Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Interferon-λ3 Promotes Epithelial Defense and Barrier Function Against Cryptosporidium parvum Infection

doi: 10.1016/j.jcmgh.2019.02.007

Figure Lengend Snippet: IECs up-regulate numerous ISGs in response to C parvum infection. Intestinal epithelium was harvested from ileum mucosa of neonatal piglets at the time of peak infection (days 3–5 after infection) (n = 8) and from age-matched uninfected controls (n = 4) for performance of gene expression analysis using microarrays. ( A ) Representative photomicrograph of ileum mucosa from an uninfected control and C parvum –infected piglet used for microarray analysis. H&E stain. Scale bar : 20 μm. ( B ) Villus height and crypt depth (μm) and the percentage of total villus IECs that were infected with C parvum in control (n = 4) and C parvum –infected (n = 8) piglets used for microarray analysis. Each data point represents the average of 5 measurements per piglet. Scale bars : means ± SD. ** P < .01, Student t test comparison between uninfected and C parvum –infected piglets. Heat map of significantly ( C ) up-regulated and ( D ) down-regulated genes in infected ( C parvum ) and control (Uninf) exfoliated ileum villus epithelial cells. Each control and infected biological replicate is represented. Gene IDs:fold change are listed to the right of the heat map. Known ISGs are highlighted in red. ( E ) qRT-PCR analysis of porcine ileum mucosal total cellular mRNA for the presence of ISG15 mRNA. Samples were obtained from piglets used for microarray analysis at peak C parvum infection (days 3–5, n = 5) and age-matched controls (n = 5). For each sample, the Ct value for ISG15 was normalized to expression of the housekeeping gene cyclophilin (ΔCt). Fold-change differences between each sample were compared with a representative uninfected sample using the 2 -ΔΔCt method. Scale bars : means ± SD. ** P < .01, Student t test comparison between uninfected and C parvum –infected piglets. ( F ) Fluorescence in situ hybridization showing ISG15 mRNA (red fluorescence) in villus epithelial cells of ileum mucosa from piglets at peak C parvum infection and absent in villus epithelium of control piglets. Photomicrograph representative of results of 2 independent experiments. Scale bar : 50 μm.

Article Snippet: The fragmented complementary RNA was diluted in hybridization buffer (2 N -morpholino-ethanesulfonic acid, NaCl, EDTA, Tween 20, herring sperm DNA, acetylated bovine serum albumin) containing biotin-labeled OligoB2 and Eukaryotic Hybridization Controls (Affymetrix).

Techniques: Infection, Gene Expression, Control, Microarray, Staining, Comparison, Quantitative RT-PCR, Expressing, Fluorescence, In Situ Hybridization

Journal: Emerging Infectious Diseases

Article Title: Identifying Influenza Viruses with Resequencing Microarrays

doi: 10.3201/eid1204.051441

Figure Lengend Snippet: Hybridization images of the respiratory pathogen microarray (RPM) version 1 prototype regions for 3 influenza virus isolates and trivalent FluMist vaccine. A) A/H1N1, B) A/H3N2, C) influenza B, and D) trivalent FluMist vaccine. In A, B, and C, only the influenza-specific tiled prototype regions of RPM version 1 are shown. Hybridization-positive identifications are shown on the right. In D, the image of the entire RPM version when hybridized with FluMist vaccine is shown. The single influenza prototype region that was hybridization negative is denoted on the right. E) Magnification of a portion of profile B showing an example of the primary sequence data generated by the hybridization of randomly amplified targets to the RPM version 1 HA3 probe set. The primary sequence generated can be read from left to right. HA, hemagglutinin; NA, neuraminidase; IQEX, internal positive hybridization control (Affymetrix); M, matrix.

Article Snippet: After the prehybridization step, 167.5 μL of hybridization cocktail master mix (3 mol/L tetramethylammonium chloride, 10 mmol/L Tris, pH 7.8, 0.01% Tween 20, 0.5 mg/mL bovine serum albumin, 0.1 mg/mL herring sperm DNA [Promega, Madison, WI, USA], 50 pmol/L Oligo B2 [Affymetrix Inc.]) and biotin-labeled DNA fragments were heated for 5 min at 95°C, equilibrated for 5 min at 45°C, and added to RPM version 1.

Techniques: Hybridization, Microarray, Virus, Sequencing, Generated, Amplification, Control

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: Biology of Reproduction

Article Title: Yes-associated protein 1 is required for proliferation and function of bovine granulosa cells in vitro ^†

doi: 10.1093/biolre/ioz139

Figure Lengend Snippet: Characteristics of antibodies used for western blotting and microscopy.

Article Snippet: Phospho-YAP1 (Ser127) , 1:400 1 /1:1000 2 /1:200 3 , 4 , Mouse , Rabbit pAB , Cell Signaling , 4911S.

Techniques: Western Blot, Microscopy

Journal: Biology of Reproduction

Article Title: Yes-associated protein 1 is required for proliferation and function of bovine granulosa cells in vitro ^†

doi: 10.1093/biolre/ioz139

Figure Lengend Snippet: Expression of Hippo signaling family members in the bovine ovary. Microarray analysis was used to determine the expression of Hippo signaling family members in bovine follicular (theca and granulosa) and luteal (small and large) cells. (A) Microarray analysis of Hippo signaling family members in bovine GCs (n = 4; open blue circle), TCs (n = 3; open blue box), SLCs (n = 3; closed red box) and LLCs (n = 3; closed red circle). Relative mRNA units are shown as symbols with means ± SEM shown as black lines, in some cases, the black lines are obscured by the symbols. Significant differences were identified as changes greater than 1.5-fold and supported by unpaired t-tests with P < 0.01. *Significant difference between GC and LLC or TC and SLC. Serine/threonine kinase 4 (STK4; MST1); serine/threonine kinase 3 (STK3, MST2); salvador family WW domain containing protein 1 (SAV1); large tumor suppressor kinase 1 (LATS1); large tumor suppressor kinase 2 (LATS2); MOB kinase activator 1A (MOB1A); Yes-associated protein 1 (YAP1); WW domain containing transcription regulator 1 (TAZ; WWTR1); and beta-actin (ACTB). (B) Comparison of the expression of MST1 versus MST2 and LATS1 versus LATS2. Levels of mRNA for each transcript in GC, TC, SLC, and LLC were pooled and analyzed as a single group. Data are means ± SEM. Differences in means in MST1 versus MST2 and LATS1 versus LATS2 were compared by t-test. ***P < 0.001.

Article Snippet: Phospho-YAP1 (Ser127) , 1:400 1 /1:1000 2 /1:200 3 , 4 , Mouse , Rabbit pAB , Cell Signaling , 4911S.

Techniques: Expressing, Microarray, Comparison

Journal: Biology of Reproduction

Article Title: Yes-associated protein 1 is required for proliferation and function of bovine granulosa cells in vitro ^†

doi: 10.1093/biolre/ioz139

Figure Lengend Snippet: Localization of Hippo signaling family members in the bovine ovary. Immunohistochemistry and western blotting were used to determine the expression and localization of YAP1, phospho-YAP1(Ser127), and TAZ in bovine follicles and the corpus luteum. (A) Representative immunohistochemistry micrographs showing expression of YAP1 in GCs and TCs (b) and corpus luteum (d), phospho-YAP1(Ser127) in GC and TC (c) and corpus luteum (e); and TAZ in GC and TC (g) and corpus luteum (h). Micron bar represents 1 mm, negative controls (a and f). Large arrows point to luteal endothelial cells. (B) Western blot analysis of the expression of YAP1 and TAZ in GC from follicles of increasing size (2–5, 5–10, >10 mm) and enriched SLCs and LLCs. Expression of aromatase (CYP19A1), steroidogenic acute regulatory protein (STARD1), 3beta-Hydroxysteroid dehydrogenase (3BHSD), Cholesterol side-chain cleavage enzyme (CYP11A1), and Beta-actin (ACTB; loading control) are shown.

Article Snippet: Phospho-YAP1 (Ser127) , 1:400 1 /1:1000 2 /1:200 3 , 4 , Mouse , Rabbit pAB , Cell Signaling , 4911S.

Techniques: Immunohistochemistry, Western Blot, Expressing, Control

Journal: Biology of Reproduction

Article Title: Yes-associated protein 1 is required for proliferation and function of bovine granulosa cells in vitro ^†

doi: 10.1093/biolre/ioz139

Figure Lengend Snippet: Expression of YAP1 and WW domain-containing transcription regulator 1 (TAZ; WWTR1) in the bovine ovary. Western blotting was used to determine the localization of YAP1, phospho-YAP1(Ser127), and TAZ in bovine follicles and the corpus luteum. (A) Representative western blots of YAP1, phospho-YAP1(Ser127) and TAZ in cytoplasmic and nuclear fractions obtained from cultured GC and enriched SLC. Nuclear protein: DNA topoisomerase II alpha (TOP2A); cytosolic protein: nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1A); beta-actin (ACTB; loading control). (B–D) Densitometry of YAP1, phospho-YAP1(Ser127), and TAZ expression in nuclear and cytoplasmic fractions. Data represent the percentage of each protein within each fraction. Bars are means ± SEM, n = 5 experiments. **P < 0.01.

Article Snippet: Phospho-YAP1 (Ser127) , 1:400 1 /1:1000 2 /1:200 3 , 4 , Mouse , Rabbit pAB , Cell Signaling , 4911S.

Techniques: Expressing, Western Blot, Cell Culture, Control

Journal: Biology of Reproduction

Article Title: Yes-associated protein 1 is required for proliferation and function of bovine granulosa cells in vitro ^†

doi: 10.1093/biolre/ioz139

Figure Lengend Snippet: Effects of cell density on nuclear localization of YAP1 in bovine GCs. Granulosa cells were seeded overnight on coverslips in six-well dishes at increasing cell densities from 0.125–0.50 × 106 cells/well. (A) Representative micrograph of GC plated at 0.125 × 106 cells/well; YAP1 (a), alpha-tubulin (b), colocalization of YAP1 and alpha-tubulin (c), 20× magnification of colocalization of YAP1 and alpha-tubulin (d), YAP1 (e), DAPI (e), colocalization of YAP1 and DAPI (g), 20× magnification of colocalization of YAP1 and alpha-tubulin (h). (B) Representative micrograph of GC plated at 0.25 × 106 cells/well. (C) Representative micrograph of GC plated at 0.50 × 106 cells/well. (D) Quantitative analysis of colocalization of YAP1 with alpha-tubulin and DAPI. Data are represented as means ± SEM, n = 3 experiments. **Significant difference as compared to 0.125 × 106 cells/well, P < 0.05. Micron bar represents 20 μm (63×) and 50 μm (20×).

Article Snippet: Phospho-YAP1 (Ser127) , 1:400 1 /1:1000 2 /1:200 3 , 4 , Mouse , Rabbit pAB , Cell Signaling , 4911S.

Techniques:

Journal: Biology of Reproduction

Article Title: Yes-associated protein 1 is required for proliferation and function of bovine granulosa cells in vitro ^†

doi: 10.1093/biolre/ioz139

Figure Lengend Snippet: Effects of cell density on nuclear localization of phosphorylated YAP1 (Ser127) in bovine GCs. Granulosa cells were seeded overnight on coverslips in six-well dishes at increasing cell densities from 0.125 to 0.50 × 106 cells/well. (A) Representative micrograph of GC plated at 0.125 × 106 cells/well; phosphorylated Yes-associated protein (p-YAP1 (Ser127)) (a), YAP1 (b), colocalization of p-YAP1 (Ser127) and YAP1 (c), 20x magnification of colocalization of p-YAP1 (Ser127) and YAP1 (d), p-YAP1 (Ser127) (e), alpha-tubulin (f), colocalization of p-YAP1 (Ser127) and alpha-tubulin (g), 20× magnification of colocalization of p-YAP1 (Ser127) and alpha-tubulin (h), p-YAP1 (Ser127) (i), DAPI (j), colocalization of p-YAP1 (Ser127) and DAPI (k), 20× magnification of colocalization of p-YAP1 (Ser127) and alpha-tubulin (l). (B) Representative micrograph of GC plated at 0.25 × 106 cells/well. (C) Representative micrograph of GC plated at 0.50 × 106 cells/well. (D) Quantitative analysis of colocalization of p-YAP1 with YAP1, alpha-tubulin, and DAPI. Data are represented as means ± SEM, n = 3 experiments. **Significant difference as compared to 0.125 × 106 cells/well, P < 0.05. Micron bar represents 20 μm (63×) and 50 μm (20×).

Article Snippet: Phospho-YAP1 (Ser127) , 1:400 1 /1:1000 2 /1:200 3 , 4 , Mouse , Rabbit pAB , Cell Signaling , 4911S.

Techniques:

Journal: Biology of Reproduction

Article Title: Yes-associated protein 1 is required for proliferation and function of bovine granulosa cells in vitro ^†

doi: 10.1093/biolre/ioz139

Figure Lengend Snippet: Effects of the YAP1 inhibitor verteporfin on GC proliferation. Bovine GC were plated at a cell density of 80 × 103 cells/well in 12-well dishes and treated with increasing concentrations of verteporfin (0–5 μM). Cell proliferation was determined as described in the Methods. (A) Representative western blot analysis showing Cyclin D1 expression in cells treated with follicle-stimulating hormone (30 ng/ml; FSH) or transforming growth factor-alpha (100 ng/ml; TGFα) in the presence or absence of verteporfin (5 μM). (B) Densitometry of cyclin D1 expression in cells treated with follicle-stimulating hormone (30 ng/mL; FSH) or transforming growth factor-alpha (100 ng/ml; TGFα). (C) DNA synthesis in cells treated with increasing concentrations of verteporfin (0, 1, 2.5. and 5 μM) following treatment of TGFα (100 ng/ml). Data are represented as means ± SEM (n = 4) of the average fold change from control in each experiment. **Significant difference as compared to control, P < 0.05.

Article Snippet: Phospho-YAP1 (Ser127) , 1:400 1 /1:1000 2 /1:200 3 , 4 , Mouse , Rabbit pAB , Cell Signaling , 4911S.

Techniques: Western Blot, Expressing, DNA Synthesis, Control

Journal: Biology of Reproduction

Article Title: Yes-associated protein 1 is required for proliferation and function of bovine granulosa cells in vitro ^†

doi: 10.1093/biolre/ioz139

Figure Lengend Snippet: Effects of siRNA-mediated knockdown of YAP1 and TAZ on TGFα-induced proliferation in GCs. YAP1 and TAZ mRNA were silenced using siYAP1 and siTAZ in bovine GCs. Following knockdown, GCs were treated with or without transforming growth factor-alpha (50 ng/mL; TGFα) to promote cell proliferation. (A) Representative western blot showing phospho-YAP1(Ser127) and YAP1 protein expression in siGlo (siCTL) or siYAP1 knockdown GCs, following treatment with or without TGFα. (B) Densitometric analyses of YAP1 protein expression obtained from siCTL (open bars) and siYAP1 (closed bars). Bars represent means ± SEM, n = 5. (C) Cell counts following knockdown of YAP1 cells and treatment with TGFα (closed bars) or without TGFα (open bars). Bars are means ± SEM, n = 3. (D) Representative western blot showing YAP1 and TAZ protein expression in GCs following 48 h treatment with siCTL, siYAP1, siTAZ, or a combination of siYAP1 and siTAZ (siY + siT). Beta-actin (ACTB; loading control). (E) Quantitative analysis showing DNA synthesis for siCTL, siYAP1, siTAZ, or siY + siT knockdown cells treated with control (open bars) or TGFα (black bars). Data are represented as means ± SEM (n = 3) of the average fold change from control in each experiment. **Significant differences between treatment groups, as compared to siCTL, P < 0.05.

Article Snippet: Phospho-YAP1 (Ser127) , 1:400 1 /1:1000 2 /1:200 3 , 4 , Mouse , Rabbit pAB , Cell Signaling , 4911S.

Techniques: Knockdown, Western Blot, Expressing, Control, DNA Synthesis

Journal: Oncology Reports

Article Title: HOXA11 plays critical roles in disease progression and response to cytarabine in AML

doi: 10.3892/or.2021.8101

Figure Lengend Snippet: Overexpression of HOXA11 in AML carrying MLL -t, oncogenic KRAS or PTPN11 mutations. (A-C) Meta-analysis of HOXA11 expression in AML using leukemia database deposited by Valk (n=285), Wouters (n=503), Haferlach (n=542) and Balgobind (n=237, childhood AML) in Oncomine™. Box plots are data of HOXA11 (reporter ID. 208493) based on cDNA microarray data in patients with AML (A) with 11q23-r or No 11q23-r, (B) patients with KRASm or KRASwt, and (C) patients with PTPN11m or PTPN11wt. Numbers listed at the bottom are case numbers. Center line in box plot represents median value, box limits are 10 and 90th percentiles, and dots represent minimum and maximum values. P-values were determined using an unpaired two-sample Student's t-test. *P<0.05 and **P<0.01. (D and E) Reverse transcription-quantitative PCR analyses were performed to determine relative HOXA11 expression level in patients with AML carrying (D) MLL/AF10 or (E) MLL/AF9 with or without oncogenic KRASm or PTPN11m mutations. Assays were performed in triplicate and data are representative of three independent experiments. Error bars indicate SD of mean. AML, acute myeloid leukemia; MLL, lysine methyltransferase 2A; MLL -t, MLL translocations; PTPN11 , tyrosine-protein phosphatase non-receptor type 11; 11q23-r, MLL rearrangements; No 11q23-r, wild-type MLL ; KRASm, oncogenic KRAS mutations; KRASwt, wild-type KRAS ; PTPN11m, oncogenic PTPN11 mutations; PTPN11wt, wild-type PTPN11 .

Article Snippet: The membrane was blocked in 5% bovine serum albumin at 4°C (Sigma-Aldrich; Merck KGaA) for 1 h, and then incubated with primary antibodies at 4°C overnight against mouse Hoxa11 (1:5,000; cat. no. NBP1-80228; Novus Biologicals, Ltd.), β-actin or Gapdh (1:10,000; cat. nos. sc-47778 or sc-32233; Santa Cruz Biotechnology, Inc.), followed by incubation for 2 h at room temperature with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (1:5,000; cat. nos.

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

Journal: Oncology Reports

Article Title: HOXA11 plays critical roles in disease progression and response to cytarabine in AML

doi: 10.3892/or.2021.8101

Figure Lengend Snippet: Expression levels of Hox clusters A, B, C, D and Meis1 genes in paired MLL/AF10 leukemia cells harboring wild-type or oncogenic RAS pathway mutations. (A-C) Heat maps of genes based on cDNA microarray data between paired cell lines: (A) 12G and AK3G, (B) AKw1G and AK3G, and (C) APw-1 and APm-1. Raw values are log2-transformed. Red and black indicate high and low levels of gene expression, respectively. Color bar depicts log2-transformed value of genes. (D) Reverse transcription-quantitative PCR analysis was performed to determine the level of Hoxa11 expression in 12G, AKw1G, AK3G, AK2G, APw-1 and APm-1 cells. Assays were performed in triplicate and data are representative of three independent experiments. Error bars indicate SD. (E) Western blotting was performed to determine the protein level of Hoxa11 in cells of the different cell lines. The detection of β-actin and Gapdh served as a loading control for immunoblot analysis. MLL, lysine methyltransferase 2A; 12G, cells with MLL/AF10(OM-LZ) alone; AK3G, cells with MLL/AF10(OM-LZ) and oncogenic KRAS G12C; AKw1G, cells with MLL/AF10(OM-LZ) and wild-type KRAS ; APw-1, cells with MLL/AF10(OM-LZ) and wild-type PTPN11 ; APm-1, cells with MLL/AF10(OM-LZ) and oncogenic PTPN11 G503A; PTPN11 , tyrosine-protein phosphatase non-receptor type 11.

Article Snippet: The membrane was blocked in 5% bovine serum albumin at 4°C (Sigma-Aldrich; Merck KGaA) for 1 h, and then incubated with primary antibodies at 4°C overnight against mouse Hoxa11 (1:5,000; cat. no. NBP1-80228; Novus Biologicals, Ltd.), β-actin or Gapdh (1:10,000; cat. nos. sc-47778 or sc-32233; Santa Cruz Biotechnology, Inc.), followed by incubation for 2 h at room temperature with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (1:5,000; cat. nos.

Techniques: Expressing, Microarray, Transformation Assay, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Oncology Reports

Article Title: HOXA11 plays critical roles in disease progression and response to cytarabine in AML

doi: 10.3892/or.2021.8101

Figure Lengend Snippet: Expression of Hoxa11 affects survival of MLL/AF10 leukemia mice. (A and C) Reverse transcription-quantitative PCR analyses were performed to determine Hoxa11 expression in (A) Hoxa11 -knockdown APm-1 (APm-1-shH11-1, APm-1-shH11-2) and control (APm-1-shV) cell lines, or in (C) Hoxa11 -overexpression 12G (12G-H11-1, 12G-H11-2) and control (12G-V1 and 12G-V3) cell lines. Assays were performed in triplicate and data shown are representative of three independent experiments. Error bars indicate SD. (B and D) Survival curves of mice i.p. injected with (B) APm-1-shV or APm-1-shH11-2, and (D) 12G-V1 or 12G-H11-1 cells. Survival analysis was conducted according to the Kaplan-Meier method. **P<0.01 and ***P<0.001 vs. APm-1-shV or 12G-V1 cells. (E) Flow cytometry analyses was performed to determine Ki-67 and Mac-1 expression in the BM cells obtained from 12G-V1 and 12G-H11-1 leukemia mice at moribund stage. Data shown are representative of three mice with similar results. (F) Competitive engraftment and clonal expansion ability between 12G-V1 and 12G-H11-1 cells in vivo . 12G-V1 and 12G-H11-1 cells were mixed in a 1:1 ratio and i.p. injected into recipient mice (n=9). The mice were sacrificed at 43 and 57 days post-transplantation (n=2 and n=7, respectively). Allele burden of the 12G-H11-1 clone in BM (left column) or spleen (right column) was determined by PCR-DNA sequencing. MLL, lysine methyltransferase 2A; APm-1, cells with MLL/AF10(OM-LZ) and oncogenic PTPN11 G503A; sh, short hairpin RNA; 12G, cells with MLL/AF10(OM-LZ) alone; i.p. intraperitoneally; Mac-1, macrophage-1 antigen; BM, bone marrow; PTPN11 , tyrosine-protein phosphatase non-receptor type 11.

Article Snippet: The membrane was blocked in 5% bovine serum albumin at 4°C (Sigma-Aldrich; Merck KGaA) for 1 h, and then incubated with primary antibodies at 4°C overnight against mouse Hoxa11 (1:5,000; cat. no. NBP1-80228; Novus Biologicals, Ltd.), β-actin or Gapdh (1:10,000; cat. nos. sc-47778 or sc-32233; Santa Cruz Biotechnology, Inc.), followed by incubation for 2 h at room temperature with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (1:5,000; cat. nos.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Knockdown, Control, Over Expression, Injection, Flow Cytometry, In Vivo, Transplantation Assay, DNA Sequencing, shRNA

Journal: Oncology Reports

Article Title: HOXA11 plays critical roles in disease progression and response to cytarabine in AML

doi: 10.3892/or.2021.8101

Figure Lengend Snippet: Phenotypic characteristics of the mice transplanted with the Hoxa11 -knockdown APm-1 and Hoxa11 -overexpression 12G leukemia cells.

Article Snippet: The membrane was blocked in 5% bovine serum albumin at 4°C (Sigma-Aldrich; Merck KGaA) for 1 h, and then incubated with primary antibodies at 4°C overnight against mouse Hoxa11 (1:5,000; cat. no. NBP1-80228; Novus Biologicals, Ltd.), β-actin or Gapdh (1:10,000; cat. nos. sc-47778 or sc-32233; Santa Cruz Biotechnology, Inc.), followed by incubation for 2 h at room temperature with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (1:5,000; cat. nos.

Techniques: Knockdown, Over Expression

Journal: Oncology Reports

Article Title: HOXA11 plays critical roles in disease progression and response to cytarabine in AML

doi: 10.3892/or.2021.8101

Figure Lengend Snippet: Hoxa11 expression affects chemotherapy drug resistance. (A-D) Viability assays of (A and C) Hoxa11 -knockdown APm-1 (APm-1-shH11-2) and control (APm-1-shV) cell lines or (B and D) Hoxa11 -overexpression 12G (12G-H11-1) and control (12G-V1) cell lines treated with Ara-C at indicated concentrations for 24 h. The viability of leukemia cells was determined by a Cell Counting Kit-8 assay. Assays were performed in triplicate and data are representative of three independent experiments. The error bars indicate SD, and P-values were determined with an unpaired two-sample Student's t-test. (E) The expression levels of the apoptosis-related genes, Nfkbia, Rela and Trp53 in Hoxa11 -knockdown or Hoxa11 -overexpression MLL/AF10 leukemia cells were evaluated via reverse transcription-quantitative PCR. *P<0.05, **P<0.01, ***P<0.001 vs. APm-1-shV or 12G-V1 cells. (F) Meta-analysis of HOXA11 expression in AML using a leukemia database deposited by Heuser (n=33) in Oncomine™. Box plots are the HOXA11 (reporter ID. AA598674) expression levels based on cDNA microarray data in patients with AML grouped by (F-a) chemotherapy responsiveness and (F-b) AML induction/consolidation response status. Numbers listed at the bottom are the number of cases. The center line in the box plot represents the median, the box limits indicate the 10 and 90th percentiles, and dots represent minimum and maximum values. MLL, lysine methyltransferase 2A; APm-1, cells with MLL/AF10(OM-LZ) and oncogenic PTPN11 G503A; sh, short hairpin RNA; 12G, cells with MLL/AF10(OM-LZ) alone; PTPN11 , tyrosine-protein phosphatase non-receptor type 11; Ara-C, cytarabine; Nfkbia , NF-κB inhibitor α; Rela , transcription factor p65; Trp53 , transformation-related protein p53; AML, acute myeloid leukemia; CR, complete remission; Res, responder; Non-Res, non-responder.

Article Snippet: The membrane was blocked in 5% bovine serum albumin at 4°C (Sigma-Aldrich; Merck KGaA) for 1 h, and then incubated with primary antibodies at 4°C overnight against mouse Hoxa11 (1:5,000; cat. no. NBP1-80228; Novus Biologicals, Ltd.), β-actin or Gapdh (1:10,000; cat. nos. sc-47778 or sc-32233; Santa Cruz Biotechnology, Inc.), followed by incubation for 2 h at room temperature with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (1:5,000; cat. nos.

Techniques: Expressing, Knockdown, Control, Over Expression, Cell Counting, Reverse Transcription, Real-time Polymerase Chain Reaction, Microarray, shRNA, Transformation Assay