Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: (A) Flow cytometry dot plot showing CD45 phosphatase activity versus CD27 expression on gated CD19 + human peripheral B cells. (B and C) CD45 phosphatase activity (B) and CD45 surface expression (C) of CD27 − (blue) and CD27 + B cells (red). Numbers in histograms represent CD45 activity (pCAP-SP1) (B) or CD45 surface expression (C) as the mean fluorescence intensity (MFI) ratio of CD27 + /CD27 − B cells. Bottom graphs: pCAP-SP1 or CD45 surface expression (MFI) in CD27 + relative to CD27 − B cells. (D) CD45 expression versus CD45 phosphatase activity in gated CD27 + MBCs; CD45 hi and CD45 lo expression gates are shown. (E) CD45 phosphatase activity and (F) CD45 expression in CD27 + MBCs expressing low (blue open histogram) or high (red open histogram) levels of surface CD45 compared to CD27 − B cells (filled blue histogram). Graphs show pCAP-SP1 or CD45 MFI relative to CD27 − B cells. n = 12. Related to . ****p < 0.0001.

Article Snippet: FITC Mouse monoclonal anti human CD27 (clone MT271) , Miltenyi Biotec , Cat# 130–093-184; RRID:AB_1036205.

Techniques: Flow Cytometry, Activity Assay, Expressing, Fluorescence

Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: FITC Mouse monoclonal anti human CD27 (clone MT271) , Miltenyi Biotec , Cat# 130–093-184; RRID:AB_1036205.

Techniques: Negative Control, Recombinant, Purification, Staining, Gene Expression, Lysis, Immunoprecipitation, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Cell Isolation, Software, Microscopy

Journal: STAR Protocols

Article Title: Protocol for quantifying drug sensitivity in 3D patient-derived ovarian cancer models

doi: 10.1016/j.xpro.2024.103274

Figure Lengend Snippet:

Article Snippet: CD326 (EpCAM) antibody, anti-human, PE, REAfinity (flow cytometry 1:200) , Miltenyi Biotec , Cat# 130-110-999; RRID: AB_2657495.

Techniques: Immunofluorescence, Flow Cytometry, Control, Recombinant, Red Blood Cell Lysis, Saline, Software, Cell Culture, Plasmid Preparation, Microscopy

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: UFL1 and C53 interact with γTuRC proteins. ( A ) Immunoprecipitation experiments. Extracts from the membranous fraction (P2) of U2OS cells were precipitated with immobilized Abs specific to UFL1 301–389 , C53, γ-tubulin (γ-Tb), or GCP2. The blots were probed with Abs to UFL1, C53, γ-tubulin (γ-Tb), GCP2, GCP4, or calcineurin (Calcin.; negative control). Load ( lane 1 ), immobilized Abs without cell extracts ( lane 2 ), precipitated proteins ( lane 3 ), and Ab-free carriers incubated with cell extracts ( lane 4 ). ( B ) Isotype controls. Extracts from the membranous fraction (P2) of U2OS cells were precipitated with immobilized rabbit Ab to myosin or mouse mAb to MAP2 (IgG2b). Blots were probed with Abs to UFL1, C53, γ-tubulin (γ-Tb), GCP2, or GCP4. Load ( lane 1 ), immobilized Abs not incubated with cell extracts ( lane 2 ), precipitated proteins ( lane 3 ), and carriers without Abs incubated with cell extracts ( lane 4 ). ( C ) The size distribution of UFL1, C53, γ-tubulin (γ-Tb), GCP2, and actin in U2OS whole-cell extracts fractionated on the Superose 6 column. The calibration standards (in kDa) are indicated on the top. The numbers at the bottom denote individual fractions. ( D ) Pooled fractions (Nos. 15–16) and (Nos. 23–24) from fractionation shown in panel ( C ) were precipitated with Ab to γ-tubulin. The blots were probed with Abs to UFL1, C53, and γ-tubulin (γ-Tb). Load ( lane 1 ), immobilized Abs without cell extracts ( lane 2 ), precipitated proteins ( lane 3 ), and Ab-free carriers incubated with cell extracts ( lane 4 ).

Article Snippet: To prepare C-terminally EGFP-tagged human CDK5RAP3 ( CDK5RAP3 ; Ref ID: NM_176096.3), the coding sequence without stop codon was amplified by PCR from the Myc-DDK-tagged CDK5RAP3 (tv3) plasmid (OriGene Technologies; Catalog No. RC209901).

Techniques: Immunoprecipitation, Negative Control, Incubation, Fractionation

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: Subcellular localization of UFL1 and C53. ( A ) U2OS cells expressing TagRFP-tagged proteins were fixed and stained with Ab to γ-tubulin. Localization of C53-TagRFP ( a , d ) and γ-tubulin ( b , e ). Superposition of images ( c , f ) C53-TagRFP, red; γ-tubulin, green; DAPI, blue). Localization of UFL1-TagRFP ( g ) and γ-tubulin ( h ). Superposition of images ( i , UFL1-TagRFP, red; γ-tubulin, green; DAPI, blue). Arrows indicate the same positions. Fixation D/F/M. Scale bar, 20 μm ( a – c , g – i ), 5 μm ( d – f ). ( B ) Association of proteins with purified centrosomes. Centrosomes enriched by centrifugation onto a Ficoll cushion were further purified by sucrose gradient centrifugation. The gradient was fractionated from the bottom. Individual fractions are indicated on the top, sucrose density in the fractions is shown at the bottom. Blots were probed with Abs to pericentrin, CDK5RAP2, ODF2, γ-tubulin (γ-Tb), C53, UFL1, PKCα, histone H1.4, and calnexin.

Article Snippet: To prepare C-terminally EGFP-tagged human CDK5RAP3 ( CDK5RAP3 ; Ref ID: NM_176096.3), the coding sequence without stop codon was amplified by PCR from the Myc-DDK-tagged CDK5RAP3 (tv3) plasmid (OriGene Technologies; Catalog No. RC209901).

Techniques: Expressing, Staining, Purification, Centrifugation, Gradient Centrifugation

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: Characterization of cells lacking UFL1 or C53. ( A ) Changes in the expression of UFL1, C53, and DDRGK1. The blots from whole-cell lysates were probed with Abs to UFL1, C53, DDRGK1, γ-tubulin (γ-Tb), GCP2, and actin (loading control). Densitometric quantification of immunoblots is shown on the right. Relative intensities of corresponding proteins normalized to control cells and the amount of actin in individual samples. Values indicate mean ± SD ( n = 3). One-way ANOVA with Sidak’s multiple comparisons test was performed to determine statistical significance. **, p < 0.01, ****, p < 0.0001. ( B ). Distribution of ER in control ( a ), UFL1_KO ( b ), and C53_KO ( c ) cells visualized by ER-Tracker in live cells. A higher magnification view of the boxed area is shown ( a’ – c’ ). The images ( a – c ) were collected and processed in the same manner. Scale bars, 20 µm ( c ) and 10 µm ( c’ ). ( C ) ER area quantification in cells stained with ER-Tracker. ER area coefficients (area occupied by ER/area free of ER) were calculated from fluorescence intensities for ER-Tracker as described in the . The distributions of ER area coefficients (arbitrary units [AU]) are shown as box plots (three independent experiments, ≥ 15 cells counted for each experimental condition). Box plot of area coefficients in UFL1_KO ( n = 85) and C53_KO cells ( n = 89) relative to control cells ( n = 93). The bottom and top of the box represent the 25th and 75th percentiles. Whiskers below and above the box indicate the 10th and 90th percentiles. One-way ANOVA with Dunnett’s multiple comparisons test was performed to determine statistical significance. ****, p < 0.0001.

Article Snippet: To prepare C-terminally EGFP-tagged human CDK5RAP3 ( CDK5RAP3 ; Ref ID: NM_176096.3), the coding sequence without stop codon was amplified by PCR from the Myc-DDK-tagged CDK5RAP3 (tv3) plasmid (OriGene Technologies; Catalog No. RC209901).

Techniques: Expressing, Western Blot, Staining, Fluorescence

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: Deletion of UFL1 or C53 increases centrosomal microtubule nucleation. Centrosomal microtubule nucleation was evaluated by quantification of microtubule regrowth in fixed cells ( A – F ) and by measuring the microtubule nucleation rate in live cells ( G , H ). ( A , B , D , E ) The distribution of α-tubulin or γ-tubulin fluorescence intensities (arbitrary units [AU]) in 2-μm ROI at 2.0 min of microtubule regrowth are shown as box plots (three independent experiments, >58 cells counted for each experimental condition). ( A , B ) Box plot of α-tubulin ( A ) and γ-tubulin ( B ) fluorescence intensities in UFL1_KO1 cells ( n = 239) relative to control cells ( n = 237). ( D , E ) Box plot of α-tubulin ( D ) and γ-tubulin ( E ) fluorescence intensities in C53_KO1 cells ( n = 257) relative to control cells ( n = 274). The bold and thin lines within the box represent mean and median (the 50th percentile), respectively. The bottom and top of the box represent the 25th and 75th percentiles. Whiskers below and above the box indicate the 10th and 90th percentiles. ( C , F ) Labeling of α-tubulin and γ-tubulin in the microtubule regrowth experiment in the control and UFL1_KO cells ( C ) or the control and C53_KO1 cells ( F ). Cells were fixed (F/Tx/M) at 2.0 min of microtubule regrowth. The pairs of images (α-Tb), (γ-Tb) were collected and processed in the same manner. Scale bars, 5 μm. ( G ) Microtubule nucleation rate (EB3 comets/min) in UFL1_KO1 cells relative to controls. Three independent experiments (at least 10 cells counted in each experiment). Control ( n = 30), UFL1_KO1 ( n = 30). The bold and thin lines within the dot plot represent mean ± SD. ( H ) Microtubule nucleation rate (EB3 comets/min) in C53_KO1 cells relative to controls. Three independent experiments (at least 10 cells counted in each experiment). Control ( n = 35), C53_KO1 ( n = 35). The bold and thin lines within the dot plot represent mean ± SD. Two-tailed, unpaired Student’s t -test was performed to determine statistical significance. ****, p ˂ 0.0001.

Article Snippet: To prepare C-terminally EGFP-tagged human CDK5RAP3 ( CDK5RAP3 ; Ref ID: NM_176096.3), the coding sequence without stop codon was amplified by PCR from the Myc-DDK-tagged CDK5RAP3 (tv3) plasmid (OriGene Technologies; Catalog No. RC209901).

Techniques: Fluorescence, Labeling, Two Tailed Test

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: Deletion of UFL1 or C53 induces UPR and expansion of ER. ( A ) Immunoblot analysis of calnexin and PDI in whole-cell lysates of U2OS cells lacking UFL1 or C53. GAPDH served as a loading control. Densitometric quantification of immunoblots is shown on the right. Relative intensities of corresponding proteins normalized to control cells and the amount of GAPDH in individual samples. Values indicate mean ± SD ( n = 4 for calnexin; n = 3 for PDI). ( B ) Immunofluorescence microscopy. ( a – d ) Control U2OS cells, ( e – h ) UFL1-deficient cells (UFL1_KO) and ( i – l ) C53-deficient cells (C53_KO). Cells were fixed and double-labeled for calnexin ( a , e , i ) and β-tubulin ( b , f , j ; Microtubules). Higher magnification views of the regions delimited by rectangles are shown on the right of images from control ( c , d ), UFL1_KO ( g , h ), and C53_KO ( k , l ) cells. The images ( a , e , i ) and ( c , g , k ) were collected and processed in the same manner. Fixation F/Tx. Scale bars, 20 μm ( j ), and 5 µm ( l ). ( C ) ER area quantification in fixed cells stained with Ab to calnexin. ER area coefficients (area occupied by ER/area free of ER) were calculated from fluorescence intensities for calnexin. The distributions of ER area coefficients (arbitrary units [AU]) are shown as box plots (four independent experiments, ≥16 cells counted for each experimental condition). Box plot of area coefficients in UFL1_KO ( n = 111) and C53_KO cells ( n = 127) relative to control cells ( n = 149). The bottom and top of the box represent the 25th and 75th percentiles. Whiskers below and above the box indicate the 10th and 90th percentiles. ( A , C ) One-way ANOVA with Dunnett’s multiple comparisons test was performed to determine statistical significance. *, p < 0.05, ***, p < 0.001, ****, p < 0.0001.

Article Snippet: To prepare C-terminally EGFP-tagged human CDK5RAP3 ( CDK5RAP3 ; Ref ID: NM_176096.3), the coding sequence without stop codon was amplified by PCR from the Myc-DDK-tagged CDK5RAP3 (tv3) plasmid (OriGene Technologies; Catalog No. RC209901).

Techniques: Western Blot, Immunofluorescence, Microscopy, Labeling, Staining, Fluorescence

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: C53 is sufficient to attenuate centrosomal microtubule nucleation and ER expansion in UFL1 knockout cells. Phenotypic rescue experiment with control cells expressing TagRFP (Control+RFP), UFL1_KO cells expressing TagRFP (UFL1_KO+RFP), and UFL1_KO cells rescued by C53-TagRFP (UFL1_KO+C53-RFP). ( A ) Immunoblot analysis of C53 and UFL1 in whole-cell lysates. GAPDH served as the loading control. Black and empty arrowheads and asterisk denote, respectively, endogenous C53, C53-TagRFP, and its fragment. ( B , C ) The distributions of α-tubulin or γ-tubulin fluorescence intensities (arbitrary units [AU]) in 2-μm ROI at 2.0 min of microtubule regrowth are shown as box plots (three independent experiments, >37 cells counted for each experimental condition). Box plot of α-tubulin ( B ) and γ-tubulin ( C ) fluorescence intensities in UFL1_KO+RFP ( n = 133) and UFL1_KO+C53-RFP ( n = 152) relative to Control+RFP ( n = 174). ( D ) Immunoblot analysis of calnexin in whole-cell lysates. GAPDH served as the loading control. Densitometric quantification of immunoblots is shown on the right. Relative intensities of corresponding proteins normalized to control cells and the amount of GAPDH in individual samples. Values indicate mean ± SD ( n = 4). ( E ) Immunofluorescence microscopy of fixed cells stained with Ab to calnexin to mark ER ( a – c ) and AlexaFluor 647-conjugated WGA to delineate cell boundary ( d – f ). The images ( a – c ) were collected and processed in the same manner. Fixation F/Tx. Scale bar, 10 μm. ( F ) ER area quantification. The distributions of ER area coefficients (area occupied by ER/area free of ER; arbitrary units [AU]) are shown as box plots (three independent experiments, ≥20 cells counted for each experimental condition). Box plot of area coefficients in UFL1_KO+RFP ( n = 91) and UFL1_KO+C53-RFP ( n = 94) relative to Control+RFP ( n = 147). ( B , C , F ) Bold and thin lines within the box represent mean and median (the 50th percentile), respectively. The bottom and top of the box represent the 25th and 75th percentiles. Whiskers below and above the box indicate the 10th and 90th percentiles. ( B – D , F ) One-way ANOVA with Sidak’s multiple comparisons test was performed to determine statistical significance. **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.

Article Snippet: To prepare C-terminally EGFP-tagged human CDK5RAP3 ( CDK5RAP3 ; Ref ID: NM_176096.3), the coding sequence without stop codon was amplified by PCR from the Myc-DDK-tagged CDK5RAP3 (tv3) plasmid (OriGene Technologies; Catalog No. RC209901).

Techniques: Knock-Out, Expressing, Western Blot, Fluorescence, Immunofluorescence, Microscopy, Staining

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: Tunicamycin affects the distribution of C53. Cells were treated with 1 µg/mL tunicamycin (+Tunicam.) or DMSO carrier (Control) for 24 h. ( A , B ) C53_KO cells expressing C53-TagRFP (C53_KO+C53-RFP). ( A ) Immunofluorescence microscopy of fixed cells stained for γ-tubulin. ( a – c ) Control cells. C53-TagRFP ( a ), γ-tubulin ( b ), superposition of images ( c , C53-TagRFP, red; γ-tubulin, green). ( d – f ) Tunicamycin-treated cells. C53-TagRFP ( d ), γ-tubulin ( e ), superposition of images ( f , C53-TagRFP, red; γ-tubulin, green). Images ( a , d ) and ( b , e ) were collected and processed in the exact same manner. Fixation Tx/F/M. Scale bar, 20 µm. ( B ) Immunoblot analysis of whole-cell lysates with Abs to RFP, UFL1, γ-tubulin (γ-Tb), and GAPDH (loading control). Densitometric quantification of immunoblots is shown on the right. Relative intensities of corresponding proteins normalized to control cells and the amount of GAPDH in individual samples. Values indicate mean ± SD ( n = 4). ( C , D ) C53-KO cells expressing TagRFP (C53_KO+RFP). ( C ) Immunofluorescence microscopy of fixed control cells ( a ) and tunicamycin-treated cells ( b ). Images ( a , b ) were collected and processed in exactly the same manner. Fixation F/Tx. Scale bar, 20 µm. ( D ) Immunoblot analysis of whole-cell lysates with Abs to RFP, γ-tubulin (γ-Tb), and GAPDH (loading control). Densitometric quantification of immunoblots is shown on the right. Relative intensities of corresponding proteins normalized to control cells and the amount of GAPDH in individual samples. Values indicate mean ± SD ( n = 3). ( E ) Distribution of proteins in fractions after differential centrifugation of the cell homogenate. Cell fractions were prepared as described in the . Cell homogenate ( lane 1 ), pellet P1 ( lane 2 ). Immunoblot analysis with Abs to C53, pericentrin, and actin (loading control). Densitometric quantification of immunoblots is shown on the right. Intensities of corresponding proteins in P1 normalized to loads (relative intensity 1.0). Values indicate mean ± SD ( n = 4). ( F ) Association of proteins with purified centrosomes. Immunoblot analysis with Abs to C53 and centrosomal protein ODF2 (loading control). Densitometric quantification of immunoblots is shown on the right. Relative intensity of C53 normalized to control cells and the amount of ODF2. Values indicate mean ± SD ( n = 7). A two-tailed, unpaired Student’s t -test was performed to determine statistical significance. **, p < 0.01; **** p < 0.0001.

Article Snippet: To prepare C-terminally EGFP-tagged human CDK5RAP3 ( CDK5RAP3 ; Ref ID: NM_176096.3), the coding sequence without stop codon was amplified by PCR from the Myc-DDK-tagged CDK5RAP3 (tv3) plasmid (OriGene Technologies; Catalog No. RC209901).

Techniques: Expressing, Immunofluorescence, Microscopy, Staining, Western Blot, Centrifugation, Purification, Two Tailed Test

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