Journal:

Article Title: RPA is an initiation factor for human chromosomal DNA replication

doi:

Figure Lengend Snippet: RPA is recruited to DNA replication foci and becomes phosphorylated in vitro. (A) Comparison of bound RPA in G1 and S phase nuclei in vivo. Nuclear extracts (25 µg protein per lane) from mimosine- arrested late G1 phase and from early S phase cells were analysed by western blot using antibodies specific for MCM5 as a control and polyclonal antibody pAb-RPA1. (B) Nuclear binding and phosphorylation of RPA during initiation of DNA replication in vitro. G1 phase nuclei isolated from in vitro incubations done in replication buffer (lane 1), 100 µg S100 cytosolic extract (lane 2), 30 and 100 ng rhRPA (lanes 3 and 4) and 100 ng rhRPA plus 35 µg of fraction QB (lane 5). The samples shown in the right-hand panel were treated with λ phosphatase before loading onto the same gel. The Rpa70 and Rpa32 subunits were visualised with pAb-RPA1. Phosphorylated Rpa32 is indicated as pRpa32. (C) Confocal microscopy. G1 phase nuclei were incubated in buffer (top row), in 100 ng of rhRPA (second row), in 100 ng rhRPA supplemented with 35 µg QB (third row) and in 100 µg of unfractionated S100 (bottom row). High-resolution micrographs are shown of RPA foci (stained with pAb-RPA1, red), replication foci (dig-UTP, green) and merged images of the same set of nuclei.

Article Snippet: Phosphatase treatments were performed after the PBS wash by resuspending the nuclei in 20 µl of λ phosphatase buffer containing 2 mM MnCl 2 and incubating for 30 min at 30°C in the presence of 200 U λ phosphatase (NEB).

Techniques: In Vitro, In Vivo, Western Blot, Binding Assay, Isolation, Confocal Microscopy, Incubation, Staining

Journal: The Journal of Biological Chemistry

Article Title: Chemical Proteomics Identifies Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1 as the Molecular Target of Quercetin in Its Anti-cancer Effects in PC-3 Cells *

doi: 10.1074/jbc.M114.553248

Figure Lengend Snippet: Schematic depiction of the workflow used to identify and characterize quercetin-binding proteins. Quercetin-specific binding proteins were captured by quercetin-Sepharose beads, and eluted fractions were resolved by SDS-PAGE. Distinct proteins in gel-eluted bands were identified using MS and validated by immunoblotting analyses and surface plasmon resonance binding assays. Specific targets were further characterized using a series of approaches, including confocal microscopy, IP, RIP, RT-qPCR, and immunoblotting analysis. Q, quercetin; T, total cell lysates; W, proteins that did not bind quercetin; E, bound proteins eluted.

Article Snippet: Quercetin was from Sigma-Aldrich, and CNBr-Sepharose 4B was obtained from GE Healthcare.

Techniques: Binding Assay, SDS Page, Western Blot, SPR Assay, Confocal Microscopy, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Location of missense and nonsense mutations in SLC9A6/NHE6 variants of patients with Christianson syndrome. A, schematic planar drawing of the predicted membrane topology of the longest splice-variant of mammalian NHE6 and location of the mutations (yellow circles). Two consensus N-linked glycosylation sites (128NVT and 145NVS) within extracellular loop 2 have been verified experimentally (data not shown) and are illustrated in the drawing. B, phylogenetic comparison of the primary sequence of segments containing the various mutations in NHE6. The affected residues are shaded in black.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Membrane, Variant Assay, Glycoproteomics, Comparison, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Structure homology modeling of transmembrane NHE6 variants associated with Christianson syndrome. A, front (membrane aspect, left panel) and top (extracellular aspect, right panel) views of a 3D structure homology model of dimeric human NHE6 based on the crystal structure of the bacterial T. thermophilus Na+/H+ antiporter NapA (TtNapA) (Protein Data Bank accession code 5bz3; 2.30 Å, 15% identity, 27% similarity), which provided the broadest coverage, highest resolution, and best spatial fit compared with other crystallized bacterial Na+/H+ antiporters. The proposed structure includes only the membrane-spanning helices (M2–M12; amino acids 74–540) that aligned with homologous segments of TtNapA. The top view includes the locations of the transmembrane-localized residues Leu-188 and Gly-383 mutated in CS. B, molecular dynamics simulation of structural changes predicted to occur in TM4 (highlighted in cyan) upon substitution of Leu-188 with Pro (L188P). The monomeric forms of NHE6 WT and L188P are illustrated. C and D, structural perturbations predicted to occur in the intramembranous re-entrant (R) loop between helices M8 and M9 upon substitution of Gly-383 with Asp (G383D). C, upper and lower panels show front and top views, respectively, of Gly-383, which is packed tightly against amino acids Phe-373, Ala-376, and Glu-377 (top left). Mutation of Gly-383 to Asp would result in steric clashes between these residues and interfere with the packing between these segments of the R-loop. D, G383D substitution would also disrupt interactions of R-loop residues Trp-379, Phe-381, and especially Thr-382 with residues Asp-92 and Ile-296 in helix M7 and residues Ile-330, Phe-331, and Ser-334 in helix M8.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Membrane, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the biosynthetic maturation of NHE6 variants. A, AP-1 cells transiently expressing NHE6GFP WT or CS-linked variants were lysed at the indicated time points (6–48 h) post-transfection. Equal amounts of proteins (20 μg) were subjected to SDS-PAGE and immunoblotting with a polyclonal anti-GFP antibody. NHE6 migrates as multiple bands: higher molecular weight bands represent the fully-glycosylated (fg) and core-glycosylated (cg) dimeric (d) forms of the exchanger that do not fully dissociate under SDS-PAGE conditions, whereas lower molecular weight bands represent fully-glycosylated and core-glycosylated forms of the dissociated monomeric (m) protein. The blots were stripped and reprobed with a mouse monoclonal anti-GAPDH antibody to control for protein loading. B, ratios of fully-glycosylated protein (monomer and dimer)/total NHE6 protein (fg/total) were quantified by densitometry of X-ray films exposed in the linear range and analyzed using ImageJ software. Values represent the mean ± S.D. of three different experiments. Significance was determined by two-way ANOVA with a post hoc Tukey test. The NHE6 variants clustered into two groups: 1) WT, A9S, and R568Q and 2) L188P, G383D, E547*, and W570*, with variants within each cluster yielding similar statistical values. Population means of the NHE6 variants are significantly different (F value = 26.4, p value = 1.5 × 10−16). Population means as a function of time are significantly different (F value = 48.6, p value = 1.9 × 10−19). § indicates significance (p < 0.01) of the means of NHE6 variants within a cluster relative to the 12-h time point. Asterisks indicate significance (★, p < 0.05, and ★★, p < 0.01) of the means between clusters of NHE6 variants at the indicated time points.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Expressing, Transfection, SDS Page, Western Blot, Molecular Weight, Control, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the protein stability of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT of CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide (CHX) for the indicated time points and lysed, and equal amounts of protein (20 μg) were analyzed by Western blotting using a mouse monoclonal anti-HA antibody. Blots were reprobed with a mouse monoclonal anti-GAPDH antibody to control for loading. Blots are representative images from four separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Effect of proteasomal and lysosomal inhibitors on cellular clearance of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT or CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide (CHX) for the indicated time points in the presence of diluent (DMSO) and the proteasomal inhibitor MG-132 (40 μm) or the lysosomal inhibitor leupeptin/pepstatin (LeuP, 100 μg/ml). Total-cell lysates were analyzed by Western blotting with a mouse monoclonal HA antibody. Membranes were also probed for β-tubulin expression as a loading control. The immunoblots are representative of three separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Effect of the lysosomotropic agent chloroquine on cellular clearance of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT or CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide for the indicated time points in the presence of diluent (H2O) or chloroquine (CQ, 500 μm). Total-cell lysates were analyzed by Western blotting with a mouse monoclonal HA antibody. Membranes were also probed for β-tubulin expression as a loading control. The immunoblots are representative of four separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Subcellular detection of NHE6 variants in recycling endosomes in transfected AP-1 cells. A, AP-1 cells were transiently transfected with mCherry fluorescent protein-tagged NHE6 (NHE6ChFP) WT or CS-linked variants. Forty eight hours post-transfection, cells were incubated with the recycling endosomal marker Alexa Fluor 488–conjugated transferrin (Tf-AF488, 10 μg/ml) for 45 min, fixed in 4% paraformaldehyde, mounted onto glass slides, and examined by confocal microscopy. Images show each channel individually, with merged images of the NHE6ChFP and Tf-AF488 channels. Scale bars represent 10 μm. B, quantitation of the degree of NHE6 overlapping with Tf-AF488 as determined by calculating the thresholded Mander's coefficient (M1) using ImageJ software and the JACoP plugin. Data are plotted as a box chart, with the central white square indicating the mean, the box representing the S.E., and the error bars showing the S.D. (n = 6–8 cells). Significance from WT was determined by one-way repeated measures ANOVA (F value = 6479.8, p value = 5.6 × 10−9), with a post hoc Dunnett's test, ★★★, p < 0.001.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Incubation, Marker, Confocal Microscopy, Quantitation Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Subcellular localization of certain CS variants in the endoplasmic reticulum in transfected AP-1 cells. A, AP-1 cells were transiently transfected with mCherry fluorescent protein-tagged NHE6 (NHE6ChFP) WT or CS-linked variants. Forty eight hours post-transfection, cells were immunostained for endogenous CANX, fixed in 4% paraformaldehyde, mounted onto glass slides, and examined by confocal microscopy. Images show each channel individually, with merged images of the NHE6ChFP and CANX channels. Scale bars represent 10 μm. B, quantitation of the degree of NHE6 overlapping with CANX as determined by calculating the thresholded Mander's coefficient (M1) using ImageJ software and the JACoP plugin. Data are plotted as a box chart, with the central white square indicating the mean, the box representing the S.E., and the error bars showing the S.D. (n = 6–8 cells). Significance from WT was determined by one-way repeated measures ANOVA (F value = 3012.9, p value = 1.7 × 10−10), with a post hoc Dunnett's test, ★★★, p < 0.001.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Confocal Microscopy, Quantitation Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the functional properties of NHE6 variants. A, biochemical determination of plasma membrane trafficking of NHE6GFP WT or CS-linked variants using a cell-surface biotinylation assay. Cell-surface proteins were labeled with N-hydroxysulfosuccinimidyl–SS–biotin in AP-1 cells expressing the NHE6GFP constructs after 48 h. Total-cell lysates (left panel; protein loading ranged from 10 to 50 μg of protein per sample as indicated below the blot) and biotinylated fractions (right panel; representing 20–100% of plasma membrane proteins extracted per sample) were examined by Western blotting with polyclonal anti-GFP and monoclonal anti-GAPDH antibodies. Representative blots from three experiments are shown. B and C, surface expression and endocytosis of external triple flag tag–labeled NHE6 (3FNHE6HA) constructs in transiently transfected (48 h) AP-1 cells using a cell-based ELISA. Mean intensity fluorescence (M.I.F.) units were determined as a function of the cellular protein concentration and then normalized as percentage (M.I.F. units for WT (100%): 25,100 ± 6,348, n = 4). The surface expression of each construct at time 0 min (before the start of internalization) is charted in B (n = 3–4 experiments). Significance from WT-expressing cells was determined using a one-way repeated measures ANOVA (F value = 463.3, p value = 0.0022), with a post hoc Dunnett's test; *, p < 0.05. Percentage internalization of NHE6 constructs normalized to the zero time point are presented in C and represent the mean ± S.D. (n = 3–4 experiments). The NHE6 variants clustered into two groups: 1) WT, A9S, and R568Q, and 2) L188P, G383D, E547*, and W570*, with variants within each cluster yielding similar statistical values. Significance from WT cells at the 5- and 15-min time points was determined using a one-way ANOVA (F value = 9.43, p value = 4.48 × 10−5), with a post hoc Tukey test; ★, p < 0.05. D, transferrin uptake in HeLa cells transiently transfected (48 h) with GFP or NHE6GFP constructs. The initial uptake (5 min) of Alexa 633–conjugated transferrin (Tf-AF633) was measured in 1 × 104 GFP-positive HeLa cells per experiment by flow cytometry (M.I.F. units for GFP control: 10,204 ± 1554, n = 4). Data were normalized as a percentage and displayed as percent change from GFP control cells. Significance from control cells was determined using a one-way repeated measures ANOVA (F value = 320.7, p value = 3.8 × 10−4), with a post hoc Dunnett's test; ★★, p < 0.001. E, recycling endosomal pH (pHe) was measured in AP-1 cells in the absence or presence of transiently transfected (48 h) NHE6ChFP constructs by fluorescence ratio image analysis of the internalized pH-sensitive probe FITC-conjugated human transferrin (Tf–FITC). Data represent the average endosomal pHe per cell pooled from three separate experiments (8–12 cells per construct/experiment; n = 24–36). Significance was determined by one-way ANOVA (F value = 40.02, p value = 0), with a post-hoc Tukey test; ★★, p < 0.001. Data in B, D, and E are plotted as box charts, with the central white square indicating the mean; the box representing the S.E.; and the error bars showing the S.D. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Functional Assay, Clinical Proteomics, Membrane, Cell Surface Biotinylation Assay, Labeling, Expressing, Construct, Western Blot, FLAG-tag, Transfection, In-Cell ELISA, Fluorescence, Protein Concentration, Flow Cytometry, Control

Journal: Scientific reports

Article Title: A novel NONO variant that causes developmental delay and cardiac phenotypes.

doi: 10.1038/s41598-023-27770-6

Figure Lengend Snippet: Figure 2. Functional evaluation of NONO variant in Drosophila. (a) Western blotting confirmed the expression of the NONO WT and the Pro459Ala variant. Myc-NONO WT or Myc-NONO Pro459Ala was expressed throughout Drosophila bodies and detected with anti-Myc antibody. Anti-α-tubulin was used as a loading control where indicated. (b) Representative bright-field microscope images of fly eyes displaying eye-specific ectopic expression of Myc-NONO WT and Myc-NONO Pro459Ala using the GMR-Gal4 driver, reared at 20 °C. (c) Quantification results of the phenotypic scores in control (n = 29), Myc-NONO WT (n = 32), and Myc-NONO Pro459Ala (n = 23). Data represent the mean ± SD. Statistical comparisons were conducted using nonparametric ANOVA (Kruskal–Wallis test) followed by Dunn’s multiple comparisons test. n.s.: not significant. ***P < 0.001, ****P < 0.0001. (d) The fly eyes displaying eye-specific overexpression of nonA using the GMR-Gal4 driver, reared at 29 °C. (e) Quantification results of the phenotypic scores in control (n = 21) and nonA (n = 19). Data represent the mean ± SD. Statistical comparisons were conducted using parametric test (unpaired t test with Welch’s correction). n.s.: not significant. ****P < 0.0001.

Article Snippet: Finally, it was washed three times with 0.3% PBT, mounted with Vectorshield (H-1000; Funakoshi, Japan), and scanned with a confocal microscope (FV3000; Olympus).

Techniques: Functional Assay, Variant Assay, Western Blot, Expressing, Control, Microscopy, Over Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Protective effects of Gryllus bimaculatus (Gb) extract on abnormal expression levels of glutamatergic and GABAergic synaptic proteins in the valproic acid (VPA)-induced autism spectrum disorder (ASD) mouse brain tissues. Immunoblot analyses for GRM5, vGluT1, NMDA R1, GABA R1α, and VGAT proteins were performed on prefrontal cortex (PFC) tissue lysates collected at embryonic day 15 (E15) (A) , postnatal day 3 (P3) (B) , and P40 (C) from mice subjected to various treatment combinations. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant).

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Expressing, Western Blot, Saline, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Regulatory effects of Gryllus bimaculatus (Gb) extract on excitatory and inhibitory neuronal activity in primary cortical neurons from valproic acid (VPA)-treated embryonic mice. (A) Schematic representation of primary cortical neuron cultures derived from embryonic mouse brains. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). (B,D) Immunoblot analyses of NMDA R1, vGluT1, GRM5, GABA R1α, VGAT, NLGN3, NRXN1, and Tuj-1 in cultured primary cortical neuron lysates. Equal amounts of protein were loaded per lane, with β-tubulin used as a loading control. The bars represent fold-changes in the densitometric values of individual protein bands relative to the corresponding β-tubulin band densities. Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant). (C) Confocal microscopy images of cortical neurons from various experimental groups. Cells were cultured for 7 days, fixed, and subsequently immunostained for vGluT1 (red), with nuclei counterstained using DAPI (blue). Scale bar: 50 μm.

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Activity Assay, Derivative Assay, Saline, Western Blot, Cell Culture, Control, Confocal Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Crucial role of astrocytes in excitatory and inhibitory (E/I) neurotransporter activities in Gryllus bimaculatus (Gb) extract-treated mixed cultures from valproic acid (VPA)-treated mouse brain. (A) Schematic representation of three different types of mixed culture systems derived from embryonic and postnatal mouse brains: Type 1, astrocytes from each treatment group combined with neurons from untreated mice; Type 2, astrocytes from untreated mice combined with neurons from each treatment group; Type 3, astrocytes and neurons both derived from the same treatment group. Astrocytes from postnatal day 3 mouse brains were seeded for 7 days, followed by the addition of cortical neurons from embryonic day 15 mouse brains onto astrocytes monolayers for an additional 7 days. (B–D) Confocal microscopy images of the different types of mixed cultures. Cells were fixed and immunostained for Tuj-1 (green) and GFAP (purple), with nuclei counterstained using DAPI (blue). Scale bar: 50 μm. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). (E) Western blots analysis of type III mixed culture. Cell lysates were immunoblotted for Tuj-1, GFAP, synaptophysin, NMDA receptor 1 (NMDA R1), GABA receptor 1α (GABA R1α), EAAT1, and EAAT2. Equal amounts of protein were loaded per each lane, with β-actin serving as the loading control. Bars represent fold-changes in the densitometric values of the bands relative to the corresponding β-actin densities. Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant).

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Derivative Assay, Confocal Microscopy, Saline, Western Blot, Control

Journal: Journal of Translational Medicine

Article Title: ALDH1L1 reverses CD8 + T cell exhaustion in the oral squamous cell carcinoma microenvironment by reprogramming L-glutamate metabolism

doi: 10.1186/s12967-026-07812-z

Figure Lengend Snippet: ALDH1L1 modulates CD8⁺ T-cell proliferation through the IL-15 signaling pathway. ( A ) Flow cytometric analysis of CD8⁺ T cell infiltration in clinical OSCC tissues ( n = 6 per group). ( B ) Expression distribution of ALDH1L1 across cell subtypes in OSCC from the TISCH2 database ( GSE172577 dataset). ( C ) Immunohistochemical staining of ALDH1L1 and CD8 in OSCC sections (ALDH1L1 low , n = 23; ALDH1L1 high , n = 29). ( D ) Immunohistochemical staining of ALDH1L1 and CD8 in xenograft tumor sections ( n = 6 per group). ( E ) Quantitative analysis of CD8⁺ T cell proportions in xenograft tissues by flow cytometry ( n = 6). ( F - G ) Correlation analysis between ALDH1L1 and chemokine mRNA expression levels in clinical OSCC samples (qRT-PCR, n = 12). H. qRT-PCR analysis of IL-15 mRNA expression in NC and ALDH1L1-KD groups of CAL 27 and SCC-25 cells ( n = 3). I. Extracellular IL-15 concentration in OSCC cell cultures with different ALDH1L1 expression levels ( n = 3). ( J ) Flow cytometric analysis of IL-15-mediated CD8⁺ T-cell proliferation in vitro, ( n = 3). (NC, negative control; OE, overexpression; KD, knockdown, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: Sections were then incubated overnight at 4 °C with ALDH1L1 primary antibody (Santa Cruz, sc-100497, 1:200 dilution for human and mouse), CD8α primary antibody (Abcam, ab217344, 1:1000 dilution for mouse; ab245118, 1:500 dilution for human), or Ki-67 primary antibody (CST, 12202; 1:500 dilution for mouse), followed by 30-min incubation with enhanced enzyme-labeled goat anti-mouse/rabbit IgG polymer secondary antibody (ZSGB-BIO, Beijing, PV9000) at 37 °C for 30 min. Antibody binding was visualized using 3,3’-diaminobenzidine (DAB) chromogen, followed by quantitative analysis under light microscopy.

Techniques: Expressing, Immunohistochemical staining, Staining, Flow Cytometry, Quantitative RT-PCR, Concentration Assay, In Vitro, Negative Control, Over Expression, Knockdown

Journal: Journal of Translational Medicine

Article Title: ALDH1L1 reverses CD8 + T cell exhaustion in the oral squamous cell carcinoma microenvironment by reprogramming L-glutamate metabolism

doi: 10.1186/s12967-026-07812-z

Figure Lengend Snippet: ALDH1L1 modulates CD8⁺ T-cell function through metabolic reprogramming. ( A ) Quantification of PD-1⁺ CD8⁺ T cells in clinical OSCC specimens by flow cytometry ( n = 6 per group). ( B ) Quantification of PD-1⁺ CD8⁺ T cells in mouse xenografts by flow cytometry ( n = 6 per group). ( C ) Top 20 enriched KEGG pathways identified from transcriptomic analysis. ( D ) Chord diagram illustrating 29 key metabolites identified by metabolomic profiling. ( E - H ) Flow cytometric evaluation of CD8⁺ T cell marker expression after stimulation with metabolites derived from ALDH1L1-KD or control CAL 27 cells and SCC-25 cells ( n = 3). (NC, negative control; OE, overexpression; KD, knockdown; ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: Sections were then incubated overnight at 4 °C with ALDH1L1 primary antibody (Santa Cruz, sc-100497, 1:200 dilution for human and mouse), CD8α primary antibody (Abcam, ab217344, 1:1000 dilution for mouse; ab245118, 1:500 dilution for human), or Ki-67 primary antibody (CST, 12202; 1:500 dilution for mouse), followed by 30-min incubation with enhanced enzyme-labeled goat anti-mouse/rabbit IgG polymer secondary antibody (ZSGB-BIO, Beijing, PV9000) at 37 °C for 30 min. Antibody binding was visualized using 3,3’-diaminobenzidine (DAB) chromogen, followed by quantitative analysis under light microscopy.

Techniques: Cell Function Assay, Flow Cytometry, Metabolomic, Marker, Expressing, Derivative Assay, Control, Negative Control, Over Expression, Knockdown

Journal: Journal of Translational Medicine

Article Title: ALDH1L1 reverses CD8 + T cell exhaustion in the oral squamous cell carcinoma microenvironment by reprogramming L-glutamate metabolism

doi: 10.1186/s12967-026-07812-z

Figure Lengend Snippet: L-glutamate accumulation mediates CD8⁺ T cell dysfunction in ALDH1L1-downregulated microenvironment. ( A - C ) Integrated multi-omics analysis reveals ALDH1L1-regulated metabolic signatures: Red boxes highlight co-enriched metabolic pathways from transcriptomic ( A ) and metabolomic ( B ) analysis, and key metabolites ( C ). ( D ) Extracellular L-glutamate concentration in conditioned media from CAL 27 and SCC-25 cell culture supernatants ( n = 3). ( E - F ) Flow cytometric analysis of L-glutamate effects on CD8⁺ T cell functional markers: ( E ) Proportion of IFN-γ⁺ CD8⁺ T cells; ( F ) Proportion of PD-1⁺ CD8⁺ T cells ( n = 3). ( G ) Cytotoxic activity of CD8⁺ T cells pretreated with conditioned medium from ALDH1L1-KD or NC OSCC cells medium with exogenous L-glutamate ( n = 3). (NC, negative control; KD, knockdown; Ctrl, control; Glu, L-glutamate; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: Sections were then incubated overnight at 4 °C with ALDH1L1 primary antibody (Santa Cruz, sc-100497, 1:200 dilution for human and mouse), CD8α primary antibody (Abcam, ab217344, 1:1000 dilution for mouse; ab245118, 1:500 dilution for human), or Ki-67 primary antibody (CST, 12202; 1:500 dilution for mouse), followed by 30-min incubation with enhanced enzyme-labeled goat anti-mouse/rabbit IgG polymer secondary antibody (ZSGB-BIO, Beijing, PV9000) at 37 °C for 30 min. Antibody binding was visualized using 3,3’-diaminobenzidine (DAB) chromogen, followed by quantitative analysis under light microscopy.

Techniques: Biomarker Discovery, Metabolomic, Concentration Assay, Cell Culture, Functional Assay, Activity Assay, Negative Control, Knockdown, Control

Journal: Journal of Translational Medicine

Article Title: ALDH1L1 reverses CD8 + T cell exhaustion in the oral squamous cell carcinoma microenvironment by reprogramming L-glutamate metabolism

doi: 10.1186/s12967-026-07812-z

Figure Lengend Snippet: ALDH1L1 transcriptionally regulates GLUL and interacts with GLUL to sustain L-glutamate metabolism. ( A ) Integrated multi-omics analysis of ALDH1L1-regulated metabolic network. Green circles: downregulated metabolites; Green squares: downregulated metabolic enzymes; Red squares: upregulated metabolic enzymes. ( B - C ) GLUL mRNA ( B ) and protein ( C ) expression in CAL 27 and SCC-25 cells assessed by qRT-PCR and western blot, respectively. ( D ) Protein-protein interaction (PPI) network between ALDH1L1 and glutamate-metabolizing enzymes predicted by the STRING database. ( E ) Three-dimensional structural model of ALDH1L1 (green)-GLUL (blue) interaction. Key residues are shown in stick representation; hydrogen bonds indicated by yellow dashed lines. ( F ) Co-immunoprecipitation analysis of endogenous interactions among ALDH1L1 and GLUL in CAL 27 cells. ( G ) ALDH1L1 enhances GLUL enzymatic activity in a dose-dependent manner in vitro ( n = 3). ( H ) Western blot validation of GLUL overexpression in plasmid-transfected CAL 27 cells. ( I ) Extracellular L-glutamate concentration following GLUL upregulation in ALDH1L1-KD CAL 27 cells ( n = 3). (NC: negative control; KD: ALDH1L1 knockdown; OE: GLUL overexpression; ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: Sections were then incubated overnight at 4 °C with ALDH1L1 primary antibody (Santa Cruz, sc-100497, 1:200 dilution for human and mouse), CD8α primary antibody (Abcam, ab217344, 1:1000 dilution for mouse; ab245118, 1:500 dilution for human), or Ki-67 primary antibody (CST, 12202; 1:500 dilution for mouse), followed by 30-min incubation with enhanced enzyme-labeled goat anti-mouse/rabbit IgG polymer secondary antibody (ZSGB-BIO, Beijing, PV9000) at 37 °C for 30 min. Antibody binding was visualized using 3,3’-diaminobenzidine (DAB) chromogen, followed by quantitative analysis under light microscopy.

Techniques: Biomarker Discovery, Expressing, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Activity Assay, In Vitro, Over Expression, Plasmid Preparation, Transfection, Concentration Assay, Negative Control, Knockdown

Journal: Journal of Translational Medicine

Article Title: ALDH1L1 reverses CD8 + T cell exhaustion in the oral squamous cell carcinoma microenvironment by reprogramming L-glutamate metabolism

doi: 10.1186/s12967-026-07812-z

Figure Lengend Snippet: Stevioside enhances anti-PD-1 immunotherapy by targeting ALDH1L1. ( A ) Virtual screening workflow for identifying potential ALDH1L1-targeting compounds. ( B ) Two-dimensional chemical structures of the top 12 candidate compounds ranked by binding affinity to ALDH1L1 protein. ( C ) Western blot analysis demonstrating the effect of stevioside treatment on ALDH1L1 protein expression levels in CAL 27 cells (Ctrl, control). ( D ) Dose-response curve of stevioside (IC₅₀ = 195.3 µM). ( E ) Molecular docking analysis reveals the binding site of stevioside on ALDH1L1 protein. (Blue ribbon structure represents ALDH1L1 protein; green stick model represents stevioside molecule; stick structures indicate key interacting amino acid residues; yellow dashed lines denote hydrogen bond interactions.) ( F ) In vivo experiments confirm that stevioside combined with anti-PD-1 antibody treatment significantly enhances antitumor efficacy ( n = 5 per group). ( G - H ) Immunohistochemical staining of Ki-67 ( G ) and CD8 ( H ) in xenograft tumor sections ( n = 5 per group). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: Sections were then incubated overnight at 4 °C with ALDH1L1 primary antibody (Santa Cruz, sc-100497, 1:200 dilution for human and mouse), CD8α primary antibody (Abcam, ab217344, 1:1000 dilution for mouse; ab245118, 1:500 dilution for human), or Ki-67 primary antibody (CST, 12202; 1:500 dilution for mouse), followed by 30-min incubation with enhanced enzyme-labeled goat anti-mouse/rabbit IgG polymer secondary antibody (ZSGB-BIO, Beijing, PV9000) at 37 °C for 30 min. Antibody binding was visualized using 3,3’-diaminobenzidine (DAB) chromogen, followed by quantitative analysis under light microscopy.

Techniques: Binding Assay, Western Blot, Expressing, Control, In Vivo, Immunohistochemical staining, Staining

Journal: Journal of Translational Medicine

Article Title: ALDH1L1 reverses CD8 + T cell exhaustion in the oral squamous cell carcinoma microenvironment by reprogramming L-glutamate metabolism

doi: 10.1186/s12967-026-07812-z

Figure Lengend Snippet: Schematic model illustrating the mechanism by which ALDH1L1 reverses CD8⁺ T cell exhaustion in OSCC

Article Snippet: Sections were then incubated overnight at 4 °C with ALDH1L1 primary antibody (Santa Cruz, sc-100497, 1:200 dilution for human and mouse), CD8α primary antibody (Abcam, ab217344, 1:1000 dilution for mouse; ab245118, 1:500 dilution for human), or Ki-67 primary antibody (CST, 12202; 1:500 dilution for mouse), followed by 30-min incubation with enhanced enzyme-labeled goat anti-mouse/rabbit IgG polymer secondary antibody (ZSGB-BIO, Beijing, PV9000) at 37 °C for 30 min. Antibody binding was visualized using 3,3’-diaminobenzidine (DAB) chromogen, followed by quantitative analysis under light microscopy.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: The ST6Gal I Sialyltransferase Selectively ModifiesN-Glycans on CD45 to Negatively Regulate Galectin-1-induced CD45 Clustering, Phosphatase Modulation, and T Cell Death

doi: 10.1074/jbc.m209595200

Figure Lengend Snippet: FIG. 4. A, expression of the ST6Gal I in the T200 cell line, which does not express CD45, did not result in increased SNA binding. Nine clones expressing ST6Gal I mRNA were examined, but none demon- strated increased SNA binding by flow cytometry; clone SNA.T1 is shown for example. C.T1 is one of nine control clones transfected with vector alone. B, SNA blotting of whole cell extracts of C.T1 and SNA.T1 cells did not demonstrate any differences in staining between the two clones. C, T200 clones express ST6Gal I mRNA and protein. RT-PCR and immunoblot analysis of nine clones demonstrated ST6Gal I expres- sion, as shown for the SNA.T1 clone, with no ST6Gal I expression in any of the controls, as shown for the C.T1 clone. The samples are represent- ative of all 18 clones examined. The expressed protein is enzymatically active, as demonstrated by the addition of sialic acid to asialofetuin. Asialofetuin was incubated with lysates of C.T1 or SNA.T1 cells and precipitated with anti-fetuin, and 2,6-linked sialic acid was detected by SNA blotting. Weak SNA reactivity of fetuin incubated with extract of C.T1 cells may reflect the addition of 2,6-linked sialic acid to O-glycans, because no SNA reactivity was detected with the asialofe- tuin acceptor substrate alone (not shown). Densitometric analysis of the SNA-binding bands was performed; the ratio of SNA binding to fetuin incubated with SNA.T1 extract compared with C.T1 extract was 6.3:1.

Article Snippet: PNGase F Digestion of CD45—Cell lysates (106 cells) were separated by SDS-PAGE, blotted to nitrocellulose and probed with polyclonal goat anti-mouse CD45 (M-20) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Binding Assay, Clone Assay, Flow Cytometry, Control, Transfection, Plasmid Preparation, Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot, Incubation

Journal: Journal of Biological Chemistry

Article Title: The ST6Gal I Sialyltransferase Selectively ModifiesN-Glycans on CD45 to Negatively Regulate Galectin-1-induced CD45 Clustering, Phosphatase Modulation, and T Cell Death

doi: 10.1074/jbc.m209595200

Figure Lengend Snippet: FIG. 3. The ST6Gal I preferentially sialylates N-glycans on CD45. A, total SNA-binding glycoproteins were precipitated from con- trol clones transfected with vector alone (lanes C.2 and C.4) or from the SNA.1 clone expressing the ST6Gal I. Precipitated glycoproteins were probed with biotinylated SNA. The only significant difference in the profile of SNA binding glycoproteins was an increase in a band with a mass of 200 kDa (arrow). B, the SNA reactive band is CD45. The cells were cultured in 2 mM DMNJ, as above, or in medium alone. The cell lysates were precipitated with CD45 antibody or SNA (indicated below) and probed with CD45 antibody. The band with increased SNA staining reacts with both SNA and antibody to CD45. In addition, the increased SNA binding to CD45 is abolished by pretreatment with DMNJ, which blocks synthesis of complex N-glycans. In both blots, the width of the CD45 band is diminished in DMNJ-treated cells compared with cells expressing the ST6Gal I, as a result of decreased complexity of glyco- sylation. C, increased SNA binding to CD45 results from sialylation of N-glycans. CD45 was detected in whole cell lysates of SNA.9 cells by immunoblotting (top panel). The CD45 bands were excised and incu- bated with or without PNGase F, as indicated, and reprobed with SNA-biotin. Removal of N-glycans from CD45 by PNGase F treatment reduced SNA binding.

Article Snippet: PNGase F Digestion of CD45—Cell lysates (106 cells) were separated by SDS-PAGE, blotted to nitrocellulose and probed with polyclonal goat anti-mouse CD45 (M-20) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Binding Assay, Clone Assay, Transfection, Plasmid Preparation, Expressing, Cell Culture, Staining, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The ST6Gal I Sialyltransferase Selectively ModifiesN-Glycans on CD45 to Negatively Regulate Galectin-1-induced CD45 Clustering, Phosphatase Modulation, and T Cell Death

doi: 10.1074/jbc.m209595200

Figure Lengend Snippet: FIG. 5. ST6Gal I expression inhibits galectin-1-induced segre- gation of CD45. C.4 or SNA.9 cells were treated with galectin-1 or buffer control and fixed, and cell surface CD45 was detected by immu- nofluorescence. A, cells were analyzed by confocal microscopy to detect CD45 segregation. B, the percentage of cells demonstrating segregation of CD45 was scored by counting 50 cells in six fields. The cells were treated with buffer control (open bars) or galectin-1 (shaded bars). The SNA.9 cells demonstrated a marked reduction in CD45 segregation, compared with CD45 segregation in control C.4 cells. The percentage of cell death for a parallel sample is indicated by the numbers above each bar.

Article Snippet: PNGase F Digestion of CD45—Cell lysates (106 cells) were separated by SDS-PAGE, blotted to nitrocellulose and probed with polyclonal goat anti-mouse CD45 (M-20) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Control, Confocal Microscopy

Journal: Journal of Biological Chemistry

Article Title: The ST6Gal I Sialyltransferase Selectively ModifiesN-Glycans on CD45 to Negatively Regulate Galectin-1-induced CD45 Clustering, Phosphatase Modulation, and T Cell Death

doi: 10.1074/jbc.m209595200

Figure Lengend Snippet: FIG. 6. ST6Gal I expression inhibits galectin-1-induced modu- lation of CD45 protein-tyrosine phosphatase activity. A, CD45 is the major PTP in PhaR2.1 cells. Whole cell lysates of the CD45 paren- tal cell line, PhaR2.1, and the CD45 derivative T200, were assayed for PTP activity in the presence (solid bar) or absence (open bar) of the PTP inhibitor bp V (phen). PTP activity was measured by the release of p-nitrophenol, detected at 415 nm. B, ST6Gal I expression abrogates the decrease in PTP activity triggered by binding of galectin-1. C.2 cells (open symbols) and SNA.9 cells (closed symbols) were incubated with 30 g of galectin-1 for the indicated times at 37 °C. At the indicated times, the cells were lysed, and PTP activity in whole cell lysates was meas- ured as described under “Experimental Procedures,” in the presence (squares) or absence (circles) of the PTP inhibitor bpV (phen). C.2 cells demonstrate a 40% reduction in PTP activity 1 min after galectin-1 binding. SNA.9 cells demonstrate no change in PTP activity after ga- lectin-1 binding.

Article Snippet: PNGase F Digestion of CD45—Cell lysates (106 cells) were separated by SDS-PAGE, blotted to nitrocellulose and probed with polyclonal goat anti-mouse CD45 (M-20) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Activity Assay, Binding Assay, Incubation

Journal: PloS one

Article Title: Live-cell imaging of the association of STAT6-GFP with mitochondria.

doi: 10.1371/journal.pone.0055426

Figure Lengend Snippet: Figure 3. Immunoimaging assays showing colocalization of STAT6 with mitochondria in the cytoplasm of digitonin- washed Hep3B, HPAEC and HPASMC cells. The indicated cells were grown in 6-well plates for one day, washed 4x with the digitonin (50 mg/ml)-0.3M sucrose buffer and then fixed followed by immuno- fluoresence analyses as indicated using either the anti-STAT6 pAb (green) and F1-ATPase mAb (red). Insets are shown at high magnification within each panel. Scale bars = 10 mm. Quantitative colocalization analyses using the Pearson’s and Costes’ plugins in Image J confirmed colocalization between STAT6 and F1-ATPase immunofluorescence at a setting of P,0.05 in Panels A, B and C. doi:10.1371/journal.pone.0055426.g003

Article Snippet: PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e55426 Antibodies and competing peptides Rabbit polyclonal antibodies to STAT6 (sc-621x), glucose-6phosphate dehydrogenase (sc-67165), the Flag tag (sc-807) and the respective relevant competing peptide to STAT6 (sc-621P) and the irrelevant OctA peptide (sc-835P) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Immunofluorescence

Journal: PloS one

Article Title: Live-cell imaging of the association of STAT6-GFP with mitochondria.

doi: 10.1371/journal.pone.0055426

Figure Lengend Snippet: Figure 4. Characterization of the specificityof the anti-STAT6 immunofluoresence and its organellar localization by immu- nogold electron microscopy. Panel A. Anti-STAT6 immunofluore- sence assayed using the rabbit pAb colocalizing with F1-ATPase in the cytoplasm of digitonin-washed Hep3B cells was compared to that of an irrelevant pAb (G6PD). Scale bar = 10 mm. Panel B. Peptide competition characterization of the anti-STAT6 pAb immunofluorescence using either the relevant STAT6 peptide that had been used as the immunogen or the irrelevant OctA peptide. Scale bar = 10 mm. Panel C. Immunogold EM localization of the anti-STAT6 pAb immunoreactiv- ity. Cryo-thin sections of HPAECs were probed using ant-STAT6 pAb and 18-nm-tagged anti-rabbit IgG as indicated in ref. 23. Insets are shown at high magnification. Scale bars = 500 nm. Similar data were obtained using Hep3B cells (not shown). doi:10.1371/journal.pone.0055426.g004

Article Snippet: PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e55426 Antibodies and competing peptides Rabbit polyclonal antibodies to STAT6 (sc-621x), glucose-6phosphate dehydrogenase (sc-67165), the Flag tag (sc-807) and the respective relevant competing peptide to STAT6 (sc-621P) and the irrelevant OctA peptide (sc-835P) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Electron Microscopy, Immunofluorescence

Journal: PloS one

Article Title: Live-cell imaging of the association of STAT6-GFP with mitochondria.

doi: 10.1371/journal.pone.0055426

Figure Lengend Snippet: Figure 5. STAT6 peptides in Hep3B and STAT6SH2-/-SH2- MEF -derived cell fractions. Panel A. Western blot analyses of cell extracts (duplicate lanes) prepared from Hep3B cultures (90 mm plates) without and after washing 4x with the digitonin-sucrose buffer and the 7.5K mitochondria- enriched cell pellet derived from STAT6SH2-/-SH2- MEFs (rightmost single lane). The amount of cell extracts used in the Hep3B lanes corresponds to that derived from equal numbers of cells. Left and right blots show immunoblotting after competition with the unrelated OctA peptide or the relevant STAT6-peptide. Panel B. The 2x washed P7.5K pellet prepared from duplicate batches of Hep3B cultures (five confluent 90 mm cultures/group) were subjected to anti-TOM22-mAb magnetic bead immunoisolation chromatography. The flow-through (FT) and the Bound fractions were resedimented, checked microscopically for enrichment of mitochondria in the Bound fraction and then respective aliquots (20% of each fraction) used for Western blotting. The full-length STAT6 peptide is shown. Panel C. Hep3B cells (ten 90 mm cultures/group) were harvested and used to prepare the 2x washed P7.5K membrane pellet fraction. This was then sedimented through a Percoll (30%)-0.25 M sucrose gradient as indicated in ‘‘Materials and Methods,’’ the respective gradient fractions collected and prepared for Western blotting (half of each fraction for each blot) using the indicated probes. doi:10.1371/journal.pone.0055426.g005

Article Snippet: PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e55426 Antibodies and competing peptides Rabbit polyclonal antibodies to STAT6 (sc-621x), glucose-6phosphate dehydrogenase (sc-67165), the Flag tag (sc-807) and the respective relevant competing peptide to STAT6 (sc-621P) and the irrelevant OctA peptide (sc-835P) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Derivative Assay, Western Blot, Chromatography, Membrane

Journal: PloS one

Article Title: Live-cell imaging of the association of STAT6-GFP with mitochondria.

doi: 10.1371/journal.pone.0055426

Figure Lengend Snippet: Figure 6. Targeting of STAT6-GFP to mitochondria in Hep3B cells assayed in live-cells using MitoTracker as the mitochon- drial vital stain. Panels A and B. Hep3B cells in 35–mm plates were transfected with the STAT6-GFP expression plasmid and 2 days later the cultures were exposed to MitoTracker Red CMXRos at 100 nM for 15 min. After washing with PBS the live cells were imaged using a 40x water-immersion objective in green (STAT6-GFP) and red (MitoTracker). Insets are shown at high magnification within each panel. Scale bars = 10 mm. Quantitative colocalization analyses using the Pearson’s and Costes’ plugins in Image J confirmed colocalization between STAT6-GFP and MitoTracker fluorescence at a setting of P,0.05 in Panels A and B. doi:10.1371/journal.pone.0055426.g006

Article Snippet: PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e55426 Antibodies and competing peptides Rabbit polyclonal antibodies to STAT6 (sc-621x), glucose-6phosphate dehydrogenase (sc-67165), the Flag tag (sc-807) and the respective relevant competing peptide to STAT6 (sc-621P) and the irrelevant OctA peptide (sc-835P) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Staining, Transfection, Expressing, Plasmid Preparation, Fluorescence

Journal: PloS one

Article Title: Live-cell imaging of the association of STAT6-GFP with mitochondria.

doi: 10.1371/journal.pone.0055426

Figure Lengend Snippet: Figure 7. Targeting of STAT6-GFP but not of N1-GFP to mitochondria in Hep3B cells assayed in live-cells using TMRE as the mitochondrial vital stain. Hep3B cells in 35–mm plates were transfected with expression plasmids for N1-GFP (Panel A) or STAT6-GFP (Panel B) and 2 days later the cultures were exposed to TMRE at 5 nM for 15 min. After washing with PBS the live cells were imaged using a 40x water-immersion objective by two-color fluorescence in green (N1- GFP or STAT6-GFP) and red (TMRE). Insets are shown at high magnification within each panel. Scale bars = 10 mm except in Panel B, insets = 5 mm. doi:10.1371/journal.pone.0055426.g007

Article Snippet: PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e55426 Antibodies and competing peptides Rabbit polyclonal antibodies to STAT6 (sc-621x), glucose-6phosphate dehydrogenase (sc-67165), the Flag tag (sc-807) and the respective relevant competing peptide to STAT6 (sc-621P) and the irrelevant OctA peptide (sc-835P) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Staining, Transfection, Expressing, Fluorescence

Journal: PloS one

Article Title: Live-cell imaging of the association of STAT6-GFP with mitochondria.

doi: 10.1371/journal.pone.0055426

Figure Lengend Snippet: Figure 8. Targeting of STAT61–459-GFP to mitochondria in Hep3B cells. Panels A and B. Hep3B cells in 35–mm plates were transfected with expression constructs for either the full-length or the 1-459 truncated version of STAT6-GFP. Two days later the plates were either fixed and immunostained for F1-ATPase (Panel A) or exposed to MitoTracker Red CMXRos at 100 nM for 15 min Panel B) and imaged as indicated in Materials and Methods. Scale bars = 10 mm. doi:10.1371/journal.pone.0055426.g008

Article Snippet: PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e55426 Antibodies and competing peptides Rabbit polyclonal antibodies to STAT6 (sc-621x), glucose-6phosphate dehydrogenase (sc-67165), the Flag tag (sc-807) and the respective relevant competing peptide to STAT6 (sc-621P) and the irrelevant OctA peptide (sc-835P) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Transfection, Expressing, Construct

Journal: PloS one

Article Title: Live-cell imaging of the association of STAT6-GFP with mitochondria.

doi: 10.1371/journal.pone.0055426

Figure Lengend Snippet: Figure 9. STAT6 associated with mitochondria in MEFs derived from wildtype (wt) or STAT6SH2-/SH2- mice. Panel A. MEFs derived from wt and STAT6SH2-/SH2- grown in 35–mm plates were washed using the digitonin-sucrose buffer and then immunostained for STAT6 and F1-ATPase. The cells were imaged using a 100x oil immersion objective. Insets represent the boxed regions within each frame. Scale bar = 10 mm. Panels B and C. Whole-cell extracts of MEFs derived from wt or STAT6SH2-/SH2-mice were evaluated (each in duplicate lane per variable) using Western blotting without (Panel B), and with peptide-competition (Panel C). doi:10.1371/journal.pone.0055426.g009

Article Snippet: PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e55426 Antibodies and competing peptides Rabbit polyclonal antibodies to STAT6 (sc-621x), glucose-6phosphate dehydrogenase (sc-67165), the Flag tag (sc-807) and the respective relevant competing peptide to STAT6 (sc-621P) and the irrelevant OctA peptide (sc-835P) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Derivative Assay, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 controls ploidy in Y235T cells. A The presented bar graph illustrates the GJB3 mRNA amounts across various human tissues, with detailed information available in the Materials and Methods section. Urothelial cells (UC#1 and UC#2) were isolated from ureters from two separate patients who underwent nephrectomy at Ulm University. The mRNA levels were normalized to GAPDH . n = 3 independent experiments were performed. Error bars represent mean ± SEM. B The representative pictures display the HE, GJB3 and IgG staining in human ureter tissues (U#1 and U#2, respectively). C The representative Western blot result indicates GJB3 protein levels in Y235T cells with shGJB3. α-tubulin is used as a loading control. n = 3 independent experiments were performed. D The bar graphs depict the effectiveness of GJB3 knockdowns at the mRNA level in Y235T cells, with the measurements reference to the GAPDH mRNA level. n = 3 independent experiments were performed. Error bars represent mean ± SEM. E Representative pictures showing metaphase spreads of Y235T cells with shControl and shGJB3#2. Chromosomes are visualized by 4',6-diamidino-2-phenylindole (DAPI) staining. Control cells showing 46 chromosomes in most metaphase spreads. Exemplary pictures demonstrating the induction of aneuploidy in Y235T cells subsequent to GJB3 knockdown. The images show a metaphase spread of Y235T-shGJB3#2 cells with 51 chromosomes. F Chromosomes numbers of metaphase spreads from Y235T cells that were knockdown GJB3. n = numbers of (Each counting is indicated within the graph). Results are pooled from three independent sets of experiments. Mean ± SEM values are shown in the dot plot, and significance was determined by using Fisher’s exact test. G Representative pictures showing micronuclei of Y235T cells with shGJB3#1. Cell nuclei are stained with DAPI, and phalloidin Alexa Fluor 488 was used for F-actin visualization. White arrows indicate micronuclei. H Quantitation of cells with micronuclei upon knockdown of GJB3. Results from n = 3 separate series of experiments. The bar graph displays the mean ± SEM values, and the two-tailed Student's t-test was used to assess the significance. I Immunofluorescence results indicate the multinucleation of Y235T shGJB3#1 cell. Cell nuclei is visualized by DAPI, and F-actin is visualized by Alexa Fluor 488. J Quantitation of cells with multinucleation with knockdown of GJB3. Results from n = 3 independent sets of experiments. Mean ± SEM values are shown in the bar graph, and the significance was determined by two-tailed Student’s t -test. K Figures depict of mitotic abnormalities in metaphase and anaphase. DAPI (blue) indicates chromosomes, Cy5 (red) indicates α-tubulin, and Alexa Fluor 488 (green) labeling illustrates γ-tubulin. White arrows are used to indicate chromatid mislocation or multipolar centrosomes. L – M Quantitative evaluation of mitotic abnormalities. Results from n = 3 distinct experiments. The bar graph displays mean ± SEM data, and a two-tailed Student’s t -test was used to assess significance. Scale bars: 200 μm ( B main panels) 50 μm ( B insets) 20 μm ( E , G , I ) and 2 μm ( K ). Images are shot at total magnification of 100x ( B main panels), 630x ( B insets, E , G , I , K )

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Isolation, Staining, Western Blot, Control, Knockdown, Quantitation Assay, Two Tailed Test, Immunofluorescence, Labeling

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 controls spindle orientation and microtubule dynamics. A Exemplary pictures illustrating the disorientation of Y235T-shGJB3#1 cells. The chromosomes are indicated by DAPI, γ-tubulin is visualized by Alexa Fluor 488, and α-tubulin is visualized by Cy5. B Quantitative assessment of the spindle pole displacement factor (SPDF) in Y235T cells. n = 256 (Y235T-shScr), 253 (Y235T-shGJB3#1), 263 (Y235T-shGJB3#2), C , Representative images showing reorientation of UMUC3 cells with ectopic GJB3. The chromosomes are indicated by DAPI, γ-tubulin is visualized by Alexa Fluor 488, and α-tubulin is visualized by Cy5. D Quantitative assessment of the spindle pole displacement factor (SPDF) in UMUC3 cells. n = 155 (UMUC3-EV), and 140 (UMUC3-GJB3). Experiments present combined data from three separate sets of independent experiments. The two-tailed Student’s t -test was used to evaluate significance, and the mean ± SEM data are displayed in the dot plot. To boost the proportion of prometaphase cells, cells were treated to dimerthylenastron for four hours prior to labeling. E Examples of images demonstrating microtubule growth in Y235T cells expressing shGJB3#2. F Rates of mitotic microtubule plus-end assembly in Y235T-shGJB3 cells. n = 60 cells are pooled from three independent sets of experiments. G Example of images demonstrating growth of microtubules in UMUC3-GJB3 cells. H Rates of mitotic microtubule plus-end assembly in UMUC3-GJB3 cells. n = 60 cells are combined from three separate sets of experiments. The two-tailed Student’s t -test was used to evaluate significance, and the mean ± SEM data are displayed in the dot plot. GJB3 interacts with α-tubulin. The deletion of GJB3 in UROtsa cells using the CRISPR-cas9 method was detailed in the main article. I Western blot displaying GJB3 protein levels in UROtsa cells with a control guide RNAs directed against green flourescent protein or two distinct gRNAs targeting GJB3 (gGJB3#1 and GJB3#2). α-tubulin serves as loading control. n = 3 separate experiments were conducted. J Exemplary pictures displaying GJB3 and α-tubulin colocalization in UROtsa cells during metaphase. GJB3 is visualized by Alexa Fluor 488 and α-tubulin is visualized by Cy5. Yellow signal indicates the overlap of GJB3 and α-tubulin. K Quantitation of GJB3 and α-tubulin colocalization in UROtsa cells by Pearson’s correlation coefficient. n = 49 (UROtsa-gControl), 58 (UROtsa-gGJB3#1), 53(UROtsa-gGJB3#2) are pooled from three to four independent experiments. L GJB3 protein level in UMUC3 cells with ectopic GJB3 was detected by Western blot. α-tubulin is used as a loading control. n = 3 independent experiments were performed. M Representative images displaying the colocalization of GJB3 and α-tubulin in UMUC3 cells during metaphase. GJB3 is visualized by Alexa Fluor 488 and α-tubulin is visualized by Cy5. Yellow signal indicates the overlap of GJB3 and α-tubulin. N Quantitation of GJB3 and α-tubulin colocalization in UMUC3 cells by Pearson’s correlation coefficient. n = 105 (UMUC3-EV), and 64 (UMUC3-GJB3) are pooled from three to four independent experiments. Mean ± SEM values are shown in the bar graph, and significance was determined by two-tailed Student’s t-test ( M , N ). O – P GJB3 bundle microtubule (MT) filament level was detected by Western blot. 5 × 10 11 MT/ml and 5–10 μm in length MTs were incubated with increasing concentrations of GJB3 (relative GJB3 amount is indicated by + or + +). Supernatant (S) and pellet (P) were subjected to 10% SDS-PAGE after high-speed centrifugation at 100,000 g . ( O ), Flag-GJB3, indicated by red arrowheads and ( P ), microtubules, indicated by red arrows, are visualized by specific antibodies. n = 3 independent experiments were performed. Scale bars: 5 μm ( A , C) and 1 μm ( E , G) and 2 μm ( J , M ). Images were captured at total magnification of 630x

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Two Tailed Test, Labeling, Expressing, CRISPR, Western Blot, Control, Quantitation Assay, Incubation, SDS Page, Centrifugation

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 as tumor suppressors in bladder cancer. A–B ( A ) RT-qPCR was used to measure the GJB3 mRNA expression , and ( B ) Western blot was employed to ascertain protein amounts. The relative mRNA expression was quantified with respect to GAPDH . The qPCR shows results of n = 4 technical repeats. Error bars represent Mean ± SEM. α-tubulin serves as loading control in Western blot. n = 3 independent experiments were performed. C Comparison of GJB3 mRNA levels in the CNUH (GSE13507) cohort of NMIBC ( n = 103) and MIBC ( n = 62) human bladder tumors. Statistical differences were defined by two-way Fisher’s ANOVA test * = p ≤ 0.05. D The IHC images represent the GJB3 staining (red arrows) in human normal bladder and bladder cancer tissues. E Quantitation of GJB3-IHC staining scores in human normal bladder and bladder cancer groups. F The IHC images display Gjb3 staining (red arrows) in mouse normal bladder and bladder cancer tissues during BBN-induced BC progression. G Quantitation of Gjb3-IHC staining scores in normal bladder and bladder cancer tissues during the BBN-induced BC progression in mice. The expression of Gjb3 gradually diminished after the BBN treatment, in contrast to the control mice (black dots), which were given water (orange dots). The bar graph displays mean ± SEM data, and a two-tailed Student's t-test was used to assess significance. Scale bars: 200 μm ( D and F , main panels) and 50 μm ( D and F insets). Images were captured at total magnification of 100 × ( D and F main panels), and 630 × ( D and F insets)

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Control, Comparison, Staining, Quantitation Assay, Immunohistochemistry, Two Tailed Test

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 inhibits cells invasion and migration. A – B Western blots displaying the GJB3 protein quantity assessments in RT4 and in UMUC3 cells with experimental modifications. The loading control was provided by α-tubulin levels. The Western blots were repeated for three times. C Cell migratory capacity in RT4 cell line with shGJB3#1 was detected by Wound healing/scratch. The exemplary imaged were captured at 24 and 120 h. D Normalized cell free area was used to quantify the impact of GJB3 knockdown on RT4 cells by Wound healing assay. n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student's t-test was used to assess significance. E Cell migratory capacity in UMUC3 cell line with ectopic GJB3 was detected by Wound healing/scratch. The representative pictures captured at 12 and 24 h in case of UMUC3 cells. F Quantitation of normalized cell free area of UMUC3 cells with ectopic GJB3 performed by Wound healing assay n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student’s t -test was used to assess significance. G The invasion capacity of RT4 cell line with shGJB3#1 was detected by Boyden chamber. The exemplary images depict cell invasion through the Boyden chamber, stained at 144 h post-seeding. H Quantitation of invasive capacity of RT4 cells expressing the indicated shRNAs targeting GJB3. n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student’s t -test was used to assess significance. I The invasion capacity of UMUC3 cell line with ectopic of GJB3 was detected by Boyden chamber. The exemplary images depict cell invasion through the Boyden chamber, stained at 48 h post-seeding. J, Quantitation of invasive capacity of UMUC3 cells with ectopic GJB3 expression. n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student's t-test was used to assess significance. K Representative images of hematoxylin/eosin stainings showing the invasion capacity of RT4 cell line with shGJB3#2 by porcine bladder ex vivo organ culture method (the invasive capacity of BC cells in the ex vivo organ culture model was quantified as shown in Fig. S4). The cells were seeded on the surface of the de-epithelized porcine bladder for 21 days. L Quantitation graphs displaying the impact of GJB3 alteration on the invasive capacity of RT4 cells in ex vivo organ culture approach. n = 4 (RT4-shScr), n = 3 (RT4-shGJB3#1), n = 3 (RT4-shGJB3#2) M Representative images of hematoxylin/eosin stainings showing the invasion capacity of UMUC3 cell line with ectopic GJB3 by porcine bladder ex vivo organ culture approach. The cells were seeded on the surface of the de-epithelized porcine bladder for or 14 days. Insets: enlarged images of the areas shown by black boxes. Black arrows indicate the cells that have spread the farthest from the surface. n = 3 (UMUC3-EV), n = 4 (UMUC3-GJB3) independent experiments were performed. Mean ± SEM values are shown in the bar graph, and significance was determined by two-tailed Student’s t -test. Scale bars: 200 μm ( C , E , G , I , K main panels, M main panels) and 100 μm ( K insets, M insets). Images were captured at total magnification of 50 × ( C , E ), 100 × ( G , I , K main panels, M main panels), and 200 × ( K insets, M insets). O - V Morphological changes and actin-enriched protrusions in UMUC3, and RT4 cells with altered GJB3 expression. Cells with actin-enriched protrusions are marked with white arrows. O - P Brightfield microscopy images depict cells exhibiting a transition towards a round morphology ( O ) of UMUC3 cells with ectopic GJB3 expression. P RT4 cells with GJB3 knockdown demonstrate an elongated shape. Magnifications indicate 100 × or 400 × , respectively. The scale bars refer to 100 μm (left), and 50 µm (right). Q - T Quantification of round or elongated morphology on fixed cells. A cell with elongated or round morphology is identified by the ratio of longest and shortest diameter of the cell from images captured randomly at 630 × magnification using a Zeiss TCS SP5 confocal microscope. Scale bars: 20 μm. The ratio is calculated as the longest diameter of the cell dividing by the shortest diameter of the cell. The ratio is calculated as longest diameter dividing by shortest diameter. A cells with Ratio ≤ 2 is identified as round morphology, while ratio > 2 is elongated morphology. Q , R Immunofluorescence staining photos illustrate the round morphology shifting of ( Q ) UMUC3 cells with GJB3 overexpression compared to cells transfected with empty vector (EV). R RT4 cells with GJB3 knockdown display a transition towards an elongated morphology compared to cells transfected with shScramble (shScr). Cell nuclei are stained with DAPI, and F-actin is labeled with Phalloidin-AF488. S , T The bar graphs reveals percentages of cells with different morphology in total in each group, shown above in Q and R . The percentage was calculated as number of cells with elongated (or rounded) morphology divide cell numbers in total. The percentage values in different groups are marked above or in the bars. Black bars indicate percentages of cells with elongated morphology, and gray bars indicate the percentage of cells with rounded morphology. The statistical significance is calculated by using chi-square statistic. U , V The graphs show the fraction of cells with actin-enriched protrusions in response to GJB3 alterations ( U ) in RT4 or ( V ) in UMUC3 cells. For each picture, the percentage of cells with actin-enriched protrusions is calculated by the number of the cells with actin-enriched protrusions divided by total number of cells. (n) indicates the number of pictures taken in the group

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Migration, Western Blot, Control, Knockdown, Wound Healing Assay, Two Tailed Test, Quantitation Assay, Staining, Expressing, Ex Vivo, Organ Culture, Microscopy, Immunofluorescence, Over Expression, Transfection, Plasmid Preparation, Labeling

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 interactics with F-actin and influences invadopodia formation via actin dynamics. A Representative images demonstrating invadopodia formation of RT4 cells with shGJB3#2. B Quantitation of the invadopodia number was performed in RT4 cells. n = 959 (RT4-shScr), n = 718 (RT4-shGJB3#1), n = 481 (RT4-shGJB3#2). Cells are pooled from three independent sets of experiments. C, Representative pictures showing invadopodia formation of UMUC3 cells upon ectopic GJB3 expression. D Quantitation of the invadopodia number was performed in UMUC3 cells. n = 110 (UMUC3-EV), n = 137 (UMUC3-GJB3). Cells are pooled from three independent sets of experiments. F-actin is visualized by Alexa Fluor 488-phalloidin and Cortactin is visualized by Cy5, respectively. Yellow spots displaying cortactin and F-actin colocalization identify the invadopodia structures. The regions indicated by white boxes are magnified in the insets. The invadopodia are marked with white arrows. The dot plot displays the mean ± SEM data, and the two-tailed Student's t-test was used to assess significance. E Representative pictures indicate the gelatin degradation by RT4 cells upon GJB3 knockdown. F Gelatin degradation capacity of the cells was quantified by measuring the degradation area per RT4 cell. n = 2809 (RT4-shScr), n = 2447 (RT4-shGJB3#1), n = 3544 (RT4-shGJB3#2). G Representative pictures indicate the gelatin degradation by UMUC3-GJB3 cells. H Gelatin degradation capacity of the cells was quantified by measuring the degradation area per UMUC3 cell. n = 1048 (UMUC3-EV), and n = 1246 (UMUC3-GJB3) are pooled from three to four independent experiments. The dot plot displays the mean ± SEM data, and the two-tailed Student's t-test was used to assess significance. I The LifeAct–Ruby signal recovery duration in UMUC3 cells with or without GJB3 is depicted in representative images after LifeAct–Ruby signal bleaching. The white arrows indicate the areas of bleaching. J , The quantitation of bleaching recovery experiments with UMUC3 cells. n = 29 (UMUC3-EV), n = 31 (UMUC3-GJB3). Three different groups of separate experiments' cells are combined. The graphs' data points correspond to the mean ± SEM. P values were calculated using the two-tailed Student's t -test at t = 49 s. K Exemplary pictures displaying the colocalization of GJB3 with F-actin in control UROtsa cells. Insets: enlarged image of the areas shown by white box. L , Quantitation of GJB3 and F-actin colocalization in UROtsa by Pearson’s correlation coefficient. n = 161 (UROtsa-gControl), n = 206 (UROtsa-gGJB3#1), n = 276 (UROtsa-gGJB3#2). M Exemplary pictures displaying the GJB3/F-actin colocalization in UMUC3-GJB3 cells. Insets: enlarged image of the areas shown by white box. N Quantitation of GJB3 and F-actin colocalization in UMUC3 cells by Pearson’s correlation coefficient. n = 672 (UMUC3-EV), and n = 831 (UMUC3-GJB3) are combined from 3 separate experiments. The two-tailed Student's t-test was used to establish significance, and the bar graph displays mean ± SEM results. Alexa Fluor 647 illustrates the F-actin, and Alexa Fluor 488 illustrates GJB3. Yellow highlights denote GJB3 and F-actin overlap. Insets: enlarged images of the colocalized areas shown by white boxes. O , P GJB3 binds bundle actin filaments in a dose-dependent manner by Western blot. Actin (2.5 mg/ml) concentrations of GJB3 (relative GJB3 amount is indicated by + or + +). Supernatant (S) and pellet (P) were subjected to 10% SDS-PAGE after high-speed centrifugation at 100,000 g . Red arrowheads indicate the GJB3, and the red arrow indicates actin filaments visualized by western blot with specific antibodies. n = 3 independent experiments were performed. Scale bars: 10 μm ( A , C , E , G , K and M main panels), 1 μm ( A , C insets), 2 μm ( E , G , K and M insets) and 1 μm ( I ). Images were captured at total magnification of 630 × ( A , C , I , K , M ) and 400 × ( E , G )

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Quantitation Assay, Expressing, Two Tailed Test, Knockdown, Control, Western Blot, SDS Page, Centrifugation

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: IFNγ treatment induces STAT1 activation in BV2 cells. BV2 cells were exposed to 20 ng/mL of IFNγ at different time points and activation of STAT1 was evaluated through different experiments. ( A ) Total protein extracts of IFNγ-treated BV2 cells at different time points were analyzed by Western Blot with anti-phospho-Tyr701 STAT1 antibody, and after stripping, the same blot was analyzed with anti-STAT1 antibody. Densitometric analysis revealed a STAT1 phosphorylation upon IFNγ treatment, peaking at 15 min compared with untreated BV2 cells (CTR). ( B ) RT-qPCR of iNOS and COX2, two STAT1-dependent genes, revealed an increase in their mRNA levels after 18 and 24 h of IFNγ treatment in BV2 cells. Data are expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01 vs. untreated cells (CTR). Single data points are shown as red dots.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Activation Assay, Western Blot, Stripping Membranes, Phospho-proteomics, Quantitative RT-PCR

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: GEE pre-treatment counteracts IFNγ-dependent STAT1 activation in BV2 cells. ( A ) Total protein extracts from BV2 cells pre-treated overnight with GEE and subsequently exposed to 20 ng/mL IFNγ for 18 h were analyzed by Western Blot using a phospho-Tyr701 STAT1 antibody. The same blot was analyzed with anti-Tubulin antibody to check the amount of loaded proteins. Densitometric analysis showed decreased STAT1 phosphorylation in GEE pre-treated cells compared to CTR. * p < 0.05, ** p < 0.01 vs. untreated BV2 cells (CTR). # p < 0.05 vs. IFNγ-treated cells. ( B ) RT-qPCR analysis revealed that iNOS and COX2 mRNA levels were reduced in BV2 cells pre-treated overnight with GEE and then exposed to 20 ng/mL IFNγ for 24 h. Data are expressed as mean ± SD (n = 3). ** p < 0.01 vs. CTR, ## p < 0.01 vs. IFNγ-treated cells. Single data points are shown as red dots.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Quantitative RT-PCR

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: GEE pre-treatment inhibits IFNγ-induced STAT1 phosphorylation and nuclear translocation in BV2 cells. The cells were immunostained with phospho-Tyr701 STAT1 (red) and analyzed by confocal microscopy (lens 60×). Nuclei were stained with DAPI (cyan). The orthogonal sections (ZX and ZY) of merge image reveal that pTYR 701 STAT1 protein is inside the nuclei. Scale bars indicate 10 μm. Images are representative of three separate experiments.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Phospho-proteomics, Translocation Assay, Confocal Microscopy, Staining

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: IFNγ treatment induces S-glutathionylation of STAT1. ( A ) Total protein extracts of BV2 cells left untreated (CTR) or treated with 20 ng/mL IFNγ for the indicated times were subjected to immunoprecipitation with anti-STAT1 antibody. Immunoprecipitated STAT1 (IP: STAT1) was analyzed by Western Blot under non-reducing conditions using anti-SSG antibody and, after membrane stripping, re-probed with anti-STAT1 antibody. Densitometric analysis revealed increased STAT1 S-glutathionylation after 5 and 15 min of IFNγ treatment compared to untreated BV2 cells. * p < 0.05, ** p < 0.01 vs. untreated BV2 cells (CTR). ( B ) Total protein lysates from the same untreated and treated BV2 cells were reserved before pull-down (input) and analyzed by Western Blot with anti-phospho-Tyr701 STAT1 antibody and, after membrane stripping, re-probed with anti-STAT1 antibody. Images are representative of four independent experiments. Single data points are shown as red dots.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Immunoprecipitation, Western Blot, Membrane, Stripping Membranes

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: FNγ induces M1 polarization in BV2 cells through oxidative stress and STAT1 activation. ( A ) The expression of the pro-inflammatory cytokines IL-6 and TNFα in the supernatant of BV2 cells was evaluated by ELISA after 18 h of IFNγ stimulation, with or without GEE pretreatment. GEE markedly reduced the IFNγ-induced secretion of both cytokines, confirming the involvement of oxidative stress in M1 polarization. Data are expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01 vs. CTR. # p < 0.05 vs. IFNγ. ( B ) Parental and STAT1 knockdown in BV2 cells were treated with 20 ng/mL of IFNγ for 18 h. Total protein extracts were analyzed by Western Blot with anti-iNOS and anti-Actin antibodies. Densitometric analysis revealed reduced iNOS induction in STAT1 knockdown in BV2 cells compared with parental cells upon IFNγ treatment. Parental and STAT1 knockdown in BV2 cells cultured without IFNγ were used as controls (CTR). Images are representative of three independent experiments. Single data points are shown as red dots. * p < 0.05, ** p < 0.01 vs. CTR. # p < 0.05 vs. IFNγ.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Knockdown, Western Blot, Cell Culture