Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 is expressed in the soma and neurites of developing hippocampal neurons. A Expression of Rasal1 in coronal section of adult mouse brain. Allen Mouse Brain Atlas, http://mouse.brain-map.org/experiment/show/75774111 . B Western blot analyses with Rasal1 antibody in the absence (left) and presence (right) of Rasal1 immunizing peptide. Lane 1: mouse hippocampus; lane 2: mouse prefrontal cortex; lane 3: rat hippocampus all at P8. C Endogenous Rasal1 immunoreactivity on DIV2, DIV4, DIV6, DIV12 and DIV14 in neurons stained for Rasal1 (red) and Tubulin (blue) and imaged with confocal microscopy. DIV 2 scale bar: 5 μm, DIV 4–10 scale bar: 10 μm

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Expressing, Western Blot, Staining, Confocal Microscopy

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 translocates to the plasma membrane in response to Ca 2+ in hippocampal neurons. A1-3 Hippocampal neuron expressing Rasal1-EGFP stained to show the distribution of Rasal1-EGFP (green) compared to total Rasal1 immunoreactivity (red) and MAP2 (blue) under basal conditions. Scale bar: 10 µm. B1-3 Depolarized hippocampal neuron expressing Rasal1-EGFP stained to show Rasal1-EGFP (green) distribution compared to total Rasal1 (red) and MAP2 (blue). C Live confocal imaging of neuron expressing Rasal1-EGFP following perfusion of depolarizing buffer (Depolarizing B) C1-4 . Followed by perfusion with basal buffer (Basal B) at 8 s (C5-7) followed by second perfusion with depolarizing buffer at 18 s (D-18s) (C8). Scale bar: 10 µm. D – G Rasal1-EGFP live imaging in two additional neurons perfused in basal buffer D , F and depolarizing buffer ( E , G ). Scale bar: 10 µm. H Time-dependent change in fluorescence ratio (R) in the membrane after addition of depolarizing buffer, basal buffer at 8 s and depolarizing buffer again at 12 s (addition of buffers indicated by dashed lines). Data presented as percentages of the original fluorescence at 0 s. Mean ± SEM are plotted, n = 20

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Membrane, Expressing, Staining, Imaging, Fluorescence

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 promotes CaMKII phosphorylation. A Representative confocal images of blocked control hippocampal neurons treated with TTX, CNQX and APV for at least 8 h to block CaMKII phosphorylation ( A1 ), EGFP-control hippocampal neurons treated for 5 min with 10 µM glycine to acutely induce CaMKII phosphorylation ( A2 ), glycine (10 µM) treated Rasal1-OE transduced hippocampal neurons ( A3 ) and glycine (10 µM) treated Rasal1-shRNA transduced hippocampal neurons ( A4 ) stained for phosphorylated (red) or total CaMKII (cyan). Transfected neurons (green) indicated by arrows were compared to neighboring non-transfected neurons. Scale bar: 20 µm. B Quantification of the ratio of phosphorylated CaMKII fluorescence intensity of transfected /non-transfected EGFP-control (n = 20), Rasal1-OE (n = 25), and Rasal1-shRNA (n = 25) transduced hippocampal neurons. C Quantification of total CaMKII levels in EGFP-control (n = 20), Rasal1-OE (n = 25), and Rasal1-shRNA (n = 25) transduced hippocampal neurons as ratio of fluorescence intensity in transfected/non-transfected neurons. D Fluorescent western blot of p-CaMKII (red) of glycine (10 µM) treated EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons. E Fluorescent western blot of CaMKII (green) and p-CaMKII (red) of glycine (10 µM) treated EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons. F Quantification of p-CaMKII/actin levels in fluorescent western blot across of glycine (10 µM) treated EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons (n = 8). G Quantification of total CaMKII/actin levels in fluorescent western blot across of glycine (10 µM) treated EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons. (n = 8). Data information: In ( B , C , F , G ) *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired student t-test

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Control, Blocking Assay, shRNA, Staining, Transfection, Fluorescence, Western Blot, Two Tailed Test

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 binds to CaMKII, PKC, α- and β-tubulin. Confirmation of suspected Rasal1 binding partners using pull-down assay (n = 3) with GST tagged Rasal1 fragments; N-terminus (GST-n), middle fragment (GST-m) or C-terminal fragment (GST-c) validated by Co-IP. A Western blot of pull-down assay (left) and Co-IP (right) of GST-tagged Rasal1 fragments stained for CaMKII in hippocampal and whole brain lysate. B Western blot of pull-down assay (left) and Co-IP (right) of GST-tagged Rasal1 fragments stained for PKC in hippocampal and whole brain lysate. C Western blot of pull-down assay (left) and Co-IP (right) of GST-tagged Rasal1 fragments stained for β-tubulin in hippocampal and whole brain lysate

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Binding Assay, Pull Down Assay, Co-Immunoprecipitation Assay, Western Blot, Staining

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 inhibits neurite growth. A Neurite tracing using SynD at DIV 6, 8, 10, 12 and 14 of mouse hippocampal neurons transduced with either Rasal1-shRNA-EGFP, Rasal1-OE, or EGFP-control. Scale bar: 50 µm. B Neurite length (µm) of EGFP-control, Rasal1-shRNA, and Rasal1-OE expressing mouse hippocampal neurons at DIV 6, 8, 10, 12, and 14. Data pooled from 4 independent experiments (n = 122). Mean ± SEM are plotted, *p < 0.05, **p < 0.01, ***p < 0.001, unpaired student t-test, two tailed

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Transduction, shRNA, Control, Expressing, Two Tailed Test

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 inhibits dendrite length and branching. A Representative confocal images of EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced mouse hippocampal neurons stained for the dendritic marker MAP2 (red) and axonal marker Tau1 (green) (left). Tracings of dendrites (middle) and axonal neurites (right) were done using SynD. Scale bar: 20 µm. B Total axon and dendrite length (µm) of EGFP-control, Rasal1-OE, and Rasal-shRNA expressing neurons. C Number of primary and secondary dendritic branches in neurons expressing EGFP-control, Rasal1-OE, and Rasal1-shRNA. Data information: In B , C Mean ± SEM are plotted, *p < 0.05, **p < 0.01, ***p < 0.001, unpaired student t-test, two tailed. n = 60 for each group, pooled from 3 repeated experiments

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Control, shRNA, Staining, Marker, Expressing, Two Tailed Test

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 increases tubulin detyrosination. A EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons stained for tyrosinated tubulin (red) and detyrosinated tubulin (green) at DIV6. Scale bar: 20 μm unless stated otherwise. B , C Quantification of fluorescence intensity measured in the mid region of the longest neurite of detyrosinated ( B ) and tyrosinated ( C ) tubulin as a proportion of total tubulin. n = 50 for each group, pooled from 5 repeated experiments, mean plus SEM are plotted, *** = p < 0.001, unpaired student t-test, two tailed

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Control, shRNA, Staining, Fluorescence, Two Tailed Test

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 promotes synapse development and NMDA-mediated synaptic transmission. A Representative ratiometric measurements of Fura-2 fluorescence, indicative of intracellular Ca 2+ concentrations, in cultured EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons following perfusion with depolarizing buffer. B Quantification of Fura-2 ratios in EGFP-control (n-41), Rasal1-OE (n = 47), and Rasal1-shRNA (n = 40) transduced hippocampal neurons following perfusion with depolarizing buffer (40 mM KCl). Mean ± SEM plotted. ****p < 0.0001. C Fura-2 color map of loaded neuron. (D1) Punctate expression of endogenous Rasal1 and perinuclear expression in a mature Div16 neuron. (D2-4) Confocal images of synapse morphology stained for FM43 (red) and Rasal1 (cyan) in depolarized EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons at DIV 16 day. Scale bar: 10 µM. E Quantification of fluorescent FM43 puncta as a measure of functional synapses per µM of dendrite in EGFP-control (n = 45), Rasal1-OE (n = 50), and Rasal1-shRNA (n = 50) transduced hippocampal neurons perfused in depolarizing medium (60 mM KCL). Quantified using SynD software. Data presented as Mean ± SEM. *p < 0.05. F The frequency plot (left) and Gaussian distribution (right) of miniature EPSCs (mEPSCs) recorded from EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons. G Representative traces of isolated NMDA-mediated mEPSCs in EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons. H NMDA-mediated mEPSC amplitude (pA) of EGFP-control (n = 28), Rasal1-OE (n = 29), and Rasal1-shRNA (n = 19) transduced hippocampal neurons. Mean ± SEM. *p < 0.05. (I) NMDA-mediated mEPSC frequency (Hz) of EGFP-control (n = 28), Rasal1-OE (n = 29), and Rasal1-shRNA (n = 19) transduced hippocampal neurons. Mean ± SEM. *p < 0.05, ***p < 0.001. Data information: In ( B , E , G , H ) data was analyzed using two-tailed unpaired student t-test

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Transmission Assay, Fluorescence, Cell Culture, Control, shRNA, Expressing, Staining, Functional Assay, Software, Isolation, Two Tailed Test

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Working model of proposed mechanistic activities of Rasal1 in neurites and synapses

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques:

Journal: Molecular Cancer

Article Title: Intercellular TIMP-1-CD63 signaling directs the evolution of immune escape and metastasis in KRAS-mutated pancreatic cancer cells

doi: 10.1186/s12943-024-02207-4

Figure Lengend Snippet: Identify DUSPs expressing epithelial subsets and gene expression pathways. Schematic showing early and advanced PDAC were collected after surgery and processed for 10x scRNA-seq (left). UMAP plots of distinct populations are clustered by the average gene expression in early and advanced PDAC tumors. Each dot represents the transcriptome of a single cell, with color coding defining clusters of cells having similar transcriptional identities (right). ( B ) UMAP plots of distinct populations from early and advanced PDAC (left). Percentage frequency of cell populations in the scRNA-seq data between early and advanced PDAC are shown (right). ( C ) A dot-plot showing the relative expression of a subset of epithelial/ ductal marker genes (left). The color of each dot represents the average expression across the cluster, the size of each dot represents the percentage of cells in the cluster expressing the gene. ( D ) Hallmark pathway enrichment analysis of DEGs in three epithelial subsets (C1-Epi, C14-Epi, and C15-Epi) detected in a Stage II PDAC tumor. ( E ) A dot-plot showing the relative expression of KRAS, DUSP1, DUSP2, DUSP4, DUSP5, and DUSP6 in clusters of different cell type. ( F ) Violin plots showing the distribution of expression levels DUSP1, DUSP2, DUSP4, and DUSP6 in three subsets of epithelial populations in early and advanced tumors. ( G ) DUSP2 regulates ERK1/2 activity in PANC-1 cells. PANC-1 cells were transfected with GFP, DUSP2-GFP, and phosphatase dead DUSP2-GFP (PD) for 24 h. Western blotting was performed to determine pERK1/2. β-actin is the loading control. GFP was used for the detection of exogenous DUSP2 expression (left). Western blotting was performed to determine pERK1/2 and total EKR1/2 (tERK1/2) in control and DUSP2-KD PANC-1 cells (right). ( H ) Representative (left) and quantification (right) of immunohistochemical staining images show expression of cleaved caspase-3 in the lesion of KC (Kras LSL−G12D/+ , Pdx1 Cre/+ ) and KDC (Kras LSL−G12D/+ , Dusp2 fl/fl , Pdx1 Cre/+ ) transgenic mouse

Article Snippet: Antibodies against Phospho-p44/42 MAPK (Cell Signaling Technology #4370, RRID: AB_2315112), p44/42 MAPK (Cell Signaling Technology #4696, RRID: AB_390780), DUSP2 (Santa Cruz sc32776, RRID: AB_2094883), E-cadherin (Cell Signaling Technology #3195, RRID: AB_2291471), VEGF-C (Genetex 113574, RRID: AB_10620764), PD-L1/CD274 (Proteintech: 66248-1-Ig, RRID: AB_2756526), GFP (Genetex, GTX113617, RRID: AB_1950371), tissue inhibitor of metallopeptidase 1 (TIMP-1, R&D, AF970), CD63 (ABclonal, A19023), β-actin (Sigma A1978, RRID: AB_476692), GAPDH (Genetex, GTX100118, RRID: AB_1080976) were used for Western blotting.

Techniques: Expressing, Gene Expression, Marker, Activity Assay, Transfection, Western Blot, Control, Immunohistochemical staining, Staining, Transgenic Assay

Journal: Molecular Cancer

Article Title: Intercellular TIMP-1-CD63 signaling directs the evolution of immune escape and metastasis in KRAS-mutated pancreatic cancer cells

doi: 10.1186/s12943-024-02207-4

Figure Lengend Snippet: Macrophage induces ERK phosphorylation in pancreatic cancer cells. ( A ) The proportion of macrophages, fibroblasts, and other types of cells in scRNA sequencing performed in mouse and human pancreatic tissue. Chi-square analysis was performed to compare the number of macrophages in mouse and human scRNA-seq data in their distribution between early and advanced disease statuses. ( B ) Western blot (upper) result shows the expression of ERK1/2 phosphorylation (pERK), total ERK1/2 (tERK), DUSP2, and β-actin in PANC-1 cells treated with control medium (Ctrl) or conditioned medium (CM) from human pancreatic stellate cells (PSC), U937 (undifferentiated naïve monocyte Mo), and macrophages (Mac) for 48 h. RT-qPCR (lower) for the expression of DUSP2 in control and macrophage-CM (MCM) treated PANC-1 cells. ( C ) Illustration of the co-culture system of PANC1 cells with monocytes (U937) by Transwell for 72 h (upper). The expression of DUSP2, pERK, tERK, and β-actin was detected in PANC-1 co-cultured with different ratio of U937. ( D ) Attachment of CD14 + PBMCs to tissue culture plate after co-culture with PANC-1 cells. Quantification of number of attached CD14 + PBMCs cells after co-culture with PANC-1 cells for 3 days (left). qRT-PCR for gene expression of IL1B and IL10 in CD14 + PBMCs cells and CD14 + PBMCs co-cultured with PANC-1 cells for 3 days (right). ( E ) Western blot result shows the expression levels of pERK1/2, tERK1/2, and DUSP2 in control and MCM treated PANC-1 cells in different time points. ( F ) PANC-1 cell morphology of control, MCM treated, and MCM plus ERK inhibitor (SCH772984) treatment (left). Western blot (right, upper) and RT-qPCR (right, lower) showed the expression of DUSP2 is reversed if treated with ERK inhibitor (SCH772984). ( G ) Representative histology and immunohistochemical staining images (serial section) show expression of phosphorylated ERK1/2 (pERK), monocyte/macrophage (Mac) and macrophage (F4/80) in the lesion of KC (Kras LSL−G12D/+ , Pdx1 Cre/+ ), and KDC (Kras LSL−G12D/+ , Dusp2 fl/fl , Pdx1 Cre/+ ) transgenic mouse compared to pancreata in control mice

Article Snippet: Antibodies against Phospho-p44/42 MAPK (Cell Signaling Technology #4370, RRID: AB_2315112), p44/42 MAPK (Cell Signaling Technology #4696, RRID: AB_390780), DUSP2 (Santa Cruz sc32776, RRID: AB_2094883), E-cadherin (Cell Signaling Technology #3195, RRID: AB_2291471), VEGF-C (Genetex 113574, RRID: AB_10620764), PD-L1/CD274 (Proteintech: 66248-1-Ig, RRID: AB_2756526), GFP (Genetex, GTX113617, RRID: AB_1950371), tissue inhibitor of metallopeptidase 1 (TIMP-1, R&D, AF970), CD63 (ABclonal, A19023), β-actin (Sigma A1978, RRID: AB_476692), GAPDH (Genetex, GTX100118, RRID: AB_1080976) were used for Western blotting.

Techniques: Phospho-proteomics, Sequencing, Western Blot, Expressing, Control, Quantitative RT-PCR, Co-Culture Assay, Cell Culture, Gene Expression, Immunohistochemical staining, Staining, Transgenic Assay

Journal: Molecular Cancer

Article Title: Intercellular TIMP-1-CD63 signaling directs the evolution of immune escape and metastasis in KRAS-mutated pancreatic cancer cells

doi: 10.1186/s12943-024-02207-4

Figure Lengend Snippet: MCM epigenetically suppresses E-cadherin expression and promotes metastasis. ( A ) Control and MCM treated PANC-1 cells were plated in petri dish and recorded by live cell imaging (JuLiBr, supplementary movie 1). The images were taken every 10 min in a total of 24 h. The track of sequence image was measured and quantified by ImageJ (right panel). Analysis was derived from results of three independent experiments with six cells of each time. ( B ) Analysis of gene sets (GSE109110) comparing PANC-1-co-cultured TAMs vs. PANC-1-alone control indicates the enrichment of extracellular/structure signature. ( C ) Representative Western blot results show the expression of E-cadherin, ZO-1, Vimentin and β-actin in control, MCM or UCM treated PANC-1 cells. ( D ) PANC-1 cells were pre-treated with MEK inhibitor (U0126) for 15 min and then treated with MCM for 24 and 48 h. Representative western blot results show the expression of pERK, E-cadherin, ERK and β-actin. ( E ) Proximity ligation assays (PLA) was performed to detect the HDAC1-CtBP and HDAC2-CtBP interaction Representative image (left) and quantification (right) was shown. ( F ) Suppressed E-cadherin is restored if HDAC inhibitor (B369 and B390) was employed (left). DUSP2-KD PANC-1 cells were treated with HDAC or ERK inhibitor and MCM for 24 h. Whole cell lysate was collected for the detection of E-cadherin and GAPDH. ( G ) Control, MCM, and MCM plus HDAC inhibitor treated PANC-1 cells were plated in low-attachment plate for 24 h. Annexin V positive cells were measured by flow cytometry. ( H ) Illustration of the isolation of KPPC cells and experimental design. KPPC cells were injected into the pancreas of immunocompetent mice. After one month of injection, pancreas, liver, and ascites were collected for further analysis. ( I ) H&E stain and immunohistochemistry staining for E-cadherin in pancreas, liver, and ascites blocks of mice injected with KPPC cells. Mice harbor KPPC tumors develop ascites which were pelleted and processed for cell block. H&E stain and immunohistochemistry staining for E-cadherin and Ck19 in the ascites cell block. ( J ) KPPC cells treated with MCM showed morphology change (upper), and decreased Dup2 and E-cadherin, and increased pERK measured by Western blotting (lower). ( K ) KPPC cells labeled with luciferase were injected into mice via portal vein. IVIS imaging was used to track the development of liver metastases in mice. After one month, mice were sacrificed and livers were taken (left). H&E stain and immunohistochemistry staining for Ck19 expression in the liver of mice injected with control or MCM-treated KPPC cells (right)

Article Snippet: Antibodies against Phospho-p44/42 MAPK (Cell Signaling Technology #4370, RRID: AB_2315112), p44/42 MAPK (Cell Signaling Technology #4696, RRID: AB_390780), DUSP2 (Santa Cruz sc32776, RRID: AB_2094883), E-cadherin (Cell Signaling Technology #3195, RRID: AB_2291471), VEGF-C (Genetex 113574, RRID: AB_10620764), PD-L1/CD274 (Proteintech: 66248-1-Ig, RRID: AB_2756526), GFP (Genetex, GTX113617, RRID: AB_1950371), tissue inhibitor of metallopeptidase 1 (TIMP-1, R&D, AF970), CD63 (ABclonal, A19023), β-actin (Sigma A1978, RRID: AB_476692), GAPDH (Genetex, GTX100118, RRID: AB_1080976) were used for Western blotting.

Techniques: Expressing, Control, Live Cell Imaging, Sequencing, Derivative Assay, Cell Culture, Western Blot, Ligation, Flow Cytometry, Isolation, Injection, Staining, Immunohistochemistry, Blocking Assay, Labeling, Luciferase, Imaging

Journal: Molecular Cancer

Article Title: Intercellular TIMP-1-CD63 signaling directs the evolution of immune escape and metastasis in KRAS-mutated pancreatic cancer cells

doi: 10.1186/s12943-024-02207-4

Figure Lengend Snippet: Macrophage exacerbates ERK active DUSP2 low mediated lymphangiogenesis and immune escape. ( A ) Cellchat analysis for cellular communication among distinct cell populations in early and late KIC pancreas. Circle plot was served as visualization outputs and different colors represent different cell groups. ( B ) Picture of pancreatic tumors developed from SCID mice orthotopically injected with control or MCM-selected PANC1. The dotted lines show tumor mass. H&E stain of mouse liver from MCM-selected PANC-1 group. The asterisk indicates tumor mass. Numbers of mice with metastatic lesions and total numbers of mice in MCM-selected PANC-1 group is indicated at the bottom of micrographs (upper). IHC staining showed the expression of E-cadherin in control or MCM-selected pancreatic tumors (lower). ( C ) All the significant signaling pathways in early and late KIC were ranked based on their differences of overall information flow. The overall information flow of a signaling network is calculated by summarizing all the communication probabilities in that network. The top signaling pathways colored by red are more enriched in late KIC, and the pathways colored by blue were more enriched in the early KIC pancreas. ( D ) IHC staining for LYVE-1 and CD31 in control or MCM-selected pancreatic tumors (left). Quantification of LYVE-1 and CD31 in control and MCM-selected pancreatic tumors (right). ( E ) The expression of VEGF-C in control, MCM, MCM plus ERK inhibitor treated PANC-1 cells measured by RT-qPCR (upper) and Western blotting (lower). ( F ) RT-qPCR for VEGF-C expression in control and DUSP2-KD PANC-1 cells co-cultured with macrophages for 2 days. ( G ) RT-qPCR for the expression of PD-L1 ( CD274 ) in pancreatic cancer cells. U937 cells were first differentiated into macrophages and co-cultured with control or DUSP2-KD PANC-1 cells for 2 days. ( H ) KPPC cells co-cultured with U937 for 5 days showed increased ERK phosphorylation (left). Immunocytochemistry for PD-L1 expression in KPPC and KPPC which has been co-cultured with U937 (right). ( I ) Total flux was determined to represent cell numbers of KPPC cells which are luciferase labeled. KPPC cells were first co-cultured with U937 for 5 days and then incubated with freshly isolated mouse splenocytes for 2 days

Article Snippet: Antibodies against Phospho-p44/42 MAPK (Cell Signaling Technology #4370, RRID: AB_2315112), p44/42 MAPK (Cell Signaling Technology #4696, RRID: AB_390780), DUSP2 (Santa Cruz sc32776, RRID: AB_2094883), E-cadherin (Cell Signaling Technology #3195, RRID: AB_2291471), VEGF-C (Genetex 113574, RRID: AB_10620764), PD-L1/CD274 (Proteintech: 66248-1-Ig, RRID: AB_2756526), GFP (Genetex, GTX113617, RRID: AB_1950371), tissue inhibitor of metallopeptidase 1 (TIMP-1, R&D, AF970), CD63 (ABclonal, A19023), β-actin (Sigma A1978, RRID: AB_476692), GAPDH (Genetex, GTX100118, RRID: AB_1080976) were used for Western blotting.

Techniques: Injection, Control, Staining, Immunohistochemistry, Expressing, Protein-Protein interactions, Quantitative RT-PCR, Western Blot, Cell Culture, Phospho-proteomics, Immunocytochemistry, Luciferase, Labeling, Incubation, Isolation

Journal: Molecular Cancer

Article Title: Intercellular TIMP-1-CD63 signaling directs the evolution of immune escape and metastasis in KRAS-mutated pancreatic cancer cells

doi: 10.1186/s12943-024-02207-4

Figure Lengend Snippet: Decipher the signaling interaction between macrophage and ERK active DUSP2 low epithelial cells. ( A ) Circle plot showing the inferred signaling networks from C15-Epi subset toward subsets of other epithelial, macrophage and T cells in early and advanced stages. The round loops along with cell type represent the interactions within the same cell type. ( B ) Circle plots showing the outgoing signals from each macrophage subset (C4, C9, and C12) in the early PDAC. ( C ) Hallmark pathway enrichment analysis of DEGs in C9-Mac in early and advanced PDAC tumor. ( D ) Cytokine array (RayBiotech) was used to identify potential factors secreted from macrophage (MCM) compared to RPMI which contains 10% FBS. ( E ) The expression of TIMPs in macrophages. Macrophages were first co-cultured with PANC-1 for 2 days and then cultured alone by serum-free RPMI medium for an additional day. Conditioned medium was collected and concentrated to measure the expression of TIMP-1 and TIMP-2 by Western blotting. ( F ) PANC-1 cells were initially treated with MCM for 1 h, after which they were cultured in serum-free medium with or without recombinant TIMP-1 (Peprotech) for 24 h. Western blotting was then performed to assess the expression of DUSP2, pERK, E-cadherin, and β-actin. ( G ) The expression of CD63, pERK, tERK, DUSP2, and internal control β-actin in control and CD63 knockdown (KD) PANC-1 cells after being treated with MCM for 1 h. ( H ) Control and CD63-KD PANC-1 cells were first treated with or without MCM for 24 h and the medium was replaced by serum-free RPMI for an additional 24 h. The expression of CD63, pERK, tERK, DUSP2, and internal control β-actin was determined by Western blotting. ( I ) The volcano plot illustrates the number of genes that are significantly upregulated or downregulated in MCM-treated cells compared to untreated cells under both Ctrl-KD and CD63-KD conditions, with a fold change greater than 2. ( J ) GO analysis was conducted on differentially expressed genes comparing MCM-treated Ctrl-KD and MCM-treated CD63-KD cells, focusing on their involvement in biological processes and molecular functions, as illustrated in cnetplots and dotplots. ( K ) The expression of CD63, pERK, and internal control β-actin in PANC-1 cells that have been co-cultured with monocyte (co-U) for 3 days, with macrophage (co-M) for 2 days, or treated with MCM for 2 days. ( L ) The expression of CD63 and β-actin in control and DUSP2-KD PANC-1 cells treated with DMSO or ERK inhibitor (SCH772984)

Article Snippet: Antibodies against Phospho-p44/42 MAPK (Cell Signaling Technology #4370, RRID: AB_2315112), p44/42 MAPK (Cell Signaling Technology #4696, RRID: AB_390780), DUSP2 (Santa Cruz sc32776, RRID: AB_2094883), E-cadherin (Cell Signaling Technology #3195, RRID: AB_2291471), VEGF-C (Genetex 113574, RRID: AB_10620764), PD-L1/CD274 (Proteintech: 66248-1-Ig, RRID: AB_2756526), GFP (Genetex, GTX113617, RRID: AB_1950371), tissue inhibitor of metallopeptidase 1 (TIMP-1, R&D, AF970), CD63 (ABclonal, A19023), β-actin (Sigma A1978, RRID: AB_476692), GAPDH (Genetex, GTX100118, RRID: AB_1080976) were used for Western blotting.

Techniques: Expressing, Cell Culture, Western Blot, Recombinant, Control, Knockdown

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 is expressed in the soma and neurites of developing hippocampal neurons. A Expression of Rasal1 in coronal section of adult mouse brain. Allen Mouse Brain Atlas, http://mouse.brain-map.org/experiment/show/75774111 . B Western blot analyses with Rasal1 antibody in the absence (left) and presence (right) of Rasal1 immunizing peptide. Lane 1: mouse hippocampus; lane 2: mouse prefrontal cortex; lane 3: rat hippocampus all at P8. C Endogenous Rasal1 immunoreactivity on DIV2, DIV4, DIV6, DIV12 and DIV14 in neurons stained for Rasal1 (red) and Tubulin (blue) and imaged with confocal microscopy. DIV 2 scale bar: 5 μm, DIV 4–10 scale bar: 10 μm

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Expressing, Western Blot, Staining, Confocal Microscopy

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 translocates to the plasma membrane in response to Ca 2+ in hippocampal neurons. A1-3 Hippocampal neuron expressing Rasal1-EGFP stained to show the distribution of Rasal1-EGFP (green) compared to total Rasal1 immunoreactivity (red) and MAP2 (blue) under basal conditions. Scale bar: 10 µm. B1-3 Depolarized hippocampal neuron expressing Rasal1-EGFP stained to show Rasal1-EGFP (green) distribution compared to total Rasal1 (red) and MAP2 (blue). C Live confocal imaging of neuron expressing Rasal1-EGFP following perfusion of depolarizing buffer (Depolarizing B) C1-4 . Followed by perfusion with basal buffer (Basal B) at 8 s (C5-7) followed by second perfusion with depolarizing buffer at 18 s (D-18s) (C8). Scale bar: 10 µm. D – G Rasal1-EGFP live imaging in two additional neurons perfused in basal buffer D , F and depolarizing buffer ( E , G ). Scale bar: 10 µm. H Time-dependent change in fluorescence ratio (R) in the membrane after addition of depolarizing buffer, basal buffer at 8 s and depolarizing buffer again at 12 s (addition of buffers indicated by dashed lines). Data presented as percentages of the original fluorescence at 0 s. Mean ± SEM are plotted, n = 20

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Clinical Proteomics, Membrane, Expressing, Staining, Imaging, Fluorescence

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 promotes CaMKII phosphorylation. A Representative confocal images of blocked control hippocampal neurons treated with TTX, CNQX and APV for at least 8 h to block CaMKII phosphorylation ( A1 ), EGFP-control hippocampal neurons treated for 5 min with 10 µM glycine to acutely induce CaMKII phosphorylation ( A2 ), glycine (10 µM) treated Rasal1-OE transduced hippocampal neurons ( A3 ) and glycine (10 µM) treated Rasal1-shRNA transduced hippocampal neurons ( A4 ) stained for phosphorylated (red) or total CaMKII (cyan). Transfected neurons (green) indicated by arrows were compared to neighboring non-transfected neurons. Scale bar: 20 µm. B Quantification of the ratio of phosphorylated CaMKII fluorescence intensity of transfected /non-transfected EGFP-control (n = 20), Rasal1-OE (n = 25), and Rasal1-shRNA (n = 25) transduced hippocampal neurons. C Quantification of total CaMKII levels in EGFP-control (n = 20), Rasal1-OE (n = 25), and Rasal1-shRNA (n = 25) transduced hippocampal neurons as ratio of fluorescence intensity in transfected/non-transfected neurons. D Fluorescent western blot of p-CaMKII (red) of glycine (10 µM) treated EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons. E Fluorescent western blot of CaMKII (green) and p-CaMKII (red) of glycine (10 µM) treated EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons. F Quantification of p-CaMKII/actin levels in fluorescent western blot across of glycine (10 µM) treated EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons (n = 8). G Quantification of total CaMKII/actin levels in fluorescent western blot across of glycine (10 µM) treated EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons. (n = 8). Data information: In ( B , C , F , G ) *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired student t-test

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Phospho-proteomics, Control, Blocking Assay, shRNA, Staining, Transfection, Fluorescence, Western Blot, Two Tailed Test

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 binds to CaMKII, PKC, α- and β-tubulin. Confirmation of suspected Rasal1 binding partners using pull-down assay (n = 3) with GST tagged Rasal1 fragments; N-terminus (GST-n), middle fragment (GST-m) or C-terminal fragment (GST-c) validated by Co-IP. A Western blot of pull-down assay (left) and Co-IP (right) of GST-tagged Rasal1 fragments stained for CaMKII in hippocampal and whole brain lysate. B Western blot of pull-down assay (left) and Co-IP (right) of GST-tagged Rasal1 fragments stained for PKC in hippocampal and whole brain lysate. C Western blot of pull-down assay (left) and Co-IP (right) of GST-tagged Rasal1 fragments stained for β-tubulin in hippocampal and whole brain lysate

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Binding Assay, Pull Down Assay, Co-Immunoprecipitation Assay, Western Blot, Staining

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 inhibits neurite growth. A Neurite tracing using SynD at DIV 6, 8, 10, 12 and 14 of mouse hippocampal neurons transduced with either Rasal1-shRNA-EGFP, Rasal1-OE, or EGFP-control. Scale bar: 50 µm. B Neurite length (µm) of EGFP-control, Rasal1-shRNA, and Rasal1-OE expressing mouse hippocampal neurons at DIV 6, 8, 10, 12, and 14. Data pooled from 4 independent experiments (n = 122). Mean ± SEM are plotted, *p < 0.05, **p < 0.01, ***p < 0.001, unpaired student t-test, two tailed

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Transduction, shRNA, Control, Expressing, Two Tailed Test

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 inhibits dendrite length and branching. A Representative confocal images of EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced mouse hippocampal neurons stained for the dendritic marker MAP2 (red) and axonal marker Tau1 (green) (left). Tracings of dendrites (middle) and axonal neurites (right) were done using SynD. Scale bar: 20 µm. B Total axon and dendrite length (µm) of EGFP-control, Rasal1-OE, and Rasal-shRNA expressing neurons. C Number of primary and secondary dendritic branches in neurons expressing EGFP-control, Rasal1-OE, and Rasal1-shRNA. Data information: In B , C Mean ± SEM are plotted, *p < 0.05, **p < 0.01, ***p < 0.001, unpaired student t-test, two tailed. n = 60 for each group, pooled from 3 repeated experiments

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Control, shRNA, Staining, Marker, Expressing, Two Tailed Test

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 increases tubulin detyrosination. A EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons stained for tyrosinated tubulin (red) and detyrosinated tubulin (green) at DIV6. Scale bar: 20 μm unless stated otherwise. B , C Quantification of fluorescence intensity measured in the mid region of the longest neurite of detyrosinated ( B ) and tyrosinated ( C ) tubulin as a proportion of total tubulin. n = 50 for each group, pooled from 5 repeated experiments, mean plus SEM are plotted, *** = p < 0.001, unpaired student t-test, two tailed

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Control, shRNA, Staining, Fluorescence, Two Tailed Test

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 promotes synapse development and NMDA-mediated synaptic transmission. A Representative ratiometric measurements of Fura-2 fluorescence, indicative of intracellular Ca 2+ concentrations, in cultured EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons following perfusion with depolarizing buffer. B Quantification of Fura-2 ratios in EGFP-control (n-41), Rasal1-OE (n = 47), and Rasal1-shRNA (n = 40) transduced hippocampal neurons following perfusion with depolarizing buffer (40 mM KCl). Mean ± SEM plotted. ****p < 0.0001. C Fura-2 color map of loaded neuron. (D1) Punctate expression of endogenous Rasal1 and perinuclear expression in a mature Div16 neuron. (D2-4) Confocal images of synapse morphology stained for FM43 (red) and Rasal1 (cyan) in depolarized EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons at DIV 16 day. Scale bar: 10 µM. E Quantification of fluorescent FM43 puncta as a measure of functional synapses per µM of dendrite in EGFP-control (n = 45), Rasal1-OE (n = 50), and Rasal1-shRNA (n = 50) transduced hippocampal neurons perfused in depolarizing medium (60 mM KCL). Quantified using SynD software. Data presented as Mean ± SEM. *p < 0.05. F The frequency plot (left) and Gaussian distribution (right) of miniature EPSCs (mEPSCs) recorded from EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons. G Representative traces of isolated NMDA-mediated mEPSCs in EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons. H NMDA-mediated mEPSC amplitude (pA) of EGFP-control (n = 28), Rasal1-OE (n = 29), and Rasal1-shRNA (n = 19) transduced hippocampal neurons. Mean ± SEM. *p < 0.05. (I) NMDA-mediated mEPSC frequency (Hz) of EGFP-control (n = 28), Rasal1-OE (n = 29), and Rasal1-shRNA (n = 19) transduced hippocampal neurons. Mean ± SEM. *p < 0.05, ***p < 0.001. Data information: In ( B , E , G , H ) data was analyzed using two-tailed unpaired student t-test

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Transmission Assay, Fluorescence, Cell Culture, Control, shRNA, Expressing, Staining, Functional Assay, Software, Isolation, Two Tailed Test

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Working model of proposed mechanistic activities of Rasal1 in neurites and synapses

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques:

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 is expressed in the soma and neurites of developing hippocampal neurons. A Expression of Rasal1 in coronal section of adult mouse brain. Allen Mouse Brain Atlas, http://mouse.brain-map.org/experiment/show/75774111 . B Western blot analyses with Rasal1 antibody in the absence (left) and presence (right) of Rasal1 immunizing peptide. Lane 1: mouse hippocampus; lane 2: mouse prefrontal cortex; lane 3: rat hippocampus all at P8. C Endogenous Rasal1 immunoreactivity on DIV2, DIV4, DIV6, DIV12 and DIV14 in neurons stained for Rasal1 (red) and Tubulin (blue) and imaged with confocal microscopy. DIV 2 scale bar: 5 μm, DIV 4–10 scale bar: 10 μm

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Expressing, Western Blot, Staining, Confocal Microscopy

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 translocates to the plasma membrane in response to Ca 2+ in hippocampal neurons. A1-3 Hippocampal neuron expressing Rasal1-EGFP stained to show the distribution of Rasal1-EGFP (green) compared to total Rasal1 immunoreactivity (red) and MAP2 (blue) under basal conditions. Scale bar: 10 µm. B1-3 Depolarized hippocampal neuron expressing Rasal1-EGFP stained to show Rasal1-EGFP (green) distribution compared to total Rasal1 (red) and MAP2 (blue). C Live confocal imaging of neuron expressing Rasal1-EGFP following perfusion of depolarizing buffer (Depolarizing B) C1-4 . Followed by perfusion with basal buffer (Basal B) at 8 s (C5-7) followed by second perfusion with depolarizing buffer at 18 s (D-18s) (C8). Scale bar: 10 µm. D – G Rasal1-EGFP live imaging in two additional neurons perfused in basal buffer D , F and depolarizing buffer ( E , G ). Scale bar: 10 µm. H Time-dependent change in fluorescence ratio (R) in the membrane after addition of depolarizing buffer, basal buffer at 8 s and depolarizing buffer again at 12 s (addition of buffers indicated by dashed lines). Data presented as percentages of the original fluorescence at 0 s. Mean ± SEM are plotted, n = 20

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Clinical Proteomics, Membrane, Expressing, Staining, Imaging, Fluorescence

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 promotes CaMKII phosphorylation. A Representative confocal images of blocked control hippocampal neurons treated with TTX, CNQX and APV for at least 8 h to block CaMKII phosphorylation ( A1 ), EGFP-control hippocampal neurons treated for 5 min with 10 µM glycine to acutely induce CaMKII phosphorylation ( A2 ), glycine (10 µM) treated Rasal1-OE transduced hippocampal neurons ( A3 ) and glycine (10 µM) treated Rasal1-shRNA transduced hippocampal neurons ( A4 ) stained for phosphorylated (red) or total CaMKII (cyan). Transfected neurons (green) indicated by arrows were compared to neighboring non-transfected neurons. Scale bar: 20 µm. B Quantification of the ratio of phosphorylated CaMKII fluorescence intensity of transfected /non-transfected EGFP-control (n = 20), Rasal1-OE (n = 25), and Rasal1-shRNA (n = 25) transduced hippocampal neurons. C Quantification of total CaMKII levels in EGFP-control (n = 20), Rasal1-OE (n = 25), and Rasal1-shRNA (n = 25) transduced hippocampal neurons as ratio of fluorescence intensity in transfected/non-transfected neurons. D Fluorescent western blot of p-CaMKII (red) of glycine (10 µM) treated EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons. E Fluorescent western blot of CaMKII (green) and p-CaMKII (red) of glycine (10 µM) treated EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons. F Quantification of p-CaMKII/actin levels in fluorescent western blot across of glycine (10 µM) treated EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons (n = 8). G Quantification of total CaMKII/actin levels in fluorescent western blot across of glycine (10 µM) treated EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons. (n = 8). Data information: In ( B , C , F , G ) *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired student t-test

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Phospho-proteomics, Control, Blocking Assay, shRNA, Staining, Transfection, Fluorescence, Western Blot, Two Tailed Test

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 binds to CaMKII, PKC, α- and β-tubulin. Confirmation of suspected Rasal1 binding partners using pull-down assay (n = 3) with GST tagged Rasal1 fragments; N-terminus (GST-n), middle fragment (GST-m) or C-terminal fragment (GST-c) validated by Co-IP. A Western blot of pull-down assay (left) and Co-IP (right) of GST-tagged Rasal1 fragments stained for CaMKII in hippocampal and whole brain lysate. B Western blot of pull-down assay (left) and Co-IP (right) of GST-tagged Rasal1 fragments stained for PKC in hippocampal and whole brain lysate. C Western blot of pull-down assay (left) and Co-IP (right) of GST-tagged Rasal1 fragments stained for β-tubulin in hippocampal and whole brain lysate

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Binding Assay, Pull Down Assay, Co-Immunoprecipitation Assay, Western Blot, Staining

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 inhibits neurite growth. A Neurite tracing using SynD at DIV 6, 8, 10, 12 and 14 of mouse hippocampal neurons transduced with either Rasal1-shRNA-EGFP, Rasal1-OE, or EGFP-control. Scale bar: 50 µm. B Neurite length (µm) of EGFP-control, Rasal1-shRNA, and Rasal1-OE expressing mouse hippocampal neurons at DIV 6, 8, 10, 12, and 14. Data pooled from 4 independent experiments (n = 122). Mean ± SEM are plotted, *p < 0.05, **p < 0.01, ***p < 0.001, unpaired student t-test, two tailed

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Transduction, shRNA, Control, Expressing, Two Tailed Test

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 inhibits dendrite length and branching. A Representative confocal images of EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced mouse hippocampal neurons stained for the dendritic marker MAP2 (red) and axonal marker Tau1 (green) (left). Tracings of dendrites (middle) and axonal neurites (right) were done using SynD. Scale bar: 20 µm. B Total axon and dendrite length (µm) of EGFP-control, Rasal1-OE, and Rasal-shRNA expressing neurons. C Number of primary and secondary dendritic branches in neurons expressing EGFP-control, Rasal1-OE, and Rasal1-shRNA. Data information: In B , C Mean ± SEM are plotted, *p < 0.05, **p < 0.01, ***p < 0.001, unpaired student t-test, two tailed. n = 60 for each group, pooled from 3 repeated experiments

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Control, shRNA, Staining, Marker, Expressing, Two Tailed Test

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 increases tubulin detyrosination. A EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons stained for tyrosinated tubulin (red) and detyrosinated tubulin (green) at DIV6. Scale bar: 20 μm unless stated otherwise. B , C Quantification of fluorescence intensity measured in the mid region of the longest neurite of detyrosinated ( B ) and tyrosinated ( C ) tubulin as a proportion of total tubulin. n = 50 for each group, pooled from 5 repeated experiments, mean plus SEM are plotted, *** = p < 0.001, unpaired student t-test, two tailed

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Control, shRNA, Staining, Fluorescence, Two Tailed Test

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Rasal1 promotes synapse development and NMDA-mediated synaptic transmission. A Representative ratiometric measurements of Fura-2 fluorescence, indicative of intracellular Ca 2+ concentrations, in cultured EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons following perfusion with depolarizing buffer. B Quantification of Fura-2 ratios in EGFP-control (n-41), Rasal1-OE (n = 47), and Rasal1-shRNA (n = 40) transduced hippocampal neurons following perfusion with depolarizing buffer (40 mM KCl). Mean ± SEM plotted. ****p < 0.0001. C Fura-2 color map of loaded neuron. (D1) Punctate expression of endogenous Rasal1 and perinuclear expression in a mature Div16 neuron. (D2-4) Confocal images of synapse morphology stained for FM43 (red) and Rasal1 (cyan) in depolarized EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons at DIV 16 day. Scale bar: 10 µM. E Quantification of fluorescent FM43 puncta as a measure of functional synapses per µM of dendrite in EGFP-control (n = 45), Rasal1-OE (n = 50), and Rasal1-shRNA (n = 50) transduced hippocampal neurons perfused in depolarizing medium (60 mM KCL). Quantified using SynD software. Data presented as Mean ± SEM. *p < 0.05. F The frequency plot (left) and Gaussian distribution (right) of miniature EPSCs (mEPSCs) recorded from EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons. G Representative traces of isolated NMDA-mediated mEPSCs in EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons. H NMDA-mediated mEPSC amplitude (pA) of EGFP-control (n = 28), Rasal1-OE (n = 29), and Rasal1-shRNA (n = 19) transduced hippocampal neurons. Mean ± SEM. *p < 0.05. (I) NMDA-mediated mEPSC frequency (Hz) of EGFP-control (n = 28), Rasal1-OE (n = 29), and Rasal1-shRNA (n = 19) transduced hippocampal neurons. Mean ± SEM. *p < 0.05, ***p < 0.001. Data information: In ( B , E , G , H ) data was analyzed using two-tailed unpaired student t-test

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: Transmission Assay, Fluorescence, Cell Culture, Control, shRNA, Expressing, Staining, Functional Assay, Software, Isolation, Two Tailed Test

Journal: Cell & Bioscience

Article Title: Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

doi: 10.1186/s13578-024-01193-w

Figure Lengend Snippet: Working model of proposed mechanistic activities of Rasal1 in neurites and synapses

Article Snippet: We compared several Rasal1 antibodies; Rasal1 sc-68771 Santa Cruz, Rasal1 NBP1-32776 Novus Biologicals and Rasal1 from GenScript (Piscataway, NJ).

Techniques: