Journal: Cell Reports

Article Title: Distinct Polymorphisms in HLA Class I Molecules Govern Their Susceptibility to Peptide Editing by TAPBPR

doi: 10.1016/j.celrep.2019.09.074

Figure Lengend Snippet:

Article Snippet: The beads were washed three times in wash buffer (One Lambda, Inc., CA, USA) to remove any excess of soluble TAPBPR and were then first incubated with PeTe4 antibody for 30 min, washed and then incubated with a PE-conjugated goat anti-mouse IgG (Abcam, UK) for another 30 min at 22°C.

Techniques: Recombinant, Mutagenesis, Plasmid Preparation, Software

Journal: Cell reports

Article Title: Distinct RORγt-dependent Th17 immune responses are required for autoimmune pathogenesis and protection against bacterial infection

doi: 10.1016/j.celrep.2024.114951

Figure Lengend Snippet: (A–E) RNA-seq analysis of WT and RORγt K256R/K256R CD4 + T cells polarized under Th17 conditions. (A) The number of differentially expressed genes (DEGs, black) including upregulated (red) and downregulated (blue) genes with a cutoff at p < 0.05 and fold change (FC) >1.9. (B) Volcano plot shows DEGs between indicated Th17 cells (log 10 p on y axis and log 2 FC on x axis). Top downregulated (left) and upregulated (right) candidates in RORγt K256R/K256R cells are indicated. Red font: the top three downregulated candidates. (C) A Venn diagram illustrates the overlapping 32 genes between 223 pathogenic Th17-specific genes and 746 downregulated genes in RORγt K256R/K256R Th17 cells (GEO: GSE39820). , , , (D) Heatmap of the 32 overlapping genes described in (C). (E) qPCR analysis of relative mRNA levels of Lgals3 in Th0 and Th17 cells ( n = 4). (F and G) qPCR analysis of Lgals3 mRNA levels (F) and flow cytometric analysis of Lgals3 protein (G, left panels) and percentage (G, right panel) of Lgals3 + cells among indicated CD4 + T cells polarized under Th17 conditions ( n = 6). (H and I) Representative flow cytometric analysis (H) and percentage (I) of Lgals3 + cells among CD4 + T cells from the spleens of indicated untreated mice (left two panels) or from the CNS of indicated EAE-induced mice (right two panels) as described in ( n = 7). (J) Representative flow cytometric analysis (left three panels) and percentage of Lgals3 + cells (right panel) among CD4 + T cells in the colons of C. rodentium -infected indicated mice described in . Lgals3 −/− group is a negative staining control. (K) Mean EAE clinical scores at different days of Rag1 −/− mice adoptively transferred with sorted 1 × 10 5 indicated CD4 + GFP + T cells retrovirally expressing GFP alone (EV) or with Ccr6 and polarized under Th17 conditions ( n = 8, two independent experiments). (L) Number of CD4 + T cells recovered from the CNS of EAE-induced mice described in (K). (M) Mean EAE clinical scores of Rag1 −/− -recipient mice adoptively transferred with sorted 1 × 10 5 Tg TCR2D GFP + CD4 + T cells retrovirally expressing GFP together with scrambled short hairpin RNA (shRNA) or shRNA targeting Lgals3 (shLgals3) and polarized under Th17 conditions ( n = 6, two independent experiments). (N) Number of CD4 + T cells recovered from the CNS of EAE-induced mice described in (M). Data are presented as mean ± SEM. Statistical significance is indicated as **p < 0.05; ***p < 0.001; ns, not significant (two-tailed unpaired Student’s t test). Also see .

Article Snippet: Lgals3 antibodies (M3/38) were obtained from Miltenyi Biotec for flow cytometric analysis or Santa Cruz Biotechnology for the Western blotting.

Techniques: RNA Sequencing, Infection, Negative Staining, Control, Expressing, shRNA, Two Tailed Test

Journal: Cell reports

Article Title: Distinct RORγt-dependent Th17 immune responses are required for autoimmune pathogenesis and protection against bacterial infection

doi: 10.1016/j.celrep.2024.114951

Figure Lengend Snippet: (A) Representative flow cytometric analysis of IL-17A expression in WT and Lgals3 −/− CD4 + T cells polarized under Th17 conditions in vitro for 3 days ( n = 4). (B) Mean clinical score of indicated mice on different days after EAE induction ( n = 6, two independent experiments). (C) The number of CD4 + T cells infiltrated into the CNS of mice described in (B). (D) Mean clinical score of Rag1 −/− recipients adoptively transferred with 3 × 10 6 naive CD4 + T cells from indicated mice, followed by induction of EAE with MOG 35–55 ( n = 7–8, two independent experiments). (E) The number of CD4 + T cells infiltrated into the CNS of Rag1 −/− mice described in (D). (F) Body weight of indicated mice on different days after oral infection with 2 × 10 9 C. rodentium ( n = 8–9, two independent experiments). (G–I) Bacterial load (G), colon length (H), and the number of CD4 + T cells in the colons (I) of mice described in (F) at day 21 post infection. (J) Representative flow cytometric analysis (left panels) and percentage (right panel) of IL-17A + cells among CD4 + T cells recovered from the colons of indicated mice shown in (F). Data are presented as mean ± SEM. Statistical significance is indicated as *p < 0.01; **p < 0.05; ***p < 0.001; ns, not significant (two-tailed unpaired Student’s t test). Also see .

Article Snippet: Lgals3 antibodies (M3/38) were obtained from Miltenyi Biotec for flow cytometric analysis or Santa Cruz Biotechnology for the Western blotting.

Techniques: Expressing, In Vitro, Infection, Two Tailed Test

Journal: Cell reports

Article Title: Distinct RORγt-dependent Th17 immune responses are required for autoimmune pathogenesis and protection against bacterial infection

doi: 10.1016/j.celrep.2024.114951

Figure Lengend Snippet: (A) Scheme of potential Runx1 DNA-binding sites. Region 1 (Rgn1) and Rgn2 are two conserved Runx1-binding sites identified from ChIP-seq for Runx1 (GEO: GSE158093). E, exon. (B) ChIP signals with anti-FLAG antibody in in vitro differentiated WT Th17 cells retrovirally transduced with GFP alone (EV) or with FLAG-tagged Runx1 ( n = 4). (C) Schematic representation of indicated luciferase reporter constructs. P, Lgals3 promoter; I, Lgals3 intron; TK, minimal thymidine kinase gene promoter; Δ, deletion; Luc, luciferase. (D and E) Relative luciferase activity from indicated reporter shown in (C) transfected into 293T cells together with expression plasmid for Runx1 or control empty plasmid (Ctrl). Basic is a promoterless reporter ( n = 4). (F and G) Flow cytometric analysis of Lgal3 expression (left panels) and percentage of Lgals3 + cells among Cas9-expressing CD4 + T cells retrovirally transduced with nontargeting (NonT) sgRNA controls or sgRNA targeting to delete Rgn1 (F) or Rgn2 (G) and polarized under Th17 conditions ( n = 4). Gray: controls unstained (Unst) with the Lgals3 antibody. (H) Number of CD4 + T cells infiltrating the CNS of Rag1 −/− adoptively transferred with Tg Tcr2D Th17 cells retrovirally transduced with NonT, sgRgn1, or sgRgn2 followed by induction of EAE with MOG 35–55 ( n = 5). (I) The endpoint clinical score of mice as described in (H). (J) Flow cytometric analysis of Lgals3 in indicated CD4 + T cells expressing GFP alone or together with Runx1. Data are presented as mean ± SEM. Statistical significance is indicated as *p < 0.01; **p < 0.05; ***p < 0.001; ns, not significant (two-tailed unpaired Student’s t test). Also see .

Article Snippet: Lgals3 antibodies (M3/38) were obtained from Miltenyi Biotec for flow cytometric analysis or Santa Cruz Biotechnology for the Western blotting.

Techniques: Binding Assay, ChIP-sequencing, In Vitro, Transduction, Luciferase, Construct, Activity Assay, Transfection, Expressing, Plasmid Preparation, Control, Two Tailed Test

Journal: Cell reports

Article Title: Distinct RORγt-dependent Th17 immune responses are required for autoimmune pathogenesis and protection against bacterial infection

doi: 10.1016/j.celrep.2024.114951

Figure Lengend Snippet: (A) Representative flow cytometric analysis (left panels) and percentage (right panel) of Ccr6 + cells among indicated CD4 + T cells polarized under Th17 conditions for 3 days ( n = 6). (B) The number (top panels) and percentage (bottom panels) of indicated types of cells in the CNS of indicated mice immunized with MOG 35–55 . (C) Representative flow cytometric analysis (top panels), percentage (two bottom-left panels), and number (two bottom-right panels) of monocytes/macrophages (Mono/Mac) and neutrophils in the CNS of Rag1 −/− mice adoptively transferred with indicated CD4 + GFP + T cells retrovirally expressing GFP alone (EV) or with Lgals3 and polarized under Th17 conditions, followed by EAE induction as described in . (D) Representative flow cytometric analysis (left panels) and percentage (right panel) of BMDMs migrated to the bottom wells containing indicated CD4 + GFP + T cells retrovirally expressing GFP alone (EV) or with Lgals3 and differentiated under Th17 cells conditions in a Transwell migration assay ( n = 4 independent experiments). (E) Immunoblot (IB) analysis of FLAG-Lgals3 immunoprecipitated (IP) from the supernatants of CD4 + T cells transduced with empty retrovirus (EV) or virus expressing 3xFLAG-Lgals3 and polarized under Th17 condition for 3 days ( n = 4). Bottom two lanes are the immunoblot analysis of Lgals3 in the whole-cell lysates (WCLs) with anti-FLAG or anti-Lgals3 antibody. (F) Representative flow cytometric analysis (left panels) and percentage (right panel) of Ccr6 + cells among in vitro differentiated RORγt K256R/K256R Th17 cells co-cultured without (None) or with BMDMs for 18 h ( n = 4). (G) Representative flow cytometric analysis (left panels) and percentage (right panel) of Ccr6 + cells among CD4 + T cells recovered from the CNS of Rag1 −/− mice adoptively transferred with indicated CD4 + GFP + T cells retrovirally expressing GFP alone (EV) or with Lgals3 and polarized under Th17 conditions followed by EAE induction as described in ( n = 7–8). Analysis was conducted on day 10 post immunization. (H) Percentage of Ccr6 + cells among CD4 + T cells infiltrated into the CNS of WT or Lgals3 −/− mice immunized with MOG 35–55 as described in ( n = 6). (I) Percentage of Ccr6 + cells among in vitro differentiated Th17 cells co-cultured without (None) or with BMDMs for 18 h in the absence or presence of indicated neutralizing antibodies, analyzed by flow cytometry ( n = 4). (J) Percentage of Ccr6 + cells among in vitro differentiated RORγt K256R/K256R Th17 cells treated with vehicle or recombinant IL-1β ( n = 5). (K) Representative flow cytometric analysis (left panels) and percentage (right panel) of IL-1β + cells among Mono/Mac cells in the spleens (left two panels) or the CNS (middle two panels) of indicated mice immunized with MOG 35–55 (n = 8–9). (L) Number of IL-1β + cells among lymphocytes recovered from CNS of indicated EAE-induced mice. Data are presented as mean ± SEM. Statistical significance is indicated as *p < 0.01; **p < 0.05; ***p < 0.001; ns, not significant (two-tailed unpaired Student’s t test). Also see .

Article Snippet: Lgals3 antibodies (M3/38) were obtained from Miltenyi Biotec for flow cytometric analysis or Santa Cruz Biotechnology for the Western blotting.

Techniques: Expressing, Transwell Migration Assay, Western Blot, Immunoprecipitation, Transduction, Virus, In Vitro, Cell Culture, Flow Cytometry, Recombinant, Two Tailed Test

Journal: Cell reports

Article Title: Distinct RORγt-dependent Th17 immune responses are required for autoimmune pathogenesis and protection against bacterial infection

doi: 10.1016/j.celrep.2024.114951

Figure Lengend Snippet:

Article Snippet: Lgals3 antibodies (M3/38) were obtained from Miltenyi Biotec for flow cytometric analysis or Santa Cruz Biotechnology for the Western blotting.

Techniques: Virus, Recombinant, Modification, Protease Inhibitor, Lysis, Radio Immunoprecipitation, Isolation, cDNA Synthesis, SYBR Green Assay, Emulsion, Reporter Assay, Microarray, CRISPR, Luciferase, Control, Software

Journal: PLoS ONE

Article Title: Snorkel: An Epitope Tagging System for Measuring the Surface Expression of Membrane Proteins

doi: 10.1371/journal.pone.0073255

Figure Lengend Snippet: Plasmid constructs were transiently transfected into HEK293 cells, grown for 22h before analysis in flow cytometry. A. Flow cytometry with CD20-snorkel cells. Transfected cells stained with anti-HA antibody (grey solid line) or with anti-CD20 (black dashed line); Grey dashed line (control); untransfected cells stained with an anti-HA antibody. B. same as A except with a CD20-stop construct. C CD20-snorkel dual stained with anti-HA (FITC labeled) and anti-CD20 (PE labeled). D. CD20-snorkel stained with CD20 antibody and the Alexa647 goat anti-mouse antibody (grey line), FRET measurement with the CD20-snorkel construct stained with anti-HA (Alexa488 labeled) and anti-CD20 and goat anti-mouse (Alexa647 labeled, black line). E same as D but plotted with both Alexa488 and 647 fluorescence for FRET pair.

Article Snippet: Cells were then washed three times at 4 degrees and incubated an additional 30 minutes with 15nM PE labeled streptavidin (Jackson Immunoresearch, West Grove, PA).

Techniques: Plasmid Preparation, Construct, Transfection, Flow Cytometry, Staining, Control, Labeling, Fluorescence

Journal: Oncotarget

Article Title: Interleukin-32α induces migration of human melanoma cells through downregulation of E-cadherin

doi: 10.18632/oncotarget.11669

Figure Lengend Snippet: A. G361-vector and G361-IL-32α cell lines were detached using enzyme-free dissociation buffer. Flow cytometry assays were performed using the PE-conjugated mouse anti-human E-cadherin antibody. B. E-cadherin, β-catenin, phospho-β-catenin and GSK-3β expression was evaluated in G361-vector and G361-IL-32α cell lines. C. Total RNA was isolated from G361-vector and G361-IL-32α cells. After reverse transcription, PCR was performed with primers for β-catenin or β-actin. D. G361-vector and G361-IL-32α cells were attached to coverslips then fixed and permeabilized as described in the Materials and Methods. After permeabilization, the coverslips were blocked with 1% BSA in PBS for 1 hour and incubated at 4°C overnight with rabbit anti-human β-catenin antibody. Coverslips were then incubated with FITC-conjugated goat anti-rabbit IgG antibody. A laser scanning confocal microscope was used for analyses. E. G361-vector and G361-IL-32α cells were incubated on coverslips. Cells attached to the coverslips were fixed and permeabilized as mentioned in Materials and Methods. F-actin staining was performed using phalloidin-conjugated Alexa Fluor 647. Confocal microscopy assays were performed as described. These data represent one of three independent experiments.

Article Snippet: Cells (5×10 5 ) were washed twice with PBS and incubated for 30 minutes on ice with the PE-conjugated mouse anti-human E-cadherin antibody (R&D Systems, Minneapolis, MN).

Techniques: Plasmid Preparation, Flow Cytometry, Expressing, Isolation, Reverse Transcription, Incubation, Microscopy, Staining, Confocal Microscopy

Journal: Oncotarget

Article Title: Interleukin-32α induces migration of human melanoma cells through downregulation of E-cadherin

doi: 10.18632/oncotarget.11669

Figure Lengend Snippet: A. Erk1/2 phosphorylation levels in G361-vector and G361-IL-32α cells were examined by western blot. Cells were lysed in lysis buffer containing phosphatase inhibitors. Erk1/2 phosphorylation was confirmed using the rabbit anti-human Erk1/2 antibody. B. PD98059-mediated inhibition of Erk1/2 phosphorylation was confirmed by western blot. The selective MEK inhibitor PD98059 was added to cells for 24 hours to inhibit Erk1/2 phosphorylation. DMSO was used as a solvent control. After treatment, western blot assays were performed using the rabbit anti-human Erk1/2 antibody. C. E-cadherin levels in DMSO- or PD98059-treated cells were examined using flow cytometry. Cells were detached using enzyme-free cell dissociation buffer and incubated with the PE-conjugated mouse anti-human E-cadherin antibody. A representative experiment of three independent experiments is shown. D. IL-32α-induced migration was inhibited by Erk1/2 inhibition. G361-IL-32α cells were treated for 24 hours with PD98059, U0126 or SB203580 and collected for transwell migration assays. Cells (5×10 4 ) were placed in upper chambers with serum-free DMEM. Lower chambers contained DMEM with 5% FBS. After 24 hours, membranes with migrated cells were fixed and stained as described. Images of membranes were analyzed by microscopy. A representative experiment of three independent experiments is shown.

Article Snippet: Cells (5×10 5 ) were washed twice with PBS and incubated for 30 minutes on ice with the PE-conjugated mouse anti-human E-cadherin antibody (R&D Systems, Minneapolis, MN).

Techniques: Phospho-proteomics, Plasmid Preparation, Western Blot, Lysis, Inhibition, Solvent, Control, Flow Cytometry, Incubation, Migration, Staining, Microscopy

Journal: bioRxiv

Article Title: Development of a monoclonal antibody against duck IFN-γ protein and the application for Intracellular Cytokine Staining

doi: 10.1101/2024.12.30.630717

Figure Lengend Snippet: Characterization of mAb against duck IFN- γ protein. (A) Morphologic observation of 24H4 hybridoma cell. Scale bar, 100 μm. (B) Western-blot identification of mAb. Lane M: protein molecular weight standard; Lane 1: supernatant of 293F cells infected with the pEE12.4 vector; Lane 2: Purified duIFN-γ-Fc protein. The purified duIFN-γ mAb was used as the primary antibody, and the goat anti-mouse IgG antibody was used as the secondary antibody. (C) IFN-γ antibody sensitivity was determined by indirect ELISA using duIFN-γ-Fc protein as the antigen and mAb 24H4 as the primary antibody. S/P ratio = sample OD 450 / NC OD 450 , S/P > 2 was considered positive. (D) Subclass of the mAb 24H4 was identified using mouse antibody homotype ELISA kit; **** on the bar means extremely significant difference (P < 0.0001).

Article Snippet: After that, cells were stained with mouse anti-duck IFN-γ for 30 min and then PE-conjugated Goat Anti-Mouse IgG3 (SouthernBiotech, Birmingham, USA) for 30 min. After washing, Samples were analyzed by flow cytometry (CytoFLEX; Beckman Coulter, Brea, CA).

Techniques: Western Blot, Molecular Weight, Infection, Plasmid Preparation, Purification, Indirect ELISA, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Effects of smoking on the tissue regeneration-associated functions of human endometrial stem cells via a novel target gene SERPINB2

doi: 10.1186/s13287-022-03061-1

Figure Lengend Snippet: Cigarette smoke exposure promoted SERPINB2 expression levels in endometrial stem cells. We postulated that SERPINB2 functions as a key regulator of smoking-induced toxic response ( A ). Cells were treated with cigarette smoke extract at 0.5, 1, 3, or 5% for 72 h. Real-time PCR and western blotting were conducted to investigate the effects of cigarette smoke exposure on the mRNA and protein levels of SERPINB2 ( B ). GEO respiratory metadata were analyzed to investigate interactions between elevated SERPINB2 expression and exposures to toxic agents, such as radiation, heavy metals, air pollution, and toxic substances ( C ). Differentially activated genes in toxicant exposed lung cells and nonexposed lung cells were analyzed using IPA software to investigate the activation statuses (intermediate, inactive, or activate) of SERPINB2 (GSE62564)-associated signaling molecules/transcription factors ( D ). Differentially activated genes in toxicant exposed liver cells and nonexposed liver cells were analyzed using IPA software to investigate the activation status of SERPINB2 (GSE62564)-associated signaling molecules/transcription factors ( E ). β-actin was used as the internal control, and PPIA as the housekeeping gene for real-time PCR. All experiments were performed in triplicate. Data are presented as means ± SDs. *, p < 0.05; **, p < 0.005; and ***, p < 0.001 (two-sample t test)

Article Snippet: Antibodies to the following proteins were used: PE-conjugated CD34 (MACS; Miltenyi Biotec, 30-081-002), CD44 (MACS; Miltenyi Biotec, 130-095-180), CD45 (MACS; Miltenyi Biotec, 130-080-201), CD73 (MACS; Miltenyi Biotec, 130-095-182), CD105 (MACS; Miltenyi Biotec, 130-094-941), CD140b (MACS; Miltenyi Biotec, 130-105-279), and W5C5 (MACS; Miltenyi Biotec, 130-111-641).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Software, Activation Assay

Journal: Stem Cell Research & Therapy

Article Title: Effects of smoking on the tissue regeneration-associated functions of human endometrial stem cells via a novel target gene SERPINB2

doi: 10.1186/s13287-022-03061-1

Figure Lengend Snippet: SERPINB2 mediated the harmful effects of cigarette smoke exposure on various tissue regeneration-associated functions of endometrial stem cells. Schematic diagram describing the roles of SERPINB2 as a key regulator that mediates cigarette smoke-induced harmful effects in endometrial stem cells ( A ). Cells were transfected with shRNA specifically targeting SERPINB2 and then exposed or not to 1% cigarette smoke extract for 72 h. Changes in cell viability were determined using an MTT assay ( B ). SERPINB2 knockdown attenuated cigarette smoke-mediated inhibition of endometrial stem cells migration as determined by transwell assay ( C ) and western blotting using MMP-2 and MMP-9 antibodies ( D ). Cells were transfected with a SERPINB2 shRNA and then treated with or without 1% cigarette smoke extract; subsequent changes in adipocyte ( E ) and osteoblast ( F ) differentiation abilities were analyzed by oil red O and alizarin red staining, respectively. Relative calcium mineral contents and lipid droplet formation within differentiated cells were assessed by measuring absorbance at 500 and 570 nm, respectively. Blocking of the effects of SERPINB2 knockdown on the cigarette smoke-induced inhibitions of pluripotency-associated factors C-MYC, KLF4, NANOG, OCT4, and SOX2 was investigated by real-time PCR ( G ). β-actin was used as the internal control, and PPIA as the housekeeping gene for real-time PCR analysis. All experiments were performed in triplicate. Data are presented as means ± SDs. *, p < 0.05; **, p < 0.005; and ***, p < 0.001 (two-sample t test)

Article Snippet: Antibodies to the following proteins were used: PE-conjugated CD34 (MACS; Miltenyi Biotec, 30-081-002), CD44 (MACS; Miltenyi Biotec, 130-095-180), CD45 (MACS; Miltenyi Biotec, 130-080-201), CD73 (MACS; Miltenyi Biotec, 130-095-182), CD105 (MACS; Miltenyi Biotec, 130-094-941), CD140b (MACS; Miltenyi Biotec, 130-105-279), and W5C5 (MACS; Miltenyi Biotec, 130-111-641).

Techniques: Transfection, shRNA, MTT Assay, Inhibition, Migration, Transwell Assay, Western Blot, Staining, Blocking Assay, Real-time Polymerase Chain Reaction

Journal: Stem Cell Research & Therapy

Article Title: Effects of smoking on the tissue regeneration-associated functions of human endometrial stem cells via a novel target gene SERPINB2

doi: 10.1186/s13287-022-03061-1

Figure Lengend Snippet: Effects of SERPINB2 overexpression on various tissue regeneration-associated functions of endometrial stem cells. Schematic showing the regulatory roles of SERPINB2 on various tissue regeneration-associated functions of endometrial stem cells ( A ). Transfection of endometrial stem cells with a specific retroviral expression vector for SERPINB2 significantly inhibition inhibited cell proliferation as compared with control vector transfection ( B ). Increased levels of caspase-3 activities following SERPINB2 overexpression were analyzed by western blotting ( C ), and SERPINB2 overexpression-induced apoptotic DNA fragmentations in endometrial stem cells were also analyzed by DAPI staining ( D ). The inhibitory effects of SERPINB2 overexpression on the migration capacity of endometrial stem cells were assessed by transwell assay ( E ) and western blotting using specific MMP-2 and MMP-9 antibodies ( F ). The inhibitory effects of SERPINB2 overexpression on osteogenic ( G ) and adipogenic ( H ) differentiation were analyzed by alizarin red or oil red O staining, respectively. Relative quantifications of calcium mineral contents and lipid droplet formation were determined by measuring absorbance at 570 or 500 nm, respectively. The inhibitory effects of SERPINB2 overexpression on the expressions of pluripotency-associated genes C-MYC, KLF4, NANOG, OCT4, and SOX2 were analyzed by real-time PCR ( I ). β-actin was used as an internal control, and PPIA as the housekeeping gene for real-time PCR analysis. All experiments were performed in triplicate. Data are presented as means ± SDs. *, p < 0.05; **, p < 0.005; and ***, p < 0.001 (two-sample t test)

Article Snippet: Antibodies to the following proteins were used: PE-conjugated CD34 (MACS; Miltenyi Biotec, 30-081-002), CD44 (MACS; Miltenyi Biotec, 130-095-180), CD45 (MACS; Miltenyi Biotec, 130-080-201), CD73 (MACS; Miltenyi Biotec, 130-095-182), CD105 (MACS; Miltenyi Biotec, 130-094-941), CD140b (MACS; Miltenyi Biotec, 130-105-279), and W5C5 (MACS; Miltenyi Biotec, 130-111-641).

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Inhibition, Western Blot, Staining, Migration, Transwell Assay, Real-time Polymerase Chain Reaction

Journal: Stem Cell Research & Therapy

Article Title: Effects of smoking on the tissue regeneration-associated functions of human endometrial stem cells via a novel target gene SERPINB2

doi: 10.1186/s13287-022-03061-1

Figure Lengend Snippet: Cigarette smoke exposure markedly suppressed various tissue regeneration-associated functions of endometrial stem cells in vivo. Schematic of the in vivo experimental procedure used, which is described in detail in Materials and Methods ( A ). Mice were intraperitoneally administrated cigarette smoke extract 10 times at 0.5 or 1 mg/kg mg/kg for or vehicle. Mice endometrial stem cells were isolated from uterine tissues, and the inhibitory effects of cigarette smoke on cell viability were assessed using an MTT assay ( B ). The changes in stem cell migratory abilities were analyzed using a transwell assay ( C ) and by western blotting using MMP-2 and MMP-9 antibodies ( D ). The inhibitory effects of cigarette smoke exposure on adipocyte ( E ) and osteoblast ( F ) differentiation of mouse endometrial stem cells in vivo were analyzed by oil red O and alizarin red staining, respectively. Relative quantifications of calcium mineral content and lipid droplet formation within differentiated cells were evaluated by measuring absorbance at 500 and 570 nm, respectively. The inhibitory effects of cigarette smoke exposure on the expression of various pluripotency-associated genes, C-MYC, KLF4, NANOG, OCT4, and SOX2 were analyzed by real-time PCR ( G ). β-actin was used as the internal control, and PPIA as the housekeeping gene for real-time PCR. All experiments were performed in triplicate. Data are presented as means ± SDs. *, p < 0.05; **, p < 0.005; and ***, p < 0.001 (two-sample t test)

Article Snippet: Antibodies to the following proteins were used: PE-conjugated CD34 (MACS; Miltenyi Biotec, 30-081-002), CD44 (MACS; Miltenyi Biotec, 130-095-180), CD45 (MACS; Miltenyi Biotec, 130-080-201), CD73 (MACS; Miltenyi Biotec, 130-095-182), CD105 (MACS; Miltenyi Biotec, 130-094-941), CD140b (MACS; Miltenyi Biotec, 130-105-279), and W5C5 (MACS; Miltenyi Biotec, 130-111-641).

Techniques: In Vivo, Isolation, MTT Assay, Transwell Assay, Western Blot, Staining, Expressing, Real-time Polymerase Chain Reaction

Journal: Stem Cell Research & Therapy

Article Title: Effects of smoking on the tissue regeneration-associated functions of human endometrial stem cells via a novel target gene SERPINB2

doi: 10.1186/s13287-022-03061-1

Figure Lengend Snippet: Cigarette smoke exposure significantly reduced the proliferations and migrations of endometrial stem cells and induced cellular senescence. We postulated that cigarette smoke exposure suppresses various tissue regeneration-associated functions of endometrial stem cells, such as proliferation, senescence, migration potential, and differentiation capacity, and pluripotency/stemness ( A ). The inhibitory effects of cigarette smoke extract (0.5, 1, 3, 5%) on the self-renewal capacity of endometrial stem cells were analyzed using a 72-h MTT assay. Proliferative potentials (%) were calculated by expressing numbers of viable cells after treatment with cigarette smoke extract (0.5, 1, 3, 5%) as percentages of numbers after treatment with vehicle ( B ). Effects of cigarette smoke exposure on the senescence (aging) of endometrial stem cells were assessed by analyzing senescence-associated β-galactosidase (SA-β-Gal) activity in cells consecutively exposed to 1% cigarette smoke extract treatment for 72 h ( C ). Effects of cigarette smoke exposure on the mRNA levels of various senescence (aging) markers (p16, p18, p21, and IL-6) were determined by real-time PCR ( D ). The GEO (Gene Expression Omnibus) metadata respiratory ( https://www.ncbi . nlm.nih.gov/geo/) was analyzed to assess relationships between cigarette smoking and levels of various senescence (aging) markers ( E ). Endometrial stem cells were exposed to 1% cigarette smoke extract for 72 h, and inhibitory effects on migration were determined using transwell assays ( F ). The relative protein levels of key regulators of cell migration (MMP-2/9) after exposure or not to cigarette smoke exposure were analyzed by western blotting ( G ). β-actin was used as the internal control for western blotting, and PPIA was used as the housekeeping gene real-time PCR. All experiments were performed in triplicate. Data are presented as means ± standard deviations (SDs). *, p < 0.05; **, p < 0.005; and ***, p < 0.001 (two-sample t test)

Article Snippet: Antibodies to the following proteins were used: PE-conjugated CD34 (MACS; Miltenyi Biotec, 30-081-002), CD44 (MACS; Miltenyi Biotec, 130-095-180), CD45 (MACS; Miltenyi Biotec, 130-080-201), CD73 (MACS; Miltenyi Biotec, 130-095-182), CD105 (MACS; Miltenyi Biotec, 130-094-941), CD140b (MACS; Miltenyi Biotec, 130-105-279), and W5C5 (MACS; Miltenyi Biotec, 130-111-641).

Techniques: Migration, MTT Assay, Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Stem Cell Research & Therapy

Article Title: Effects of smoking on the tissue regeneration-associated functions of human endometrial stem cells via a novel target gene SERPINB2

doi: 10.1186/s13287-022-03061-1

Figure Lengend Snippet: Cigarette smoke exposure significantly reduced differentiation capacity and the pluripotency/stemness of endometrial stem cells. Human endometrial stem cells were pretreated with or without 1% cigarette smoke extract and then cultured in adipocyte or osteoblast differentiation medium. The suppressive effects of cigarette smoke exposure on adipocyte ( A ) and osteoblast ( B ) differentiation were analyzed by oil red O and alizarin red staining, respectively. The relative quantification of calcium mineral contents and lipid droplet formation within differentiated cells was determined by measuring absorbance at 500 nm and 570 nm, respectively. The suppressive effects of cigarette smoke exposure on the mRNA levels of various pluripotency-associated genes (C-MYC, KLF4, NANOG, OCT4, and SOX2) were analyzed by real-time PCR ( C ). The GEO metadata respiratory was analyzed to investigate the relationships between cigarette smoke exposure and levels of various pluripotency-associated genes ( D ). PPIA was used as the housekeeping gene for real-time PCR. All experiments were performed in triplicate, and data are presented as means ± SDs. *, p < 0.05; **, p < 0.005; and ***, p < 0.001 (two-sample t test)

Article Snippet: Antibodies to the following proteins were used: PE-conjugated CD34 (MACS; Miltenyi Biotec, 30-081-002), CD44 (MACS; Miltenyi Biotec, 130-095-180), CD45 (MACS; Miltenyi Biotec, 130-080-201), CD73 (MACS; Miltenyi Biotec, 130-095-182), CD105 (MACS; Miltenyi Biotec, 130-094-941), CD140b (MACS; Miltenyi Biotec, 130-105-279), and W5C5 (MACS; Miltenyi Biotec, 130-111-641).

Techniques: Cell Culture, Staining, Real-time Polymerase Chain Reaction

Journal: Stem Cell Research & Therapy

Article Title: Effects of smoking on the tissue regeneration-associated functions of human endometrial stem cells via a novel target gene SERPINB2

doi: 10.1186/s13287-022-03061-1

Figure Lengend Snippet: Cigarette smoke exposure significantly increased metabolic activity (mitochondrial oxidative phosphorylation and glycolysis) in endometrial stem cells. To investigate the effect of cigarette smoke exposure on metabolic activity (energetic status) in endometrial stem cells, mitochondrial oxidative phosphorylation and glycolysis were measured after exposing or not exposing cells to cigarette smoke extract. Oxidative phosphorylation patterns of endometrial stem cells exposed or not to 1% cigarette smoke extract for 72 h were analyzed by measuring twelve consecutive oxygen consumption rates (OCR) using an XF analyzer (Seahorse Bioscience). A schematic of real-time analysis of mitochondrial oxidative phosphorylation using a Seahorse XF analyzer ( A ). Endometrial stem cells were seeded in multiplates at a density of 2 × 10. 4 cells per well, incubated overnight in complete growth medium containing 10% FBS, and gently washed 3 times with seahorse XF medium. Cells were then treated with ATP synthase blocker oligomycin (a complex V inhibitor, 1.5 μM) to prevent coupled respiration (respiration linked to ATP production), FCCP (a potent uncoupler of mitochondrial oxidative phosphorylation, 2 μM) to reduce the electrochemical proton gradient (Δψm), rotenone (a complex I blocker of the electron transport chain, 0.5 uM), and antimycin A (a complex III blocker of the electron transport chain, 0.5 uM) to completely prevent mitochondrial oxidative phosphorylation. These blockers were added automatically into the multiplate, and OCR was analyzed every 15 min. Overall mitochondrial respiratory responses of endometrial stem cells were significantly elevated by cigarette smoke exposure and showed increased levels of nonmitochondrial oxygen consumption ( B ). Cigarette smoke exposure markedly enhanced the basal respiration rate ( C ), reserve respiratory capacity ( D ), and maximal respiratory potential ( E ). ATP synthesis was markedly increased by cigarette smoke exposure in mitochondria and cytosol ( F ). A schematic of cytosolic glycolysis analysis in real time using a Seahorse XF analyzer ( G ). The Seahorse XF glycolytic rate assay was used to measure OCR and ECAR (extracellular acidification rate) in real-time to determine glycolytic proton efflux rates (glycoPER) of endometrial stem cells, which were cultured in glucose-free medium after treatment with antimycin A (1.67 μM), rotenone, and 50 mM 2-deoxyglucose (2-DG, glycolysis blocker). These agents were added automatically to multiplates, and ECAR was analyzed at 15 min intervals. The overall percentage (%) of PER from glycolytic rate indicates the contribution made by glycolysis to total ECAR ( G ). Compensatory glycolysis is the glycolytic rate in cells after blocking oxidative phosphorylation and supporting compensatory energy synthesis through glycolysis to meet energy requirements. Cigarette smoke exposure also markedly increased basal glycolysis ( H ) and compensatory glycolysis ( I ). ECAR values were normalized with respect to cell counts in each well. Bar graphs represent the averages of three independent experiments. Significant differences are presented as * p < 0.05, ** p < 0.005, and *** p < 0.001 (two-sample t test)

Article Snippet: Antibodies to the following proteins were used: PE-conjugated CD34 (MACS; Miltenyi Biotec, 30-081-002), CD44 (MACS; Miltenyi Biotec, 130-095-180), CD45 (MACS; Miltenyi Biotec, 130-080-201), CD73 (MACS; Miltenyi Biotec, 130-095-182), CD105 (MACS; Miltenyi Biotec, 130-094-941), CD140b (MACS; Miltenyi Biotec, 130-105-279), and W5C5 (MACS; Miltenyi Biotec, 130-111-641).

Techniques: Activity Assay, Incubation, Cell Culture, Blocking Assay