis, Transwell Migration Assay, Flow Cytometry, FACS, Fluorescence, Expressing, Western Blot, Stable Transfection, Plasmid Preparation, Positive Control, Incubation, Staining

Journal: bioRxiv

Article Title: Glucose metabolism distinguishes TE from ICM fate during mammalian embryogenesis

doi: 10.1101/826875

Figure Lengend Snippet: ( A-D ) AP-2γ translation is controlled by PPP function. Phosphorylation of threonine 37/46 on 4E-BP1 (p-4E-BP1), a target of mTOR is expressed at high levels in the 8-cell embryo ( A ). This expression is lost in –G ( B ) or when PPP is inhibited ( C ). Quantitation in ( D ) also shows that blocking HBP does not affect p-4E-BP1. ( E, F ) Western blot analysis of members of mTOR signaling pathway and its activation. ( E ) Western blot analysis shows that total mTOR, TSC2, phosphorylation level of serine 473 on AKT1/2 (p-AKT) and serine 127 on YAP1 (p-YAP1) all remain unchanged in embryos grown without glucose when compared to +G control. In contrast, phosphorylated mTOR targets 4E-BP1 and RPS6 (p-RPS6) levels are significantly reduced in –G embryos. ( F ) p-RPS6 is eliminated when S1PR 2 (JTE), Rac1 (NSC) or mTOR (INK) is inhibited. ( G-J ) AP-2γ expression requires mTOR and S1P signaling. The expression of AP-2γ ( G ) is eliminated in the presence of mTOR inhibitors, Torin2 and INK ( H, I ) and S1PR 2 inhibitor JTE ( J ). An identical effect is seen on CDX2 expression . ( K ) Quantitation of AP-2γ expression in embryos in which the mTOR and S1P pathways are inhibited. ( L ) Quantitation shows that the morula to blastocyst transition is acutely sensitive to mTOR inhibitors (Torin2, INK128), but is not affected by any of the seven PI3K inhibitors tested (BLY719, BKM120, GDC0941, TGX221, BX912, GSK2334470, MK2201). However, blastocyst formation is extremely sensitive to inhibitors of the S1P pathway (JTE013, SKI-II) and its downstream component Rac1 (NSC23766). ( M-T ) Role of glucose and S1P signaling in controlling Rac1 expression. In control, Rac1-GTP staining is observed only on the apical surface of the polar cells ( M ). Inhibition of Rac1 ( N ), S1P receptor ( P ), and S1P biosynthesis ( Q ) cause a significant reduction in Rac1-GTP staining. In contrast, inhibition of mTOR ( O ), lack of glucose during culture ( R ), or inhibition of specific arms of glucose metabolism (PPP and HBP) ( S-T ) has no effect on Rac1-GTP levels or localization, suggesting a S1P dependent and glucose independent requirement for apical Rac1-GTP localization and mTOR activation.

Article Snippet: Fluorescein Phalloidin (Thermo Fisher #F432, at 1:2000 dilution), mouse anti-CDX2 (Biogenex #MU392A-UC), goat anti-OCT3/4 (Santa Cruz #sc-8628), rabbit anti-NANOG (ReproCELL #RCAB0002P-F), mouse anti-YAP1 (Abnova #H00010413-M01), rabbit anti-AP-2γ (Santa Cruz #sc-8977), mouse anti-AP-2γ (Santa Cruz #sc-12762), rabbit anti-phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (CST #3141), rabbit anti-SBNO1 (Abcam #ab122789), rabbit anti-GATA3 (Santa Cruz #sc-9009), rabbit anti-PARD6B (Santa Cruz #sc-67393), mouse anti-O-Linked N-Acetylglucosamine [RL2] (Abcam #ab2739), rabbit anti-phospho-4E-BP1 (Thr37/46) (CST #2855), rabbit anti-phospho-S6 Ribosomal Protein (Ser235/236) (CST #4858), rabbit anti-mTOR (CST #2983), rabbit anti-TSC2 (CST #4308), rabbit anti-phospho-AKT (Ser473) (CST #4060), rabbit anti-phospho-YAP (Ser127) (CST #4911), rabbit anti-phospho-ULK1 (Ser757) (CST #6888), and mouse anti-Rac1-GTP (NewEast #26903, at 1:500 dilution).

Techniques: Expressing, Quantitation Assay, Blocking Assay, Western Blot, Activation Assay, Staining, Inhibition

Journal: Oncogene

Article Title: Microphthalmia-Associated Transcription Factor Suppresses Invasion by Reducing Intracellular GTP Pools

doi: 10.1038/onc.2016.178

Figure Lengend Snippet: (A) SK-Mel-28 and 501Mel cells transduced with control vector, or two different shRNAs to MITF were collected as described in Material and Methods and tested in the GTPase pull-down assay followed by immunoblotting with RAC1 antibodies (representative images and quantification of at least 2 independent experiments are shown). (B) SK-Mel-28 cells as in (A) were collected as described in Material and Methods and tested in the GTPase pull-down assay followed by immunoblotting with RHO-A and RHO-C antibodies (representative images and quantification of at least 2 independent experiments are shown). (C) SK-Mel-28 cells as in (A) were treated with the RAC1 inhibitor NSC23766 (25µM) and tested for invasion through matrigel. The data represents the average −/+ SEM of at least two independent. * p <0.05; ** p <0.01 compared to control; # p <0.05 compared to untreated. Statistics performed by Student t-Test.

Article Snippet: The following antibodies were used: mouse monoclonal to MITF (clone D5, Cat# M3621, Dako, Carpinteria, CA); mouse monoclonal to GAPDH (Cat. # AM4300, Ambion, Austin,TX); rabbit polyclonal to GMPR (Cat # SAB1101144 Sigma-Aldrich, St. Louis, MO); rabbit polyclonal to RAC1 (Cell Signaling, Cat# 2465); rabbit monoclonal to RHO-A (Cell Signaling, Cat# 2117); and rabbit monoclonal to RHO-C (Cell Signaling, Cat# 3430).

Techniques: Transduction, Control, Plasmid Preparation, Pull Down Assay, Western Blot

Journal: Oncogene

Article Title: Microphthalmia-Associated Transcription Factor Suppresses Invasion by Reducing Intracellular GTP Pools

doi: 10.1038/onc.2016.178

Figure Lengend Snippet: (A) SK-Mel-28 and A375 human melanoma cells were transduced with an empty vector or an overexpression vector encoding for MITF and probed in invasion assay; cells were supplemented or not with 100µM guanosine for 24hrs prior to and throughout the invasion assay. (B) Cells as in (A) were collected as described in Material and Methods and collected for nucleotides quantification by HPLC. (C) Cells as in (A) were collected as described in Material and Methods and tested in the GTPase pull-down assay followed by immunoblotting with RAC1 antibodies (representative images and quantification of at least two independent experiments are shown). (D) SK-Mel-28 cells as in (A) were collected as described in Material and Methods and tested in the GTPase pull-down assay followed by immunoblotting with RHO-A and RHO-C antibodies (representative images and quantification of at least two independent experiments are shown). The data represents the average −/+ SEM of at least two independent experiments performed in duplicates * p <0.05; ** p <0.001 compared to control; # p <0.05 compared to MITF-overexpressing control. Statistics performed by Student t-Test.

Article Snippet: The following antibodies were used: mouse monoclonal to MITF (clone D5, Cat# M3621, Dako, Carpinteria, CA); mouse monoclonal to GAPDH (Cat. # AM4300, Ambion, Austin,TX); rabbit polyclonal to GMPR (Cat # SAB1101144 Sigma-Aldrich, St. Louis, MO); rabbit polyclonal to RAC1 (Cell Signaling, Cat# 2465); rabbit monoclonal to RHO-A (Cell Signaling, Cat# 2117); and rabbit monoclonal to RHO-C (Cell Signaling, Cat# 3430).

Techniques: Transduction, Plasmid Preparation, Over Expression, Invasion Assay, Pull Down Assay, Western Blot, Control

Journal: bioRxiv

Article Title: BIN1 genetic risk factor for Alzheimer is sufficient to induce early structural tract alterations in entorhinal-hippocampal area and memory-related hippocampal multi-scale impairments

doi: 10.1101/437228

Figure Lengend Snippet: BIN1 is expressed in postsynaptic dendritic spines. d-STORM acquisition of BIN1 (red) and Phalloïdin (green) on DIV21 WT and hBIN1 hippocampal neurons. A. Percentage of BIN1 molecules detected on pre-synaptic and post-synaptic compartment on Wild-type neurons. Scale bar=0,5µm. B. Quantification of BIN1 molecules detected using d-STORM in pre-synaptic compartment in WT (n=118) and in hBIN1 (n=170) neurons from n= 3 WT and n=4 hBIN1 embryos. *** p < 0.0005. C. Quantification of BIN1 molecules detected using d-STORM in post-synaptic compartment in WT (n=118) and in hBIN1 (n=170) neurons from n= 3 WT and n=4 hBIN1 embryos. *** p < 0.0005. D. Cytoscape network of BIN1 interactions obtained by yeast-two-hybrid tests (TIAM1, KALRN, DLG1) and from Biogrid (RAC1). E. In situ PLA (red) using anti-BIN1 and anti-TIAM1 or anti-TIAM1 and anti-DLG1 antibodies co-labelled with actin (Phalloïdin, green) on WT mice primary hippocampal neurons 21DIV. Scale bars=5μm. F. Western blot analyses of GTP-Rac1 pull-down bound with GST-PAK1-PDB protein fusion and total Rac1 from WT (n=8) and hBIN1 (n=4) hippocampi, showing an increase of Rac1 active forms (*p<0,05).

Article Snippet: Active Rac1 Detection was performed using Active Rac1 Detection Kit of Cell Signalling Technology.

Techniques: In Situ, Western Blot

Journal: Journal of Functional Foods

Article Title: Oxyresveratrol from mulberry (Morus alba L.) ameliorates post-inflammatory hyperpigmentation in vitro by anti-melanogenesis, inhibiting melanosome transfer, and providing photoprotection

doi: 10.1016/j.jff.2024.106557

Figure Lengend Snippet: Fig. 4. The inhibitory effects of OXY, RSV, and GLA on melanin transport in human melanocytes. (a) 15.67 μg/mL OXY, RSV, and GLA were cultured in human melanocytes for 48 h. The mRNA levels of melanin transport genes RAC1, RAB27A, and CDC42 were determined by qRT-PCR. (b) The protein expression level of melanin transport genes RAC1, RAB27A, and CDC42 was determined by western blotting using GAPDH as a loading control. Values are means ± SD of three in dependent experiments run in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to the control.

Article Snippet: Primary antibodies against CDC42 (10155–1- AP), RAB27A (17817–1-AP), RAC1 (24072–1-AP), and GAPDH (60004–1-Ig), beta-actin polyclonal antibody (β-actin, 20536–1-AP) were purchased from ProteinTech (Wuhan, China).

Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: Journal of Functional Foods

Article Title: Oxyresveratrol from mulberry (Morus alba L.) ameliorates post-inflammatory hyperpigmentation in vitro by anti-melanogenesis, inhibiting melanosome transfer, and providing photoprotection

doi: 10.1016/j.jff.2024.106557

Figure Lengend Snippet: Fig. 5. Interaction of OXY with RAB27A, RAC1, and CDC42. The molecular docking results between OXY and RAB27A (a), RAC1 (b), and CDC42 (c) were visualized by PyMOL. The protein models of RAB27A (ID: 3BC1), RAC1 (ID: 3TH5), and CDC42 (ID: 1E0A) were downloaded from the RCSB Protein Data Bank. The ligand, metal ion and protein were labeled in red, yellow, and blue, respectively. The hydrogen bonds between the ligand and protein were repre sented by dotted yellow lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies against CDC42 (10155–1- AP), RAB27A (17817–1-AP), RAC1 (24072–1-AP), and GAPDH (60004–1-Ig), beta-actin polyclonal antibody (β-actin, 20536–1-AP) were purchased from ProteinTech (Wuhan, China).

Techniques: Labeling

Journal: Journal of Biological Chemistry

Article Title: AKAP-Lbc Anchors Protein Kinase A and Nucleates Gα12-selective Rho-mediated Stress Fiber Formation

doi: 10.1074/jbc.m106629200

Figure Lengend Snippet: FIG. 4. AKAP-Lbc associates with Rho in vivo. A, AKAP-Lbc was immunoprecipitated from transfected HEK293 cells. Immunoprecipitates were used in an in vitro interaction assay to pull down purified GST-RhoA, GST-Rac1, or GST-Cdc42 preloaded with either GDP or GTPS or nucleotide-depleted (no nucl.). RhoA (lanes 4–6), Rac1 (lanes 7–9), and Cdc42 (lanes 10–12) were detected with anti-GST antibody. B, lysates from untransfected HeLa-S3 cells were subjected to immunoprecipitation with preimmune serum (Pre-i) or affinity-purified anti-AKAP-Lbc antibody. Immunoprecipitates as well as HeLa-S3 cell extracts (Ex.) were separated by SDS-PAGE (4–15%) and electrotransferred to nitrocellulose. RhoA, Rac1, and Cdc42 were detected by immunoblot using monoclonal antibodies against RhoA (lanes 1–3; upper panel), Rac1 (lanes 4–6), or Cdc42 (lanes 7–9), respectively. AKAP-Lbc was detected with an affinity-purified anti-AKAP-Lbc polyclonal antibody (lower panel). C, HEK293 cells were transfected with AKAP-Lbc in combination with either wild type (WT), constitutively active (V14), or dominant negative (N19) RhoA. AKAP-Lbc immunoprecipitates as well as cell extracts (Ex.) were separated by SDS-PAGE (4–15%) and electrotransferred to nitrocellulose. AKAP-Lbc and RhoA were detected by immunoblot using an affinity-purified anti-AKAP-Lbc antibody (lower panel) and a monoclonal anti-RhoA antibody (upper panel). D, HEK293 cells were transfected with the AKAP-Lbc Y2153F mutant in combination with the cDNA encoding wild-type RhoA. Lysates were subjected to immunoprecipitation with preimmune serum (Pre-i) or anti-AKAP-Lbc antibody. RhoA and AKAP-Lbc were detected by immunoblot using a monoclonal anti-RhoA antibody (upper panel) or anti-AKAP-Lbc (lower panel). All of the results are representative of three independent experiments.

Article Snippet: The following affinity-purified primary antibodies were used for immunoblotting: rabbit polyclonal antibody to RhoA (200 g/ml, 1:250 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); mouse monoclonal antibodies to RhoA, Rac1, or Cdc42 (200 g/ml, 1:250 dilution; Santa Cruz Biotechnology); mouse monoclonal antibody to PKA catalytic subunit (clone 5B, 1:1000 dilution; Transduction Laboratories); mouse monoclonal to PKA type II regulatory subunit (1:250 dilution; Transduction Laboratories); mouse monoclonal antibody to the GST tag (1:500 dilution; Santa Cruz Biotechnology); mouse monoclonal antibody to the FLAG tag (1:2000 dilution; Sigma); rabbit polyclonal antibody to the GFP tag (1:100 dilution; Invitrogen); rabbit polyclonal antibody to G 12 (1:500 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to G 13 (1:250 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to G q (1:500 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to G 11 (1:500 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to G i2 (1:500 dilution; Santa Cruz Biotechnology); and rabbit polyclonal antibody to G s (1:500 dilution; Santa Cruz Biotechnology).

Techniques: In Vivo, Immunoprecipitation, Transfection, In Vitro, Purification, Affinity Purification, SDS Page, Western Blot, Bioprocessing, Dominant Negative Mutation, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: AKAP-Lbc Anchors Protein Kinase A and Nucleates Gα12-selective Rho-mediated Stress Fiber Formation

doi: 10.1074/jbc.m106629200

Figure Lengend Snippet: FIG. 6. AKAP-Lbc is a Rho-specific guanine nucleotide exchange factor. A, model depicting the guanine nucleotide exchange reaction mediated by AKAP- Lbc. B, time course for the binding of [35S]GTPS to purified RhoA. Purified RhoA was preloaded with GDP and then added to reaction incubations containing [35S]GTPS together with aliquots from HEK293 cell lysates overexpressing AKAP-Lbc (solid circles) or the AKAP-Lbc Y2153F mutant (open circles). C, time course for the dissociation of [3H]GDP from purified RhoA. Purified RhoA was preloaded with [3H]GDP and then added to reaction incubations containing 1 mM GTP together with aliquots from HEK293 cell lysates overexpressing AKAP-Lbc (solid circles) or the AKAP-Lbc Y2153F mutant (open circles). D, effect of AKAP- Lbc on [3H]GDP dissociation from either RhoA Rac1 or Cdc42. Each GTP-binding protein was preloaded with [3H]GDP and then mixed in reaction buffer with ex- tracts overexpressing AKAP-Lbc (solid bars) or the AKAP-Lbc Tyr2153 mutant (gray bars) for 20 min before termination of reactions. Results are expressed as mean S.E. of three independent experiments.

Article Snippet: The following affinity-purified primary antibodies were used for immunoblotting: rabbit polyclonal antibody to RhoA (200 g/ml, 1:250 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); mouse monoclonal antibodies to RhoA, Rac1, or Cdc42 (200 g/ml, 1:250 dilution; Santa Cruz Biotechnology); mouse monoclonal antibody to PKA catalytic subunit (clone 5B, 1:1000 dilution; Transduction Laboratories); mouse monoclonal to PKA type II regulatory subunit (1:250 dilution; Transduction Laboratories); mouse monoclonal antibody to the GST tag (1:500 dilution; Santa Cruz Biotechnology); mouse monoclonal antibody to the FLAG tag (1:2000 dilution; Sigma); rabbit polyclonal antibody to the GFP tag (1:100 dilution; Invitrogen); rabbit polyclonal antibody to G 12 (1:500 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to G 13 (1:250 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to G q (1:500 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to G 11 (1:500 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to G i2 (1:500 dilution; Santa Cruz Biotechnology); and rabbit polyclonal antibody to G s (1:500 dilution; Santa Cruz Biotechnology).

Techniques: Binding Assay, Purification, Mutagenesis