git1 shrnas  (Millipore)

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: A. In vitro phosphorylation of brain homogenate by recombinant active JNK1 revealed phosphorylation of GIT1 on S371 and S692. Mass spectrometry-identified sequences are shown for GIT1 (accession NP_001078923.1). Β. Representative autoradiograph and Coomassie Brilliant Blue (CBB) stained gel image are shown for recombinant GST-GIT1- wild-type (WT), -S371A, -S692A or S371A/S692A (SAA) variants after phosphorylation by GST-JNK1. JNK1 phosphorylated GIT1-WT and GIT1–S692A but not GIT1-S371A, indicating that S371 is the preferred JNK1 phosphorylation site. Phosphorylation of GIT1 by JNK1 was prevented by JNK inhibitor 10 µM SP600125. C. Quantitative data from 4 repeats of experiment shown in B . D. Domain map of human GIT1 (UniProt identifier: Q9Y2X7-3) shows predicted JNK1 phosphorylation sites (yellow circles). Abbreviations: Arf GTPase activating protein domain (GAP), 3x Ankyrin repeats (ANK), Spa-Homology domain (SHD), Synapse Localization Domain (SLD) and Paxillin Binding Domain (PBD). E. Quantitative LC-MS/MS data of GIT1-S371 phosphorylation in wild-type (WT) and Jnk1-/- whole brains. F. Quantitative LC-MS/MS data of GIT1-S371 phosphorylation in brains from DJNKI-1- infused adult mice. G. Active Arf1 was detected from pull downs using GST-GAT-GGA3 immobilized on glutathione beads. Cells expressed GIT1-variants together with Arf1-WT or constitutively active Arf1-Q71L. H. Quantitative data from 3 repeats of experiment described in G. Phosphorylation-site mutants of GIT1 did not alter Arf1 activity. Error bars represent standard error of the mean (SEM) and P-values were determined using Student’s t test.

Article Snippet: For immunoblotting dilutions were as follows: 1:1000 paxillin (Bioscience), 1:500 GABAβ3 (Neuromab), 1:2000 GIT1 (Santa-Cruz), 1:1000 β-PIX (Bioscience), 1:1000 14-3-3ζ (Santa-Cruz), 1:2000 FLAG (Sigma Aldrich), 1:10,000 EGFP (JL-8, Clontech), 1: 2000 HA (Cell Signaling), 1:1000 JNK1 (Bioscience), 1:50,000 anti-rabbit and anti-mouse secondary HRP antibodies (Millipore).

Techniques: In Vitro, Recombinant, Mass Spectrometry, Autoradiography, Staining, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: A. Representative maximum projection images (25x optical slices, z=0.173 µm) depict EGFP-GIT1-WT and mRub-Lifeact in 16-DIV hippocampal neurons. Regions of interest (R.O.I.s; circles) depict where in the dendrite, spine and background mean intensity signals were acquired. B, D. Relative enrichment of EGFP-GIT1 variants in spines or dendrites, was calculated as a ratio of the mean intensity from the spine R.O.I. to the mean intensity from the R.O.I in the dendrite, after background subtraction, for the given treatments Jnk1-/- or TAT/DJNKI-1 (20 µM), 8 hours. C, E. Relative enrichment of mRuby-Lifeact fluorescence in spines was measured as for B and E. Mean data +/- S.E.Ms from a total of ∼80 spines from several neurons per condition are shown. Neurons originated from at least two independent mouse litters. P-values were calculated from Student’s two-tailed t test. F, G. We next checked the overall effect of DJNKI-1 inhibitor or Jnk1 knockout on GABAAR expression at the cell surface in the dendritic compartment. Both conditions increased general surface expression of GABAAR. H. In order to test the requirement for GIT1 in GABAAR trafficking, we characterised shRNA targeting GIT1. I. Representative maximum projection images of GABAAR β3 staining from 25 x optical sections (z=0.173 μm) are shown for WT and Jnk1-/- hippocampal neurons at 16 DIV. Staining was performed in non-permeabilized cells expressing mRuby, with non-targeting (NT)-shRNA or GIT1-shRNA and EGFP-GIT1 variants, as shown. White outlines were traced from mRuby fluorescence. Surface staining of GABAAR β3 is shown in pseudocolour. Representative images of total GABAAR β3 staining from an independent set of permeabilized cells is shown for comparison. J, K . Quantitative data from these experiments is shown for spines and dendrites. Mean data +/- S.E.M are shown from ∼70 dendritic spines per condition, in cells from at least two independent mouse litters. L, M. Cell surface expression of GABAAR β3 was measured from biotinylated samples. Representative blots show GABAAR β3 of lysates from before streptavidin-biotin enrichment (total) or after streptavidin-biotin enrichment (surface) are shown. Mean data from 3 repeats +/- S.E.M are shown. P-values were calculated from Student’s two-tailed t test.

Article Snippet: For immunoblotting dilutions were as follows: 1:1000 paxillin (Bioscience), 1:500 GABAβ3 (Neuromab), 1:2000 GIT1 (Santa-Cruz), 1:1000 β-PIX (Bioscience), 1:1000 14-3-3ζ (Santa-Cruz), 1:2000 FLAG (Sigma Aldrich), 1:10,000 EGFP (JL-8, Clontech), 1: 2000 HA (Cell Signaling), 1:1000 JNK1 (Bioscience), 1:50,000 anti-rabbit and anti-mouse secondary HRP antibodies (Millipore).

Techniques: Fluorescence, Two Tailed Test, Knock-Out, Expressing, shRNA, Staining, Comparison

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: A. Representative maximum projection images (32 x optical sections of 0.173 μm) of neurons expressing mRuby to visualize morphology. Neurons at 16 DIV were immunostained for endogenous GIT1 (green) and paxillin (PXN), 14-3-3ζ or β-PIX (magenta). B. Quantitative data show mean intensities of GIT1, PXN, 14-3-3ζ and β-PIX in spines. DJNKI- 1 increases GIT1 in dendritic spines compared to control (TAT), and reduces β-PIX in the same cell compartment. C. Mean intensity ratios of GIT1, PXN, 14-3-3ζ and β-PIX in dendritic spine/shaft. Mean data +/- SEMs from ∼42 spines per condition.

Article Snippet: For immunoblotting dilutions were as follows: 1:1000 paxillin (Bioscience), 1:500 GABAβ3 (Neuromab), 1:2000 GIT1 (Santa-Cruz), 1:1000 β-PIX (Bioscience), 1:1000 14-3-3ζ (Santa-Cruz), 1:2000 FLAG (Sigma Aldrich), 1:10,000 EGFP (JL-8, Clontech), 1: 2000 HA (Cell Signaling), 1:1000 JNK1 (Bioscience), 1:50,000 anti-rabbit and anti-mouse secondary HRP antibodies (Millipore).

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: A. We examined whether GABAAR β3, paxillin, β-PIX or 14-3-3ζ co-immunoprecipitated with GIT1 using cortex homogenates from WT and Jnk1-/- adult mice. +/- denotes addition or not of antibody. Representative immunoblots of input and GIT1 immunoprecipitates probed with indicated antibodies are shown. B. Quantitative data show the extent of GABAAR β3, PXN, β-PIX and 14-3-3ζ co-purification with GIT1 from brain. For 14-3-3ζ quantification, the background bands present in samples without GIT1 antibody were subtracted from bands in samples with GIT1 antibody. There was increased interaction between GIT1 and GABAAR β3, PXN and 14-3-3ζ in Jnk1-/- brain compared to WT. In contrast, interaction between GIT1 and β-PIX decreased. Histogram bars represent mean data from 4 experimental repeats +/- SEM. C. The effect of TAT or DJNKI-1 on GIT1 interaction with binding partners was determined. Cortical neurons at 16 DIV were treated with 20 µM TAT or DJNKI-1 for 8 hours. Representative immunoblots of GIT1 co-precipitations are shown. D. Band quantification from blots in . DJNKI-1-treated neurons show increased interaction between GIT1 and GABAAR β3, paxillin and 14-3-3ζ, whereas β-PIX interaction with GIT1 reduced. Bars show mean data from 5 repeats ± SEM. E. We tested whether GIT1-S371 phosphorylation site mutants altered these interactions. HEK-293 cells were transfected with EGFP-GIT1-WT, EGFP-GIT1-S371A or EGFP-GIT1-S371D and either Flag (as a control), Flag-PXN-WT or Flag-PXN-S178A, as indicated. Representative blots of input and co-immunoprecipitations are shown. F. Quantitative band intensities from E are shown using log10 scale. PXN-S178A interacted most highly with GIT1-S371A. Mean data ± SEM from 4 repeats are shown. Student’s t-test p-values above bars are compared to samples with EGFP-GTI1-WT and Flag-PXN-WT. G. The same approach was used to test endogenous 14-3-3ζ and Flag-β-PIX interaction with GIT1. Representative blots are shown. H. Quantitative data for Flag-β−PIX interaction with EGFP-GIT1 variants. I. Quantitative data for 14-3-3ζ interaction with EGFP-GIT1 variants. Endogenous 14-3-3ζ interacts most highly with EGFP-GIT1-S371A. Bars ± SEM represent means from 3 repeats. p-values were calculated from Student’s two-tailed t-test and are compared to conditions where WT is expressed. IB: immunoblot, IP: immunoprecipitation.

Article Snippet: For immunoblotting dilutions were as follows: 1:1000 paxillin (Bioscience), 1:500 GABAβ3 (Neuromab), 1:2000 GIT1 (Santa-Cruz), 1:1000 β-PIX (Bioscience), 1:1000 14-3-3ζ (Santa-Cruz), 1:2000 FLAG (Sigma Aldrich), 1:10,000 EGFP (JL-8, Clontech), 1: 2000 HA (Cell Signaling), 1:1000 JNK1 (Bioscience), 1:50,000 anti-rabbit and anti-mouse secondary HRP antibodies (Millipore).

Techniques: Immunoprecipitation, Western Blot, Copurification, Binding Assay, Transfection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: A. Representative maximum projection images (22 x optical sections of 0.173 μm) showing staining of GABAAR β3 in non-permeabilised 16-DIV hippocampal neurons (magenta). Cells co-express mRuby with NT-shRNA or GIT1shRNA, as indicated. Cell tracing was based on mRuby images. Cells were treated with 10 µM R18 for 24 h and 20 µM TAT or DJNKI-1 for 8 h. B. Quantification of GABAAR β3 enrichment in dendritic spines. R18 prevented DJNKI-1-induced GABAAR β3 increase at the dendritic spines. C. R18 prevented GIT1-S371A-induced increase in GABAAR β3 at the cell surface. Histogram bars represent means from ∼51 spines per condition from several neurons. P-values were calculated from Student’s two-tailed t-tests were compared to control (TAT) unless otherwise indicated. D. Neurons were co-transfected at 7 DIV with EGFP-Dynamin 2-WT or EGFP-Dynamin 2- K44A and treated with 20 µM TAT or DJNKI-1 for 8 h. Representative images are shown. E. Mean intensities for GABAAR β3 are shown from non-permeabilised cells in mushroom spines. GABAAR surface expression increased in DJNKI-1-treated neurons, even when the K44A mutant is expressed. Mean data +/- SEMs from ∼70 spines per condition are shown. p- values (calculated from Student’s two-tailed t test) were compared to control (TAT) unless otherwise indicated by brackets.

Article Snippet: For immunoblotting dilutions were as follows: 1:1000 paxillin (Bioscience), 1:500 GABAβ3 (Neuromab), 1:2000 GIT1 (Santa-Cruz), 1:1000 β-PIX (Bioscience), 1:1000 14-3-3ζ (Santa-Cruz), 1:2000 FLAG (Sigma Aldrich), 1:10,000 EGFP (JL-8, Clontech), 1: 2000 HA (Cell Signaling), 1:1000 JNK1 (Bioscience), 1:50,000 anti-rabbit and anti-mouse secondary HRP antibodies (Millipore).

Techniques: Staining, shRNA, Two Tailed Test, Control, Transfection, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: We propose that when JNK1 active it phosphorylates GIT1 on S371. This reduces levels of GABAAR at the cell surface, both at excitatory synapses and at extrasynapatic sites. Conversely when JNK is inhibited or Jnk1 genetically deleted, GIT1 associates with a paxillin and 14-33-containing receptor complex at the membrane. GIT1 phosphorylation by JNK on S371 dissociates it from binding to this complex, leading to run-down of receptor surface expression. The consequence for neuronal physiology is increased inhibitor post synaptic currents and increased tonic inhibitory current.

Article Snippet: For immunoblotting dilutions were as follows: 1:1000 paxillin (Bioscience), 1:500 GABAβ3 (Neuromab), 1:2000 GIT1 (Santa-Cruz), 1:1000 β-PIX (Bioscience), 1:1000 14-3-3ζ (Santa-Cruz), 1:2000 FLAG (Sigma Aldrich), 1:10,000 EGFP (JL-8, Clontech), 1: 2000 HA (Cell Signaling), 1:1000 JNK1 (Bioscience), 1:50,000 anti-rabbit and anti-mouse secondary HRP antibodies (Millipore).

Techniques: Membrane, Binding Assay, Expressing

Journal: Nature Communications

Article Title: GIT1 protects against breast cancer growth through negative regulation of Notch

doi: 10.1038/s41467-022-28631-y

Figure Lengend Snippet: a , b Immunostaining of GIT1 and ERα in one ER(+) and one ER(−) patient breast tumour section ( a ) and the quantitative analysis of GIT1 immunofluorescence relative to DAPI ( b ; ER(+) ( n = 45) versus ER(−) ( n = 30), P = 0.0006, t test). Scale bar, 10 μm. c Mass spectrometry data from CPTAC of the GIT1 protein levels in ER(+) and ER(−) patients (ER(+) ( n = 51) versus ER(−) ( n = 11), P = 0.024, t test). d , e , Western blots of GIT1 in various human breast cancer cells ( d ) and the quantitative analysis ( e ; n = 7 independent biological replicates, 184A1 versus BT474, P = 0.0004; MCF7, P = 0.0003; MDA-MB-134-VI, P = 0.015; MDA-MB-361, P < 0.0001; HCC1954, P = 0.017; MDA-MB-157, P = 0.023; MDA-MB-231, P = 0.0067; t tests). ER, oestrogen receptor. PR, progesterone receptor. HER2, human epidermal growth factor receptor 2. f Kaplan–Meier plot of relapse-free survival of the breast cancer patients from the TCGA database stratified by GIT1 expression ( n = 292, P = 0.0084, log-rank test). g – i Kaplan–Meier plots of relapse-free survival of all breast cancer (BC) patients ( g ; n = 3,310, P < 0.0001, log-rank test), ER(+) patients ( h ; n = 2527, P = 0.032, log-rank test), and ER(−) patients ( i ; n = 779, P = 0.0007, log-rank test) from KM-plotter stratified by GIT1 expression. Hazard ratios (HRs) and their 95% confidence intervals (95%CI) are indicated. j , Forest plot of HRs for survival analysis of all BC, ER(+), and ER(−) patients stratified by GIT1 expression (ER(+) ( n = 2527) versus ER(−) ( n = 779), P = 0.047, one-sided unpaired t test). Error bars show 95%CI. All data are shown as the mean ± s.e.m. For the box plots, the centre line shows the median, the plus sign shows the mean, the upper and lower boundaries of the box show the upper and lower quartiles, and the whiskers show the minimum and maximum values. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by two-sided unpaired t tests. Source data are provided as a Source Data file.

Article Snippet: Lentiviral transduction was performed with control shRNA (LV-Ctrl-shRNA, Cat. SHC002V, Sigma-Aldrich) and GIT1 shRNAs (LV-GIT1-shRNA3, Cat. TRCN0000008401; LV-GIT1-shRNA4, Cat. TRCN0000008403; Sigma-Aldrich) for one day. mRFP and mRFP-GIT1 lentiviral transduction particles were produced using the lentiviral package system: pCDF1-MC2_EF1Puro (Cat. CD110B-SBI, BioCat GmbH, Heidelberg, Germany), p.MD2.G (Cat. 12259, AddGene, Watertown, MA, USA), and ps.PAX2 (Cat. 12260, AddGene).

Techniques: Immunostaining, Immunofluorescence, Mass Spectrometry, Western Blot, Expressing

Journal: Nature Communications

Article Title: GIT1 protects against breast cancer growth through negative regulation of Notch

doi: 10.1038/s41467-022-28631-y

Figure Lengend Snippet: a Immunostaining of GIT1 and Notch1 ICD (N1ICD) in one ER(+) and one ER(−) breast cancer sample. Nuclei were detected using DAPI. Images from representative micrographs; the experiment was repeated n = 6 times for ER(+) samples and n = 4 times for ER(−) samples with similar results. Scale bar, 10 μm. b – d Luciferase reporter assays of 12xCSL-Luc ( b ; n = 5, Ctrl-siRNA versus DAPT, P < 0.0001; Ctrl-siRNA versus GIT1-siRNA1, P = 0.0038; one-way ANOVA, F 2,12 = 64.17, P < 0.0001 (shaded area); mRFP versus GIT1-mRFP, P < 0.0001; t tests) and western blots of Hey1 ( c ) and the quantitative analysis ( d ; n = 6, Ctrl-shRNA versus GIT1-shRNA2, P = 0.030; mRFP versus GIT1-mRFP; P = 0.0002; t tests) in MDA-MB-231 cells treated as indicated. e , f Volcano plot of differentially expressed genes for high and low GIT1 e ; n = 2509 patients) and a correlation analysis between GIT1 and ALDH1A1 mRNA expression ( f ; n = 2509 patients, Spearman ρ = −0.45, P < 0.0001) in breast cancer samples from the METABRIC database . Breast cancer stemness genes are indicated. g , h Flow cytometric analysis of Aldefluor-assayed MDA-MB-231 cells treated as indicated ( g ) and the quantitative analysis ( h ; n = 5, vehicle versus DAPT, P = 0.0014, t test; Ctrl-shRNA versus GIT1-shRNA2, P = 0.031; GIT1-shRNA2 versus GIT1-shRNA2 + DAPT, P = 0.040; Ctrl-shRNA + DAPT versus GIT1-shRNA2 + DAPT, P = 0.44; one-way ANOVA, F 3,16 = 7.216, P = 0.0028 (shaded area); mRFP versus GIT1-mRFP, P = 0.025, t test). SSC, side scatter. i Clonogenic assay of MDA-MB-231 cells treated as indicated ( n = 6, vehicle versus DAPT, P < 0.0001, t test; Ctrl-shRNA versus GIT1-shRNA2, P < 0.0001; GIT1-shRNA2 versus GIT1-shRNA2 + DAPT, P < 0.0001; Ctrl-shRNA + DAPT ( n = 3) versus GIT1-shRNA2 + DAPT ( n = 3), P = 1.00; one-way ANOVA, F 3,14 = 57.84, P < 0.0001 (shaded area); mRFP ( n = 5) versus GIT1-mRFP ( n = 6), P = 0.010, t test). j , k Quantitative analyses of Cyclin A ( j ; n = 3, vehicle versus DAPT, P = 0.036, t test; Ctrl-shRNA versus GIT1-shRNA2, P = 0.0032; GIT1-shRNA2 versus GIT1-shRNA2 + DAPT, P < 0.0001; Ctrl-shRNA + DAPT versus GIT1-shRNA2 + DAPT, P = 0.84; one-way ANOVA, F 3,8 = 32.06, P < 0.0001 (shaded area); mRFP versus GIT1-mRFP, P = 0.028, t test) and Cyclin B1 ( k ; n = 3, vehicle versus DAPT, P = 0.024, t test; Ctrl-shRNA versus GIT1-shRNA2, P = 0.049; GIT1-shRNA2 versus GIT1-shRNA2 + DAPT, P = 0.0022; Ctrl-shRNA + DAPT versus GIT1-shRNA2 + DAPT, P = 0.94; one-way ANOVA, F 3,8 = 15.92, P = 0.0010 (shaded area); mRFP versus GIT1-mRFP, P = 0.043, t test) from western blots of MDA-MB-231 cells treated as indicated. All data are shown as the mean ± s.e.m. n denotes the number of biologically independent replicates, unless stated otherwise. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant by two-sided unpaired t tests or one-way ANOVA with Tukey’s post hoc comparison. Source data are provided as a Source Data file.

Article Snippet: Lentiviral transduction was performed with control shRNA (LV-Ctrl-shRNA, Cat. SHC002V, Sigma-Aldrich) and GIT1 shRNAs (LV-GIT1-shRNA3, Cat. TRCN0000008401; LV-GIT1-shRNA4, Cat. TRCN0000008403; Sigma-Aldrich) for one day. mRFP and mRFP-GIT1 lentiviral transduction particles were produced using the lentiviral package system: pCDF1-MC2_EF1Puro (Cat. CD110B-SBI, BioCat GmbH, Heidelberg, Germany), p.MD2.G (Cat. 12259, AddGene, Watertown, MA, USA), and ps.PAX2 (Cat. 12260, AddGene).

Techniques: Immunostaining, Luciferase, Western Blot, shRNA, Expressing, Clonogenic Assay

Journal: Nature Communications

Article Title: GIT1 protects against breast cancer growth through negative regulation of Notch

doi: 10.1038/s41467-022-28631-y

Figure Lengend Snippet: a , b Co-IP of GIT1 with Notch1 from lysates of the indicated mouse organs ( a ) and human breast cell lines ( b ). c Co-IP of the Notch1-2 ICDs with GIT1 from lysates of the MDA-MB-231 cells treated as indicated. d , Schematic diagrams of recombinant fusion protein fragments. e , f GIT1 pulled down by GST Notch fragments from MDA-MB-231 cell lysates. g Pulldown of purified GIT1-His by purified GST-N1ICD/N. CB, Coomassie Blue. Images from representative blots; the experiment was repeated n = 3 times with similar results. *, indicates recombinant GST-fused peptides. Source data are provided as a Source Data file.

Article Snippet: Lentiviral transduction was performed with control shRNA (LV-Ctrl-shRNA, Cat. SHC002V, Sigma-Aldrich) and GIT1 shRNAs (LV-GIT1-shRNA3, Cat. TRCN0000008401; LV-GIT1-shRNA4, Cat. TRCN0000008403; Sigma-Aldrich) for one day. mRFP and mRFP-GIT1 lentiviral transduction particles were produced using the lentiviral package system: pCDF1-MC2_EF1Puro (Cat. CD110B-SBI, BioCat GmbH, Heidelberg, Germany), p.MD2.G (Cat. 12259, AddGene, Watertown, MA, USA), and ps.PAX2 (Cat. 12260, AddGene).

Techniques: Co-Immunoprecipitation Assay, Recombinant, Purification

Journal: Nature Communications

Article Title: GIT1 protects against breast cancer growth through negative regulation of Notch

doi: 10.1038/s41467-022-28631-y

Figure Lengend Snippet: a – c Quantitative analyses of Notch1 ( a ; n = 4, vehicle versus DAPT, P = 0.032; Ctrl-shRNA versus GIT1-shRNA2, P = 0.26; mRFP versus GIT1-mRFP, P = 0.40; t tests) and the Notch1 ICD (N1ICD) ( b ; n = 4, vehicle versus DAPT, P < 0.0001; Ctrl-shRNA versus GIT1-shRNA2, P = 0.67; mRFP versus GIT1-mRFP, P = 0.46; t tests) western blots ( c ) from lysates of MDA-MB-231 cells treated as indicated. d – f Quantitative analyses of cytosolic ( d ; n = 7, vehicle versus DAPT, P = 0.013; Ctrl-shRNA versus GIT1-shRNA2, P = 0.0045; mRFP versus GIT1-mRFP, P = 0.035; t tests) and nuclear ( e ; n = 7, vehicle versus DAPT, P = 0.0001; Ctrl-shRNA versus GIT1-shRNA2, P = 0.0042; mRFP versus GIT1-mRFP, P = 0.0025; t tests) subcellular fractionation assays with western blots ( f ) from MDA-MB-231 cells treated as indicated. g , h Confocal images ( g ) and quantitative analysis of EGFP intensities in the cytoplasm and nucleus ( h ; Notch1ΔE−EGFP ( n = 150 cells) versus: Notch1ΔE−EGFP + DAPT ( n = 79 cells), P < 0.0001; Notch1ΔE-mutNLS1+2−EGFP ( n = 180 cells), P < 0.0001; Notch1ΔE-mutNLS1+2−EGFP + DAPT ( n = 63 cells), P < 0.0001; one-way ANOVA, F 3,468 = 42.82, P < 0.0001) of MDA-MB-231 cells treated as indicated. Nuclei were detected using DAPI. i , j Luciferase reporter assays of Notch1 ICD (UAS-Luc) in MDA-MB-231 cells with knockdown or overexpression of GIT1 ( i ; n = 4, vehicle versus DAPT, P = 0.0019; Ctrl-siRNA versus GIT1-siRNA1, P = 0.020; mRFP versus GIT1-mRFP, P = 0.027; t tests) or mutated NLS1 and NLS2 ( j ; n = 7, Notch1ΔE-GVP versus: Notch1ΔE-GVP + DAPT, P < 0.0001; Notch1ΔE-mutNLS1+2-GVP, P < 0.0001; Notch1ΔE-mutNLS1+2-GVP + DAPT, P < 0.0001; one-way ANOVA, F 3,24 = 1828, P < 0.0001). Scale bar, 10 μm. All data are shown as the mean ± s.e.m. n denotes the number of biologically independent replicates, unless stated otherwise. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant by two-sided unpaired t tests or one-way ANOVA with Tukey’s post hoc comparison. Source data are provided as a Source Data file.

Article Snippet: Lentiviral transduction was performed with control shRNA (LV-Ctrl-shRNA, Cat. SHC002V, Sigma-Aldrich) and GIT1 shRNAs (LV-GIT1-shRNA3, Cat. TRCN0000008401; LV-GIT1-shRNA4, Cat. TRCN0000008403; Sigma-Aldrich) for one day. mRFP and mRFP-GIT1 lentiviral transduction particles were produced using the lentiviral package system: pCDF1-MC2_EF1Puro (Cat. CD110B-SBI, BioCat GmbH, Heidelberg, Germany), p.MD2.G (Cat. 12259, AddGene, Watertown, MA, USA), and ps.PAX2 (Cat. 12260, AddGene).

Techniques: shRNA, Western Blot, Fractionation, Luciferase, Over Expression

Journal: Nature Communications

Article Title: GIT1 protects against breast cancer growth through negative regulation of Notch

doi: 10.1038/s41467-022-28631-y

Figure Lengend Snippet: a – c , k Kaplan–Meier plots showing the percentage of tumour-free mice out of ten mice transplanted with MDA-MB-231 ( a , k ) or HCC1395 cells ( b , c ) stably expressing the indicated plasmids. All injection sites were assessed independently, and a tumour was defined as >100 mm 3 (MDA-MB-231) and >500 mm 3 (HCC1395). Comparisons of Kaplan–Meier curves, MDA-MB-231 implants: Ctrl-shRNA versus GIT1-mRFP, P < 0.0001; Ctrl-shRNA versus GIT1-shRNA2, P = 0.12 ( a ); HCC1395 implants: LV-mRFP versus LV-GIT1-mRFP, P = 0.012 ( b ); LV-Ctrl-shRNA versus LV-GIT1-shRNA3, P = 0.025 ( c ); MDA-MB-231 implants: Ctrl-shRNA versus DNMM1, P = 0.0038; Ctrl-shRNA versus GIT1-shRNA2 + DNMM1, P = 0.030; DNMM1 versus GIT1-shRNA2 + DNMM1, P = 0.58 ( k ); log-rank tests. d Scatter plot of tumour volume over time in the mice transplanted with HCC1395 cells expressing LV-GIT1-shRNA3 and LV-GIT1-mRFP. Time is days from the tumour volume >100 mm 3 . Solid line, exponential regression. Shaded area, 95% confidence bands. Comparison of growth rate constants k (Methods) for LV-GIT1-shRNA3 ( n = 290 tumour measurements, k = 0.057) and LV-GIT1-mRFP ( n = 440 tumour measurements, k = 0.023), P < 0.0001; F -test. e , f Tumour doubling time ( e ) and volume change ( f ) for LV-GIT1-shRNA3 versus LV-Ctrl-shRNA and LV-GIT1-mRFP versus LV-mRFP. Solid line, exponential regression. Shaded area, 95% confidence interval. g , j Flow cytometric analysis of ALDH1A1+ cells ( g ; n = 4, Ctrl-shRNA versus GIT1-shRNA2, P = 0.0090, t test; Ctrl-shRNA versus GIT1-shRNA2, P = 0.019; Ctrl-shRNA versus DNMM1, P = 0.018; GIT1-shRNA2 + DNMM1 versus DNMM1, P = 1.00; one-way ANOVA, F 2,9 = 7.877, P = 0.011 (shaded area)) and Hey1+ cells ( j , n = 4, Ctrl-shRNA versus GIT1-shRNA2, P = 0.012, t test; Ctrl-shRNA versus GIT1-shRNA2, P = 0.0063; Ctrl-shRNA versus DNMM1, P = 0.0059; GIT1-shRNA2 + DNMM1 versus DNMM1, P = 1.00; one-way ANOVA, F 2,9 = 11.61, P = 0.0032 (shaded area)) in MDA-MB-231 xenograft tumours. h , i Confocal images of the Notch1 ICD (N1ICD)-immunolabelled MDA-MB-231 xenograft tumours ( h ) and quantitative analysis ( i ; n = 8 tumours, P = 0.0004, t test). Nuclei were detected using DAPI. Scale bar, 5 μm. All data are shown as the mean ± s.e.m. n denotes the number of biologically independent replicates, unless stated otherwise. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant by two-sided unpaired t test, F -test, or one-way ANOVA with Tukey’s post hoc comparison. Source data are provided as a Source Data file.

Article Snippet: Lentiviral transduction was performed with control shRNA (LV-Ctrl-shRNA, Cat. SHC002V, Sigma-Aldrich) and GIT1 shRNAs (LV-GIT1-shRNA3, Cat. TRCN0000008401; LV-GIT1-shRNA4, Cat. TRCN0000008403; Sigma-Aldrich) for one day. mRFP and mRFP-GIT1 lentiviral transduction particles were produced using the lentiviral package system: pCDF1-MC2_EF1Puro (Cat. CD110B-SBI, BioCat GmbH, Heidelberg, Germany), p.MD2.G (Cat. 12259, AddGene, Watertown, MA, USA), and ps.PAX2 (Cat. 12260, AddGene).

Techniques: Stable Transfection, Expressing, Injection, shRNA

Journal: Nature Communications

Article Title: GIT1 protects against breast cancer growth through negative regulation of Notch

doi: 10.1038/s41467-022-28631-y

Figure Lengend Snippet: a , b Immunostaining of GIT1 and ERα in one ER(+) and one ER(−) patient breast tumour section ( a ) and the quantitative analysis of GIT1 immunofluorescence relative to DAPI ( b ; ER(+) ( n = 45) versus ER(−) ( n = 30), P = 0.0006, t test). Scale bar, 10 μm. c Mass spectrometry data from CPTAC of the GIT1 protein levels in ER(+) and ER(−) patients (ER(+) ( n = 51) versus ER(−) ( n = 11), P = 0.024, t test). d , e , Western blots of GIT1 in various human breast cancer cells ( d ) and the quantitative analysis ( e ; n = 7 independent biological replicates, 184A1 versus BT474, P = 0.0004; MCF7, P = 0.0003; MDA-MB-134-VI, P = 0.015; MDA-MB-361, P < 0.0001; HCC1954, P = 0.017; MDA-MB-157, P = 0.023; MDA-MB-231, P = 0.0067; t tests). ER, oestrogen receptor. PR, progesterone receptor. HER2, human epidermal growth factor receptor 2. f Kaplan–Meier plot of relapse-free survival of the breast cancer patients from the TCGA database stratified by GIT1 expression ( n = 292, P = 0.0084, log-rank test). g – i Kaplan–Meier plots of relapse-free survival of all breast cancer (BC) patients ( g ; n = 3,310, P < 0.0001, log-rank test), ER(+) patients ( h ; n = 2527, P = 0.032, log-rank test), and ER(−) patients ( i ; n = 779, P = 0.0007, log-rank test) from KM-plotter stratified by GIT1 expression. Hazard ratios (HRs) and their 95% confidence intervals (95%CI) are indicated. j , Forest plot of HRs for survival analysis of all BC, ER(+), and ER(−) patients stratified by GIT1 expression (ER(+) ( n = 2527) versus ER(−) ( n = 779), P = 0.047, one-sided unpaired t test). Error bars show 95%CI. All data are shown as the mean ± s.e.m. For the box plots, the centre line shows the median, the plus sign shows the mean, the upper and lower boundaries of the box show the upper and lower quartiles, and the whiskers show the minimum and maximum values. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by two-sided unpaired t tests. Source data are provided as a Source Data file.

Article Snippet: Transfection of pRL-TK vector (Cat. E2241, Promega, Madison, WI, USA), GIT1 siRNA (Cat. HSS178932, Invitrogen), control siRNA (Cat. 45–2001, Invitrogen), GIT1 shRNA (Cat. sc-35477-SH, Santa Cruz Biotechnology), and control shRNA (Cat. sc-108066, Santa Cruz Biotechnology) was performed as described previously .

Techniques: Immunostaining, Immunofluorescence, Mass Spectrometry, Western Blot, Expressing

Journal: Nature Communications

Article Title: GIT1 protects against breast cancer growth through negative regulation of Notch

doi: 10.1038/s41467-022-28631-y

Figure Lengend Snippet: a Immunostaining of GIT1 and Notch1 ICD (N1ICD) in one ER(+) and one ER(−) breast cancer sample. Nuclei were detected using DAPI. Images from representative micrographs; the experiment was repeated n = 6 times for ER(+) samples and n = 4 times for ER(−) samples with similar results. Scale bar, 10 μm. b – d Luciferase reporter assays of 12xCSL-Luc ( b ; n = 5, Ctrl-siRNA versus DAPT, P < 0.0001; Ctrl-siRNA versus GIT1-siRNA1, P = 0.0038; one-way ANOVA, F 2,12 = 64.17, P < 0.0001 (shaded area); mRFP versus GIT1-mRFP, P < 0.0001; t tests) and western blots of Hey1 ( c ) and the quantitative analysis ( d ; n = 6, Ctrl-shRNA versus GIT1-shRNA2, P = 0.030; mRFP versus GIT1-mRFP; P = 0.0002; t tests) in MDA-MB-231 cells treated as indicated. e , f Volcano plot of differentially expressed genes for high and low GIT1 e ; n = 2509 patients) and a correlation analysis between GIT1 and ALDH1A1 mRNA expression ( f ; n = 2509 patients, Spearman ρ = −0.45, P < 0.0001) in breast cancer samples from the METABRIC database . Breast cancer stemness genes are indicated. g , h Flow cytometric analysis of Aldefluor-assayed MDA-MB-231 cells treated as indicated ( g ) and the quantitative analysis ( h ; n = 5, vehicle versus DAPT, P = 0.0014, t test; Ctrl-shRNA versus GIT1-shRNA2, P = 0.031; GIT1-shRNA2 versus GIT1-shRNA2 + DAPT, P = 0.040; Ctrl-shRNA + DAPT versus GIT1-shRNA2 + DAPT, P = 0.44; one-way ANOVA, F 3,16 = 7.216, P = 0.0028 (shaded area); mRFP versus GIT1-mRFP, P = 0.025, t test). SSC, side scatter. i Clonogenic assay of MDA-MB-231 cells treated as indicated ( n = 6, vehicle versus DAPT, P < 0.0001, t test; Ctrl-shRNA versus GIT1-shRNA2, P < 0.0001; GIT1-shRNA2 versus GIT1-shRNA2 + DAPT, P < 0.0001; Ctrl-shRNA + DAPT ( n = 3) versus GIT1-shRNA2 + DAPT ( n = 3), P = 1.00; one-way ANOVA, F 3,14 = 57.84, P < 0.0001 (shaded area); mRFP ( n = 5) versus GIT1-mRFP ( n = 6), P = 0.010, t test). j , k Quantitative analyses of Cyclin A ( j ; n = 3, vehicle versus DAPT, P = 0.036, t test; Ctrl-shRNA versus GIT1-shRNA2, P = 0.0032; GIT1-shRNA2 versus GIT1-shRNA2 + DAPT, P < 0.0001; Ctrl-shRNA + DAPT versus GIT1-shRNA2 + DAPT, P = 0.84; one-way ANOVA, F 3,8 = 32.06, P < 0.0001 (shaded area); mRFP versus GIT1-mRFP, P = 0.028, t test) and Cyclin B1 ( k ; n = 3, vehicle versus DAPT, P = 0.024, t test; Ctrl-shRNA versus GIT1-shRNA2, P = 0.049; GIT1-shRNA2 versus GIT1-shRNA2 + DAPT, P = 0.0022; Ctrl-shRNA + DAPT versus GIT1-shRNA2 + DAPT, P = 0.94; one-way ANOVA, F 3,8 = 15.92, P = 0.0010 (shaded area); mRFP versus GIT1-mRFP, P = 0.043, t test) from western blots of MDA-MB-231 cells treated as indicated. All data are shown as the mean ± s.e.m. n denotes the number of biologically independent replicates, unless stated otherwise. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant by two-sided unpaired t tests or one-way ANOVA with Tukey’s post hoc comparison. Source data are provided as a Source Data file.

Article Snippet: Transfection of pRL-TK vector (Cat. E2241, Promega, Madison, WI, USA), GIT1 siRNA (Cat. HSS178932, Invitrogen), control siRNA (Cat. 45–2001, Invitrogen), GIT1 shRNA (Cat. sc-35477-SH, Santa Cruz Biotechnology), and control shRNA (Cat. sc-108066, Santa Cruz Biotechnology) was performed as described previously .

Techniques: Immunostaining, Luciferase, Western Blot, shRNA, Expressing, Clonogenic Assay, Comparison

Journal: Nature Communications

Article Title: GIT1 protects against breast cancer growth through negative regulation of Notch

doi: 10.1038/s41467-022-28631-y

Figure Lengend Snippet: a , b Co-IP of GIT1 with Notch1 from lysates of the indicated mouse organs ( a ) and human breast cell lines ( b ). c Co-IP of the Notch1-2 ICDs with GIT1 from lysates of the MDA-MB-231 cells treated as indicated. d , Schematic diagrams of recombinant fusion protein fragments. e , f GIT1 pulled down by GST Notch fragments from MDA-MB-231 cell lysates. g Pulldown of purified GIT1-His by purified GST-N1ICD/N. CB, Coomassie Blue. Images from representative blots; the experiment was repeated n = 3 times with similar results. *, indicates recombinant GST-fused peptides. Source data are provided as a Source Data file.

Article Snippet: Transfection of pRL-TK vector (Cat. E2241, Promega, Madison, WI, USA), GIT1 siRNA (Cat. HSS178932, Invitrogen), control siRNA (Cat. 45–2001, Invitrogen), GIT1 shRNA (Cat. sc-35477-SH, Santa Cruz Biotechnology), and control shRNA (Cat. sc-108066, Santa Cruz Biotechnology) was performed as described previously .

Techniques: Co-Immunoprecipitation Assay, Recombinant, Purification

Journal: Nature Communications

Article Title: GIT1 protects against breast cancer growth through negative regulation of Notch

doi: 10.1038/s41467-022-28631-y

Figure Lengend Snippet: a – c Quantitative analyses of Notch1 ( a ; n = 4, vehicle versus DAPT, P = 0.032; Ctrl-shRNA versus GIT1-shRNA2, P = 0.26; mRFP versus GIT1-mRFP, P = 0.40; t tests) and the Notch1 ICD (N1ICD) ( b ; n = 4, vehicle versus DAPT, P < 0.0001; Ctrl-shRNA versus GIT1-shRNA2, P = 0.67; mRFP versus GIT1-mRFP, P = 0.46; t tests) western blots ( c ) from lysates of MDA-MB-231 cells treated as indicated. d – f Quantitative analyses of cytosolic ( d ; n = 7, vehicle versus DAPT, P = 0.013; Ctrl-shRNA versus GIT1-shRNA2, P = 0.0045; mRFP versus GIT1-mRFP, P = 0.035; t tests) and nuclear ( e ; n = 7, vehicle versus DAPT, P = 0.0001; Ctrl-shRNA versus GIT1-shRNA2, P = 0.0042; mRFP versus GIT1-mRFP, P = 0.0025; t tests) subcellular fractionation assays with western blots ( f ) from MDA-MB-231 cells treated as indicated. g , h Confocal images ( g ) and quantitative analysis of EGFP intensities in the cytoplasm and nucleus ( h ; Notch1ΔE−EGFP ( n = 150 cells) versus: Notch1ΔE−EGFP + DAPT ( n = 79 cells), P < 0.0001; Notch1ΔE-mutNLS1+2−EGFP ( n = 180 cells), P < 0.0001; Notch1ΔE-mutNLS1+2−EGFP + DAPT ( n = 63 cells), P < 0.0001; one-way ANOVA, F 3,468 = 42.82, P < 0.0001) of MDA-MB-231 cells treated as indicated. Nuclei were detected using DAPI. i , j Luciferase reporter assays of Notch1 ICD (UAS-Luc) in MDA-MB-231 cells with knockdown or overexpression of GIT1 ( i ; n = 4, vehicle versus DAPT, P = 0.0019; Ctrl-siRNA versus GIT1-siRNA1, P = 0.020; mRFP versus GIT1-mRFP, P = 0.027; t tests) or mutated NLS1 and NLS2 ( j ; n = 7, Notch1ΔE-GVP versus: Notch1ΔE-GVP + DAPT, P < 0.0001; Notch1ΔE-mutNLS1+2-GVP, P < 0.0001; Notch1ΔE-mutNLS1+2-GVP + DAPT, P < 0.0001; one-way ANOVA, F 3,24 = 1828, P < 0.0001). Scale bar, 10 μm. All data are shown as the mean ± s.e.m. n denotes the number of biologically independent replicates, unless stated otherwise. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant by two-sided unpaired t tests or one-way ANOVA with Tukey’s post hoc comparison. Source data are provided as a Source Data file.

Article Snippet: Transfection of pRL-TK vector (Cat. E2241, Promega, Madison, WI, USA), GIT1 siRNA (Cat. HSS178932, Invitrogen), control siRNA (Cat. 45–2001, Invitrogen), GIT1 shRNA (Cat. sc-35477-SH, Santa Cruz Biotechnology), and control shRNA (Cat. sc-108066, Santa Cruz Biotechnology) was performed as described previously .

Techniques: shRNA, Western Blot, Fractionation, Luciferase, Knockdown, Over Expression, Comparison

Journal: Nature Communications

Article Title: GIT1 protects against breast cancer growth through negative regulation of Notch

doi: 10.1038/s41467-022-28631-y

Figure Lengend Snippet: a – c , k Kaplan–Meier plots showing the percentage of tumour-free mice out of ten mice transplanted with MDA-MB-231 ( a , k ) or HCC1395 cells ( b , c ) stably expressing the indicated plasmids. All injection sites were assessed independently, and a tumour was defined as >100 mm 3 (MDA-MB-231) and >500 mm 3 (HCC1395). Comparisons of Kaplan–Meier curves, MDA-MB-231 implants: Ctrl-shRNA versus GIT1-mRFP, P < 0.0001; Ctrl-shRNA versus GIT1-shRNA2, P = 0.12 ( a ); HCC1395 implants: LV-mRFP versus LV-GIT1-mRFP, P = 0.012 ( b ); LV-Ctrl-shRNA versus LV-GIT1-shRNA3, P = 0.025 ( c ); MDA-MB-231 implants: Ctrl-shRNA versus DNMM1, P = 0.0038; Ctrl-shRNA versus GIT1-shRNA2 + DNMM1, P = 0.030; DNMM1 versus GIT1-shRNA2 + DNMM1, P = 0.58 ( k ); log-rank tests. d Scatter plot of tumour volume over time in the mice transplanted with HCC1395 cells expressing LV-GIT1-shRNA3 and LV-GIT1-mRFP. Time is days from the tumour volume >100 mm 3 . Solid line, exponential regression. Shaded area, 95% confidence bands. Comparison of growth rate constants k (Methods) for LV-GIT1-shRNA3 ( n = 290 tumour measurements, k = 0.057) and LV-GIT1-mRFP ( n = 440 tumour measurements, k = 0.023), P < 0.0001; F -test. e , f Tumour doubling time ( e ) and volume change ( f ) for LV-GIT1-shRNA3 versus LV-Ctrl-shRNA and LV-GIT1-mRFP versus LV-mRFP. Solid line, exponential regression. Shaded area, 95% confidence interval. g , j Flow cytometric analysis of ALDH1A1+ cells ( g ; n = 4, Ctrl-shRNA versus GIT1-shRNA2, P = 0.0090, t test; Ctrl-shRNA versus GIT1-shRNA2, P = 0.019; Ctrl-shRNA versus DNMM1, P = 0.018; GIT1-shRNA2 + DNMM1 versus DNMM1, P = 1.00; one-way ANOVA, F 2,9 = 7.877, P = 0.011 (shaded area)) and Hey1+ cells ( j , n = 4, Ctrl-shRNA versus GIT1-shRNA2, P = 0.012, t test; Ctrl-shRNA versus GIT1-shRNA2, P = 0.0063; Ctrl-shRNA versus DNMM1, P = 0.0059; GIT1-shRNA2 + DNMM1 versus DNMM1, P = 1.00; one-way ANOVA, F 2,9 = 11.61, P = 0.0032 (shaded area)) in MDA-MB-231 xenograft tumours. h , i Confocal images of the Notch1 ICD (N1ICD)-immunolabelled MDA-MB-231 xenograft tumours ( h ) and quantitative analysis ( i ; n = 8 tumours, P = 0.0004, t test). Nuclei were detected using DAPI. Scale bar, 5 μm. All data are shown as the mean ± s.e.m. n denotes the number of biologically independent replicates, unless stated otherwise. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant by two-sided unpaired t test, F -test, or one-way ANOVA with Tukey’s post hoc comparison. Source data are provided as a Source Data file.

Article Snippet: Transfection of pRL-TK vector (Cat. E2241, Promega, Madison, WI, USA), GIT1 siRNA (Cat. HSS178932, Invitrogen), control siRNA (Cat. 45–2001, Invitrogen), GIT1 shRNA (Cat. sc-35477-SH, Santa Cruz Biotechnology), and control shRNA (Cat. sc-108066, Santa Cruz Biotechnology) was performed as described previously .

Techniques: Stable Transfection, Expressing, Injection, shRNA, Comparison