Journal: Acta biochimica et biophysica Sinica

Article Title: PGAM5 deacetylation mediated by SIRT2 facilitates lipid metabolism and liver cancer proliferation.

doi: 10.3724/abbs.2023155

Figure Lengend Snippet: Figure 2. SIRT2 mediates PGAM5-K191 deacetylation (A) Flag-tagged PGAM5 was stably expressed in PLC cells treated with TSA and/or NAM, followed by immunoprecipitation using an anti-Flag antibody and western blot analysis of the lysine acetylation (K-Ac) of PGAM5. (B) Localization of sirtuin family deacetylases in cells. (C) Immunoprecipitation was performed using anti-Flag antibody or IgG in PLC cells. Flag-PGAM5 and HA- SIRT2 expression were determined by western blot analysis. (D) PLC cells stably expressing Flag-PGAM5 were further transfected with vectors expressing EV or HA-SIRT2. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. The acetylation of Flag-PGAM5 was detected by western blot analysis. (E) PLC cells stably expressing Flag-PGAM5 were further transfected with vectors expressing NTC or SIRT2 shRNA. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. The acetylation of Flag-PGAM5 was determined by western blot analysis. (F) PLC cells with stable knockdown of endogenous PGAM5 were further transfected with vectors expressing Flag-PGAM5- WT, Flag-PGAM5-K191R and Flag-PGAM5-K191Q. The above cells were subsequently infected with viruses expressing NTC or SIRT2 shRNA. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. The acetylation of Flag-PGAM5 was detected by western blot analysis. (G) Diagram of the mechanism by which SIRT2 regulates the deacetylation of PGAM5-K191.

Article Snippet: Primary antibodies against the following proteins were used: PGAM5 (Cat# sc-515880; Santa Cruz, Santa Cruz, USA), PGAM5 (Cat# 28445-1-AP; Proteintech, Rosemont, USA), SIRT2 (Cat# 19655-1-AP; Proteintech), SIRT2 (Cat# sc-28298; Santa Cruz), ME1 (Cat# 16619-1-AP; Proteintech), acetylated-lysine (Cat# 9441; CST, Beverly, USA), acetylated-lysine (Cat# PTM-105; Jingjie PTM BioLab, Hangzhou, China), phosphorylation-serine (Cat# sc-81514; Santa Cruz), HA-HRP (Cat# 2999; CST), HA (Cat# 51064-2-AP; Proteintech), mouse IgG1 isotype control (Cat# 66360-1-Ig; Proteintech), Flag (Cat# F1804; Sigma-Aldrich), and β-actin (Cat# 66009-1-Ig; Proteintech).

Techniques: Stable Transfection, Immunoprecipitation, Western Blot, Expressing, Transfection, shRNA, Knockdown, Infection

Journal: Acta biochimica et biophysica Sinica

Article Title: PGAM5 deacetylation mediated by SIRT2 facilitates lipid metabolism and liver cancer proliferation.

doi: 10.3724/abbs.2023155

Figure Lengend Snippet: Figure 3. The deacetylation of PGAM5-K191 mediated by SIRT2 facilitates ME1 dephosphorylation and activity (A) Flag-PGAM5 and HA-ME1 were co-overexpressed in PLC cells. Immunoprecipitation was performed using an anti-Flag antibody or IgG, followed by western blot analysis of HA-ME1 and Flag-PGAM5. (B) PLC cells stably expressing Flag-PGAM5 were further transfected with vectors expressing NTC or SIRT2 shRNA. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. ME1 and Flag-PGAM5 were determined by western blot analysis. (C) PLC cells stably expressing Flag-PGAM5 were further transfected with empty vector (EV) or vectors expressing HA-SIRT2. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. ME1 and Flag-PGAM5 were determined by western blot analysis. (D) PLC cells with stable knockdown of endogenous PGAM5 were further transfected with EV or vectors expressing Flag-wild-type- PGAM5, Flag-PGAM5-K191R and Flag-PGAM5-K191Q. The above cells were subsequently infected with viruses expressing HA-ME1. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. Flag-PGAM5 and HA-ME1 were determined by western blot analysis. (E) PLC cells with stable knockdown of endogenous PGAM5 were further transfected with EV or vectors expressing HA-PGAM5, HA-PGAM5-K191R and HA-PGAM5-K191Q. The above cells were knocked down with ME1 shRNA and were subsequently infected with viruses expressing Flag-ME1-WT or Flag-ME1-S336A. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. Phosphorylation of ME1, Flag-ME1 and HA-PGAM5 was determined by western blot analysis. (F) PLC cells with stable knockdown of endogenous PGAM5 were further transfected with vectors expressing PGAM5-WT, PGAM5-K191R and PGAM5-K191Q. The above cells were knocked down with ME1 shRNA and were subsequently infected with viruses expressing Flag-ME1-WT or Flag-ME1-S336A. Meanwhile, the cells were infected with the viruses expressing NTC or SIRT2 shRNA. Immunoprecipitation was performed using an anti-Flag antibody or IgG in the above cells. Phosphorylation of ME1, Flag-ME1, HA-PGAM5 and SIRT2 was detected by western blot analysis. (G) The metabolic enzyme activity of ME1 was detected by a kit assay in the PLC cells used in (F). The enzyme activity data are expressed as the mean±SD (n=3) and compared with the corresponding groups. *P<0.05, ns, no significant difference.

Article Snippet: Primary antibodies against the following proteins were used: PGAM5 (Cat# sc-515880; Santa Cruz, Santa Cruz, USA), PGAM5 (Cat# 28445-1-AP; Proteintech, Rosemont, USA), SIRT2 (Cat# 19655-1-AP; Proteintech), SIRT2 (Cat# sc-28298; Santa Cruz), ME1 (Cat# 16619-1-AP; Proteintech), acetylated-lysine (Cat# 9441; CST, Beverly, USA), acetylated-lysine (Cat# PTM-105; Jingjie PTM BioLab, Hangzhou, China), phosphorylation-serine (Cat# sc-81514; Santa Cruz), HA-HRP (Cat# 2999; CST), HA (Cat# 51064-2-AP; Proteintech), mouse IgG1 isotype control (Cat# 66360-1-Ig; Proteintech), Flag (Cat# F1804; Sigma-Aldrich), and β-actin (Cat# 66009-1-Ig; Proteintech).

Techniques: De-Phosphorylation Assay, Activity Assay, Immunoprecipitation, Western Blot, Stable Transfection, Expressing, Transfection, shRNA, Plasmid Preparation, Knockdown, Infection, Phospho-proteomics

Journal: Science Advances

Article Title: IgM and IgG1 B cell receptors differentially affect B cell fates and dictate the pathogenesis of mature B cell lymphomas

doi: 10.1126/sciadv.adp9391

Figure Lengend Snippet: ( A ) IGHM (left) and IGHG1 (right) expression as transcripts per million (TPM) in IgM high ( n = 111) or IgG1 high ( n = 63) samples. ( B ) Kaplan-Meir curves displaying progression-free survival (probability) in IgM and IgG1 high samples. ( C ) Distribution of distinct DLBCL subtypes in IgM and IgG1 high groups. ( D to F ) Kaplan-Meir curves for progression-free survival (probability) in IgM high and IgG1 high in BN2 (D), EZB (E), and Other (F) genetic subtypes. sgRNA, single guide RNA. ( G ) Engineering of switched isogenic human lymphoma cell lines using CRISPR-Cas9. ( H ) IgM and IgG1 expression in BJAB, OCI-Ly7. and HBL-1 cells before and after CRISPR-Cas9 editing. ( I ) Tumor growth of IgM or IgG1 cells (1 million per mouse) subcutaneously injected into NSG mice ( n = 5 mice). ( J ) Tumor growth curves of IgM or IgG1 expressing BJAB subcutaneously injected into NSG mice. ( K ) Surface expression of Ig-kappa light chain (Igκ) in IgM BJAB cells (gray), IgM tumors (gray dashed), and IgG1 BJAB cells (red), tumors which grew from IgG1 BJAB cells (red dashed) and BJAB IgM cells stained with secondary Ab (no Ab control, black). ( L ) Cotransplantation of IgG1 and IgM lymphoma cells mixed at 1:1. Bottom; Representative plots of IgM and IgG1 at days 0 and 21 posttransplant. ( M ) Quantification of IgM and IgG1 cell frequency in tumors after 21 days ( n = 4 for Mino IgG1, Mino IgM, and BJAB IgG1; n = 5 for BJAB IgM). ( N and O ) Bar graphs quantifying percent IgM and IgG1 BJAB (N) and Mino (O) cells after mixing (day 0) and upon coculture for 5 or 8 days. Statistical significance calculated by log-rank test [(B) and (D) to (F)], t test (I), and two-way ANOVA [(M) to (O)]. Error bars represent ± SEM; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. [(G) and (L)] Created in BioRender. Shukla, V. (2025) https://BioRender.com/1mfdxwl . See also fig. S1.

Article Snippet: For stimulation experiments, the isolated IgG1 and IgM B cells were then stimulated for 12 hours with anti-kappa light chain antibody (1 μg/ml; 75861S, Cell Signaling Technology) in supplemented RPMI as previously described at a density of –2 million cells/ml.

Techniques: Expressing, CRISPR, Injection, Staining, Control

Journal: Science Advances

Article Title: IgM and IgG1 B cell receptors differentially affect B cell fates and dictate the pathogenesis of mature B cell lymphomas

doi: 10.1126/sciadv.adp9391

Figure Lengend Snippet: ( A ) Schematic for SRBC immunization. ( B ) Representative plots for gating GC B cells (left). Quantification of cleaved caspase-3 from IgM and IgG1 GC B cells (right). Connecting lines show data points within each mouse. ( C ) Schematic of iGB cocultures. ( D ) Representative plots for IgM and IgG1 frequency from CD19+CD138−, 12 hours poststimulation (BCR) with anti-kappa antibody (1 μg/ml). ( E ) Quantification of IgG1 and IgM B cell frequency with or without BCR stimulation. IgM and IgG1 are normalized to respective frequencies under untreated conditions ( n = 7, three independent experiments). ( F ) Quantification of IgM and IgG1 B cell numbers ( n = 7, three independent experiments). ( G ) Representative flow plots (left) and quantification of cleaved caspase-3 (right). Cleaved caspase-3 is normalized to IgM cells in the untreated group ( n = 17 from seven independent experiments). ( H ) Genome browser tracks of RNA-seq data in the IgH locus ( Ighg1 and Ighm genes are highlighted). Black and red tracks are from IgM and IgG1 cells from untreated and BCR-stimulated groups. Tracks are averaged from three replicates. ( I ) Volcano plot displaying DEGs in IgG1- versus IgM expressing cells from untreated (left) and BCR-stimulated conditions (right). ( J ) Up-regulated (red) and down-regulated (gray) pathways identified from DEGs in IgG1 compared with IgM using Metascape. The x axis represents the z score. ( K ) Gene set enrichment analysis (GSEA) of apoptosis and abnormal mitochondrial morphology gene sets. The y axis denotes enrichment score. NES, normalized enrichment score; P nominal P value. Statistical significance is calculated by the paired t test (B) and ordinary two-way ANOVA with Šídák multiple comparisons test [(E) and (F)]. Error bars represent ± SEM; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. Schematics in (A) and (C) created in BioRender. Shukla, V. (2025) https://BioRender.com/1mfdxwl . See also fig. S3.

Article Snippet: For stimulation experiments, the isolated IgG1 and IgM B cells were then stimulated for 12 hours with anti-kappa light chain antibody (1 μg/ml; 75861S, Cell Signaling Technology) in supplemented RPMI as previously described at a density of –2 million cells/ml.

Techniques: RNA Sequencing, Expressing

Journal: Science Advances

Article Title: IgM and IgG1 B cell receptors differentially affect B cell fates and dictate the pathogenesis of mature B cell lymphomas

doi: 10.1126/sciadv.adp9391

Figure Lengend Snippet: ( A ) Representative plots of IgM and IgG1 GC B cells (Fas+, CD38low) (left) 14 days postimmunization with SRBCs. Histograms show MitoTracker Green staining in IgM and IgG1 cells (middle). Median fluorescence intensity of MitoTracker Green in IgM and IgG1 cells (right) ( n = 8). ( B ) Representative ImageStream analysis of IgM and IgG1 GC B cells stained with DAPI (nucleus), Tomm20 (PE: mitochondria), IgG1 (FITC), and IgM (PE-Cy7). ( C ) Histograms showing MitoTracker Green in IgM and IgG1 cells from iGB cultures stimulated with anti-kappa (1 μg/ml) for 12 hours (left). Quantification of relative MitoTracker Green mean fluorescence intensity (MFI) (middle). Values are normalized to untreated IgM from respective experiments. Relative MitoTracker Green MFI also normalized to cell size (right) ( n = 6 from two independent experiments). ( D ) Flow cytometry histograms showing MitoTracker Green staining (top) and quantification of MitoTracker Green MFI (bottom) in IgM and IgG1 splenic B cells from heterozygous IgH γ1μ /+ mice ( n = 3). ( E ) Representative confocal microscopy images of splenic B cells from heterozygous IgH γ1μ /+ mice (left) and quantified as mean fluorescence intensity (right). ( F ) Experimental design with IgG1 cells isolated from expressing homozygous IgH γ1μ/γ1μ mice and treated with TAT-Cre for 2 hours and stimulated with anti-kappa antibody. ( G ) Representative flow plots of IgM and IgG1 staining of IgH γ1μ/γ1μ splenic B cells before TAT-Cre treatment and 24 and 48 hours after TAT-Cre treatment. ( H ) Histograms showing MitoTracker Green staining (left) and quantification of MitoTracker Green MFI (right) in IgM and IgG1 B cells from IgH γ1μ /γ1μ mice 48 hours post-BCR anti-kappa stimulation (2 μg/ml) ( n = 5). Statistical significance is calculated by the paired t test (A), ordinary two-way ANOVA with Šídák multiple comparisons test [(C), (D), and (H)], and unpaired t test (E). Error bars represent ± SEM; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. See also fig. S4.

Article Snippet: For stimulation experiments, the isolated IgG1 and IgM B cells were then stimulated for 12 hours with anti-kappa light chain antibody (1 μg/ml; 75861S, Cell Signaling Technology) in supplemented RPMI as previously described at a density of –2 million cells/ml.

Techniques: Staining, Fluorescence, Flow Cytometry, Confocal Microscopy, Isolation, Expressing

Journal: Science Advances

Article Title: IgM and IgG1 B cell receptors differentially affect B cell fates and dictate the pathogenesis of mature B cell lymphomas

doi: 10.1126/sciadv.adp9391

Figure Lengend Snippet: ( A ) Changes in OCR measured over time in IgM (black) and IgG1 (red) IgH γ1μ /+ B cells stimulated with anti-kappa antibody, treated with oligomycin, phenylhydrazone (FCCP), and antimycin A and rotenone. ( B ) Changes in OCR measured over time in IgM and IgG1 BJAB lymphoma cells treated with anti-Igκ antibody, oligomycin, FCCP, antimycin A, and rotenone. ( C ) Representative flow cytometry plots of IgM and IgG1 splenic GC B cells (Fas+, CD38 low) stained with MitoTracker Green ( x axis) and TMRM ( y axis). Gated population indicates dysfunctional mitochondria (left), identified as lower TMRM and positive MitoTracker Green staining. Quantification of the frequency of dysfunctional mitochondria in IgM and IgG1 GC B cells (right) ( n = 3). ( D ) Representative flow cytometry plots of IgM and IgG1 B cells from in vitro iGB cultures stained with MitoTracker Green ( x axis) and TMRM ( y axis) (left). Quantification of the frequency of dysfunctional mitochondria measured by identifying as lower TMRM and positive MitoTracker Green staining (right) ( n = 5 from two independent experiments). ( E ) Frequency of cleaved caspase-3–positive cells in IgM and IgG1 B cells from in vitro iGB cultures ( n = 5 from two independent experiments). ( F and G ) Representative flow cytometry plots of OCI-Ly7 (F) and BJAB (G) IgM and IgG1 lymphoma cells stained with MitoTracker Green and TMRM after BCR treatment for 24 hours (left). Quantification of the frequency of dysfunctional mitochondria (right) ( n = 6 from two independent experiments (F) and n = 3 (G) (right). Statistical significance is calculated using a paired t test (C) and ordinary two-way ANOVA with Šídák multiple comparisons test [(D) to (G)]. Error bars represent means ± SEM; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. Schematic in (F) created in BioRender. Shukla, V. (2025) https://BioRender.com/1mfdxwl . See also fig. S4.

Article Snippet: For stimulation experiments, the isolated IgG1 and IgM B cells were then stimulated for 12 hours with anti-kappa light chain antibody (1 μg/ml; 75861S, Cell Signaling Technology) in supplemented RPMI as previously described at a density of –2 million cells/ml.

Techniques: Flow Cytometry, Staining, In Vitro

Journal: Frontiers in Immunology

Article Title: Toll-Like Receptor 7 Enhances Rabies Virus-Induced Humoral Immunity by Facilitating the Formation of Germinal Centers

doi: 10.3389/fimmu.2019.00429

Figure Lengend Snippet: TLR7 is indispensable for optimal antibody production after RABV immunization. TLR7 −/− ( n = 12), TLR3 −/− ( n = 10), or WT mice ( n = 11–12) were immunized i.m. with 10 6 FFU RABV vaccine strain LBNSE in the hind legs. At indicated times post immunization, sera were collected for measuring RABV-specific total IgG (A) and VNA titers (B) . Error bars represent standard error of mean (SEM) ( * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Bound Ab was detected using HRP-conjugated Abs (IgG (1:1,000), IgG1 (1:1,500), IgG2a (1:1,500), IgG2b (1:2,000), IgG3 (1:1,500), or IgM (1:2,000) (Boster, Wuhan, China).

Techniques:

Journal: Frontiers in Immunology

Article Title: Toll-Like Receptor 7 Enhances Rabies Virus-Induced Humoral Immunity by Facilitating the Formation of Germinal Centers

doi: 10.3389/fimmu.2019.00429

Figure Lengend Snippet: TLR7 facilitates the formation of GCs. (A,B) TLR7 −/− and WT mice were immunized i.m. with 10 6 FFU LBNSE in the hind legs. Draining LNs were excised, and tissue sections were prepared and stained for GCs (GL-7, red; B220, blue; and IgG, green). Scale bars represent 200 μm. Representative sections are shown in (A) Numbers of GCs (GL-7 positive cell clusters labeled with white arrows) in the draining LNs are calculated and shown in (B) ( n = 3). (C) Blood samples were collected at indicated time points and the concentration of serum CXCL13 was determined by using a commercial ELISA kit ( n = 9). Error bars represent SEM ( * P < 0.05; *** P < 0.001).

Article Snippet: Bound Ab was detected using HRP-conjugated Abs (IgG (1:1,000), IgG1 (1:1,500), IgG2a (1:1,500), IgG2b (1:2,000), IgG3 (1:1,500), or IgM (1:2,000) (Boster, Wuhan, China).

Techniques: Staining, Labeling, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Toll-Like Receptor 7 Enhances Rabies Virus-Induced Humoral Immunity by Facilitating the Formation of Germinal Centers

doi: 10.3389/fimmu.2019.00429

Figure Lengend Snippet: TLR7 facilitates Th1-biased immune responses after RABV vaccination. TLR7 −/− and WT mice were immunized i.m. with 10 6 FFU LBNSE in the hind legs. Spleens were collected at 7 d.p.i. and subjected to ELISpot assay to measure RABV-specific IFN-γ (A,B) and IL-4 (C,D) producing cells ( n = 3 mice/ group). (E–G) Blood samples were collected at the indicated times p.i. for analysis of RABV G-specific IgG1 (E) , IgG2a (F) , and IgG2b (G) ( n = 10). Error bars represent SEM. ND, not detectable. NS, no significantly difference ( * P < 0.05; *** P < 0.001).

Article Snippet: Bound Ab was detected using HRP-conjugated Abs (IgG (1:1,000), IgG1 (1:1,500), IgG2a (1:1,500), IgG2b (1:2,000), IgG3 (1:1,500), or IgM (1:2,000) (Boster, Wuhan, China).

Techniques: Enzyme-linked Immunospot