Journal: Science Advances

Article Title: STAT3 haploinsufficiency is associated with autosomal dominant hyper-IgE syndrome

doi: 10.1126/sciadv.adw2464

Figure Lengend Snippet: ( A ) STAT3 expression was evaluated by Western blot in Stat3 +/+ and Stat3 +/− cells derived from the spleen, thymus, and bone marrow (BM). Tubulin was used as loading control. Each lane represents one mouse. ( B ) A quantification of the STAT3/loading control ratio after normalization is shown for independently performed experiments as in (A). The mean ± SD is plotted. ( C ) IgE serum levels in Stat3 +/+ and Stat3 +/− mice were measured by ELISA. ( D ) Flow cytometry to identify IgG1 + and IgE + cells from IgM − IgD − cells 4 days after culturing Stat3 +/+ and Stat3 +/− B220 + splenocytes with IL-4 (12.5 ng/ml) and anti-CD40 antibody (62.5 ng/ml) in the absence or presence of IL-21 (25 ng/ml). Numbers in dot plots represent cell frequencies. ( E ) Mean (±SD) frequencies of the IgG1 + and IgE + cells as gated in (D). ( F ) Levels of IgG1 and IgE in the supernatant of cell cultures as done in (D) were measured by ELISA. B220 + splenocytes from Stat3 +/+ and Stat3 +/− mice were stimulated for different time points with IL-21 (50 ng/ml) ( G ), IL-10 (50 ng/ml) ( H ), and IL-6 (50 ng/ml) ( I ), and the phosphorylation of STAT3 (p-STAT3) was evaluated by flow cytometry. Mean (±SD) of mean fluorescent intensity (MFI) is plotted. Data are representative of at least three independent experiments. P values by unpaired t test (C) or two-way ANOVA followed by Bonferroni’s or Tukey’s multiple comparisons test (B and E to I).

Article Snippet: Cells were cultured in complete B cell medium [RPMI with 10% FBS, 10 mM Hepes, GlutaMAX, penicillin-streptomycin (Gibco), and 50 μM β-mercaptoethanol (Sigma-Aldrich), supplemented with IL-4 (12.5 ng/ml; PeproTech) and anti-CD40 antibody (62.5 ng/ml; Miltenyi Biotec), in the absence or presence of IL-21 (25 ng/ml; PeproTech)].

Techniques: Expressing, Western Blot, Derivative Assay, Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Phospho-proteomics

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the phenotype of periphery innate immune cells in EAE mice at first day post‐injection. Single‐cell suspensions were obtained from blood and spleen tissue of anti‐CD52‐Ab or vehicle‐treated EAE mice at first day after the final dose injection. Cells were stained for various surface markers and analysed by flow cytometry. (A) Flow cytometry gating strategy. (B) Blood and spleen CD11c + cells were analysed for expression of MHC‐II ( n = 9), CD40 ( n = 5), CD80 ( n = 5) and CD86 ( n = 9 blood; n = 5 spleen). (C) Blood and spleen CD11b + cells were analysed for expression of MHC‐II ( n = 8), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5), CD38 ( n = 5) and CD206 ( n = 9 blood; n = 7 spleen). Unpaired two‐tailed Student's t test. * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the phenotype and function of CNS innate immune cells in EAE mice at first day post‐injection. Single‐cell suspensions were obtained from CNS (brain and spinal cord) tissue of anti‐CD52‐Ab or vehicle‐treated EAE mice at first day after the final treatment dose. Cells were stained for various surface markers and analysed by flow cytometry. (A) Flow cytometry gating strategy. (B) CD11b + CD45 lo/neg resident microglia and (C) CNS CD11b + CD45 hi infiltrating macrophages and were analysed for expression of MHC‐II ( n = 10), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5 microglia; n = 9 macrophages) and CD206 ( n = 7 microglia; n = 5 macrophages). Unpaired two‐tailed Student's t test. CD11b + cells were isolated from CNS tissue of anti‐CD52‐Ab or vehicle‐treated mice at first day after the final treatment dose. (D) Respiratory burst activity of CD11b + ‐isolated cells. (E) Phagocytosis of FITC‐coated beads by CD11b + isolated cells. Unpaired two‐tailed Student's t test. (F) Cells were left unstimulated or were stimulated with LPS or Poly(I:C)(PIC) for 24 h and cytokine production determined by ELISA. Two‐way ANOVA with Sidak multiple comparisons test, * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test, Isolation, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the phenotype of periphery and CNS innate immune cells in EAE mice at three weeks post‐injection. Single‐cell suspensions were obtained from blood, spleen and CNS (brain and spinal cord) tissue of anti‐CD52‐Ab or vehicle‐treated mice three weeks after the final treatment dose. Cells were stained for various surface markers and analysed by flow cytometry. (A) Blood and spleen CD11c + cells were analysed for expression of MHC‐II ( n = 9), CD40 ( n = 5), CD80 ( n = 5) and CD86 ( n = 9 blood; n = 5 spleen). (B) Blood and spleen CD11b + cells were analysed for expression of MHC‐II ( n = 8), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5), CD38 ( n = 5) and CD206 ( n = 9 blood; n = 7 spleen). (C) CNS CD11b + CD45 hi ‐infiltrating macrophages and CD11b + CD45 lo/neg resident microglia were analysed for expression of MHC‐II ( n = 10), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5 microglia; n = 9 macrophages) and CD206 ( n = 7 microglia; n = 5 macrophages). Unpaired two‐tailed Student's t test. * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the function of periphery and CNS innate immune cells in SCID mice at first day post‐injection. SCID mice were treated with either vehicle or 10 mg/kg murine anti‐CD52‐Ab‐Ab for five consecutive days. Single‐cell suspensions were then obtained from blood and spleen tissues first day after the final treatment dose. Cells were stained for various surface markers and analysed by flow cytometry. (A) Blood and spleen CD11c + cells were analysed for expression of MHC‐II, CD40, CD80 and CD86 (all n = 7). (B) Blood and spleen CD11b + cells were analysed for expression of MHC‐II, CD40, CD80, CD86, and CD206 (all n = 7). Unpaired two‐tailed Student's t test. * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: A non-catalytic role of TET3 promotes open chromatin and enhances global transcription

doi: 10.1101/177626

Figure Lengend Snippet: A) TET3 exon usage in germinal vesicle oocytes (data from ). RNA-seq reads were quantified per position in transcript and are shown in blue. Averages over exons are shown in red. Splice junctions were quantified as percentages of the most abundant splice junction and are shown in green for the oocyte specific transcript (Tet3) and in purple for the CXXC containing transcript (Tet3 CXXC ). The majority of transcripts start at the oocyte promoter and most are spliced directly onto exon 3 skipping the CXXC domain containing exon 2. Splicing from the oocyte exon onto exon 2 results in a frame shift. B) Experimental setup of the transdifferentiation assay: embryonic stem (ES) cell lines are cultured in trophoblast stem (TS) cell conditions (see methods) for 6 days. TS-like (TSL) cells are identified morphologically and by immunofluorescent staining for the trophoblast marker CD40. C) Percentage of CD40 positive cells at day 6 of transdifferentiation. iRas cells were used as a positive control for facilitated transdifferentiation . E14 ES cells were used as a negative control. TET3 low and TET3 high denote clonal cell lines with low and high expression levels of Tet3 respectively. **** p<0.0001, Chi-squared test. D) Tet3 expression during somatic reprogramming monitored by RNA-seq (data from . MEF: mouse embryonic fibroblasts; OSKM: ectopic expression of Oct4, Sox2, Klf4 and cMyc; iPS: induced pluripotent stem cells; reprogramming cells are negative for Thy1 and positive for SSEA1; refractory cells are positive for Thy1 and negative for SSEA1 . E) Tet3 expression during primordial germ cell (PGC) development monitored by RNA-seq (data from . E: embryonic day. Green datapoints represent levels in male embryos, orange datapoints levels in female embryos.

Article Snippet: Cells were incubated with anti-CD40 antibody (AF440, R&D systems, 1:50) for 30 min, then washed and incubated with anti-goat Alexa Fluor 647 antibody (A21447, Thermo Fisher Scientific, 1:500) and anti-THY1-PE (12-0900-81, eBioscience, 1:2500, feeder detection).

Techniques: RNA Sequencing, Cell Culture, Staining, Marker, Positive Control, Negative Control, Expressing

Journal: bioRxiv

Article Title: A non-catalytic role of TET3 promotes open chromatin and enhances global transcription

doi: 10.1101/177626

Figure Lengend Snippet: A) Expression changes of a group of genes differentially regulated by TET3 for cells that either overexpress the first half of the protein (TET3 trunc ) or the second half (CD, catalytic domain), analysed by qPCR and shown as fold changes of TET3 variant expressing versus control cells. TET3 trunc increases expression whereas the CD does not. B) Percentage of CD40 (trophoblast marker) positive cells at the end point of a transdifferentiation experiment. Trophoblast stem (TS) cells were used as a positive control for CD40 staining. Oct4 KO cells were used as a model for facilitated trans-differentiation ( , ). Control cells are GFP negative cells from the Tet3 trunc transgenic line. TET3 trunc expressing cells show a significantly higher proportion of transdifferentiated cells compared to control cells as determined by Chi-squared test (**** p<0.0001). C) Heatmap of TET3 ChIP-seq peaks identified using MACS peak calling. Peaks identified for each individual sample were combined and reads in peaks quantified in all samples. Every row represents one peak. Individual replicates are shown and grouped as denoted by horizontal lines below. Signal in peaks was extremely similar between TET3, TET3 CXXC and TET3 trunc . D) Quantitative comparison of TET3 interaction partners by Stable Isotope Labelling with Amino Acids in Cell culture (SILAC). For every protein the number of peptides found in the TET3 sample was divided by the number of peptides found in the TET3 trunc samples, and normalised for the ratio of found TET3 peptides. The list was manually curated to exclude actins, keratins and ribosomal proteins. The high log 2 ratios for JIP3 and OGT indicate that their interactions with TET3 are lost upon deletion of the catalytic domain (see also Table S4). E) Cells were transiently transfected with siRNA against OGT and then TET3 expression was induced. Cells were sorted for TET3 and a group of genes differentially regulated by TET3 analysed by qPCR. Shown are expression fold changes of TET3 positive versus TET3 negative cells for cells treated with siRNA against OGT or untransfected cells (NT). Genes normally upregulated upon TET3 expression are still upregulated in the absence of OGT. F) Expression changes of a group of genes differentially regulated by TET3 for cells that either overexpress TET3 (oocyte isoform) or TET3 CXXC , analysed by qPCR and shown as log 2 fold changes of TET3 variant expressing versus control cells. TET3 and TET3 CXXC have a similar effect on expression. G) Expression changes of a group of genes differentially regulated by TET3 for cells that either overexpress TET3, TET3 trunc , exon 4 only or GFP (see for constructs), analysed by qPCR and shown as fold change between TET3 variant expressing versus control cells. Exon 4 by itself induces the similar expression changes as TET3 and TET3 trunc . H) Zoomed out view of : Evolutionary conservation across placental mammals of Tet3 (exon 4 to end) and the corresponding regions of other Tet family members was measured using PhastCons . Exons are represented as grey boxes with exon 4 and corresponding exons highlighted in blue. Conservation scores between 0 and 1 are represented by the height of the black bars.

Article Snippet: Cells were incubated with anti-CD40 antibody (AF440, R&D systems, 1:50) for 30 min, then washed and incubated with anti-goat Alexa Fluor 647 antibody (A21447, Thermo Fisher Scientific, 1:500) and anti-THY1-PE (12-0900-81, eBioscience, 1:2500, feeder detection).

Techniques: Expressing, Variant Assay, Control, Marker, Positive Control, Staining, Transgenic Assay, ChIP-sequencing, Comparison, Cell Culture, Multiplex sample analysis, Transfection, Construct

Journal: Experimental & Molecular Medicine

Article Title: Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation

doi: 10.3858/emm.2012.44.2.009

Figure Lengend Snippet: p190RhoGEF protein levels and changes after CD40 stimulation of B cells of various maturational stages. (A) Mouse splenic B cells (1 × 10 6 /ml) and the B cell lines WEHI 231, BAL17, CH12.LX (5 × 10 5 /ml) were stimulated with isotype control Ig or anti-CD40 at a concentration of 1 µg/ml for 48 h. (B) HEK 293T cells were transfected with plasmids carrying an empty vector (EV), WT, or DN p190RhoGEF (HA). (C) WEHI 231 B cells were stably transfected with MIGR1 (EV), WT, or DN p190RhoGEF. Lysates were prepared from these cells, subjected to a SDS-PAGE (6% or 8% gels), transferred to nitrocellulose, and probed with an Ab specific for p190RhoGEF (A-C) or HA (B). Equal loading of the protein samples was demonstrated by anti-β actin IB in (A). (D, E) Mouse splenic B cells (1 × 10 6 /ml) were stimulated with control Ig or anti-CD40 at a concentration of 1 µg/ml. After 48 h of stimulation, cells were fixed, permeabilized, and stained for endogenous p190RhoGEF, CD23, and IgD. The fluorescence intensities of cells stained with an isotype control Ab or antiserum were all corrected (data not shown). The results from flow cytometry are presented as a dot plot, showing IgD versus CD23 positive cells. For the cell populations in R1, R2, and R3 as indicated in (D), the population of cells (%) and the expression level of p190RhoGEF as mean fluorescence intensity (MFI) in the absence (control) and presence of CD40 stimulation is presented in (E). The results shown are representative of three independent experiments.

Article Snippet: The rat anti-mouse CD40 mAb (clone 1C10) from R&D Systems, Inc. (Minneapolis, MN) and the hamster anti-mouse CD40 mAb (HM40-3) from BD PharMingen (San Diego, CA) were used to stimulate B cells.

Techniques: Control, Concentration Assay, Transfection, Plasmid Preparation, Stable Transfection, SDS Page, Staining, Fluorescence, Flow Cytometry, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation

doi: 10.3858/emm.2012.44.2.009

Figure Lengend Snippet: Expression of p190RhoGEF, CD138, and B220 in B cells at various maturational stages. (A, B) Mouse splenic B cells (1 × 10 6 /ml) were stimulated with control Ig or anti-CD40 at a concentration of 1 µg/ml. After 72 h of stimulation, cells were fixed, permeabilized, and stained for endogenous p190RhoGEF, B220, and CD138. The fluorescence intensities of cells stained with an isotype control Ab or antiserum were all corrected (data not shown). The results from flow cytometry are presented as a dot plot, showing the B220 positive cells versus the CD138 positive cells. The data shown are representative of three independent experiments. For the cell populations indicated in (A), expression of p190RhoGEF is presented as MFI in (B). Data are presented as the mean ( n = 4) ± SD. (C, D) The B cell lines, BAL17, CH12.LX, and NS-1 were fixed, permeabilized, and stained for endogenous p190RhoGEF, CD138, and B220. The results were obtained as described above. For the cell populations indicated in (C), expression of p190RhoGEF is presented as MFI in (D). Data are presented as the mean ( n = 3) ± SD.

Article Snippet: The rat anti-mouse CD40 mAb (clone 1C10) from R&D Systems, Inc. (Minneapolis, MN) and the hamster anti-mouse CD40 mAb (HM40-3) from BD PharMingen (San Diego, CA) were used to stimulate B cells.

Techniques: Expressing, Control, Concentration Assay, Staining, Fluorescence, Flow Cytometry

Journal: Experimental & Molecular Medicine

Article Title: Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation

doi: 10.3858/emm.2012.44.2.009

Figure Lengend Snippet: Expression changes of transcriptional regulators after CD40 stimulation and in response to p190RhoGEF and RhoA down-regulation in B cells at various maturational stages. (A) cDNA was prepared from B cells that were either untreated (control) or stimulated for 24 h with anti-CD40 (1 µg/mL). (B) cDNA was prepared from BAL17 and NS-1 cells that were stably transfected with MIGR1 (EV), p190RhoGEF (Y1003A) or RhoA (T19N). The PCR products for p190RhoGEF, RhoA, Blimp-1, XBP-1, IRF-4, and GAPDH were separated as described in "Methods." The data shown are representative of three independent experiments. Values indicate normalization for GAPDH.

Article Snippet: The rat anti-mouse CD40 mAb (clone 1C10) from R&D Systems, Inc. (Minneapolis, MN) and the hamster anti-mouse CD40 mAb (HM40-3) from BD PharMingen (San Diego, CA) were used to stimulate B cells.

Techniques: Expressing, Control, Stable Transfection, Transfection

Journal: Experimental & Molecular Medicine

Article Title: Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation

doi: 10.3858/emm.2012.44.2.009

Figure Lengend Snippet: Direct effects of manipulating the activity of p190RhoGEF and RhoA on B cell maturation and downstream transcriptional regulators. WEHI 231 B cells were stably transfected with MIGR1 (EV), WT p190RhoGEF, DN p190RhoGEF (Y1003A), CA RhoA (Q63L), DN RhoA (T19N), or a combination of WT p190RhoGEF and DN RhoA (T19N). (A) cDNA was prepared from these stably transfected cells. The PCR products for p190RhoGEF, RhoA, Blimp-1, XBP-1, IRF-4, Bcl-6, AID, and GAPDH were separated as described in "Methods." The data shown are representative of three independent experiments. Values indicate normalization for GAPDH. (B) These stably transfected cells were removed from the cultures and then washed, counted, and recultured in fresh medium for 24 h at 1 × 10 6 cells/ml. Some EV-transfected cells were also incubated with a medium alone (black bars) or with a combination of CD40 and IL4 as positive controls (white bars). Supernatants were harvested and serially diluted, and secreted Ig was measured by anti-IgM and anti-IgG ELISA as described in "Methods." Values of the optical densities at 490 nm in triplicate supernatant samples were collected in each independent experiment. Data are presented as the mean of three independent experiments ( n = 3) ± SD, * P < 0.05, ** P < 0.01, compared with cells transfected with EV. (C, D) These stably transfected cells were fixed, stained with anti-CD138 or anti-CXCR4, and analyzed by standard flow cytometry. The fluorescence intensities of cells stained with an isotype control Ab or antiserum were all corrected (data not shown).The bar graphs show the expression of CD138 (C) or CXCR4 (D), as indicated by MFI. Data are presented as the mean of three independent experiments ( n = 3) ± SEM, ** P < 0.01, *** P < 0.001, compared with cells transfected with EV.

Article Snippet: The rat anti-mouse CD40 mAb (clone 1C10) from R&D Systems, Inc. (Minneapolis, MN) and the hamster anti-mouse CD40 mAb (HM40-3) from BD PharMingen (San Diego, CA) were used to stimulate B cells.

Techniques: Activity Assay, Stable Transfection, Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Fluorescence, Control, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation

doi: 10.3858/emm.2012.44.2.009

Figure Lengend Snippet: Effects of the overexpressed DN forms of p190RhoGEF or RhoA on the expression of CD138, CXCR4, and transcriptional regulators after CD40 stimulation. WEHI 231 B cells were stably transfected with either MIGR1 (EV), a plasmid encoding DN p190RhoGEF (Y1003A), or a plasmid encoding DN RhoA (T19N). (A, B) After CD40 stimulation for 48 h, cells were fixed, stained with anti-CD138 or anti-CXCR4, and measured by flow cytometry. The fluorescence intensities of cells stained with an isotype control Ab or antiserum were all corrected (data not shown). The results show the expression intensities of CD138 (A) or CXCR4 (B). Data are presented as the mean of three independent experiments ( n = 3) ± SD. (C) cDNA was prepared from these stably transfected cells, after being stimulated for 48 h with anti-CD40 Ab (1 µg/ml). The PCR products for p190RhoGEF, RhoA, Blimp-1, XBP-1, IRF-4, and GAPDH were separated as described in "Methods." The data shown are representative of three independent experiments. Values indicate normalization for GAPDH.

Article Snippet: The rat anti-mouse CD40 mAb (clone 1C10) from R&D Systems, Inc. (Minneapolis, MN) and the hamster anti-mouse CD40 mAb (HM40-3) from BD PharMingen (San Diego, CA) were used to stimulate B cells.

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Staining, Flow Cytometry, Fluorescence, Control

Journal: Scientific Reports

Article Title: Bioinformatics analysis and validation of novel biomarkers and competitive endogenous RNA networks involved in pyroptosis in diabetic nephropathy

doi: 10.1038/s41598-025-87854-3

Figure Lengend Snippet: The 22 DEPRGs in DN samples compared to normal samples.

Article Snippet: The blots were probed with primary antibodies for GAPDH (1:5000, Proteintech, 10494-1-AP), NLRP3 (1:1500, Proteintech, 27458-1-AP), pro-casp1 (1:1500, Affinity, AF5418), cleaved-casp1 (1:1000, Affinity, AF4005), GSDMD (1:1500, Affinity, AF4012), and GSDMD-N (1:1500, Affinity, AF4012), IRF9(1:1000, Proteintech, 14167-1-AP) , TXNIP (1:1000, Proteintech, 18243-1-AP), TRAF3(1:3000, Proteintech, 18099-1-AP).

Techniques:

Journal: Scientific Reports

Article Title: Bioinformatics analysis and validation of novel biomarkers and competitive endogenous RNA networks involved in pyroptosis in diabetic nephropathy

doi: 10.1038/s41598-025-87854-3

Figure Lengend Snippet: Validation of Hub DEPRGs in DN. ( A ) Expression box plot of the 5 Hub DEPRGs in the validation dataset GSE142025. ( B ) Expression box plot of the 5 Hub DEPRGs in the validation dataset GSE96804. The blue bars represent control specimens from normal individuals, and the red bars represent specimens from DN patients. ( C–F ) ROC curves of CASP1 ( C ), TXNIP ( D ), IRF9 ( E ) and TRAF3 ( F ) in the training dataset GSE30529. ROC curves the represents receiver operator characteristic curves; AUC represents area under the curve; CI represents credibility interval.

Article Snippet: The blots were probed with primary antibodies for GAPDH (1:5000, Proteintech, 10494-1-AP), NLRP3 (1:1500, Proteintech, 27458-1-AP), pro-casp1 (1:1500, Affinity, AF5418), cleaved-casp1 (1:1000, Affinity, AF4005), GSDMD (1:1500, Affinity, AF4012), and GSDMD-N (1:1500, Affinity, AF4012), IRF9(1:1000, Proteintech, 14167-1-AP) , TXNIP (1:1000, Proteintech, 18243-1-AP), TRAF3(1:3000, Proteintech, 18099-1-AP).

Techniques: Biomarker Discovery, Expressing, Control

Journal: Scientific Reports

Article Title: Bioinformatics analysis and validation of novel biomarkers and competitive endogenous RNA networks involved in pyroptosis in diabetic nephropathy

doi: 10.1038/s41598-025-87854-3

Figure Lengend Snippet: Correlation between immune cells and Hub DEPRGs. Correlation between CASP1 ( A ), TXNIP ( B ), IRF9 ( C ), TRAF3 ( D ) and infiltrating immune cells in DN and normal sample groups.The size of the dots from small to big represents the degree of correlation coefficient from low to high and the colour of the dots from yellow to green represents the p -value from high to low. The right side of the numbers depict p -value, with red color indicating a p -value less than 0.05.

Article Snippet: The blots were probed with primary antibodies for GAPDH (1:5000, Proteintech, 10494-1-AP), NLRP3 (1:1500, Proteintech, 27458-1-AP), pro-casp1 (1:1500, Affinity, AF5418), cleaved-casp1 (1:1000, Affinity, AF4005), GSDMD (1:1500, Affinity, AF4012), and GSDMD-N (1:1500, Affinity, AF4012), IRF9(1:1000, Proteintech, 14167-1-AP) , TXNIP (1:1000, Proteintech, 18243-1-AP), TRAF3(1:3000, Proteintech, 18099-1-AP).

Techniques:

Journal: Scientific Reports

Article Title: Bioinformatics analysis and validation of novel biomarkers and competitive endogenous RNA networks involved in pyroptosis in diabetic nephropathy

doi: 10.1038/s41598-025-87854-3

Figure Lengend Snippet: Establishment of DN model rats and validation of the ceRNA networks. ( A ) UTP, SCr and BUN levels in rats. ( B ) Pathologic morphology of rat kidney tissues observed by HE, PAS and TUNEL staining. ( C ) Protein levels of NLRP3, pro-casp1, cleaved-casp1, GSDMD, GSDMD-N, IRF9, TXNIP and TRAF3 by Western blotting, and quantitative analysis in rat kidney tissues (The eight target gene bands originated from different gels, and the gels/blots were cropped. Samples derive from the same experiment and that gels/blots were processed in parallel. Original blots/gels are presented in Supplementary Fig. S2). ( D ) The relative expression of mRNAs and miRNAs in rat kidney tissues was detected by qRT-PCR. P value was shown as: * p < 0.05; ** p < 0.01.

Article Snippet: The blots were probed with primary antibodies for GAPDH (1:5000, Proteintech, 10494-1-AP), NLRP3 (1:1500, Proteintech, 27458-1-AP), pro-casp1 (1:1500, Affinity, AF5418), cleaved-casp1 (1:1000, Affinity, AF4005), GSDMD (1:1500, Affinity, AF4012), and GSDMD-N (1:1500, Affinity, AF4012), IRF9(1:1000, Proteintech, 14167-1-AP) , TXNIP (1:1000, Proteintech, 18243-1-AP), TRAF3(1:3000, Proteintech, 18099-1-AP).

Techniques: Biomarker Discovery, TUNEL Assay, Staining, Western Blot, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Bioinformatics analysis and validation of novel biomarkers and competitive endogenous RNA networks involved in pyroptosis in diabetic nephropathy

doi: 10.1038/s41598-025-87854-3

Figure Lengend Snippet: qRT-PCR primers information.

Article Snippet: The blots were probed with primary antibodies for GAPDH (1:5000, Proteintech, 10494-1-AP), NLRP3 (1:1500, Proteintech, 27458-1-AP), pro-casp1 (1:1500, Affinity, AF5418), cleaved-casp1 (1:1000, Affinity, AF4005), GSDMD (1:1500, Affinity, AF4012), and GSDMD-N (1:1500, Affinity, AF4012), IRF9(1:1000, Proteintech, 14167-1-AP) , TXNIP (1:1000, Proteintech, 18243-1-AP), TRAF3(1:3000, Proteintech, 18099-1-AP).

Techniques: