Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 1. GIV (CCDC88A) is highly expressed in spermatocytes in testis and localizes to the acrosomal cap. (A) Bar graph displays the relative fluorescence unit (RFU) of endogenous full-length GIV protein in immunoblots of organ lysates published previously using three independent anti-GIV antibodies raised against different epitopes of GIV (Anai et al., 2005). (Figure 1—source data 1)(B) RNA expression in the single-cell-type clusters identified in the human testis visualized by a UMAP plot (inset) and a bar plot. The bar plot shows RNA expression (pTPM) in each cell-type cluster.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Fluorescence, Western Blot, RNA Expression

Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 2. Transcripts of CCDC88A (GIV) are downregulated in infertile male testis and semen. (A) Schematic displays the approach used to search NCBI GEO database for testis and sperm transcriptomic datasets suitable to study correlations between the abundance of CCDC88A transcripts and male fertility. (B–E) Whisker plots show the relative abundance of CCDC88A (expressed as Log2 normalized expression; see Materials and methods for different normalization approaches used for microarray and RNA-seq datasets) in sperm or testis samples (as annotated using schematics) in samples

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Whisker Assay, Expressing, Microarray, RNA Sequencing

Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 8. Summary and working model: spatiotemporally segregated roles of GIV/Girdin during sperm capacitation. Schematic summarizes the key findings in this work and places them in the context of existing literature. GIV is likely to primarily function during capacitation of sperm, during which it fulfills two key roles as a signal transducer in a spatiotemporally segregated manner. The first role (right, top) is in the head of the sperm, where GIV’s GEM motif inhibits the AC→cAMP pathway and prevents acrosomal reaction. The second role (right, bottom) is in the mid-piece and tail region of the sperm, which involves tyrosine phosphorylation of GIV, which

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Phospho-proteomics

Journal: Age

Article Title: Global gene expression profile of normal and regenerating liver in young and old mice

doi: 10.1007/s11357-015-9796-7

Figure Lengend Snippet: Expression of cell cycle genes in old vs young mice after PH. Livers of hepatectomized young or old mice were analyzed at indicated time points

Article Snippet: The following antibodies were used: mouse monoclonal antibodies against cyclin D1 (72–13), cyclin D2, cyclin D3, and proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, CA); actin (clone AC-40) (Sigma Chem.

Techniques: Expressing, Transformation Assay, Derivative Assay

Journal: Age

Article Title: Global gene expression profile of normal and regenerating liver in young and old mice

doi: 10.1007/s11357-015-9796-7

Figure Lengend Snippet: YAP expression in old and young livers after PH. a Microarray analysis of Yap mRNA. mRNA expression was determined in old and young mice after PH. Gene expression is reported as fold difference relative to age-matched controls. b Western blot analysis of YAP and phospho-YAP proteins. Old and young mice were sacrificed 12, 24, and 36 h after 2/3 PH. Total extracts (100 to 150 mg/lane) were prepared as described in “Materials and methods” section. Appropriate loading was confirmed by staining the gel with Coomassie Blue and using anti-actin antibody as loading control. Each lane represents a pool of three livers. CO controls. c Western blot analysis of cyclin D1, PCNA, YAP, and phospho-YAP in livers from mice subjected to PH with or without TCPBOP pretreatment (TCP + PH and PH, respectively). Young mice were subjected to PH 3 days after treatment with the mitogen TCP, as described in “Materials and methods” section. Animals were sacrificed 72 h after PH. Total extracts (100 to 150 mg/lane) were prepared from the livers, and Western analysis was performed as described in “Materials and methods” section. Appropriate loading was confirmed by staining the gel with Coomassie Blue and using anti-actin antibody as loading control. Each lane represents a pool of three livers. CO controls. d Western blot analysis of nuclear YAP in TCP + PH or PH livers. Nuclear extracts for YAP (100 to 150 mg/lane) were prepared, and Western analysis was performed as described in “Materials and methods” section. Appropriate loading was confirmed by staining the gel with Coomassie Blue and using anti-albumin antibody as loading control. Each lane represents a pool of three livers. CO controls

Article Snippet: The following antibodies were used: mouse monoclonal antibodies against cyclin D1 (72–13), cyclin D2, cyclin D3, and proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, CA); actin (clone AC-40) (Sigma Chem.

Techniques: Expressing, Microarray, Gene Expression, Western Blot, Staining, Control

Journal: Cancer Research

Article Title: Expression of Interleukin-13 Receptor α2 in Glioblastoma Multiforme: Implications for Targeted Therapies

doi: 10.1158/0008-5472.can-07-1493

Figure Lengend Snippet: Figure 1. Analysis of oligonucleotide microarray data for IL13Ra2 gene expression. A, Tukey box plot comparing the natural log of the MAS5 expression values of IL13Ra2 in nonneoplastic brain control (green) and glioblastoma multiforme (GBM; red) samples. Boxes, interquartile range of expression values. The whiskers extend to the highest and lowest values of expression that are not considered outliers. Circles, outliers. B, XY scatterplot of expression levels of individual samples. X axis, individual control (green) or glioblastoma multiforme (red) sample; Y axis, natural log of the MAS5 IL13Ra2 expression level of the represented sample. The solid line indicates the mean expression level of the controls and the hashed line indicates two SDs above this value. Samples that lie above this line are considered to exhibit significantly greater expression of IL13Ra2 than the controls. C, sample density distribution plot comparing the natural log of the MAS5 expression levels of IL13Ra2 in nonneoplastic brain control (green) and glioblastoma multiforme (red) samples. For a given IL13Ra2 gene expression value (X axis), the relative density of samples is represented by the density estimate (Y axis). For all panels, MAS5 Expression denotes the expression values derived from the Affymetrix suite version 5 software.

Article Snippet: The sections were then incubated with goat anti-IL13Ra2 polyclonal antibodies (1:500 dilution; R&D Systems) followed by Alexa Flour 647–conjugated donkey anti-goat antibodies (1:500 dilution; Molecular Probes).

Techniques: Microarray, Gene Expression, Expressing, Control, Derivative Assay, Software

Journal: Cancer Research

Article Title: Expression of Interleukin-13 Receptor α2 in Glioblastoma Multiforme: Implications for Targeted Therapies

doi: 10.1158/0008-5472.can-07-1493

Figure Lengend Snippet: Figure 3. IL13Ra2 expression as determined by immunohistochemical staining. A to D, expression of IL13Ra2 in paraffin-embedded sections of nonneoplastic brain white matter (A) and paraffin-embedded sections of glioblastoma multiforme tumors (B–D). Representative glioblastoma multiforme tumor sections with high (B), mixed (C), and negative (D) IL13Ra2 expression. Anti-IL13Ra2 antibody staining (green) and DAPI staining of nuclei (blue). Bar, 50 Am.

Article Snippet: The sections were then incubated with goat anti-IL13Ra2 polyclonal antibodies (1:500 dilution; R&D Systems) followed by Alexa Flour 647–conjugated donkey anti-goat antibodies (1:500 dilution; Molecular Probes).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cancer Research

Article Title: Expression of Interleukin-13 Receptor α2 in Glioblastoma Multiforme: Implications for Targeted Therapies

doi: 10.1158/0008-5472.can-07-1493

Figure Lengend Snippet: Figure 2. QRT-PCR results for expression of IL13Ra2 in patient samples of glioblastoma multiforme (black columns) and nonneoplastic brain control (white columns). Y axis, natural log of the fold increase in IL13Ra2 expression level of a particular sample relative to the mean IL13Ra2 expression level of the nonneoplastic brain samples. Horizontal solid line, mean IL13Ra2 expression of the nonneoplastic brain samples. Samples with fold expression levels above the hashed line show significantly higher IL13Ra2 expression than the mean expression level of the nonneoplastic brain samples.

Article Snippet: The sections were then incubated with goat anti-IL13Ra2 polyclonal antibodies (1:500 dilution; R&D Systems) followed by Alexa Flour 647–conjugated donkey anti-goat antibodies (1:500 dilution; Molecular Probes).

Techniques: Quantitative RT-PCR, Expressing, Control

Journal: Epigenetics & Chromatin

Article Title: The histone and non-histone methyllysine reader activities of the UHRF1 tandem Tudor domain are dispensable for the propagation of aberrant DNA methylation patterning in cancer cells

doi: 10.1186/s13072-020-00366-4

Figure Lengend Snippet: LIG1K126me2 is read by a high-affinity interaction through the UHRF1 TTD. a Protein reader domain microarrays consisting of 308 GST-tagged domains (see Additional file : Fig. S1), each printed in duplicate, were probed by either anti-GST antibody, or Cy3-labeled LIG1 (118–130) K126me2, H3 (1–20) K9me2, or LIG1 (118–130) K126me0 (left). Reader arrays were quantified using ArrayNinja software. Data were normalized to the brightest signal for each peptide (right). Error bars represent the range from duplicate spots. Full reader array datasets are available in Additional file : Table S1. b The indicated GST-tagged reader domains were hybridized to histone peptide microarrays at 1 µM, followed by fluorescent detection and quantified by ArrayNinja. Results for interactions with H3 (1–20) K9me2 and LIG1 (118–130) K126me2 are shown. Error bars represent standard error of the mean of six printed spots. Full histone peptide array data are available in Additional file : Table S2. c Fluorescence polarization binding assays between the indicated GST-tagged reader domains and either FAM-LIG1 (118–130) K126me2 (left) or H3 (1–20) K9me2-FAM (right). Error bars represent the 95% confidence interval from triplicate measurements

Article Snippet: Antibodies and dilutions used were as follows: LIG1 (ProteinTech 18051–1-AP, 1:1000, lot # unk.), UHRF1 (Cell Signaling 12387, 1:1000, lot # 1), FLAG (Sigma 1804, 1:5000, lot # unk), beta 3 Tubulin (UniProtKB Q13509, ProteinTech 66240-1-Ig, 1:100,000, lot # unk.), histone H3 (EpiCypher 13–0001, 1:100,000, lot # 12320001), H3K9me2 (Abcam 1220, 1:5000, lot # unk.), and H3K9me3 (Active Motif 39161, 1:5000, lot # unk).

Techniques: Labeling, Software, Peptide Microarray, Fluorescence, Binding Assay

Journal: Epigenetics & Chromatin

Article Title: The histone and non-histone methyllysine reader activities of the UHRF1 tandem Tudor domain are dispensable for the propagation of aberrant DNA methylation patterning in cancer cells

doi: 10.1186/s13072-020-00366-4

Figure Lengend Snippet: Unlike binding to H3K9me2, the UHRF1 PHD and its N-terminal linker do not modulate LIG1K126me2 recognition through the TTD. a Structural models of UHRF1 TTD bound to LIG1K126me3 (PDB:5YYA), UHRF1 PBR (PDB:6B9M), H3K9me3 (PDB:2L3R), and UHRF1 TTD–PHD bound to H3K9me3 (PDB:3ASK) with the linker between TTD and PHD shown in pink. b , c FP binding assays between the indicated GST-tagged b UHRF1 domains or c TTD–PHD mutants reader domains and FAM-LIG1 (118–130) K126me2, H3 (1–20) K9me2-FAM, or FAM-H3 (1–20) K9me2. Error bars represent standard error of the mean from triplicate measurements

Article Snippet: Antibodies and dilutions used were as follows: LIG1 (ProteinTech 18051–1-AP, 1:1000, lot # unk.), UHRF1 (Cell Signaling 12387, 1:1000, lot # 1), FLAG (Sigma 1804, 1:5000, lot # unk), beta 3 Tubulin (UniProtKB Q13509, ProteinTech 66240-1-Ig, 1:100,000, lot # unk.), histone H3 (EpiCypher 13–0001, 1:100,000, lot # 12320001), H3K9me2 (Abcam 1220, 1:5000, lot # unk.), and H3K9me3 (Active Motif 39161, 1:5000, lot # unk).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Functional screening implicates miR-371-3p and peroxiredoxin 6 in reversible tolerance to cancer drugs

doi: 10.1038/ncomms12351

Figure Lengend Snippet: ( a ) Downregulation of luciferase by miR-371-3p upon erlotinib treatment from 3′UTR reporters corresponding to candidate miR-371-3p target genes * P <0.05. ( b ) Microarray analysis of mRNA expression of miR-371-3p targets in pre-miR-371-expressing PC9 cells * P <0.05. ( c ) siRNA screen data corresponding to miR-371-3p targets plotted as z scores of siRNAs in erlotinib ( y axis) versus DMSO ( x axis)-treated cells. ( d ) Validation of siRNA knockdown effects of miR-371-3p targets, PRDX6 , PLCβ4 and STX12 on DTPs from PC9 cells. * and ** indicate significant differences from DMSO- and erlotinib-treated NTC controls, respectively. ( e ) Quantitative PCR of gene expression of PRDX6 , PLCβ4 and STX12 in pre-miR-371-expressing PC9 cells. ( f ) Mutations of seed sequences of the putative miR-371-3p recognition element in target 3′UTR's prevented inhibition by miR-371-3p. ( g ) PRDX6 , PLCβ4 and STX12 mRNA, and ( h ) protein expression in PC9 parental cells and DTPs. ( i ) DTP count of PC9 cells with PRDX6 , PLCβ4 and STX12 CRISPR/Cas9 knockouts, and ( j ) PC9 cells expressing sh PRDX6 , sh PLCβ4 and sh STX12 single, double or triple knockdowns. * indicates significant differences from wild-type (WT) controls. All experiments were performed in triplicate and data are representative of at least two independent experiments. Data are represented as mean±s.e.m. * and ** P <0.05, Student's t -test. NS, not significant.

Article Snippet: Immunoblotting was performed using 1:1,000 dilutions of the following antibodies, p-PKCα (sc-12356, Santa Cruz Biotechnology), PKCα (sc-208, Santa Cruz Biotechnology), PRDX6 (13585-1-AP, ProteinTech), PLCβ4 (sc-404, Santa Cruz Biotechnology), STX12 (Syntaxin 12) (sc-368438, Santa Cruz Biotechnology) and β-actin (3700S, Cell Signaling).

Techniques: Luciferase, Microarray, Expressing, Biomarker Discovery, Knockdown, Real-time Polymerase Chain Reaction, Gene Expression, Inhibition, CRISPR

Journal: Nature Communications

Article Title: Functional screening implicates miR-371-3p and peroxiredoxin 6 in reversible tolerance to cancer drugs

doi: 10.1038/ncomms12351

Figure Lengend Snippet: ( a ) Co-treatment with bisindolylmaleimide XI reduces DTPs in PC9, COLO-205 and HCC-1954 cells. ( b ) Co-treatment with cinnamycin reduces DTPs in COLO-205, HCC-1954 and MKN-45 cells. Decreased PLA2 activity upon ( c ) siRNA knockdown of PRDX6 , PLCβ4 or STX12 and ( d ) treatment with PLA2 inhibitors cinnamycin and the PKCα inhibitor bisindolylmaleimide XI. * indicates significant differences from wild-type (WT) or DMSO controls. ( e ) PKC activity in PC9 cells and DTPs, stable cell lines expressing pre-miR-371 or PRDX6 , PLCβ4 or STX12 knockouts and upon treatment with bisindolylmaleimide XI±erlotinib. * indicates significant differences from DMSO or WT controls. ( f ) Immunoblot of PKCα activation in PC9 DTPs and PC9 cells with PRDX6 , PLCβ4 or STX12 knockouts. ( g ) si PRKCA knockdown in PC9 cells reduces DTPs. * and ** indicate significant differences from DMSO- and erlotinib-treated NTC controls respectively. ( h ) Co-treatment with the PKC activator phorbol-12-myristate13-acetate (PMA) increases DTPs in PC9 cells i , Fluorescence microscopy images of surviving PC9 DTPs co-cultured with parental cells (T: top panel), PC9 DTPs (M: middle panel) or parental cells (B: bottom panel) after 6-day treatment with cinnamycin±erlotinib, Scale bar, 50 μm and ( j ) viable cell population counts of imaged cells in f on day 6 of treatment. All experiments were performed in triplicate and data are representative of at least two independent experiments. Data are represented as mean±s.e.m. For c – e and g , * or ** P <0.05, Student's t -test, NS, not significant. For a , b and h , P represents interaction P value.

Article Snippet: Immunoblotting was performed using 1:1,000 dilutions of the following antibodies, p-PKCα (sc-12356, Santa Cruz Biotechnology), PKCα (sc-208, Santa Cruz Biotechnology), PRDX6 (13585-1-AP, ProteinTech), PLCβ4 (sc-404, Santa Cruz Biotechnology), STX12 (Syntaxin 12) (sc-368438, Santa Cruz Biotechnology) and β-actin (3700S, Cell Signaling).

Techniques: Activity Assay, Knockdown, Stable Transfection, Expressing, Western Blot, Activation Assay, Fluorescence, Microscopy, Cell Culture

Journal: Nature Communications

Article Title: Functional screening implicates miR-371-3p and peroxiredoxin 6 in reversible tolerance to cancer drugs

doi: 10.1038/ncomms12351

Figure Lengend Snippet: ( a ) DTEP formation after erlotinib treatment is reduced in PC9 cells on pre-miR-371 overexpression, single, double or triple knockdowns of PRDX6 , PLCβ4 or STX12 , knockouts of PRDX6 , PLCβ4 or STX12 and treatment with PLA2/PKCα inhibitors or phorbol-12-myristate13-acetate (PMA; n =3). ( b ) DTEP colony counts obtained in a . Data are representative of at least two independent experiments. Data are represented as mean±s.e.m. * ( P <0.05, Student's t -test) indicate significant differences from wild-type (WT) controls. ( c ) Xenograft studies with PC9+GFP and PC9+pre-miR-371 cell lines using erlotinib ( n =15). Data are represented as mean±s.e.m. Interaction P values * P =0.0288, ** P =5.17e−05, *** P =0.00408. ( d ) Change in tumour volumes during drug treatment and after treatment cessation. ( e ) Quantitative PCR for expression of PRDX6 , PLCβ4 and STX12 mRNAs in xenograft tumours ( n =5). * P <0.05, Student's t -test. ( f ) Kaplan–Meier plots of overall survival of lung adenocarcinoma, colorectal cancer, gastric cancer and breast cancer patients; patient groups were separated based on PRDX6 mRNA expression. ( g ) Model depicting proposed mechanism for regulation of DTP survival via miR-371-3p and PRDX6 .

Article Snippet: Immunoblotting was performed using 1:1,000 dilutions of the following antibodies, p-PKCα (sc-12356, Santa Cruz Biotechnology), PKCα (sc-208, Santa Cruz Biotechnology), PRDX6 (13585-1-AP, ProteinTech), PLCβ4 (sc-404, Santa Cruz Biotechnology), STX12 (Syntaxin 12) (sc-368438, Santa Cruz Biotechnology) and β-actin (3700S, Cell Signaling).

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Expressing

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 mRNA was increased in atacicept-induced plasmablasts (PBs) and plasma cells (PCs).

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Clinical Proteomics, Marker

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 mRNA was increased in LPS-induced PBs/PCs.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Microarray, Marker

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 was highly expressed in plasmablasts (PBs) and plasma cells (PCs). (A–C) Hspa13 expression was up-regulated in LPS-induced PBs/PCs. The splenocytes were separated from six 7- to 9-week-old C57BL/6 mice. B cells were sorted by B220 microbeads and stimulated for 0, 24, 48, and 72 hours (hrs) in vitro by 10 μg/ml LPS. Cells were collected and subjected to qPCR (A) , RT-PCR/agarose (B) , and western blot (C) analysis. DNA band ( B , upper panel) was verified as Hspa13 by DNA sequencing. (D,E) Hspa13 was highly expressed in PBs and PCs but not in naïve B cells or germinal center (GC) B cells. Lymphocytes were collected from splenocytes and lymph nodes (LNs) of six sheep red cell (SRC)-immunizated C57BL/6 mice described in . Naïve B cells (CD19 + B220 + IgM + IgD + ), GC B cells (CD19 + B220 + GL7 + CD38 low ), PBs (TACI + CD138 + B220 int CD19 int ), and PCs (TACI + CD138 + B220 − CD19 − ) were sorted by flow cytometry (FACS) and described in , and subjected to qPCR (D) and western blot (E) analysis. (A–E) Data represent three independent experiments with six individual mice each. (A,D) Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P < 0.01, *** P < 0.001.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Clinical Proteomics, Expressing, In Vitro, Reverse Transcription Polymerase Chain Reaction, Western Blot, DNA Sequencing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 mRNA was expressed in PBs.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: CD19 cre Hspa13 fl/fl mice were developed. (A) A construction map of Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice. This project used the principle of homologous recombination and adopted embryonic stem (ES) cell targeting to modify the Hspa13 locus (Chr16:75755190-75767276 bp) by flox modification. The brief process is as follows: The BAC clone containing the gene of interest was purchased from the Sanger Institute (UK). The ES cell targeting vector was constructed by the ET-clone method. The vector contains a 3.4 Kb 5′ homology arm, a 541 bp flox region, and a PGK-neo-polyA, 3.6 kb 3′ homology arm, plus MC1-TK-polyA negative selection marker. After the vector was linearized, JM8A3 ES cells were transfected electrically. A total of 96 resistant clones were obtained after screening with the G418 and Ganc drugs. A total of 13 positive clones with correct homologous recombination were identified by long fragment PCR. Positive ES cell clones were expanded and injected into blastocysts of C57BL/6J mice to obtain chimeric mice. A high proportion of chimeric mice were mated with C57BL/6J mice to obtain seven positive F1 mice. Hspa13 gene flox heterozygous mice showed no significant abnormalities. After mating the flox mouse with a heterologous CD19 cre mouse, the progeny of the flox homozygous, Cre-positive mouse was knocked out, resulting in a functional loss of the gene of interest in B cells. (B) Hspa13 was knocked out in PBs/PCs from CD19 cre Hspa13 fl/fl mice. PCs (TACI + CD138 + B220 − CD19 − ) were sorted from the spleens and bone marrows (BMs) of 7- to 9-week-old heterologous CD19 cre , Hspa13 fl/fl , and CD19 cre Hspa13 fl/fl mice by FACS. PCs were subjected to PCR (B) and western blot (C) analysis. PCR products: with cre activity: 1,468 bp; with no cre activity: 2,120 bp; wild type: 1,897 bp. (B,C) Data represent three independent experiments with three individual mice each.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Homologous Recombination, Modification, Plasmid Preparation, Construct, Selection, Marker, Transfection, Clone Assay, Injection, Functional Assay, Western Blot, Activity Assay

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: PBs, PCs, and antibodies were reduced in CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice. (A) Hspa13 cKO did not affect naïve B cells or germinal center (GC) B cells in mice. Splenic lymphocytes from 9-week-old wild type, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of CD19 + B220 + B cells and CD38 lo GL7 hi B220 + CD19 + GC B cells are shown. (B) Hspa13 cKO reduced PBs, early PCs, and mature PCs in mice. Lymphocytes from the spleen, LNs, and BMs of 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (C) Hspa13 cKO reduced antibodies in mice. Sera were collected from 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice, and the total IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A–C) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Staining, Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 cKO reduced the production of LPS-induced PBs, PCs, and antibodies. Splenic B cells from 9-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were sorted with B220 microbeads and were then stimulated with 10 μg/ml LPS for 0, 1, 2, and 3 days. (A) Hspa13 cKO did not affect LPS-stimulated B-cell proliferation. On days 0, 1, 2, and 3 following LPS stimulation, a CCK8 assay was used to evaluate the cell proliferation. (B,C) Hspa13 cKO did not affect LPS-stimulated B-cell activation. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse B220 and GL7 antibodies, and analyzed by FACS. The percentages (B) and the absolute numbers (C) of B220 + GL7 + B cells are shown. (D,E) Hspa13 cKO reduced LPS-induced PBs, early PCs, and mature PCs. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies, and were then analyzed by FACS. The percentages (D) and the absolute numbers (E) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (F) Hspa13 cKO reduced LPS-induced antibody secretion. On day 3 following LPS stimulation, culture supernatants were collected and the total IgM, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–F) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (A) and one-way (C,E,F) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). *** P < 0.001.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: CCK-8 Assay, Activation Assay, Staining, Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 deficiency reduced Prdm1 and Xbp1 expression.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Expressing, Marker

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 cKO reduced sheep red blood cell (SRC)-induced PB/PC and antibody production. Nine-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected intraperitoneally (i.p.) with 1 × 10 9 SRCs on days 0 and 7. (A,B) Hspa13 cKO did not affect SRC-induce GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies, and were then analyzed by FACS. The percentages (A) and the absolute numbers (B) of CD38 lo GL7 hi GC cells gated on CD19 + B220 + are shown. (C,D) Hspa13 cKO did not affect the SRC-induced dark zone (DZ) and light zone (LZ) GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with anti-mouse CD19, B220, CD38, GL7, CXCR4, and CD86 antibodies, and were then analyzed by FACS. The percentages (C) and the absolute numbers (D) of CXCR4 hi CD86 lo DZ and CXCR4 lo CD86 hi LZ GC B cells gated on CD19 + B220 + CD38 lo GL7 hi GC cells are shown. (E,F) Hspa13 cKO reduced SRC-induced IgG1-, IgG2b-, IgG2c-, and IgG3-expressing PBs/PCs. On day 21 following SRC stimulation, splenic lymphocytes were collected and intracellular staining was performed with isotype control antibodies, anti-mouse B220, IgG1, IgG2b, IgG2c, and IgG3 antibodies. The percentages (E) and the absolute numbers (F) of IgG1-, IgG2b-, IgG2c-, and IgG3-expressing B220 + PBs and B220 − PCs are shown. (G) Hspa13 cKO reduced SRC-induced antibody secretion. On day 21 following SRC stimulation, sera were collected and the total IgM, IgG, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–G) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (D) and one-way (B,F,G) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P < 0.01, *** P < 0.001.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Injection, Staining, Control, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 cKO reduced 4-hydroxy-3-nitrophenylacetyl (NP)-specific antibody production. Nine-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected i.p. with T cell-independent antigen NP-Ficoll (A) and T cell-dependent antigen NP-keyhole lymphocyte hemocyanin (KLH) (B) on days 0 and 7. On day 21 following NP-Ficoll (A) or NP-KLH (B) stimulation, sera were collected and the NP-specific IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A,B) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 cKO reduced class switch recombination (CSR), somatic hypermutation (SHM), and affinity maturation of antibodies. Nine-week-old female Hspa13 fl/fl (control) and CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice (three mice per group) were injected i.p. with 1 × 10 9 SRCs (A–C) or NP-KLH (D,E) on days 0 and 7. On day 21 following SRC stimulation, splenocytes were stained with PerCP-conjugated anti-mouse B220 antibodies and sorted by FACS. Single cells were captured using the 10 X Genomics Full Chromium platform and subjected to RNA- and VDJ-sequencing. (A) Hspa13 cKO reduced SRC-induced PBs. Of the single PBs, 27 (3.49%) and 11 (1.07%) (Ighm + , Ighg1 + , Ighg2b + , Ighg2c + , Ighg3 + , Igha + , or Ighe + Cd3d − Cd3e − Cd3gCd4 − Cd8a − Cd19 + Ptprc + Ms4a1 + Ighd − Bcl6 − Aicda − Prdm1 + Xbp1 + Sdc1 + ) within the splenic B cell population were identified by single-cell RNA-sequencing out of 774 and 1,025 single cells corresponding to CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively. (B) Hspa13 cKO reduced SRC-induced antibody CSR. Single cells expressing genes encoding IgD, IgM, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibodies were identified by single-cell VDJ-sequencing. The percentage of different antibody subtypes expressed by single cells out of 734 and 382 antibody-expressing single cells from CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively, is shown. (C) Hspa13 cKO reduced SRC-induced antibody SHM. The single antibody gene was determined by single-cell VDJ-sequencing. SHM percentages in the CDR (complementarity-determining region) of the heavy (H) and light (L) chains are based on 382 and 734 antibody genes from Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice, respectively. (D) Hspa13 cKO reduced NP-specific SHM induced by NP-KLH. The distribution of the number of mutations per unique clone (VH186.2 segment) is shown. Numbers refer to 100 individual sequences; three animals per group were analyzed. (E) Hspa13 cKO reduced NP-specific high-affinity clones induced by NP-KLH. On day 21 following NP-KLH stimulation, the percentage of NP high-affinity clones containing the W33L mutation in CDR1 in purified GC B cells of Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice was determined. Each dot corresponds to a single animal (30 unique clones/mouse; Mann-Whitney test; error bars represent s.e.m; *** P < 0.001).

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Control, Injection, Staining, Sequencing, RNA Sequencing, Expressing, Clone Assay, Mutagenesis, Purification, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 interacts with endoplasmic reticulum (ER) proteins involved in positive regulation of protein transport from the ER to the cytosol. Splenic B220 + B cells from 9-week-old C57BL/6 mice (three mice per group) were sorted using B220 microbeads and were then stimulated for 3 days with 10 μg/ml LPS. LPS-stimulated B cells were collected for anti-Hspa13 antibody co-immunoprecipitation (IP) experiments. Co-immunoprecipitated proteins were identified by mass spectrometry. (A) SDS-PAGE and silver staining results showing affinity captured interacting proteins from whole cell extracts. Putative interacting protein bands marked as 1, 2, 3, and 4 were excised for mass spectrometry analysis. (B) Anti-Hspa13 antibody specifically co-immunoprecipitated 56 proteins. We identified 393, 56, and 148 proteins that were co-immunoprecipitated by control IgG antibodies only, anti-Hspa13 antibodies only, and the two antibodies together, respectively. (C) Bar plot ranking of the top 10 cellular components (CC), based on enrichment score. Gene ontology (GO)-analysis was performed using a gene ontology website ( http://www.geneontology.org/ ). (D) Bar plot ranking of the top 10 biologic processes (BP) based on enrichment score. GO-analysis was performed based on the gene ontology website ( http://www.geneontology.org/ ). (E) A list of the 10 best hits of Hspa13 interacting partners. Detailed interacting protein data are shown in . (F) Interaction of Hspa13 and Bcap31. The recombinant plasmids expressing Hspa13-V5 and Bcap31-Flag were transiently transfected into 293T cells. At 48 hrs after transfection, cells were lysed and anti-V5 antibody was used to immunoprecipitate proteins probed with anti-Flag antibody. Data are shown for one representative experiment from three independent experiments with similar results. (G) Hspa13 cKO reduced Bcap31 mRNA expression in PBs induced by SRC. Bcap31 mRNA expression was analyzed from 10 and 8 PBs from the single-cell RNA sequencing data of SRC-primed Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice, respectively, described in . Student's t -test (two tailed). Error bars represent s.e.m. * P < 0.05.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Immunoprecipitation, Mass Spectrometry, SDS Page, Silver Staining, Control, Recombinant, Expressing, Transfection, RNA Sequencing, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 mRNA was increased in B220 + cells from patients with multiple myeloma (MM).

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Marker

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 mRNA was increased in B220 + cells from patients with systemic lupus erythematosus (SLE).

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Marker

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 cKO reduced autoantibodies and proteinuria in pristane-induced lupus and lupus-prone MRL/lpr mouse model. (A) Hspa13 cKO reduced autoantibodies in pristane-induced lupus mice. To induce lupus, 9-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice on a B6 background were injected i.p. with 0.5 ml of pristane. On day 21 following pristane stimulation, sera were collected and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (B) Hspa13 cKO reduced proteinuria in the pristane-induced lupus mice. On day 21 following pristane stimulation, urine was collected and proteinuria was measured. (C) Hspa13 cKO reduced autoantibodies in a lupus-prone MRL/lpr mouse model. Sera were collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in lupus-prone MRL/lpr mice background at 6 months of age and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (D) Hspa13 cKO reduced proteinuria in the lupus-prone MRL/lpr mouse model. Urine was collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in a lupus-prone MRL/lpr mice background at 6 months of age and proteinuria was measured. (A–D) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P < 0.01, *** P < 0.01.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Potent STING activation stimulates immunogenic cell death to enhance antitumor immunity in neuroblastoma

doi: 10.1136/jitc-2019-000282

Figure Lengend Snippet: Nanoparticle-enabled cytosolic delivery of 2’3’-cGAMP activates the STING pathway in neuroblastoma cells. (A) Integrated molecular analysis of mRNA expression of genes from the pediatric neuroblastoma TARGET dataset that have been distinguished by functional significance and clustered into evenly split tertiles based on high (upper tertile, n=47), intermediate (median tertile, n=47), and low (bottom tertile, n=47) TMEM173 mRNA expression. (B) TMEM173 mRNA expression in MYCN non-amplified and amplified samples profiled by microarray in the TARGET pediatric neuroblastoma (n=55) datasets. Data were accessed through the cBioPortal. Mann-Whitney U test (two-tailed) was used for statistical comparison.(C) Schematic representation of STING-NPs designed to enhance cytosolic delivery of cGAMP via endosomal escape, resulting in potent activation of STING signaling. (D) Neuroblastoma cell lines (Neuro-2a, 9464D, SK-N-SH, and LAN-1) were treated with vehicle (PBS), empty nanoparticles (NP), 200 nM (or 400 nM for 9464D) cGAMP, or STING-NPs for 48 hours; cells were collected for western blot analysis using anti-IRF3 and anti-phospho-IRF3 (p-IRF3) antibodies. Gel loading was normalized for equal actin; representative blots from one of two independent experiments. The relative density of bands is shown under each immunoblot, after normalization to the levels of actin. (E) qRT-PCR gene expression of IFNB1, TNF, CXCL10, and IL12 in neuroblastoma cell lines at 6, 24 and 48 hours after treatment with cGAMP or STING-NPs. (n=2, data shown as mean±SD; data was analyzed by two-way ANOVA followed by Dunnett’s posthoc test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 indicate a statistically significant difference relative to vehicle (PBS). ANOVA, analysis of variance; cGAMP, cyclic guanosine monophosphate–adenosine monophosphate; qRT-PCR, quantitative real-time PCR; STING, stimulator of interferon genes; STING-NPs, STING-activating nanoparticles.

Article Snippet: Antibodies against STING (13 647S), p-IRF3 (4997S), IRF3 (4302S), Cleaved-Caspase 3 (9664T), and p-STAT1 (9167S) were purchased from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: Expressing, Functional Assay, Amplification, Microarray, MANN-WHITNEY, Two Tailed Test, Comparison, Activation Assay, Western Blot, Quantitative RT-PCR, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: Potent STING activation stimulates immunogenic cell death to enhance antitumor immunity in neuroblastoma

doi: 10.1136/jitc-2019-000282

Figure Lengend Snippet: STING activation induces apoptosis associated with hallmarks of immunogenic cell death in neuroblastoma cell lines. (A) Neuroblastoma cells (Neuro-2a, 9464D, SK-N-SH and LAN-1) were treated with cGAMP, STING-NPs, or empty NPs for 72 hours and cell viability was measured. IC 50 values were obtained by non-linear regression curve fitting; n=2–4 biologically independent samples. (B) Neuroblastoma cells were treated with vehicle (PBS), empty NP, cGAMP, and STING-NPs for 24 hours followed by western blot analysis of caspase 3 and cleaved caspase 3 in cell lysates. Gel loading was normalized for equal actin; representative blots from one of two independent experiments. The relative density of bands is shown under each immunoblot, after normalization to the levels of actin. (C) ATP release in neuroblastoma cells following treatment with vehicle (PBS), 500 nM cGAMP, or STING-NPs for 6, 24 and 48 hours (n= 3 biologically independent samples; one-way ANOVA with Dunnett’s post hoc test). (D) Concentration of HMGB1 released into culture supernatants by Neuro-2a or 9464D cells 48 hours after treatment (n= 3 biologically independent samples; one-way ANOVA with Dunnett’s post hoc test). (E) Flow cytometric detection of cell surface calreticulin (CRT) on Neuro-2a and 9464D cells in response to PBS, NP, 500 nM cGAMP, or STING-NPs after 48 hours. Data is represented as fold increase in median fluorescent intensity over unstained control (n= 3 biologically independent samples; one-way ANOVA with Dunnett’s post hoc test). (F) Representative fluorescence micrographs demonstrating CRT surface localization in Neuro-2a and 9464D cells following treatment with PBS or STING-NPs (500 nM) for 48 hours. Cell nuclei (blue), surface membrane (green), and CRT (red) were detected by DAPI, Alexa Fluor 488-conjugated Wheat Germ Agglutinin, and Alexa Fluor 647-conjugated anti-CRT antibody staining, respectively. All data shown are mean±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; ATP, adenosine triphosphate; cGAMP, cyclic guanosine monophosphate–adenosine monophosphate; STING, stimulator of interferon genes; STING-NPs, STING-activating nanoparticles.

Article Snippet: Antibodies against STING (13 647S), p-IRF3 (4997S), IRF3 (4302S), Cleaved-Caspase 3 (9664T), and p-STAT1 (9167S) were purchased from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: Activation Assay, Western Blot, Concentration Assay, Control, Fluorescence, Membrane, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Potent STING activation stimulates immunogenic cell death to enhance antitumor immunity in neuroblastoma

doi: 10.1136/jitc-2019-000282

Figure Lengend Snippet: STING activation in neuroblastoma generates an immunogenic, tumoricidal, and T-cell inflamed tumor microenvironment. Mice with 200 mm 3 Neuro-2a tumors were administered PBS or STING-NPs (10 µg cGAMP) intratumorally three times, spaced 3 days apart, and tumors were harvested 48 hours following the last treatment. (A) qRT-PCR analysis of IFNB1, TNF, CXCL10, and IL12 gene expression in injected Neuro-2a tumors. n=5 to 7 mice per group represented as mean±SEM, *p<0.05, **p<0.01 indicate a statistically significant difference using a two-tailed Mann-Whitney U test. (B) Neuro-2a tumor lysates (n=4–5) were analyzed using western blot for phospho-STAT1 (p-STAT1), IRF3, and phospho-IRF3 (p-IRF3), caspase 3, and cleaved caspase 3. Gel loading was normalized for equal actin. The density of bands is shown under each immunoblot, after normalization to the levels of actin. (C/D) Immunohistochemical staining of tumor sections for cleaved caspase-3 (apoptosis), Ki-67 (proliferation), CD8, and FoxP3 and corresponding quantification of staining intensity using ImageJ software. Data shown as mean±SD for n=3 Neuro-2a tumors, ***p<0.001, ****p<0.0001 indicate a statistically significant difference using a Student’s t-test. cGAMP, cyclic guanosine monophosphate–adenosine monophosphate; qRT-PCR, quantitative real-time PCR; STING, stimulator of interferon genes; STING-NPs, STING-activating nanoparticles.

Article Snippet: Antibodies against STING (13 647S), p-IRF3 (4997S), IRF3 (4302S), Cleaved-Caspase 3 (9664T), and p-STAT1 (9167S) were purchased from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: Activation Assay, Quantitative RT-PCR, Gene Expression, Injection, Two Tailed Test, MANN-WHITNEY, Western Blot, Immunohistochemical staining, Staining, Software, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: Potent STING activation stimulates immunogenic cell death to enhance antitumor immunity in neuroblastoma

doi: 10.1136/jitc-2019-000282

Figure Lengend Snippet: STING activation sensitizes MYCN-amplified neuroblastoma to PD-L1 immune checkpoint blockade. (A) Schematic summary of treatment for 9464D tumors. Mice were inoculated with 1×10 6 9464D cells in both flanks, and when tumors became palpable (day 14) the right side was treated with STING-NP (10 µg cGAMP) intratumorally and/or αPD-L1 antibodies (100 µg) intraperitoneally. (B) Average tumor size and spider plots for treated and untreated tumors. (n=8–10 mice/group; tumor volumes of treated groups were compared with vehicle on day 37 by one-way ANOVA followed by Dunnett’s post hoc test ***p<0.001,****p<0.0001. (C) Kaplan-Meier survival curves of mice treated with indicated formulation using a total tumor volume >1500 mm 3 as endpoint criteria (n=8-10 mice/group; two-tailed Mantel-Cox test, *p<0.05, ***p<0.001). (D/E) Immunohistochemical staining and quantification of CD8 and FoxP3 staining on treated and untreated tumors 48 hours following the last STING-NP administration; data shown as mean±SD and treated tumors were compared with vehicle control by one-way ANOVA followed by Dunnett’s post hoc test *p<0.05, ***p<0.001, ****p<0.0001 indicate a statistically significant difference. Scale bars: 25 µm. ANOVA, analysis of variance; cGAMP, cyclic guanosine monophosphate–adenosine monophosphate; STING, stimulator of interferon genes; STING-NPs, STING-activating nanoparticles.

Article Snippet: Antibodies against STING (13 647S), p-IRF3 (4997S), IRF3 (4302S), Cleaved-Caspase 3 (9664T), and p-STAT1 (9167S) were purchased from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: Activation Assay, Amplification, Formulation, Two Tailed Test, Immunohistochemical staining, Staining, Control

Journal: Cancer Cell International

Article Title: SCUBE3 downregulation modulates hepatocellular carcinoma by inhibiting CCNE1 via TGFβ/PI3K/AKT/GSK3β pathway

doi: 10.1186/s12935-021-02402-z

Figure Lengend Snippet: Affymetrix GeneChip microarray analysis and validation. A Analysis of significant differences according to the screening criteria of fold change |> 1.5| and false discovery rate (FDR) < 0.05. Scatter plot, volcano plot, heat map were constructed based on the genes differentially expressed between the control and SCUBE3-knockdown cells. B Disease and functional enrichment analysis of differential genes by IPA. The heat map of disease and function showed the relationship between the activation and inhibition of disease and function by the change of differential gene expression level. Orange indicates that the disease or functional state is activated (Z-score > 0), blue indicates that the disease or functional state is inhibited (Z-score < 0), and grey indicates that the disease or functional state is not determined (Z-score cannot be calculated). According to the internal algorithm and standard of IPA, Z-score > 2 means that the disease or function is significantly activated, and Z-score <—2 means that the disease or function is significantly inhibited. The diseases or functions significantly activated include Organic death (Z-score = 3.247), morbidity or mortality (Z-score = 3.200), etc.The diseases or functions significantly inhibited included: viral infection (Z-score = − 3.110), invasion of cells (Z-score = − 2.933). Histogram of disease and functional enrichment analysis statistics. C Significant GO terms associated with SCUBE3 knockdown in HCC cells.Analysis of cellular component (GO-CC), Analysis of biological process (GO-BP). Analysis of molecular function (GO-MF). KEGG Pathway enrichment. D Four genes associated with cell cycle regulation were screened by the microarray Chip analysis following the screening criteria. E The expression levels of three proteins (CCNE1, CCND1, CDK6) were verified by WB

Article Snippet: Primary antibodies against CCND1 (#5550, 1:1000), CCNE1 (#20,808, 1:1000), CDK6 (#13,331, 1:1000), AKT (#4691, 1:1000), p-AKT (#4060, 1:1000), GAPDH (#5174, 1:1000), GSK3β (#5676, 1:1000), and p-GSK3β (#9322, 1:1000) were purchased from Cell Signaling Technology (USA).

Techniques: Microarray, Biomarker Discovery, Construct, Control, Knockdown, Functional Assay, Activation Assay, Inhibition, Gene Expression, Infection, Expressing

Journal: eLife

Article Title: Inhibiting USP16 rescues stem cell aging and memory in an Alzheimer’s model

doi: 10.7554/eLife.66037

Figure Lengend Snippet:

Article Snippet: Rabbit antibody to pSMAD 1/5/8 (1:100; CST 9516) and mouse antibody to beta-amyloid (1:100; Invitrogen 13–200) were diluted in the same 3% blocking buffer and incubated overnight at 4°C.

Techniques: Transgenic Assay, Mutagenesis, Transfection, Construct, Control, Isolation, Recombinant, Plasmid Preparation, Sample Prep, Software, Microarray, Staining

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific protease 7-mediated stabilization of discoidin domain receptor 1 drives progression of TP53-Mutant cancers

doi: 10.1016/j.jbc.2025.110515

Figure Lengend Snippet: TP53 mutation or depletion promotes DDR1 expression and its association with USP7. A , DDR1 protein levels were quantitatively analyzed in DLBCL cell lines with TP53-WT and TP53-MUT. Mean ± SD; Unpaired t test. B , tissue microarray (TMA): Tumor tissue samples from 28 DLBCL patients were classified into 2 groups based on in situ FISH analysis: TP53-WT (n = 22) and TP53-deletion (n = 6). DDR1 expression was assessed by immunohistochemical staining, with staining intensity (scored from 0 to 3) and positivity rate (scored from 0 to 4) evaluated separately. The composite score, ranging from 0 to 12, was calculated as the product of these 2 scores. Scale bar represents 50 μm. C , statistical analysis corresponding to the data shown in panel (B) is presented in this panel. Mean ± SD; Unpaired t test. D , HEK-293T cells were co-transfected with a DDR1 promoter-driven firefly luciferase reporter containing either wild-type (WT) or mutated (MUT) binding sites, with or without TP53, and incubated for 48 h before harvesting for luciferase activity measurement. Mean ± SD (n = 6); One-way ANOVA. E and F , TP53 was knocked down in A549 cells using puromycin-resistant shTP53-1 and shTP53-2 lentiviral constructs, with scrambled (SCR) shRNA as a non-targeting control. DDR1, TP53, and β-Actin protein levels were evaluated by western blotting with the indicated antibodies. qPCR was used to measure the mRNA levels of DDR1 and TP53, normalized to GAPDH. Mean ± SD (n = 6); One-way ANOVA. G , A549 and Pfeiffer cells were treated with NSC632839 for 16 h at concentrations of 0, 2.5, 5, and 10 μM. DDR1, TP53, MMP2, β-Actin, and GAPDH protein levels were assessed by western blotting using the specified antibodies. H , HEK-293T SCR and TP53-KD cells were transfected with Flag-tagged DDR1 and Myc-tagged USP7. Myc pulldown was performed, and DDR1 and Myc protein levels were detected in both immunoprecipitations (IPs) and cell lysates (INPUT) by western blotting using the indicated antibodies. Shown are the representative results of 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Cells were incubated with TP53 (Proteintech, 10442-1-AP) or rabbit IgG Isotype control (Proteintech, 30000-0-AP) for 2 h in RT, followed by MNase incubation at 4 °C for 1 h and fragmentation on ice for 90 min in the presence of Ca2 + .

Techniques: Mutagenesis, Expressing, Microarray, In Situ, Immunohistochemical staining, Staining, Transfection, Luciferase, Binding Assay, Incubation, Activity Assay, Construct, shRNA, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific protease 7-mediated stabilization of discoidin domain receptor 1 drives progression of TP53-Mutant cancers

doi: 10.1016/j.jbc.2025.110515

Figure Lengend Snippet: NSC632839 leads to the killing of TP53-mutant DDR1-positive PDCs both ex vivo and in vivo . A , patient-derived cells (PDCs) from DLBCL patients (n = 3) were treated with NSC632839 or 7rh at varying concentrations for 48 h. Cell proliferation was assessed using the CellTiter-Glo (CTG) assay. Mean ± SD. B , PDCs were treated with NSC632839 or 7rh for 16 h, followed by cell lysis to detect protein levels of DDR1, MMP9, MMP2, p-AKT, AKT, β-Actin, or GAPDH. The data shown are representative of 3 independent experiments. C , NSG mice were implanted subcutaneously with TP53 MUT DLBCL patient tissue (n = 8/group), 12 days later, 20 mg/kg (QD) of NSC632839 was given intraperitoneally. D , mice were treated with vehicle or NSC632839 (20 mg/kg) and sacrificed on day 16 of treatment. Tumor size measurements of PDX mice after vehicle and NSC632839 treatment. Mean ± SEM; Two-way ANOVA. E , effects of 16 days of NSC632839 treatment on the growth of the PDX model were determined. Mean ± SEM; Unpaired t test. Tumor growth inhibition (TGI) rates were thus determined for both groups using the formula: TGI = ((MTVcontrol-MTVtreated)/MTVcontrol) × 100. F , DDR1 protein levels in tumor tissue were determined by western blotting, followed by quantification and representation in a bar graph. Mean ± SEM; Unpaired t test. G , CD19 CAR-T cells were generated from human pan-T cells isolated from PBMCs using a human pan-T isolation kit. The T cells were activated with CD3/CD28 cross-linking antibody beads and transduced with CAR lentiviral constructs at an MOI of 5 to 10 on day 3. Following transduction, the cells were expanded in vitro and co-cultured with primary TP53-mutant DLBCL PDCs (with or without NSC632839 pretreatment for 16 h at concentration of 2.5 μM) at E:T ratios ranging from 1:8 to 2:1. The co-culture was maintained for 4 h. The remaining CD19 levels were analyzed by flow cytometry using the indicated antibodies. Mean ± SD (n = 3); Two-way ANOVA. Shown are the representative results of 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Cells were incubated with TP53 (Proteintech, 10442-1-AP) or rabbit IgG Isotype control (Proteintech, 30000-0-AP) for 2 h in RT, followed by MNase incubation at 4 °C for 1 h and fragmentation on ice for 90 min in the presence of Ca2 + .

Techniques: Mutagenesis, Ex Vivo, In Vivo, Derivative Assay, CTG Assay, Lysis, Inhibition, Western Blot, Generated, Isolation, Transduction, Construct, In Vitro, Cell Culture, Concentration Assay, Co-Culture Assay, Flow Cytometry