Journal: Cellular Microbiology

Article Title: Influenza A virus-mediated priming enhances cytokine secretion by human dendritic cells infected with Streptococcus pneumoniae

doi: 10.1111/cmi.12122

Figure Lengend Snippet: Preceding influenza infection leads to elevated levels of secreted IL-12p70 and IL-6 after co-infection with SP. MDDCs were infected with IAV for 4 h before infection with SP. The cells were further incubated for 18 h before the concentration of IL-12p70 (A), IL-6 (B), TNFα (C), IL-1β (D), IFN-β (E) and IL-8 (F) in the supernatants was measured by ELISA. Each dot in a graph represents the cytokine concentration from cells of one donor. The graphs show mean ± SEM of seven (A, C), three (B), five (D, E) or four (F) independent experiments with different donors. Statistical analysis was performed using paired Student’s t -test. (* P < 0.05, ** P < 0.01).

Article Snippet: Cells were pelleted and medium was replaced with R10 medium containing antibodies against human IFN-α (No. 31101-1, PBL Interferon source), IFN-β (No. 31410-1, PBL Interferon source) and IFNAR (MMHAR-2, PBL Interferon source) at concentrations of 5000 U ml −1 , 2000 U ml −1 and 20 μg ml −1 respectively.

Techniques: Infection, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Cellular Microbiology

Article Title: Influenza A virus-mediated priming enhances cytokine secretion by human dendritic cells infected with Streptococcus pneumoniae

doi: 10.1111/cmi.12122

Figure Lengend Snippet: Type I IFNs are responsible for the priming of DCs to produce IL-12p70. MDDCs were incubated with different concentrations of recombinant human IFN-α 4 h prior to infection with SP. The cells were further incubated for 18 h before the concentration of IL-12p70 in the supernatants was measured by ELISA. MDDCs were sequentially infected with IAV and SP in the presence of neutralizing antibodies against IFN-α, IFN-β and IFNAR. After 22 h incubation the cells were lysed and total RNA was isolated and assayed for the presence of IL-12p35 and IL-12p40 mRNA. Ct values were normalized against γ-actin and the relative induction of the genes was calculated using the ΔΔCt method. Values represent mean ± SEM three independent experiments with different donors (A) or one representative donor of 3 (B, C). Statistical analysis was performed using paired Student’s t -test. (* P < 0.05).

Article Snippet: Cells were pelleted and medium was replaced with R10 medium containing antibodies against human IFN-α (No. 31101-1, PBL Interferon source), IFN-β (No. 31410-1, PBL Interferon source) and IFNAR (MMHAR-2, PBL Interferon source) at concentrations of 5000 U ml −1 , 2000 U ml −1 and 20 μg ml −1 respectively.

Techniques: Incubation, Recombinant, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Isolation

Journal: The EMBO Journal

Article Title: Function of HNRNPC in breast cancer cells by controlling the dsRNA‐induced interferon response

doi: 10.15252/embj.201899017

Figure Lengend Snippet: Concentrations of IFNβ, measured by ELISA, in the culturing media of MCF7 and T47D cells 48 hours after siRNA transfections. siNC: non‐targeting siRNA as a negative control, siHN‐1: siRNA sequence 1 for HNRNPC, siHN‐2: siRNA sequence 2 for HNRNPC, siLMNA: siRNA for LMNA as another negative control. Each sample has three replicates. Data represent mean ± SD. The normal MCF7 (left) and T47D (right) cells were cultured in the media collected from the corresponding cells 48 h after siRNA‐mediated gene knock‐down. Expressions of the ISGs in these normal cells were measured with qPCR. Each sample has three replicates. Data represent mean ± SD. Different doses of the IFNβ antibody were added to the media of the siRNA‐transfected MCF7 (left) or T47D (right) cells right before the media were transferred to the wild‐type cells. The wild‐type MCF7 or T47D cells were then cultured in these media for 48 hours, and the expressions of ISGs were measured by qPCR. Each sample has three replicates. Data represent mean ± SD. Growth curves of the MCF7 (left) and T47D (right) cells cultured in the media collected from the siRNA‐transfected cells. Each sample has three replicates. Data represent mean ± SD.

Article Snippet: Ruxolitinib (2 mM) (Invivogen), 0–20 U/ml of IFNAR2 antibody (PBL Interferon Source 21385‐1), 0–500 U/ml of IFNβ antibody (PBL Interferon Source 31401‐1) were added when changing the medium 6 h after siRNA transfection.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Negative Control, Sequencing, Cell Culture, Knockdown

Journal: The EMBO Journal

Article Title: Function of HNRNPC in breast cancer cells by controlling the dsRNA‐induced interferon response

doi: 10.15252/embj.201899017

Figure Lengend Snippet: A–C In the siRNA‐transfected MCF7 (left) or T47D cells (right), the interferon signaling pathway was blocked at different stages by means of IFNβ neutralization (A), IFNAR2 neutralization (B), or JAK‐STAT inhibition (C). The expressions of the ISGs were measured by qPCR. Each sample has three replicates. Data represent mean ± SD. D, E Growth curves of the MCF7 (D) or T47D cells (E) upon HNRNPC knock‐down but with the interferon response blocked with the IFNβ antibody. Each sample has three replicates. Data represent mean ± SD. F Growth curves of the MCF7 (left) or T47D (right) cells upon HNRNPC knock‐down and JAK‐STAT inhibition with ruxolitinib (5 μM). siNC: non‐targeting siRNA as a negative control, siLMNA: siRNA for LMNA as another negative control, siHN‐1: siRNA sequence 1 for HNRNPC, siHN‐2: siRNA sequence 2 for HNRNPC. Each sample has three replicates. Data represent mean ± SD.

Article Snippet: Ruxolitinib (2 mM) (Invivogen), 0–20 U/ml of IFNAR2 antibody (PBL Interferon Source 21385‐1), 0–500 U/ml of IFNβ antibody (PBL Interferon Source 31401‐1) were added when changing the medium 6 h after siRNA transfection.

Techniques: Transfection, Neutralization, Inhibition, Knockdown, Negative Control, Sequencing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Complement 5a‐mediated trophoblasts dysfunction is involved in the development of pre‐eclampsia

doi: 10.1111/jcmm.13466

Figure Lengend Snippet: Expression of angiogenesis‐related factors in human placenta. ( A ) Relative mRNA levels of anti‐angiogenic factors ( IL ‐1β, TNF ‐α, IL ‐6, MCP ‐1 and sF lt1) and pro‐angiogenic ( PIGF , IL ‐10) in normal and pre‐eclamptic placentas. Data are represented as mean ± S.E.M. N = 4–6 in each group. * P < 0.05, ** P < 0.01, *** P < 0.001. ( B ) Immunofluorescence analysis of representative angiogenesis‐related factors (red) and Cy7 (green) in normal and PE placentas. Nuclei were counterstained with DAPI (blue). Scale bar: 60 μm.

Article Snippet: The human placenta slices were blocked in 10% normal goat serum for 30 min., then incubated with primary antibodies against CD31 (Santa Cruz), CD11b (BD Biosciences, San Jose, CA, USA), C5a (Comp Tech, A221), C5aR (Biolegend), cytokeratin 7 (Cy7, Santa Cruz), PIGF (Proteintech group, Wuhan, Hubei, China), sFlt1 (Life Technologies, Waltham, MA, USA), IL‐1β (Boster), IL‐6 (Boster) and MCP‐1 (Boster, Wuhan, Hubei, China) at 4°C overnight.

Techniques: Expressing, Immunofluorescence

Journal: Journal of Cellular and Molecular Medicine

Article Title: Complement 5a‐mediated trophoblasts dysfunction is involved in the development of pre‐eclampsia

doi: 10.1111/jcmm.13466

Figure Lengend Snippet: Trophoblasts stimulated with C5a display an anti‐angiogenic phenotype. ( A ) HTR ‐8/ SV neo cells treated with C5a showed a polarization towards an anti‐angiogenic phenotype with significantly increased mRNA levels of IL ‐1β, TNF ‐α, IL ‐6, MCP ‐1, sF lt1 and decreased mRNA level of PIGF and IL ‐10. The respective mRNA was normalized to β‐actin housekeeping gene. Data are represented as mean ± S.E.M . N = 3 in each group * P < 0.05, ** P < 0.01. ( B ) Immunofluorescence staining for sF lt1 and PIGF expression in HTR ‐8/ SV neo cells in the presence of C5a or PBS ( CON ). Scale bar: 60 μm.

Article Snippet: The human placenta slices were blocked in 10% normal goat serum for 30 min., then incubated with primary antibodies against CD31 (Santa Cruz), CD11b (BD Biosciences, San Jose, CA, USA), C5a (Comp Tech, A221), C5aR (Biolegend), cytokeratin 7 (Cy7, Santa Cruz), PIGF (Proteintech group, Wuhan, Hubei, China), sFlt1 (Life Technologies, Waltham, MA, USA), IL‐1β (Boster), IL‐6 (Boster) and MCP‐1 (Boster, Wuhan, Hubei, China) at 4°C overnight.

Techniques: Immunofluorescence, Staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Generation of Genetically Engineered Human Induced Pluripotent Stem Cells Overexpressing IFN-β for Future Experimental and Clinically Oriented Studies

doi: 10.3390/ijms252212456

Figure Lengend Snippet: IFNB-iPSCs express functional IFN-β. IFNB-iPSCs and K7-iPSCs were cultured in parallel and used for RNA isolation; the preparation of cell extracts; and the collection of cell culture supernatants. ( a ) IFNB-iPSCs display an increased expression of IFNB as compared with the parental K7-iPSC line. RNAs isolated from IFNB-iPSCs and K7-iPSCs were subjected to RT-PCR; IFNB expression values were normalized relative to GAPDH , and fold changes were calculated as relative IFNB mRNA levels in IFNB-iPSCs relative to K7-iPSCs (2 −∆∆Cq ). Dotted line, fold change = 2 (significance threshold). Data are shown as boxes and whiskers with minimal and maximal values (K7-iPSCs, LA8-iPSCs, and LC8-iPSCs, summarized results of at least 3 independent experiments; LE4-iPSCs, one experiment, technical replicates). Note that LA8-iPSCs and LC8-iPSCs bear a homozygous IFNB insertion, whereas LE4-iPSCs are heterozygous. The significance of the differences was determined using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. ( b , c ) IFNB-iPSCs express IFN-β at the protein level. ( b ) IFNB-iPSCs and K7-iPSCs were pelleted, frozen, and analyzed in a Western blot. The graph shows the relative densitometric values of IFN-β normalized to β-actin. ( c ) The supernatants were collected from IFNB-iPSCs and K7-iPSCs and analyzed using ELISA (the results of two independent experiments; in each of them, K7-iPSCs, LA8-iPSCs, and LC8-iPSCs were cultured in parallel and independently of cells of another experiment). ( d ) The supernatants of IFNB-iPSCs exhibit IFN-β-like functional activity. The supernatants were obtained from IFNB-iPSC and K7-iPSC cultures and were added to THP-1 macrophage-like cells pre-activated with PMA. Control THP-1 cells were cultured in the absence of iPSC supernatants. THP-1 RNA was isolated from all cultures, and the expressions of ISGs were analyzed in RT-qPCR using RPL27 as a housekeeping gene. Data are shown as boxes and whiskers with minimal and maximal values and individual points. The representative results of one out of two experiments are presented. Numbers on the graphs show the FDRs (Benjamini, Krieger, and Yekutieli correction for multiple comparisons). FDRs < 0.05 were considered as significant; for the comparison of IFNB-iPSCs and K7-iPSCs, FDRs > 0.05 are also shown. The differences between LA8-iPSCs and LC8-iPSCs were insignificant. PMA, phorbol 12-myristate-13-acetate.

Article Snippet: The membrane was blocked with a 5% non-fat dry milk (Cell Signaling Technology, Danvers, MA, USA) in a TNT buffer (10 mM of Tris-HCl, pH 7.5, 150 mM of NaCl, 0.1% Tween-20) and incubated with the following primary antibodies (4 °C, overnight, 1% milk): rabbit anti-human-IFN-β polyclonal antibodies (1:500, FNab10475, FineTest Biotech, Wuhan, Hubei, China), rabbit anti-human-PAX6 polyclonal antibodies (1:500, PAH446Ra01, Cloud-Clone Corp.), mouse anti-human-OCT-4 monoclonal antibody (1:1000, ab184665, Abcam, Cambridge, UK), mouse anti-human-actin-β antibodies (1:10,000, Abcam); and rabbit anti-human-HSP90 polyclonal antibodies (1:10,000, Sigma-Aldrich).

Techniques: Functional Assay, Cell Culture, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Control, Quantitative RT-PCR, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Generation of Genetically Engineered Human Induced Pluripotent Stem Cells Overexpressing IFN-β for Future Experimental and Clinically Oriented Studies

doi: 10.3390/ijms252212456

Figure Lengend Snippet: Exogenous IFN-β disrupts the expression of ectoderm-associated genes in differentiating parental K7-iPSCs. K7-iPSCs were subjected to spontaneous ( a ) or directed ( b – d ) differentiation in the absence or in the presence of recombinant human IFN-β; at the end of the differentiation, the expression of ectoderm-associated markers was analyzed using RT-PCR (housekeeping gene, RPL27 ) and compared with that observed in original K7-iPSCs. The significance of the differences was determined using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. ( a ) Changes in the expression of ectoderm-associated genes in spontaneously differentiated EBs. Gene expression was analyzed on day 20. Data are shown as boxes and whiskers with minimal and maximal values. The summarized results of two independent experiments are shown. ( b ) Light microscopy of K7-iPSCs differentiating in the absence or in the presence of IFN-β. Made on differentiation days 1 and 7. ( c ) Changes in the expression of ectoderm-associated genes following the directed differentiation of K7-iPSCs in the STEMdiff™ Trilineage Ectoderm Medium. Gene expression was analyzed on day 7 (as recommended by the manufacturer). Data are shown as boxes and whiskers with minimal and maximal values. The representative results of one out of two experiments are presented. ( d ) Changes in the expression of the IFNB gene following the directed differentiation of K7-iPSCs in the STEMdiff™ Trilineage Ectoderm Medium (the representative results of one out of two experiments).

Article Snippet: The membrane was blocked with a 5% non-fat dry milk (Cell Signaling Technology, Danvers, MA, USA) in a TNT buffer (10 mM of Tris-HCl, pH 7.5, 150 mM of NaCl, 0.1% Tween-20) and incubated with the following primary antibodies (4 °C, overnight, 1% milk): rabbit anti-human-IFN-β polyclonal antibodies (1:500, FNab10475, FineTest Biotech, Wuhan, Hubei, China), rabbit anti-human-PAX6 polyclonal antibodies (1:500, PAH446Ra01, Cloud-Clone Corp.), mouse anti-human-OCT-4 monoclonal antibody (1:1000, ab184665, Abcam, Cambridge, UK), mouse anti-human-actin-β antibodies (1:10,000, Abcam); and rabbit anti-human-HSP90 polyclonal antibodies (1:10,000, Sigma-Aldrich).

Techniques: Expressing, Recombinant, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Light Microscopy