Journal: Nature Communications

Article Title: Cathepsin D deficiency in mammary epithelium transiently stalls breast cancer by interference with mTORC1 signaling

doi: 10.1038/s41467-020-18935-2

Figure Lengend Snippet: a Pictures of Ctsd +/+ and Ctsd − /− PyMT cells cultured for 7–10 days in 10% FCS or 1% FCS medium. Bars, 50 μm. Representative of 5 independent experiments. b Staining for acidic β-galactosidase in 10% FCS and 1% FCS Ctsd +/+ and Ctsd − /− PyMT cells. Bars, 50 μm. Representative of 3 independent experiments. c Quantification of label-retaining cells among 10% FCS and 1% FCS Ctsd +/+ and Ctsd − /− PyMT cells ( n = 7 independent experiments; two-sided two-sample t -test). d Amount of Annexin-V - 7-AAD − (living) cells among 1% FCS Ctsd +/+ and Ctsd − /− PyMT cells treated with Navitoclax, relative to DMSO control and Ctsd +/+ ( n = 3 independent experiments; two-sided one-sample t -test). e Quantification of IL-6 by alphaLISA in cell-conditioned media from 10% FCS and 1% FCS Ctsd +/+ and Ctsd − /− PyMT cells ( n = 2 independent experiments). f Relative Cdkn2a/p16 and Cdkn1a/p21 expression determined by RT-PCR in 10% FCS and 1% FCS Ctsd +/+ and Ctsd − /− PyMT cells ( n = 3 independent experiments; two-sided two-sample t -test). Bar charts show all data points with mean + SD and p -value. Source data are provided as a Source Data file.

Article Snippet: CREB, phosphorylated CREB and IL-6 levels in cells and cell-conditioned media were measured with the AlphaLISA® SureFire® Ultra™ CREB Total Assay Kit (ALSU-TCREB-A500), AlphaLISA® SureFire® Ultra™ p-CREB (Ser133) Assay Kit (ALSU-PCREB-A500), and the AlphaLISA Mouse Interleukin 6 (mIL6) Kit (AL504 C) from PerkinElmer, respectively.

Techniques: Cell Culture, Staining, Control, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Cathepsin D deficiency in mammary epithelium transiently stalls breast cancer by interference with mTORC1 signaling

doi: 10.1038/s41467-020-18935-2

Figure Lengend Snippet: a Phospho-kinase antibody arrays with lysates from Ctsd +/+ and Ctsd − /− PyMT cells cultured for 7–10 days (1% FCS) or for ≥8 weeks (1% FCS LT) in 1% FCS medium. Changes in phosphorylation are plotted as log2 fold change (FC) for indicated comparisons ( n = 2 independent experiments). b , c Transcriptome analysis of 1% FCS and 1% FCS LT Ctsd +/+ and Ctsd − /− PyMT cells ( n = 3 independent experiments). b Change in expression of significantly regulated genes of the CREB gene set for indicated comparisons. Red dashed lines, borders for gene up- and downregulation (|log2 FC | > 1). c Clustering analysis of the log2 FC of CREB genes with significant upregulation in Ctsd − /− 1% FCS LT versus 1% FCS PyMT cells for indicated comparisons shown as heatmap. Source data are provided as a Source Data file.

Article Snippet: CREB, phosphorylated CREB and IL-6 levels in cells and cell-conditioned media were measured with the AlphaLISA® SureFire® Ultra™ CREB Total Assay Kit (ALSU-TCREB-A500), AlphaLISA® SureFire® Ultra™ p-CREB (Ser133) Assay Kit (ALSU-PCREB-A500), and the AlphaLISA Mouse Interleukin 6 (mIL6) Kit (AL504 C) from PerkinElmer, respectively.

Techniques: Cell Culture, Expressing

Journal: Frontiers in Immunology

Article Title: Granulocytic Myeloid-Derived Suppressor Cell Exosomal Prostaglandin E2 Ameliorates Collagen-Induced Arthritis by Enhancing IL-10 + B Cells

doi: 10.3389/fimmu.2020.588500

Figure Lengend Snippet: G-MDSC exosomal PGE2 promotes IL-10 + B cells (A) COX-2 expression in G-MDSCs was detected after treatment with the COX-2 inhibitor celecoxib. (B) The ratio of COX2 and GAPDH in each group was statistically analyzed. (C) The level of PGE2 in the G-MDSC culture supernatant treated with celecoxib was detected by ELISA. (D) The level of PGE2 in the G-exo treated with celecoxib was detected by ELISA. (E) IL-10–producing B cells after treatment with celecoxib-treated G-exo in the present of LPS were analyzed by flow cytometry. (F) The level of IL-10 in culture supernatant after treatment with celecoxib-treated G-exo was detected by ELISA. (G) The p-GSK-3β, T-GSK-3β, p-CREB, and T-CREB were detected by Western blot analysis. (H) The ratio of p-CREB and T-CREB in each group was statistically analyzed. (I) The ratio of p-GSK-3β and T-GSK-3β in each group was statistically analyzed. Bar graphs show the means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ns: indicates no significance.

Article Snippet: The membranes were then incubated with specific rabbit antibodies against phosphorylated (p)-GSK-3β (Santa Cruz Biotechnology, Texas, USA ) , total (t)- GSK-3β (Wanleibio, Shenyang, China), phosphorylated (p)-cAMP response element binding protein (CREB) (Wanleibio, Shenyang, China) and total (t)-CREB (Wanleibio, Shenyang, China) followed by an incubation with the secondary HRP-conjugated goat anti-rabbit IgG (CST, Danvers, MA, USA) according to manufacturer’s protocol.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Western Blot

Journal: Frontiers in Immunology

Article Title: Granulocytic Myeloid-Derived Suppressor Cell Exosomal Prostaglandin E2 Ameliorates Collagen-Induced Arthritis by Enhancing IL-10 + B Cells

doi: 10.3389/fimmu.2020.588500

Figure Lengend Snippet: Schematic image demonstrating that Granulocytic myeloid-derived suppressor cell exosomal prostaglandin E2 ameliorates collagen-induced arthritis by inducing IL-10 + B cells production via affecting GSK-3β and CREB phosphorylation.

Article Snippet: The membranes were then incubated with specific rabbit antibodies against phosphorylated (p)-GSK-3β (Santa Cruz Biotechnology, Texas, USA ) , total (t)- GSK-3β (Wanleibio, Shenyang, China), phosphorylated (p)-cAMP response element binding protein (CREB) (Wanleibio, Shenyang, China) and total (t)-CREB (Wanleibio, Shenyang, China) followed by an incubation with the secondary HRP-conjugated goat anti-rabbit IgG (CST, Danvers, MA, USA) according to manufacturer’s protocol.

Techniques: Derivative Assay, Phospho-proteomics

Journal: PLoS Pathogens

Article Title: Mitogen and Stress Activated Kinases Act Co-operatively with CREB during the Induction of Human Cytomegalovirus Immediate-Early Gene Expression from Latency

doi: 10.1371/journal.ppat.1004195

Figure Lengend Snippet: A ) Chromatin immunopreciptations on monocytes (Mono), immature DCs (iDC), mature DCs (mDC) or mature DCs pre-treated with ERK inhibitor were performed with an anti-CREB or isotype control antibody. CREB binding is expressed relative to IgG control. B ) Chromatin immunopreciptations on monocytes (Mono), immature DCs (iDC), mature DCs (mDC), mature DCs pre-treated with ERK or p38 inhibitors were performed with an anti-CREB, anti-phospho CREB or isotype control antibody. The relative levels of phosphorylated CREB binding was expressed a percentage of total CREB binding. S.D. shown from n = 3 (A,B). C ) Western blot for phosphor-CREB, total CREB and GAPDH was performed on immature DCs or DCs stimulated with LPS after incubation with mock, DMSO or ERK-MAPK inhibitor.

Article Snippet: DNA associated with histones was immunoprecipitated with control serum (Sigma, Poole, UK), anti-phosphor-histone H3-serine 10 antiserum (ChIP grade, 1∶200 dilution), anti-dimethyl lysine 4 histone H3 antiserum (ChIP grade, 1∶200), anti-trimethyl lysine 9 H3 antiserum (ChIP grade, 1∶200 dilution), anti-phosphor or total CREB (both 1∶250), or anti-MSK1 antibody (1∶200 dilution; Cell signalling Technology, Danvers, MA) - all antibodies Upstate Biotechnology, Charlottesville, VA unless stated.

Techniques: Control, Binding Assay, Western Blot, Incubation

Journal: PLoS Pathogens

Article Title: Mitogen and Stress Activated Kinases Act Co-operatively with CREB during the Induction of Human Cytomegalovirus Immediate-Early Gene Expression from Latency

doi: 10.1371/journal.ppat.1004195

Figure Lengend Snippet: A ) Western blot for phosphor and total CREB, phosphor and total ERK1/2, phosphor and total MSK and GAPDH was performed on immature DCs or DCs stimulated with IL-6 (30 mins) after incubation with DMSO or MSK inhibitor (2 hours). B ) Chromatin immunopreciptations on immature DCs (iDC) derived from monocytes infected with HCMV (1–3) were performed alongside IL-6 (4–12) stimulated DCs for phosphor-CREB (CR), histone H3-S10 P (S10) and histone H3-K9 3Me (K9) binding at 2 hours post stimulation. DNA was amplified in an MIEP PCR and expressed as ratio of the Input sample. S.D. of n = 2. C ) Chromatin immunopreciptations on immature DCs (iDC) stimulated with IL-6 were performed at 15 mins to 3 hours post stimulation with an anti-MSK antibody or an isotype matched control. DNA was amplified in an MIEP PCR and expressed as ratio of the Input sample. S.D. of n = 2.

Article Snippet: DNA associated with histones was immunoprecipitated with control serum (Sigma, Poole, UK), anti-phosphor-histone H3-serine 10 antiserum (ChIP grade, 1∶200 dilution), anti-dimethyl lysine 4 histone H3 antiserum (ChIP grade, 1∶200), anti-trimethyl lysine 9 H3 antiserum (ChIP grade, 1∶200 dilution), anti-phosphor or total CREB (both 1∶250), or anti-MSK1 antibody (1∶200 dilution; Cell signalling Technology, Danvers, MA) - all antibodies Upstate Biotechnology, Charlottesville, VA unless stated.

Techniques: Western Blot, Incubation, Derivative Assay, Infection, Binding Assay, Amplification, Control

Journal: PLoS Pathogens

Article Title: Mitogen and Stress Activated Kinases Act Co-operatively with CREB during the Induction of Human Cytomegalovirus Immediate-Early Gene Expression from Latency

doi: 10.1371/journal.ppat.1004195

Figure Lengend Snippet: A ) Immature DCs (iDC) derived from monocytes infected with wild type (WT), Revertant (Rev) or CRE deletion virus (ΔCRE) were left un-stimulated (1,3,5) or IL-6 treated cells (2,4,6). Then, chromatin immunoprecipitation of histone H3-S10 P was performed and samples amplified in an MIEP qPCR (2 hours post stimulation). Following normalisation to GAPDH, samples were expressed as a ratio of the Input signal. B–C ) immature (latent;1,2) and IL-6 stimulated (reactivating, 3,4) DCs (2 hours post stimulation) were subject to chromatin immunoprecipitation with an anti-histone H3-S10 P (S10P), anti-histone H3-K9 3Me (K9M) or isoptype control (IgG) antibodies and then amplified in an MIEP qPCR and signal expressed as a ratio of the Input. C ) The primary ChIPs from the reactivating samples (B) were then subject to a second ChIP with a phosphor CREB antibody or isotype matched control. The phosphor-CREB IP from H3-S10 P IP (1–4) was then expressed as a ratio of the Input signal for the MIEP or GAPDH qPCR. Alternatively, the phosphor-CREB or isotype control IPs from H3-K9 3Me (5,6) were amplified in an MIEP qPCR and expressed as a ratio of Input. S.D. of n = 2. D ) IL-6 stimulated DCs (2 hours) were subject to a primary IP with anti-histone H3-serine 10 antibody then subject to second IP with an anti-MSK1 antibody or isoptype control. Samples were amplified in an MIEP or GAPDH promoter specific PCR and signal expressed as a ratio of Input. S.D. of n = 2.

Article Snippet: DNA associated with histones was immunoprecipitated with control serum (Sigma, Poole, UK), anti-phosphor-histone H3-serine 10 antiserum (ChIP grade, 1∶200 dilution), anti-dimethyl lysine 4 histone H3 antiserum (ChIP grade, 1∶200), anti-trimethyl lysine 9 H3 antiserum (ChIP grade, 1∶200 dilution), anti-phosphor or total CREB (both 1∶250), or anti-MSK1 antibody (1∶200 dilution; Cell signalling Technology, Danvers, MA) - all antibodies Upstate Biotechnology, Charlottesville, VA unless stated.

Techniques: Derivative Assay, Infection, Virus, Chromatin Immunoprecipitation, Amplification, Control

Journal: PLoS Pathogens

Article Title: Mitogen and Stress Activated Kinases Act Co-operatively with CREB during the Induction of Human Cytomegalovirus Immediate-Early Gene Expression from Latency

doi: 10.1371/journal.ppat.1004195

Figure Lengend Snippet: A ) Western blot analysis of iDCs (1), stimulated with IL-6 (2–4) post transduction with adenoviral vectors expressing GFP (3) or dn-MEK1 (4). B ) RNA isolated from IL-6 stimulated iDCs pre infected with AD-GFP or AD-dnMEK was analysed by RT-qPCR for IE gene expression and expressed relative to IL-6 stimulated control. C ) ChIP analyses were performed with isotype control (IgG), anti-phospho-CREB (CR), anti-phospho-serine 10 histone H3 (S10) or anti-trimethylated lysine 9 histone H3 (K9) antibodies and DNA amplified in an MIEP specific qPCR. Values were expressed relative to the Input control.

Article Snippet: DNA associated with histones was immunoprecipitated with control serum (Sigma, Poole, UK), anti-phosphor-histone H3-serine 10 antiserum (ChIP grade, 1∶200 dilution), anti-dimethyl lysine 4 histone H3 antiserum (ChIP grade, 1∶200), anti-trimethyl lysine 9 H3 antiserum (ChIP grade, 1∶200 dilution), anti-phosphor or total CREB (both 1∶250), or anti-MSK1 antibody (1∶200 dilution; Cell signalling Technology, Danvers, MA) - all antibodies Upstate Biotechnology, Charlottesville, VA unless stated.

Techniques: Western Blot, Transduction, Expressing, Isolation, Infection, Quantitative RT-PCR, Gene Expression, Control, Amplification