Journal: iScience

Article Title: Roles of class III phosphatidylinositol 3-kinase, Vps34, in phagocytosis, autophagy, and endocytosis in retinal pigmented epithelium

doi: 10.1016/j.isci.2025.112371

Figure Lengend Snippet: Identification and phosphoinositide contents of autophagy-associated membrane aggregates in chloroquine-treated cultured RPE cells (A–L) In cultured RPE cells treated with 25 μg/mL chloroquine overnight, autophagy marker LC3 accumulated (A–D and H) and co-localized with WIPI2 (C), WIPI1 (D), and Atg9 (H). In these cells p62 and Atg16L co-localized with WIPI2 (E–F), and Atg9 co-localized with p62 (G). PI(3)P was observed in membranes surrounding, but not truly co-localized with, LC3 puncta (B). Probes for PI(4)P (EGFP-FAPP1, (I)), PI(5)P (DsRed-3xING2, (J)), and PI(3,4)P2 (DsRed-TAPP1, (K)) did not co-localize with LC3. A probe for PI(4,5)P2 (EGFP-PLCδ, (L)) was partially found in membranes surrounding LC3 puncta. Line profiles of intensity for the indicated puncta ( insets ) are plotted on the far right . Arrows indicate puncta visible in more than one channel for alignment and comparison in adjacent panels.

Article Snippet: Two tandem copies of Hrs, FAPP1, or TAPP1, three tandem copies of ING2, or one copy of PLCδ phosphoinositide-binding domains, or WIPI1 or WIPI2 cDNA were cloned with N-terminal EGFP or DsRed fusions in pCAG vector (a gift from Connie Cepko (Addgene plasmid #11150)) Matsuda & Cepko, using EcoRI and NotI sites. All constructs were verified by sequencing. , Addgene , #11150.

Techniques: Membrane, Cell Culture, Marker, Comparison

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A and B ) Affibody-chase experiments. Cells surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP) to stimulate α V β 6 integrin and trigger α V β 6 endocytosis, or vehicle (Control), 0- to 60-min time course. Quantitation represents cytoplasmic HER2 fluorescence intensity analysis in (A) trastuzumab-sensitive or (B) trastuzumab-resistant BT474 cells ( N = 3; 27 to 50 cells per condition), normalized to control trastuzumab-sensitive BT474 cells (0 min); scale bar, 10 μm. Two-way ANOVA with Šídák’s multiple comparison test. Image intensity increased in (B), relative to (A), due to low cell surface HER2 levels in trastuzumab-resistant cells to highlight internalization differences. ( C ) HER2 (green) and RAB5 (magenta) immunofluorescence in trastuzumab-sensitive and trastuzumab-resistant BT474 cells, treated with soluble LAP, 0 to 60 min ( N = 3; 16 to 28 cells per condition); scale bar, 10 μm. ( Ca ) HER2/RAB5 colocalization quantitation (Pearson’s coefficient ± SEM). Two-way ANOVA with Dunnett’s multiple comparison test. ( D ) Active RAB5 pull-down assays. 0- to 60-min LAP stimulation time course. Quantitation of mean RAB5 activity (pull-down eluate), relative to total RAB5 (lysate) ± SEM ( N = 3), normalized to 0-min trastuzumab-sensitive cells. One-way ANOVA with Dunnett’s multiple comparison test. ( E and F ) Affibody-chase experiments in (E) siControl Trastuzumab-Sensitive or (F) Trastuzumab-Resistant BT474 cells expressing constitutively active RAB5 (RAB5CA), dominant-negative RAB5 (RAB5DN), dominant-negative RAB7 (RAB7DN), or mCherry vector control. Cells were surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP), or vehicle control (control), for 0 or 30 min. Quantitation represents cytoplasmic HER2 fluorescence intensity ( N = 3; 81 to 87 cells per condition); scale bar, 10 μm. One-way ANOVA with Tukey’s multiple comparison test. Representative images in fig. S10 (A and B). Further HER2 internalization analyses: Supplementary Results and fig. S11 (A to D). [(A), (B), and (D) to (F)] Data are arbitrary units (AU) normalized to control means ± SEM. [(A) to (F)] Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: For protein expression, cells were transfected with DNA (1 μg/ml): constitutively active RAB5 ( ) [mcherry-RAB5CA(Q79L), Addgene plasmid #35138], dominant-negative RAB5 [mCherry-RAB5DN(S34N), Addgene plasmid #35139] , dominant-negative RAB7 [DsRed-RAB7 DN(T22N), Addgene plasmid #12662] , or empty pmCherry-C1 vector (Clontech, Addgene plasmid #3552).

Techniques: Labeling, Control, Quantitation Assay, Fluorescence, Comparison, Immunofluorescence, Activity Assay, Expressing, Dominant Negative Mutation, Plasmid Preparation

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A ) Trastuzumab-Sensitive Cells: GDI2 is recruited to sites proximal to α V β 6 IACs and coordinates HER2 and α V β 6 trafficking and signaling by locally modulating RAB5 activity. GDI2-mediated cross-talk between α V β 6 and HER2 affects membrane availability of both receptors, ultimately influencing migration, invasion, and TGFβ activation. ( B ) Trastuzumab-Resistant Cells: GDI2 is excluded from α V β 6 IACs, leading to dysregulation of RAB5 activation dynamics, followed by increased RAB7 activation. Consequently, HER2/α V β 6 cross-talk is impaired, altering receptor trafficking dynamics and disrupting bioavailability of both HER2 and α V β 6 integrin at the plasma membrane. This dysregulation further affects TGFβ activation, resulting in increased cell invasiveness and metastatic potential. Overall, these changes may increase the ability of cells to evade HER2 targeting drugs.

Article Snippet: For protein expression, cells were transfected with DNA (1 μg/ml): constitutively active RAB5 ( ) [mcherry-RAB5CA(Q79L), Addgene plasmid #35138], dominant-negative RAB5 [mCherry-RAB5DN(S34N), Addgene plasmid #35139] , dominant-negative RAB7 [DsRed-RAB7 DN(T22N), Addgene plasmid #12662] , or empty pmCherry-C1 vector (Clontech, Addgene plasmid #3552).

Techniques: Activity Assay, Membrane, Migration, Activation Assay, Clinical Proteomics

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A ) Differential gene expression data (RNA-seq) for the GDI2 / RAB5A / RAB7A / ERBB2 / ITGB6 cluster in normal breast tissue ( n = 403; light gray) and breast invasive carcinoma ( n = 1097; dark gray). Data were extracted from the TNMplot database ( tnmplot.com ). Black lines in violin blots represent the median. Mann-Whitney test. ( B ) Volcano plot showing statistical analysis (ANOVA) of RNA-seq gene expression data of patients with HER2+ breast cancer from the METABRIC cohort expressing high (Right) and low (Left) levels of ITGB6 (Q1 versus Q4). Significant genes (dark gray); nonsignificant genes (light gray); relevant genes are highlighted in purple. ( C ) Visual representation of GO terms analysis (ClueGO, cellular compartment) of genes highly and significantly expressed in tumors expressing high levels of ITGB6 (Q4). Colors represent specific merged GO term groups, node size represents the level of significance of each GO term, and clustering and edge length represent functionally grouped networks based on kappa score. ( D ) OS of patients with HER2+ breast cancer and with high (above median) expression of ITGB6 , expressing high (red) or low (black) levels of GDI2 , ERBB2 , RAB5A , and RAB7A . ( E and F ) Differential ITGB6 gene expression (gene chip) in patients with HER2+ breast cancer subdivided according to therapeutic response to trastuzumab. (E) Initial pathological complete response (responder) versus residual disease after completing therapy (nonresponder) ( n = 77 patients). (F) RFS at 5 years (responder) versus samples relapsed before 5 years (nonresponder) ( n = 24 patients). Two-sided Student’s t test. [(A), (E), and (F)] Statistical significance: * P < 0.05; **** P < 0.0001.

Article Snippet: For protein expression, cells were transfected with DNA (1 μg/ml): constitutively active RAB5 ( ) [mcherry-RAB5CA(Q79L), Addgene plasmid #35138], dominant-negative RAB5 [mCherry-RAB5DN(S34N), Addgene plasmid #35139] , dominant-negative RAB7 [DsRed-RAB7 DN(T22N), Addgene plasmid #12662] , or empty pmCherry-C1 vector (Clontech, Addgene plasmid #3552).

Techniques: Gene Expression, RNA Sequencing, MANN-WHITNEY, Expressing, Clinical Proteomics

Journal: Nature Communications

Article Title: Cre-Controlled CRISPR mutagenesis provides fast and easy conditional gene inactivation in zebrafish

doi: 10.1038/s41467-021-21427-6

Figure Lengend Snippet: a Scheme of the 3C rationale. A Cre effector construct controls the expression of a floxed Stop cassette upstream of the sequence encoding a fusion protein of Cas9 and GFP. In addition, a U6a promoter drives the constitutive expression of a gRNA targeting a gene of interest (GOI). Following exogenous or transgenic Cre supply, site-specific recombination results in the expression of Cas9-GFP. Combined with the gRNA a functional CRISPR complex is formed and mutates the target site within the gene of interest. b Scheme of the 3C gene inactivation construct targeting tyrosinase ( tyr ). The temperature-inducible hsp70l promotor drives expression of a floxed DsRed cassette. c Identification of transgenic animals expressing DsRed at 50 hpf after a heat treatment at 24 hpf. Example shown is a representative of a total of >100 heat-treated clutches from four independent 3C tyr transgenic insertions. Scale bar: 1000 µm.

Article Snippet: The fragment was subsequently ligated into pTol hsp70l:loxP-DsRed-GFP digested with SmaI and NheI replacing GFP and giving rise to pTol hsp70l:loxP-DsRed-loxP-Cas9-GFP (addgene #158962).

Techniques: Construct, Expressing, Sequencing, Transgenic Assay, Functional Assay, CRISPR

Journal: iScience

Article Title: Fate plasticity of interneuron specification

doi: 10.1016/j.isci.2025.112295

Figure Lengend Snippet:

Article Snippet: Chicken anti-RFP (Red Fluorescent Protein) , Rockland Immunochemicals , Cat# 600-901-379; RRID: AB_10704808.

Techniques: Virus, Plasmid Preparation, Recombinant, Membrane, Imaging, RNA Sequencing, Software, Cell Culture, Modification, Knock-Out, Passaging

Journal: Cell Metabolism

Article Title: Mitochondria-Endoplasmic Reticulum Contacts in Reactive Astrocytes Promote Vascular Remodeling

doi: 10.1016/j.cmet.2020.03.005

Figure Lengend Snippet: Key Resources Table

Article Snippet: rabbit polyclonal anti-RFP , Rockland , #600401379; RRID: AB_2209751.

Techniques: Microscopy, Imaging, Recombinant, Mass Spectrometry, Plasmid Preparation, Software

Journal: Frontiers in Immunology

Article Title: Impaired tissue homing by the Ikzf3 N159S variant is mediated by interfering with Ikaros function

doi: 10.3389/fimmu.2023.1239779

Figure Lengend Snippet: Overexpression of Ikaros restored CD62L expression in Ikzf3 N159S/N159S B cells. (A) Immunoblot showing expression of Ikaros and Aiolos protein in packaging Plate E cell line transfected with empty vector (EV), pMigR1-Ikaros or pMigR1-Aiolos vector. (B) Zebra plots showing CD62L expression of retrovirally transduced Ikzf3 +/+ and Ikzf3 N159S/N159S B cells. DsRED and GFP fluorescent marker expression indicate Ikaros and Aiolos expression, respectively. Lower histograms showing CD62L expression on DsRED + or GFP + transduced cells. Numerical values in each histogram indicate percentages of each region. (C) Graphs show the percentage of CD62L + in DsRED + or GFP + subset. Each circle represents data from one experiment. Data are presented as the mean ± SD, n = 3 per group. Student’ s unpaired, two-tailed t test). * P < 0.05.

Article Snippet: After confirming the sequence, the cDNA fragment was cloned into the pMigR-DsRED plasmid, which was generated by ourselves via replacing the GFP sequence with DsRED in the pMigR1 plasmid (Addgene Plasmid #27490).

Techniques: Over Expression, Expressing, Western Blot, Transfection, Plasmid Preparation, Marker, Two Tailed Test