Journal: Stem cell research

Article Title: OP9-Lhx2 stromal cells facilitate derivation of hematopoietic progenitors both in vitro and in vivo.

doi: 10.1016/j.scr.2015.08.009

Figure Lengend Snippet: Fig. 1. OP9-Lhx2 enhances generation of ESCs-derived hematopoietic cells. (A) Strategy for hematopoietic differentiation by ESC (H1 line)/stromal cell co-culture. (B) Phenotypic identi- fication of hematopoietic cells by FACS analysis. Dead cells and debris were excluded. (C) Statistical analysis of hematopoietic cells derived from co-culture system. Each column represents the mean of triplicate wells. Data are shown as means + SDs (*p b 0.05). (D), (E) Colony-forming assay of CD34+ cells derived from co-culture. The hematopoietic progenitors from OP9-Lhx2/ESCs and OP9-GFP/ESCs co-culture formed CFU-GM, CFU-M and BFU-E colonies.

Article Snippet: To generate a OP9-Lhx2 cell line that stably overexpressed mouse Lhx2, Lhx2 cDNA (OriGene) was subcloned into pMYs-IRES-EGFP (RTV-021, Cell Biolabs, INC) to generate the pMYs-Lhx2-IRES-EGFP vector.

Techniques: Derivative Assay, Co-Culture Assay

Journal: Stem cell research

Article Title: OP9-Lhx2 stromal cells facilitate derivation of hematopoietic progenitors both in vitro and in vivo.

doi: 10.1016/j.scr.2015.08.009

Figure Lengend Snippet: Fig. 2. OP9-Lhx2 facilitates derivation of murine hematopoietic progenitors in vivo via teratoma formation. (A) Strategy for producing hematopoietic cells in vivo via teratoma formation. (B) OP9-Lhx2/ESCs (C57/B6 background, CD45.2+)-formed teratomas produced a higher proportion of hematopoietic progenitors compared with OP9-GFP/ESCs teratoma controls. Teratoma-derived blood cells were defined as tdTomato+CD45.2+ cells; teratoma derived hematopoietic progenitors were defined as tdTomato+CD45.2+CD150+CD48−cells. Plots show the analysis of representative teratomas of two groups. (C) Statistic analysis of phenotypic HSCs. Teratomas from three teratoma-bearing mice from each group were analyzed. Data are shown as mean + SD (*p b 0.01). (D) Hematopoietic progenitors derived from OP9-Lhx2/mESCs teratoma are transiently transplantable. Five hundred hematopoietic progenitors (tdTomato+CD45.2+CD48−CD150+) were transplanted into each lethally irradiated recipient (CD45.1+) via an injection into the femur cavity. A flow cytometric analysis of peripheral blood (PB) detected a low engraftment of OP9-Lhx2/mESCs teratoma-derived hematopoietic cells (CD45.2+CD45.1−) four weeks after transplantation. (E) Lineages analysis of engrafted cells in peripheral blood in recipients (n = 3) one month after transplantation.

Article Snippet: To generate a OP9-Lhx2 cell line that stably overexpressed mouse Lhx2, Lhx2 cDNA (OriGene) was subcloned into pMYs-IRES-EGFP (RTV-021, Cell Biolabs, INC) to generate the pMYs-Lhx2-IRES-EGFP vector.

Techniques: In Vivo, Produced, Derivative Assay, Irradiation, Injection, Transplantation Assay

Journal: Stem cell research

Article Title: OP9-Lhx2 stromal cells facilitate derivation of hematopoietic progenitors both in vitro and in vivo.

doi: 10.1016/j.scr.2015.08.009

Figure Lengend Snippet: Fig. 3. OP9-Lhx2 facilitates the derivation of human hematopoietic progenitors in vivo via teratoma formation. (A) Flow cytometry analysis of human hematopoietic progenitors derived from OP9/hESCs and OP9-Lhx2/hESCs teratomas. An analysis was conducted 8–10 weeks after ESC injection. Human hematopoietic progenitors were phenotypically defined as mCD45−

Article Snippet: To generate a OP9-Lhx2 cell line that stably overexpressed mouse Lhx2, Lhx2 cDNA (OriGene) was subcloned into pMYs-IRES-EGFP (RTV-021, Cell Biolabs, INC) to generate the pMYs-Lhx2-IRES-EGFP vector.

Techniques: In Vivo, Flow Cytometry, Derivative Assay, ESC Injection

Journal: Communications Biology

Article Title: Hypoxia-induced circPLOD2a/b promotes the aggressiveness of glioblastoma by suppressing XIRP1 through binding to HuR

doi: 10.1038/s42003-025-07503-3

Figure Lengend Snippet: A – D Proteins pulled down by biotin-labeled circPLOD2a ( A ) or circPLOD2b ( C ) antisense probes and corresponding scrambled control probes in hypoxic U87 cells were visualized by SDS-PAGE and silver staining. Unique peptides identified by mass spectrometry in ( B ) circPLOD2a ( n = 73) or ( D ) circPLOD2b ( n = 54) compared with corresponding control lanes. E , F Potential proteins interacting with ( E ) circPLOD2a and ( F ) circPLOD2b were verified by RNA pull-down assays with corresponding antisense probes in hypoxic U87 cells. Relative integrated density normalized to β-actin was marked above each band. G – J RIP assay confirms the interaction of circPLOD2a/b and linear PLOD2 with HuR. CircPLOD2a/b and linear PLOD2 were detected by RT-PCR ( G ). The immunoprecipitation products of anti-HuR compared with IgG were treated with or without RNase R. Quantitative enrichment analysis of circPLOD2a ( H ), circPLOD2b ( I ) and linear PLOD2 ( J ) were analyzed by qRT-PCR, n = 3. K Cellular localization of circPLOD2b (red) and HuR (green) were examined by dual RNA-FISH and IF in U87 cells treated with normoxic and hypoxic conditions. Scale bar: 25 μm. L Schematic diagram of wild type and various truncated HuR with Flag-tag. (M) The interaction of circPLOD2a/b with full-length or truncated HuR protein were detected by RIP assay in U87 cells. CircPLOD2a/b were detected by RT-PCR; full-length and truncated HuR proteins were detected by western blot using anti-Flag antibody. N CircPLOD2a and circPLOD2b were immunoprecipitated by anti-Flag beads in U87 cells transfected with plasmids carrying HuR- wildtype (HuR-wt) or truncated forms (HuR-△RRM1/2/3) and detected by qRT-PCR, n = 3. Data are shown as mean ± SEM (error bars) and analyzed using Student’s t -test ( H – J ) and ANOVA ( N ).

Article Snippet: The primary antibodies used for western blotting were anti-β-actin antibody (66009-1-Ig, Proteintech, 1:2000), anti-HIF1α antibody (ab179483, Abcam, 1:1000), anti-PLOD2 antibody (66342-1-Ig, Proteintech, 1:2000), anti-ALDA antibody (11217-1-AP, Proteintech, 1:2000), anti-BIP antibody (11587-1-AP, Proteintech, 1:2000), anti-DDX5 antibody(67025-1-Ig, Proteintech, 1:2000), anti-HuR antibody (11910-1-AP, Proteintech, 1:2000), anti-IGF2BP3 antibody (14642-1-AP, Proteintech, 1:2000), anti-E-cadherin antibody (20874-1-AP, Proteintech, 1:1000), anti-N-cadherin antibody (22018-1-AP, Proteintech, 1:1000), anti-Vimentin antibody (10366-1-AP, Proteintech, 1:1000), anti-Flag antibody (20543-1-AP, Proteintech, 1:2000), anti-XIRP1 antibody(PA5-48605, Invitrogen, 1:1000), HRP Goat anti-Rabbit antibody (AS014, Abclonal, 1:5000), and HRP Goat anti-Mouse antibody (SA00001-1, Proteintech, 1:5000).

Techniques: Labeling, Control, SDS Page, Silver Staining, Mass Spectrometry, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Quantitative RT-PCR, FLAG-tag, Western Blot, Transfection

Journal: Communications Biology

Article Title: Hypoxia-induced circPLOD2a/b promotes the aggressiveness of glioblastoma by suppressing XIRP1 through binding to HuR

doi: 10.1038/s42003-025-07503-3

Figure Lengend Snippet: A , B Relative expression of ( A ) circPLOD2a and ( B ) circPLOD2b were examined by qRT-PCR in clinical samples from patients with grade II-III ( n = 10) and grade IV ( n = 9) glioma. C Relative expression of XIRP1 in tissues from circPLOD2a-high and -low groups. The GBM tissues were divided into circPLOD2a-high ( n = 10) and -low ( n = 9) groups according to the expression of circPLOD2a. D Relative expression of XIRP1 in tissues from circPLOD2b-high and -low groups. The GBM tissues were divided into circPLOD2a-high ( n = 10) and -low ( n = 9) groups according to the expression of circPLOD2b. E Relative expression of XIRP1 in GBM and non-tumor brain tissues were analyzed from Rembrandt database. F Representative immunohistochemistry images of XIRP1 in a tissue microarray containing grade I ( n = 3), II ( n = 50), III ( n = 24) and IV ( n = 94) glioma. Scale bar: 100 μm. G , H Expression levels of XIRP1 in tissues from patients with different grade glioma were evaluated by ( G ) the percentage of cells with strong/moderate/weak intensity of XIRP1 staining and ( H ) histochemistry score (H-score) of each tissue on this microarray. I Kaplan–Meier survival curves of glioma patients in XIRP1-high and low groups. Glioma patients were categorized into XIRP1-high and -low group by the expression level of XIRP1 based on H-score. J Schematic illustration of HIF1α/circPLOD2a/b/HuR/XIRP1 axis. HIF1α induces the up-regulation of linear PLOD2 , circPLOD2a and circPLOD2b under hypoxia. CircPLOD2a/b suppress XIRP1 by competitively binding to HuR and enhance GBM cell aggressiveness. Data are shown as mean ± SEM (error bars) and analyzed using Student’s t -test ( A – E ), ANOVA ( H ) and Log-rank test ( I ).

Article Snippet: The primary antibodies used for western blotting were anti-β-actin antibody (66009-1-Ig, Proteintech, 1:2000), anti-HIF1α antibody (ab179483, Abcam, 1:1000), anti-PLOD2 antibody (66342-1-Ig, Proteintech, 1:2000), anti-ALDA antibody (11217-1-AP, Proteintech, 1:2000), anti-BIP antibody (11587-1-AP, Proteintech, 1:2000), anti-DDX5 antibody(67025-1-Ig, Proteintech, 1:2000), anti-HuR antibody (11910-1-AP, Proteintech, 1:2000), anti-IGF2BP3 antibody (14642-1-AP, Proteintech, 1:2000), anti-E-cadherin antibody (20874-1-AP, Proteintech, 1:1000), anti-N-cadherin antibody (22018-1-AP, Proteintech, 1:1000), anti-Vimentin antibody (10366-1-AP, Proteintech, 1:1000), anti-Flag antibody (20543-1-AP, Proteintech, 1:2000), anti-XIRP1 antibody(PA5-48605, Invitrogen, 1:1000), HRP Goat anti-Rabbit antibody (AS014, Abclonal, 1:5000), and HRP Goat anti-Mouse antibody (SA00001-1, Proteintech, 1:5000).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Microarray, Staining, Binding Assay

Journal: Antioxidants

Article Title: Hypoxia-Induced Aquaporins and Regulation of Redox Homeostasis by a Trans-Plasma Membrane Electron Transport System in Maize Roots

doi: 10.3390/antiox11050836

Figure Lengend Snippet: Alterations of PM redox systems, ascorbate transporters, and PIPs on transcript and protein level after 24 h of hypoxia stress. Nomenclature of peroxidases according to RedoxiBase (Available online: https://peroxibase.toulouse.inra.fr/ (accessed on 4 May 2019)) [ 58 ]. Significant differences between stressed and controls, calculated with Student’s t -test, were marked at p < 0.001 (***), p < 0.01 (**), and p < 0.05 (*). fc, fold change; accession number, Acc; MW, molecular weight in kDa; pI, isoelectric point; TMH, transmembrane helices; GPI, glycosylphosphatidylinositol anchor; DoHcyt b 561, Cytochrome b 561, and DOMON domain-containing protein; MDHAR, Monodehydroascorbate reductase homolog 1.

Article Snippet: The INTERPRO and SMART domains that were found were related to redox proteins such as lipoxygenase (IPR001024, IPR020833, IPR001246, IPR000907, IPR019794, and SM00308), aquaporins (IPR022357, 000425, and 023271), and peroxidases (IPR019794).

Techniques: Molecular Weight

Journal: Antioxidants

Article Title: Hypoxia-Induced Aquaporins and Regulation of Redox Homeostasis by a Trans-Plasma Membrane Electron Transport System in Maize Roots

doi: 10.3390/antiox11050836

Figure Lengend Snippet: Gene ontology analyses of PM-bound proteins altered by hypoxia. Plasma membrane-bound proteins that were significantly altered by 24 h of hypoxia were determined by mass spectrometry (shotgun). Using DAVID, gene ontology (GO) enrichment analyses were performed. The top five GO terms and the corresponding number of genes are shown in the graph. Asterisks indicate p -values ( p < 0.05 *, p < 0.01 **, p < 0.001 ***). ANX, annexin repeats; Proteasome_A_N, proteasome subunit A N-terminal signature; LH2, lipoxygenase homology 2 (beta barrel) domain; 14_3_3, 14-3-3 homologues; Gln-synt_C, glutamine synthetase, catalytic domain; Enolase N, enolase, N-terminal domain; Enolase C, enolase, C-terminal TIM barrel domain; EF1G, elongation factor 1 gamma, conserved domain.

Article Snippet: The INTERPRO and SMART domains that were found were related to redox proteins such as lipoxygenase (IPR001024, IPR020833, IPR001246, IPR000907, IPR019794, and SM00308), aquaporins (IPR022357, 000425, and 023271), and peroxidases (IPR019794).

Techniques: Clinical Proteomics, Membrane, Mass Spectrometry