Journal: Cell stem cell

Article Title: M 6 A demethylase ALKBH5 selectively promotes tumorigenesis and cancer stem cell self-renewal in acute myeloid leukemia

doi: 10.1016/j.stem.2020.04.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Lineage Cell Depletion Kit , Miltenyi Biotec , Cat#130-090-858.

Techniques: Recombinant, Control, Virus, Magnetic Beads, Reverse Transcription, Proliferation Assay, Purification, Real-time Polymerase Chain Reaction, Software

Journal: Frontiers in Immunology

Article Title: TLR9- and CD40-Targeting Vaccination Promotes Human B Cell Maturation and IgG Induction via pDC-Dependent Mechanisms in Humanized Mice

doi: 10.3389/fimmu.2021.672143

Figure Lengend Snippet: TLR9 agonist CpG-B promotes IgG responses in humanized mice vaccinated with CD40-targeting vaccine. Humanized mice were treated with PBS control (n=3) or vaccinated with αCD40-HIV5pep (n=3) alone or vaccinated with αCD40-HIV5pep plus CpG-B (n=4) at week0, week3 and week6. At week 7, mice were sacrificed. (A, B) The expression of IgM and IgG on B cells from PBMCs and spleens was detected by FACS. (C) The total level of IgM and IgG in the plasma was detected by ELISA. (D) Splenocyte from humanized mice were cultured ex vivo in the present of R848(1μg/ml) and IL-2(10 u/ml) for 48hours, the cells were used for total IgG detection by ELISpot. (E) Antigen specific IgG level in the plasma was detected by ELISA. (F) The expression of activation-induced cytidine deaminase (AID) in spleen cells was detected by RT-PCR. Each dot represents one individual mouse, bars indicate mean. *P < 0.05, **P < 0.01, by unpaired, two-tailed Student’s t-test comparing the two vaccinated groups.

Article Snippet: Total IgM and IgG were detected by ELISA kits, purchased from Bethyl Laboratories,int.(Cat.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Ex Vivo, Enzyme-linked Immunospot, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Journal of the American Society of Nephrology

Article Title: Chordin-like 1 and Twisted Gastrulation 1 Regulate BMP Signaling following Kidney Injury

doi: 10.1681/asn.2008070768

Figure Lengend Snippet: Figure 4. CHRDL1 functions as a general BMP-signaling amplifier but specifically antagonizes BMP7 in the presence of TWSG1. (A) P19 embryonal carcinoma cells transfected with the BMP transcriptional reporter pBRE-Luc were incubated overnight with BMP4 (5 ng/ml) or BMP7 (10 ng/ml) and increasing amounts of CHRDL1 (50 to 400 ng/ml) or Chordin (100 to 800 ng/ml). Both CHRDL1 and Chordin amplify BMP4 signaling; however, in contrast to Chordin, which antagonizes BMP7 signaling, CHRDL1 amplifies BMP7 signaling in a dosage-responsive manner. (B) In the presence of TWSG1 (100 to 400 ng/ml), CHRDL1 (200 ng/ml) continues to act as an amplifier of BMP4 signaling unlike Chordin (400 ng/ml), which becomes a potent BMP4 antagonist. In contrast, CHRDL1 amplification of BMP7 signaling is converted to antagonism by addition of TWSG1 in a dosage-dependent manner. (C) In the P19 pBRE-luc reporter assay, the addition of TWSG1, CHRDL1, or Chordin (400 ng/ml) alone does not affect transcriptional activation. (D) CHRDL1 binds BMP7 and TWSG1 only in a trimolecular complex. A co-immunoprecipitation experiment in which CHRDL1 (500 ng/ml) is incubated in the presence of TWSG1 (500 ng/ml) and/or BMP7 (250 ng/ml) demonstrates that CHRDL1 binds TWSG1 and BMP7 only when all three proteins are present. No evidence of binding is detected when TWSG1 or BMP7 alone is incubated with CHRDL1. Recombinant proteins were immunoprecipitated with a goat polyclonal antibody against CHRDL1, and blots of goat IgG serve as loading controls. The last panel, a CHRDL1 immunoblot of an identical immunoprecipitation substituting an irrelevant goat antibody, demonstrates the absence of nonspecific binding by CHRDL1 to either the goat antibody or Protein G beads used in the experiment. The first lane of all blots contains recombinant protein as a positive control. (E) TWSG1 is strongly expressed in tubule epithelia of the mouse and human kidney. (Top) Strong immunohistochemical staining for TWSG1 in tubule epithelia of adult mouse kidney but weak staining in the glomerulus (G) and interstitial cell population (inset). (Right) Negative control using an antibody of the same species specific for macrophage. (Bottom) Immunofluorescent micro- graphs of an adult human kidney showing TWSG1 expression (red) in both proximal tubules (PT), marked green with Lotus lectin, and other tubules. As in the mouse, glomerular expression of TWSG1 is much weaker than seen in tubular epithelia. (Right) Negative control for TWSG1.

Article Snippet: BMP7 was detected with mouse anti- BMP7 (R&D Systems) and a sheep anti-mouse HRP conjugate (Am- ersham).

Techniques: Transfection, Incubation, Amplification, Reporter Assay, Activation Assay, Immunoprecipitation, Binding Assay, Recombinant, Western Blot, Positive Control, Immunohistochemical staining, Staining, Negative Control, Expressing

Journal: Journal of the American Society of Nephrology

Article Title: Chordin-like 1 and Twisted Gastrulation 1 Regulate BMP Signaling following Kidney Injury

doi: 10.1681/asn.2008070768

Figure Lengend Snippet: Figure 5. CHRDL1 reduces BMP7-stimulated Smad activation and ID gene expression in the presence of TWSG1 but has no effect on BMP4 signaling. (A and B) In the presence of TWSG1, CHRDL1 inhibits Smad phosphorylation by BMP7 but not by BMP4. P19 cells were serum starved for 2 h and then incubated with BMPs (10 ng/ml) and antagonists for 2 h before lysis and Western blotting. Blots were probed for phosphorylated Smads 1, 5, and 8 and -tubulin. In lanes 3, 4, and 5, Noggin (200 ng/ml), TWSG1, and CHRDL1 alone (400 ng/ml) were added. In lanes 6 through 9 CHRDL1 (400 ng/ml) was added together with increasing concentrations of TWSG1 (50, 100, 200, and 400 ng/ml). BMPs were incubated at 37 C for 1 h with or without antagonists before application. Note that the arrow points to the band corresponding to pSmad1/5/8. The top band (arrowhead) is a contaminating band specific to P19 lysates and is not present in lysates from MDCK cells (compare C and D). (C and D) The effects of CHRDL1 and TWSG1 on BMP signaling can be reproduced in the MDCK kidney cell line. (E) RT-PCR showing expression of ID1, 2, and 3 genes in HK-2 cells stimulated with BMP4 or 7 (25 ng/ml) for 6 h in the presence of varying concentrations (100 to 400 ng/ml) of CHRDL1 and/or TWSG1. ID expression by HK-2 cells in response to BMP4 is unaffected by the presence of TWSG1. In cells incubated with BMP7, the addition of TWSG1 reduces ID gene expression.

Article Snippet: BMP7 was detected with mouse anti- BMP7 (R&D Systems) and a sheep anti-mouse HRP conjugate (Am- ersham).

Techniques: Activation Assay, Gene Expression, Phospho-proteomics, Incubation, Lysis, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Journal of the American Society of Nephrology

Article Title: Chordin-like 1 and Twisted Gastrulation 1 Regulate BMP Signaling following Kidney Injury

doi: 10.1681/asn.2008070768

Figure Lengend Snippet: Figure 6. Overexpression of mouse Chrdl1 in the collecting duct substan- tially reduces BMP signaling in vivo. (A) Diagram of mouse Chrdl1 transgene construct driven by a collecting duct–specific enhancer element from intron 1 of the Bmp7 gene; quantitative PCR assay shows Chrdl1 overexpression in kidneys of two embryonic day 17.5 transgenic embryos (150 and 151) com- pared with wild-type. (B through D) Immunolocalization of phosphorylated Smads (red) in the embryonic day 17.5 wild-type kidney (B), and kidneys from transgenic embryos 150 and 151 (C and D) show that BMP signaling is substantially reduced by expression of the Chrdl1 transgene. Collecting ducts (CD) were localized by staining with the lectin Dolichos Biflorus Agglu- tinin (green), and nuclei were counterstained with DAPI (blue).

Article Snippet: BMP7 was detected with mouse anti- BMP7 (R&D Systems) and a sheep anti-mouse HRP conjugate (Am- ersham).

Techniques: Over Expression, In Vivo, Construct, Real-time Polymerase Chain Reaction, Transgenic Assay, Expressing, Staining

Journal: Endocrinology

Article Title: Peritonitis activates transcription of the human prolactin locus in myeloid cells in a humanized transgenic rat model.

doi: 10.1210/en.2011-1926

Figure Lengend Snippet: FIG. 2. Ex vivo analysis of hPRL gene promoter activation. A, qPCR of hPRL promoter activity of PE , BM cells, and PBMC in T24 and S24 rats. A minimum of four samples from each treatment group was assayed in duplicate. Data are presented as mean SEM; *, P 0.05. B, Luciferase activity of PE and BM cells isolated from S24 and T24 rats. ***, P 0.001. C, Ex vivo bioluminescent imaging of FITC-labeled, CD11b adherent cells from the PE, BM, and PBMC of S24 transgenic rats. Scale bar, 50 m. D, Comparison of bioluminescence between PE cells from a S24 rat and a T24 rat. S24, saline 24 h; T24, TG 24 h; RLU, Relative luminescence units. Scale bar, 100 m.

Article Snippet: Adherent cells were blocked with a mouse antirat CD32 antibody (BD Pharmingen, San Diego, CA), then stained with an antirat CD11b fluorescein isothiocyanate (FITC)-conjugated antibody or matched isotype control (1:10 antibody; AbD Serotec, Kidlington, UK).

Techniques: Ex Vivo, Activation Assay, Activity Assay, Luciferase, Isolation, Imaging, Labeling, Transgenic Assay, Comparison, Saline

Journal: Endocrinology

Article Title: Peritonitis activates transcription of the human prolactin locus in myeloid cells in a humanized transgenic rat model.

doi: 10.1210/en.2011-1926

Figure Lengend Snippet: FIG. 3. Identification of subpopulations of CD11b cells expressing luciferase in the PE. A, FACS analysis of cells isolated from PE of S24 rats: two-dimensional dot plot (ssc vs. fsc) showing two populations of CD11b-Alexa Fluor 647-stained cells (histograms, central panel). Blue, CD11b/sschigh; green, CD11B/ssclow. Diff-Quick staining of the corresponding sorted cell populations (right panels). Small black arrow, Mast cell, dark purple granules; small white arrow, eosinophils, red granules; large white arrow, B-cell, small cell with little cytoplasm; large black arrow, macrophage. Scale bar, 50 m. B, qPCR quantification of luciferase transcript in the S24 CD11b/ssclow population (n 5). C, FACS analysis of cells isolated from PE of T24 rats. Two-dimensional dot plot (ssc vs. fsc), representing three populations of CD11b-Alexa Fluor 647- and/or Gra-PE-stained cells (histograms, central panel). Blue, CD11b/sschigh; green, CD11b/Gra; pink, CD11b/Gra. Diff-Quick staining of the corresponding sorted cell populations (right panels). Scale bar, 50 m. D, qPCR quantification of luciferase transcript in the T24 CD11b/Gra (n 3) and CD11b/Gra (n 5) populations. E, RT-PCR for detection of extrapituitary luciferase and endogenous rPRL gene expression. In addition to the expected 122-bp product, a larger band (260 bp) was produced by alternative splicing as described previously (31). M, marker; 1, Luciferase from T24 CD11b/Gra; 2, rPRL from T24 CD11b/Gra; 3, Luciferase from T24 CD11b/Gra; 4, rPRL from T24 CD11b/Gra; 5, rPRL on pituitary (control); S24, saline 24 h; T24, TG 24 h.

Article Snippet: Adherent cells were blocked with a mouse antirat CD32 antibody (BD Pharmingen, San Diego, CA), then stained with an antirat CD11b fluorescein isothiocyanate (FITC)-conjugated antibody or matched isotype control (1:10 antibody; AbD Serotec, Kidlington, UK).

Techniques: Expressing, Luciferase, Isolation, Staining, Diff-Quik, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Produced, Alternative Splicing, Marker, Control, Saline

Journal: Endocrinology

Article Title: Peritonitis activates transcription of the human prolactin locus in myeloid cells in a humanized transgenic rat model.

doi: 10.1210/en.2011-1926

Figure Lengend Snippet: FIG. 4. Subpopulations of CD11b cells expressing luciferase in BM and PBMC. A, FACS analysis of cells isolated from BM of T24 rats. Two- dimensional dot plot (ssc vs. fsc) showing two populations of CD11b-Alexa Fluor 647- and/or Gra-PE-stained cells (histograms, central panel). Blue, CD11b/sschigh (Gra); green, CD11b/ssclow (Gra). Diff-Quick staining of corresponding sorted cell populations (right panels). Black arrow, Monocytes; white arrow, neutrophils. Scale bar, 50 m. B, qPCR quantification of luciferase transcript in the S24 (n 5) and T24 (n 6) CD11b/ssclow population in BM. C, RT-PCR for the detection of extrapituitary luciferase and endogenous rPRL gene expression. M, Marker; 1 and 3, Luciferase on T24 CD11b/sschigh; 2 and 4, rPRL on T24 CD11b/sschigh; 5, rPRL on pituitary (control). D, FACS analysis of cells isolated from PB of T24 rats. Two-dimensional dot plot (ssc vs. fsc) showing two populations of CD11b-Alexa Fluor 647- and/or Gra-PE-stained cells (histograms, central panel). Blue, CD11b/Gra and green, CD11b/Gra. E, qPCR quantification of luciferase transcript in the S24 (n 5) and T24 (n 5) CD11b/Gra and T24 CD11b/Gra (n 5 in each group). S24, saline 24 h; T24, TG 24 h.

Article Snippet: Adherent cells were blocked with a mouse antirat CD32 antibody (BD Pharmingen, San Diego, CA), then stained with an antirat CD11b fluorescein isothiocyanate (FITC)-conjugated antibody or matched isotype control (1:10 antibody; AbD Serotec, Kidlington, UK).

Techniques: Expressing, Luciferase, Isolation, Staining, Diff-Quik, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Marker, Control, Saline

Journal: Endocrinology

Article Title: Peritonitis activates transcription of the human prolactin locus in myeloid cells in a humanized transgenic rat model.

doi: 10.1210/en.2011-1926

Figure Lengend Snippet: FIG. 7. Ex vivo stimulation of PRL expression in human peripheral blood monocytes. A, Peripheral blood monocytes stained with CD11b antibody (left panel). Diff-Quick stain of CD11b cells to confirm monocytes morphology. Scale bar, 50 m. B and C, RT-PCR analysis of two distinct donors, showing the bad pattern of extrapituitary hPRL gene expression. D and E, qPCR of hPRL extrapituitary gene expression in human CD11b PBMC of two donors. Cells were treated with TNF- (TNF), LPS, and TG for 16 h, then analyzed for hPRL gene expression. CycloA, Cyclophilin A; Ctrl, no-stimulation control.

Article Snippet: Adherent cells were blocked with a mouse antirat CD32 antibody (BD Pharmingen, San Diego, CA), then stained with an antirat CD11b fluorescein isothiocyanate (FITC)-conjugated antibody or matched isotype control (1:10 antibody; AbD Serotec, Kidlington, UK).

Techniques: Ex Vivo, Expressing, Staining, Diff-Quik, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Control

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 1 Effect of IL1B on mbIL1R, mbIL1R2 and IL1RA expression in KLE cells. Confluent KLE cell cultures were treated with 0.1 ng/ml IL1B for varying periods of time (0–24 h) (A) or with different concentrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h (B). Cells were recovered to evaluate IL1R1, IL1R2 and IL1RA protein expression in total cell protein extracts by western blot. a-Tubulin was also probed on the same membranes to ensure equal protein loading. The intensity of mbIL1Rs, IL1RA and corresponding a-tubulin bands was evaluated by densitometric analysis (C and D). Values were normalized to a-tubulin band intensity and expressed as % of control (ratio of normalized mbIL1R1, mbIL1R2 or IL1RA band intensity detected following treatment with IL1B to that detected following incubation with the control culture medium alone for an equivalent period of time). *P , 0.05 and **P , 0.01 as compared with control. Data are from three different experiments. MM, minimal medium; mb, membrane-bound; s, soluble.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Expressing, Western Blot, Control, Incubation, Membrane

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 3 Immunocytofluorescence of IL1R1, IL1R2 and IL1RA in KLE cells. Cells cultured in chamber slides were incubated for 24 h with the culture medium alone or containing 0.1 ng/ml IL1B. Detection of IL1R1 (A and C), IL1R2 (E and G) and IL1RA (I and K) was performed by immunocytochemical staining using specific monoclonal mouse antibodies. Note the increase in IL1R1 (C), IL1R2 (G) and IL1RA (K) immunofluorescent signal in KLE cells exposed to IL1B by comparison with non-stimulated cells (A, E and I, respectively). No immunofluorescence was observed in the absence of primary antibodies (B, F and J) or the presence of mouse IgGs (D, H and L) (controls). Data are representative of four different experiments.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Cell Culture, Incubation, Staining, Comparison

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 4 Effect of IL1B on IL1R1, IL1R2 and IL1RA mRNA expression in KLE cells. Confluent KLE cell cultures were treated with 0.1 ng/ml IL1B for varying periods of time (0–24 h) (A, B and C, respectively) or with different con- centrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h (D, E and F, respectively). Total RNA was extracted and reverse transcribed, IL1R1, IL1R2, IL1RA and GAPDH cDNAs were amplified by Real-Time PCR as described in Materials and Methods and IL1R1, IL1R2 and IL1RA mRNA levels were normalized to GAPDH mRNA levels. Data were expressed as % of control (ratio of IL1R1, IL1R2 or IL1RA mRNA levels found in cells incubated with IL1 to those found in cells incubated with the control basal culture medium for an equivalent period of time). *P , 0.05, **P , 0.01 and ***P , 0.001 as compared with control. Data are from three different experiments.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Incubation

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 5 Effect of IL1B on IL1R1, IL1R2 and IL1RA mRNA stability and gene expression in KLE cells. Confluent KLE cell cultures were stimulated with IL1B (1 ng/mll) for 12 h. Actinomycin D (10 mg/ml) was added to stop the de novo RNA synthesis, and cells were harvested after 0, 2, 4 and 8 h of incubation with actinomycin D. Total RNA was extracted and reverse transcribed. cDNA was analyzed by real-time PCR with specific primers for IL1R1, IL1R2, IL1RA and GAPDH. Levels of IL1R1, IL1R2 and IL1RA mRNA were normalized to those of GAPDH to assess the kinetics of IL1R1 (A), IL1R2 (B) and IL1RA (C) mRNA degradation. Data were expressed as % of control (ratio of IL1R1, IL1R2 or IL1RA mRNA levels found at different periods of time following the arrest of de novo mRNA transcription to that found at the time of arrest); data are from three different experiments. To evaluate IL1R1, IL1R2 and IL1RA transcriptional activation (D), confluent KLE cell cultures were stimulated with IL1B (1 ng/ml) for 6 h, cell nuclei were isolated and nuclear mRNA transcription was analyzed by nuclear run-on as described in Materials and Methods. DNA samples immobilized onto nylon membranes were as follows: lane 1, IL1R1 cDNA; lane 2, IL1R2 cDNA; lane 3, IL1RA; lane 4, 28S cDNA; and lane 5, pBluescript plasmid DNA. Radioactive transcripts were from KLE cells stimulated in MM or with 1 ng/ml IL1B. Data are repre- sentative of four different experiments.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Gene Expression, Incubation, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Activation Assay, Isolation, Plasmid Preparation

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 6 Effect of IL1B on mbIL1R1, mbIL1R2 and IL1RA expression in endometrial epithelial cells. Confluent endometrial epithelial cell cultures were treated with different concentrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h. Cells were recov- ered to evaluate IL1R1, IL1R2 and IL1RA protein expression in total cell protein extracts by Western blot (A). a-Tubulin was probed on the same membranes to ensure equal protein loading. Culture supernatants were recovered to evaluate sIL1R2 release (C) and IL1RA secretion (D) by ELISA; data were from four different endometrial samples and expressed as % of control (ratio of sIL1R2 or IL1RA concentrations detected in the presence of IL1 to those detected in the control basal culture medium for an equivalent period of 12 h). *P , 0.05, as compared with control. The intensity of mbIL1Rs, IL1RA and corresponding a-tubulin bands was evaluated by densitometric analysis (B). Values were normalized to a-tubulin band intensity and expressed as % of control (ratio of normalized mbIL1R1, mbIL1R2 or IL1RA band intensity detected following treatment with IL1B to that detected following incubation with the control culture medium alone for an equivalent period of time). *P , 0.05 and **P , 0.01 as compared with control. Data were from three different endometrial samples. sIL1R1 and sIL1R2 release and IL1RA secretion from endometrial epithelial cell cultures was also analyzed by metabolic labeling with 35S-cysteine and immunoprecipitation using a goat anti-IL1R1, goat anti-IL1R2 or rabbit anti- IL1RA antibody (E). Immunoprecipitated proteins were then analyzed by SDS-PAGE as described in Materials and Methods (representative data from three different endometrial samples); mb, membrane-bound; s, soluble.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Incubation, Labeling, Immunoprecipitation, SDS Page, Membrane

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 7 Effect of IL1B on IL1R1, IL1R2 and IL1RA mRNA expression in endometrial epithelial cells. Confluent cultures were treated with 0.1 ng/ml IL1B with different concentrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h. Total RNA was extracted and reverse transcribed, IL1R1, IL1R2, IL1RA and GAPDH cDNAs were amplified by Real-Time PCR as described in Materials and Methods and IL1R1 (A), IL1R2 (B) and IL1RA (C) mRNA levels were normalized to GAPDH mRNA levels. Data were from three different endometrial samples and expressed as % of control (ratio of IL1R1, IL1R2 or IL1RA mRNA levels found in cells incubated with IL1 to those found in cells incubated with the control basal culture medium for an equivalent period of 12 h). *P , 0.05 and **P , 0.01 as compared with control.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Incubation

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: RETRACTED: Blockade of TNF-α signaling suppresses the AREG-mediated IL-6 and IL-8 cytokines secretion induced by anti-Ro/SSA autoantibodies.

doi: 10.1038/labinvest.2010.168

Figure Lengend Snippet: Figure 1 Semiquantitative RT-PCR and real-time PCR for Furin, TACE and AREG genes expression. (a) RT-PCR analysis of Furin, TACE and AREG mRNA extracted from SGEC treated with anti-Ro/SSA autoantibodies. M, marker; control, untreated SGEC; HIgG, SGEC treated with IgG fractions extracted from sera of healthy donors; anti-Ro, SGEC treated with anti-Ro/SSA autoantibodies. RT-PCR of GADPH was used as control. Band intensities were analyzed by densitometry (b). (c) Real-time PCR for Furin, TACE and AREG genes expression. Representative histograms of mRNA levels of Furin, TACE and AREG in untreated SGEC (control), SGEC treated with healthy IgG (HIgG), anti-Ro/SSA autoantibodies (anti-Ro). The mRNA levels of the housekeeping gene, b-2 microglobulin, were quantified between untreated control cells and variously treated cells (data represent the mean±s.e. of five independent experiments).

Article Snippet: Membranes were incubated for 90 min with rabbit anti-human Furin polyclonal antibody (pAb), goat antihuman TACE pAb (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human AREG mAb (R&D Systems, Minneapolis, MN USA), and for 30 min with the relative secondary antibodies-HRP conjugates (Santa Cruz Biotechnology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, Marker, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: RETRACTED: Blockade of TNF-α signaling suppresses the AREG-mediated IL-6 and IL-8 cytokines secretion induced by anti-Ro/SSA autoantibodies.

doi: 10.1038/labinvest.2010.168

Figure Lengend Snippet: Figure 2 Analysis of Furin, TACE and AREG expression in anti-Ro/SSA Abs-treated SGEC. (a) Flow cytometric analysis of Furin, TACE and AREG expression in SGEC after anti-Ro/SSA Abs treatment. Examples of flow cytometric images from one representative experiment. (A) Furin expression analysis in untreated and anti-Ro/SSA or HIgG-treated SGEC; (B) intracellular active TACE expression analysis in untreated and anti-Ro/SSA or HIgG-treated SGEC; (C) AREG expression analysis in untreated and anti-Ro/SSA or HIgG-treated SGEC. (b) Western blot analysis of Furin, TACE and AREG proteins expression in SGEC treated or not with anti-Ro/SSA. Immunoblotting gave rise to bands of the expected size (97 kDa for Furin, 80 kDa for active TACE and 50 kDa for AREG). b-Actin was used as protein loading control. (c) Detection of soluble AREG by ELISA. Secreted AREG was detected by ELISA in the conditioned medium. Control, untreated SGEC; HIgG, SGEC treated with IgG fractions extracted from sera of healthy donors; anti-Ro, SGEC treated with anti-Ro/SSA autoantibodies. (Data represent the mean±s.e. of four independent experiments).

Article Snippet: Membranes were incubated for 90 min with rabbit anti-human Furin polyclonal antibody (pAb), goat antihuman TACE pAb (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human AREG mAb (R&D Systems, Minneapolis, MN USA), and for 30 min with the relative secondary antibodies-HRP conjugates (Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A&B) The effect of IL-1β on the mRNA expression of chemokine receptors in Tca8113 (A) and Hep2 (B) cells. Cells were treated with 20 ng/ml IL-1β for 24 h. The mRNA levels of CXCR4, CCR6 and CCR7 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (C) Quantitative CXCR4 mRNA expression in (A). * P < 0.05 compared with the non-treated group. (D) Time course of CXCR4 mRNA expression in response to IL-1β stimulation. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. The mRNA levels of CXCR4 were detected by RT-PCR. β-actin mRNA levels were measured as loading controls. (E) The quantitative data corresponding to (D). * P < 0.05 compared with the non-treated group. (F) The effect of IL-1β on CXCR4 protein expression. Tca8113 cells were treated with the indicated concentrations of IL-1β for 24 h. CXCR4 protein expression was detected by FACS. (G) The effect of IL-1β on SDF-1α-induced cell migration. Tca8113 cells were treated with the indicated concentrations of IL-1β for 24 h. Cell migration in response to medium or 20 ng/ml SDF-1α was measured by the Transwell assay. * P < 0.05 compared with control groups. (I) The transwell assay showed cell migration in response to 20 ng/ml SDF-1α after treatment with the indicated concentrations of IL-1β for 24 h (Scale bars: 200 μM).

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Migration, Transwell Assay, Control

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A) The expression of IL-1 receptors in Tca8113 cells. Cells were treated with the indicated concentrations of IL-1β for 24 h. The mRNA levels of IL-1R1 and IL-1RII were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (B) The expression of IL-1 receptors in Hep2. Cells were treated as described in (A). The mRNA levels of IL-1R1 and IL-1R2 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (C) The effect of IL-1β on IL-1R1 expression. Tca8113 cells were treated with the indicated concentrations of IL-1β for 24 h. The protein levels of IL-1R1 were measured by western blot. β-actin protein levels were measured as loading controls. (D&E) The effect of IL-1Ra on IL-1β-induced CXCR4 mRNA up-regulation. Tca8113 cells, pre-treated with the indicated concentrations of IL-1Ra for 1 h, were treated with or without 20 ng/ml IL-1β for 1 h. The mRNA levels of CXCR4 were measured by RT-PCR (D) and qRT-PCR (E). * P < 0.05 compared with the IL-1β-treated group. (F) The effect of IL-1Ra on IL-1β-induced CXCR4 protein up-regulation. Tca8113 cells, pre-treated with the indicated concentrations of IL-1Ra for 1 h, were treated with or without 20 ng/ml IL-1β for 24 h. The protein expression of CXCR4 was measured by FACS. (G) The effect of RNA interference on the expression of IL-1R1 protein. Tca8113 cells, transfected with non-specific shRNA (Nssi) or with IL-1R1 shRNA (IL-1R1si), were treated with the indicated concentrations of IL-1β for 24 h. The expression of IL-1R1 protein was measured by western blot. β-actin protein levels were measured as loading controls. (H) The effect of IL-1R1 down-regulation on CXCR4 mRNA expression. Non-specific shRNA (Nssi) or IL-1R1 shRNA (IL-1R1si) transfected Tca8113 cells were treated with medium or 20 ng/ml IL-1β for 24. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (I) The quantitative data corresponding to (H). * P < 0.05 compared with the Nssi group.

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Transfection, shRNA

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A) The effect of IL-1β on mRNA levels of IL-1β and TNF-α. Tca8113 cells were treated with the indicated concentrations of IL-1β for 24 h. The mRNA levels of IL-1β and TNF-α were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (B) IL-1β quantitative mRNA levels in (A). * P < 0.05 compared with the non-treated control. (C) Time-course of IL-1β mRNA expression in response to IL-1β treatment. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. The mRNA levels of IL-1β were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (D) IL-1β quantitative mRNA levels in (C). * P < 0.05 compared with the non-treated group. (E) The effect of IL-1Ra on IL-1β-induced IL-1β mRNA expression. Tca8113 cells, pre-treated with the indicated concentrations of IL-1Ra for 1 h, were stimulated with 20 ng/ml IL-1β for 24 h. The mRNA levels of IL-1β were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (F) IL-1β quantitative mRNA levels in (E). * P < 0.05 compared with the non-treated group. (G) The sustained effect of IL-1β on the expression of IL-1β and CXCR4. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated number of days. The mRNA levels of IL-1β and CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (H-I) Quantitative data of IL-1β (H) and CXCR4 (I) in (G). * P < 0.05 compared with the non-treated groups.

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Expressing

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A) Time-dependent activation of Notch by IL-1β. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. Activated Notch NCID fragments were detected by western blot. β-actin protein levels were measured as loading controls. (B) Dose dependent activation of Notch by IL-1β treatment for 1 h. (C) The effect of IL-1β on Hes1 mRNA levels. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. The mRNA levels of the Notch1 targeting gene Hes1 were measured by qRT-PCR. * P < 0.05 compared with the control group. (D) The effect of Notch inhibition on CXCR4 expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of Notch inhibitor L685458 for 30 min, were treated with 20 ng/ml IL-1β for 1 h. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (E) Quantitative data of CXCR4 expression in (D). * P < 0.05 compared with IL-1β-treated alone group. (F) The effect of Notch inhibition on IL-1β expression induced by IL-1β. Cells were treated as described in (D). The mRNA levels of IL-1β were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (G) Quantitative data of IL-1β expression in (F). * P < 0.05 compared with the IL-1β-treated alone group. (H) The effect of Notch inhibition on long-term CXCR4 mRNA expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of Notch inhibitor L685458 for 30 min, were treated with 20 ng/ml IL-1β for 24 h. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (I) The effect of Notch inhibition on CXCR4 protein expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of the Notch inhibitor L685458 for 30 min, were treated with 20 ng/ml IL-1β for 24 h. The protein levels of CXCR4 were measured by western blot. β-actin protein levels were measured as loading controls.

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Control, Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A) The effect of IL-1β on the activation of MAPKs. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. The phosphorylation levels of MAPKs were measured by western blot. β-actin protein levels were measured as loading controls. (B) The effect of IL-1β on the activation of IκB-α. Tca8113 cells were treated as described in (A). IκB-α protein levels were measured by western blot. β-actin protein levels were measured as loading controls. (C) The effect of ERK inhibition on IL-1β-induced CXCR4 expression. Tca8113 cells, pre-treated with the indicated concentrations of U0126 for 30 min, were stimulated with 20 ng/ml IL-1β for 1 h. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (D) Quantitative data of (C). * P < 0.05 compared with the IL-1β-treated group. (E) The effect of ERK inhibition on IL-1β-induced IL-1β mRNA expression. Tca8113 cells were treated as described in (C). The mRNA levels of IL-1β were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (F) Quantitative data of (E). * P < 0.05 compared with the IL-1β-treated group. (G) The effect of ERK inhibition on long-term CXCR4 mRNA expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of ERK inhibitor U0126 for 30 min, were treated with 20 ng/ml IL-1β for 24 h. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (I) The effect of ERK inhibition on CXCR4 protein expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of ERK inhibitor U0126 for 30 min, were treated with 20 ng/ml IL-1β for 24 h. The protein levels of CXCR4 were measured by western blot. β-actin protein levels were measured as loading controls.

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: The FASEB Journal

Article Title: Resveratrol‐enhanced SIRT1‐mediated osteogenesis in porous endplates attenuates low back pain and anxiety behaviors

doi: 10.1096/fj.202002524R

Figure Lengend Snippet: Resveratrol attenuates inflammation and sensory innervation in endplates. A‐C, ELISA analysis of the PGE2, IL‐1β, and RT‐PCR analysis of expression of TNFα in the lumbar endplates lysates from mice in the sham, vehicle and 40 mg/kg/d resveratrol treated groups at 4 and 8 weeks post‐LSI surgery. D‐E, Representative images of the immunofluorescence and quantitative analysis of COX2+ cells (green) in endplates from mice in the sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. Scale bar: 100 μm. F‐G, Representative images of the immunofluorescence and quantitative analysis of CGRP+ sensory fibers (green) in the endplates from mice in the sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. Scale bar: 50 μm. H‐I, Representative images of the immunofluorescence and quantitative analysis of the CGRP+ sensory neurons (green) in L 2‐4 DRGs from mice in the sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. Scale bar: 100 μm. N ≥ 6 per group. * P < .05, ** P < .01. (Student’s t test)

Article Snippet: The concentrations of PGE2 and IL‐1β in the L 4‐5 endplates and the concentration of BDNF in the hippocampus were measured by a PGE2 ELISA kit (514010, Cayman Chemical, Ann Arbor, MI, USA), IL‐1β ELISA kit (BMS6002, Thermo Fisher, Waltham, MA, USA), and BDNF ELISA kit (dy248, R&D System, Minneapolis, MN), respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunofluorescence

Journal: The FASEB Journal

Article Title: Resveratrol‐enhanced SIRT1‐mediated osteogenesis in porous endplates attenuates low back pain and anxiety behaviors

doi: 10.1096/fj.202002524R

Figure Lengend Snippet: Resveratrol promotes osteogenesis in the porous endplates after LSI surgery. A‐B, Representative immunofluorescence images of the Osterix staining in the L 4‐5 endplates and the quantitative analysis of the number of osterix+ cells per section from the sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. Scale bar, 20 μm. C‐D, Representative images of the calcein double‐labeling of porous endplates with quantification of the mineral apposition rate from the sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery, distance between white arrowheads were measured. E‐F, Representative images of Trap staining of the endplates and quantitative analysis of the osteoclast number per section from the sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. Scale bar, 50 μm. G‐H, The RT‐PCR analysis of Slit3 and Runx2 expression in the L 4‐5 endplates from the sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. I, The quantitative ELISA evaluation of Netrin‐1 concentration in the L 4‐5 endplates from the sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. N ≥ 6 per group. * P < .05, ** P < .01. (Student’s t test)

Article Snippet: The concentrations of PGE2 and IL‐1β in the L 4‐5 endplates and the concentration of BDNF in the hippocampus were measured by a PGE2 ELISA kit (514010, Cayman Chemical, Ann Arbor, MI, USA), IL‐1β ELISA kit (BMS6002, Thermo Fisher, Waltham, MA, USA), and BDNF ELISA kit (dy248, R&D System, Minneapolis, MN), respectively.

Techniques: Immunofluorescence, Staining, Labeling, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: The FASEB Journal

Article Title: Resveratrol‐enhanced SIRT1‐mediated osteogenesis in porous endplates attenuates low back pain and anxiety behaviors

doi: 10.1096/fj.202002524R

Figure Lengend Snippet: Resveratrol alleviates hippocampal inflammation and enhances pCREB activation, BDNF expression in the hippocampus. A‐C, The RT‐PCR analysis of the expression of the inflammatory mediators TNFα (A), IL‐1β (B), and IL‐6 (C) in the hippocampus of resveratrol‐treated and control mice at 8 weeks post‐LSI surgery. D, Representative images of the pCREB immunostaining at the dentate gyrus of the hippocampus from sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. Scale bar, 200 μm. E‐F, Representative images of the BDNF immunostaining at the dentate gyrus of the hippocampus and the quantitative ELISA analysis of BDNF in the hippocampus from sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. Scale bar, 200 μm. * P < .05. (Student’s t test)

Article Snippet: The concentrations of PGE2 and IL‐1β in the L 4‐5 endplates and the concentration of BDNF in the hippocampus were measured by a PGE2 ELISA kit (514010, Cayman Chemical, Ann Arbor, MI, USA), IL‐1β ELISA kit (BMS6002, Thermo Fisher, Waltham, MA, USA), and BDNF ELISA kit (dy248, R&D System, Minneapolis, MN), respectively.

Techniques: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Immunostaining, Enzyme-linked Immunosorbent Assay