Journal: International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group

Article Title: Hyperthermia promotes exosome secretion by regulating Rab7b while increasing drug sensitivity in adriamycin-resistant breast cancer.

doi: 10.1080/02656736.2022.2029585

Figure Lengend Snippet: Figure 4. The validation experiment of hub genes. (A) The differences in gene expression of 10 hub genes were validated with RT-qPCR. (B) Western blot analysis of FOS, CREB5, MAPK8 and NFKB1 protein level. (C) The efficiency of siRNA to knockdown the expression of FOS and CREB5. (D) Cell proliferation in the si-FOS and si-CREB5 group was faster compared with that in the control group, using the CCK-8 assay. (E). Half-inhibition rate of adriamycin in MCF-7/ADR cells treated with si-FOS and si-CREB5. (F) Different expression of FOS and CERB5 between invasive breast cancer and normal breast tissue by TCGA database. Error bars represented the mean ± SD of at least three independent experiments, p < .05, p < .01, p < .001.

Article Snippet: The membrane was then probed with primary antibodies at 4 C overnight and with species-specific secondary antibodies at room temperature for 1 h. The primary antibodies, including FOS antibody (1:1000, Proteintech, 66590-1-Ig), NF-jB antibody (1:1000, Proteintech, 14220-1-AP), MAPK8 antibody (1:1000, Proteintech, 66210-1-Ig), CREB5 antibody (1:1000, Proteintech, 14196-1-AP), GAPDH antibody (1:5000, Proteintech, 60004-1-Ig), CD9 antibody (1:1000, Abcam, ab223052), CD63 antibody (1:1000, Abcam, ab68418), TSG101 Figure 1.

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Western Blot, Knockdown, Expressing, Control, CCK-8 Assay, Inhibition

Journal: International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group

Article Title: Hyperthermia promotes exosome secretion by regulating Rab7b while increasing drug sensitivity in adriamycin-resistant breast cancer.

doi: 10.1080/02656736.2022.2029585

Figure Lengend Snippet: Figure 5. Hyperthermia promotes the secretion of exosome in MCF-7/ADR cells. (A) The expression level of FOS and CREB5 in MCF-7/ADR exosomes after hyper- thermia. (B) Half-inhibition rate of adriamycin in MCF-7/ADR cells incubated with the exosomes which produced by MCF-7/ADR cells after hyperthermia. (C) The expression level of FOS and CREB5 in MCF-7/ADR after incubated with hyperthermia ADR/exo. (D) Particles concentration of exosomes derived from breast cancer cells analyzed by NTA. (E) Total protein amounts of exosomes derived from breast cancer cells analyzed by bicinchoninic acid (BCA) protein kit. (F) Transmission electron micrographs (TEM) of cells before or 6 h after hyperthermia. Intense extracellular vesicle shedding occurred 6 h after hyperthermia. Scale bar ¼1 lm. (G) Confocal microscope analysis of exosome uptake by MCF-7/ADR cells with or without hyperthermia treatment. Scale bar ¼20 lm. Error bars represent the mean ± SD of at least three independent experiments, p < .05, p < .01, p < .001.

Article Snippet: The membrane was then probed with primary antibodies at 4 C overnight and with species-specific secondary antibodies at room temperature for 1 h. The primary antibodies, including FOS antibody (1:1000, Proteintech, 66590-1-Ig), NF-jB antibody (1:1000, Proteintech, 14220-1-AP), MAPK8 antibody (1:1000, Proteintech, 66210-1-Ig), CREB5 antibody (1:1000, Proteintech, 14196-1-AP), GAPDH antibody (1:5000, Proteintech, 60004-1-Ig), CD9 antibody (1:1000, Abcam, ab223052), CD63 antibody (1:1000, Abcam, ab68418), TSG101 Figure 1.

Techniques: Expressing, Inhibition, Incubation, Produced, Concentration Assay, Derivative Assay, Transmission Assay, Microscopy

Journal: Cell Death & Disease

Article Title: Airway epithelial cGAS inhibits LPS-induced acute lung injury through CREB signaling

doi: 10.1038/s41419-023-06364-0

Figure Lengend Snippet: A The heat map of the differentially expressed genes (DEGs) (top 20 up-regulated and downregulate genes) in control- or cGAS-siRNA HBE cells through RNA sequencing analysis. Blue indicates a relatively low expression and red indicates a relatively high expression. B The volcano plot of differentially expressed genes (DEGs) in control- or cGAS-siRNA HBE cells through RNA sequencing analysis. C RT-qPCR of CREB in HBE cells transfected with cGAS siRNA or control siRNA for 24 h and incubated with 100 μg/ml LPS for an additional 24 h ( n = 3). Representative immunofluorescence images ( D ) and quantification of staining ( E ) of P-CREB (red) and nuclei (DAPI, blue) in HBE cells transfected with cGAS siRNA or control siRNA for 24 h and incubated with 100 μg/ml LPS for an additional 24 h. Scale bars, 10 μm. F HBE cells were transfected with cGAS siRNA or control siRNA for 24 h and were treated with LPS (100 μg/ml) for 24 h, and the cell lysates were collected for immunoblotting. G Representative lung immunofluorescence images of P-CREB (red), nuclei (DAPI, blue), and airway epithelial cells (marked with CC10, green) in mice. Scale bars, 100 μm. Two-way ANOVA with Sidaks multiple comparisons ( C , E ), *** p < 0.001, **** p < 0.0001. The data presented are one representative experiment of three independent experiments and shown as mean ± SEM.

Article Snippet: SiRNA of CREB (SR306377) was purchased from Origene (USA).

Techniques: Control, RNA Sequencing, Expressing, Quantitative RT-PCR, Transfection, Incubation, Immunofluorescence, Staining, Western Blot

Journal: Cell Death & Disease

Article Title: Airway epithelial cGAS inhibits LPS-induced acute lung injury through CREB signaling

doi: 10.1038/s41419-023-06364-0

Figure Lengend Snippet: A – D HBE cells transfected with cGAS-, CREB- or control-siRNA were incubated with 100 μg/ml LPS for 24 h. RT-qPCR ( A , B ) and ELISA ( C , D ) analysis of IL-6 and IL-8 of HBE cells and culture supernatants were performed. The data presented are one representative experiment of three independent experiments and shown as mean ± SEM and statistical analyses were calculated using two-way ANOVA with Sidaks multiple comparisons. E , F CC10-rtTA/(tetO)7-Cre/cGAS flox/flox mice (KO) and their littermate controls (WT) were fed with doxycycline in their drinking water (2 mg/ml) for 20 days. The mice were pretreated with 10 mg/kg 666-15(CREB inhibitor) or DMSO for 2 h and then intratracheally challenged with LPS (5 mg/kg) for 24 h. E The count of the inflammatory cells in BALF from mice. F RT-qPCR of IL-6, IL-1β, TNF-α, CXCL1, and CXCL2 in lung tissues from mice. In E and F , each symbol represents an individual mouse ( n = 4~7). The data shown as mean ± SEM and statistical analyses were calculated using one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: SiRNA of CREB (SR306377) was purchased from Origene (USA).

Techniques: Transfection, Control, Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: A trio of tumor suppressor miRNA downregulates CREB5 dependent transcription to modulate neoadjuvant hormonal therapy sensitivity

doi: 10.1016/j.neo.2022.100875

Figure Lengend Snippet: CREB5 is the direct target of miR-l42-3p, miR-150-5p and miR-342-3p. (A-D) The mRNA level of AR and PSA measured in LNCaP-ENZR and 22RV1 cells after transfection with indicated miRNAs mimics/inhibitor (RT-qPCR). (E) Heatmap displaying the relation of enrichment score of AR related signatures with miR-l42-3p, miR-150-5p and miR-342-3p expression according to TCGA data. The color represents the correlation coefficient and P-value are reported. The AR related signatures were downloaded from MsigDB of GSEA ( http://www.gsea-msigdb.org/gsea/msigdb/index.jsp ). Enrichment score were quantified by ssGSEA in R package GSVA. (F) A Venn diagram depicted sixteen genes that commonly targeted by indicated miRNAs, which based on four prediction algorithms (MiRWalk,miranda,RNA22 and Targetscan). (G) Heatmap displaying the expression of miRNAs target genes in Tewari cohort. (H-I) The protein level of CREB5 assessed in LNCaP cells by Western blot after transfection with indicated miRNAs mimics/inhibitor and relative control RNA. (J-K) The recognition sites for indicated miRNAs in CREB5 3′UTR region. (L-N) Luciferase reporter assay performed in HEK-293T cells. Cells were transiently co-transfected with the wild or mutated CREB5 3′UTR region together with corresponding miRNA mimics, respectively. After incubation for 48h, luciferase activities were measured. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Briefly, sections were incubated overnight with primary antibody against CREB5 (Abnova, 8A5, USA.

Techniques: Transfection, Quantitative RT-PCR, Expressing, Western Blot, Control, Luciferase, Reporter Assay, Incubation

Journal: Neoplasia (New York, N.Y.)

Article Title: A trio of tumor suppressor miRNA downregulates CREB5 dependent transcription to modulate neoadjuvant hormonal therapy sensitivity

doi: 10.1016/j.neo.2022.100875

Figure Lengend Snippet: CREB5 promotes proliferation and antiandrogen resistance in vitro . (A-D) Cell proliferation measured in LNCaP and VCaP cells. MTS assays (A-B) and EdU (C-D) assays were performed in LNCaP cells with CREB5 overexpression and in VCaP cells with CREB5 knockdown. Quantitative results of EdU assays are shown in the right panel. (E) Cell viability measured in the LNCaP cells under bicalutamide treatment. LNCaP cells were transfect with CREB5 plasmids/negative control and treated with titrated doses of bicalutamide for 48h. Cell viability was quantified by MTS assays. Data shown are means ± SD. (F) Cell proliferation determined with bicalutamide treatment. LNCaP cells were transiently transfected with CREB5 overexpression. Following treatment with 44 μM bicalutamide, the cells were subjected to MTS assays. (G-J) Relative proliferation rate of LNCaP-ENZR (G-H) and 22RV1 cells (I-J). Cells were transfected as indicated, then subjected to MTS assays treated with bicalutamide or enzalutamide. Cells were cultured in 10% CS-FBS. The absorbance at 490nm was measured after 48h treatment with bicalutamide or enzalutamide. (K-L) The mRNA levels of AR(K) and PSA(L) assessed by RT-qPCR after transfection as indicated in 22RV1 cells. * P < 0.05, ** P < 0.01, *** P < 0.001. All data represent means ± SD of at least three independent replicates.

Article Snippet: Briefly, sections were incubated overnight with primary antibody against CREB5 (Abnova, 8A5, USA.

Techniques: In Vitro, Over Expression, Knockdown, Negative Control, Transfection, Cell Culture, Quantitative RT-PCR

Journal: Neoplasia (New York, N.Y.)

Article Title: A trio of tumor suppressor miRNA downregulates CREB5 dependent transcription to modulate neoadjuvant hormonal therapy sensitivity

doi: 10.1016/j.neo.2022.100875

Figure Lengend Snippet: IL6 signaling is involved in CREB5-mediated antiandrogen resistance. (A-B) Gene sets significantly enriched in high-CREB5 tumors accordingly to the original annotation in the MsigDB Database Hallmark ( http://software.broadinstitute.org/gsea/msigdb/index.jsp ). Normalized enrichment score (NES) and P-value (P) are reported. (C-D) Correlation between the mRNA levels of CREB5 and IL6 in the GSE141551 (C) and TCGA data (D). Spearman r score and P value are shown. (E) The mRNA levels of IL6 assessed by RT-qPCR after transfection with CREB5 plasmids in LNCaP cells. (F-I) The mRNA levels of IL6 assessed by RT-qPCR after transfection with miRNAs mimics and inhibitors in 22RV1 (F-G) and LNCaP-ENZR (H-I) cells. * P < 0.05, ** P < 0.01.

Article Snippet: Briefly, sections were incubated overnight with primary antibody against CREB5 (Abnova, 8A5, USA.

Techniques: Software, Quantitative RT-PCR, Transfection

Journal: Neoplasia (New York, N.Y.)

Article Title: A trio of tumor suppressor miRNA downregulates CREB5 dependent transcription to modulate neoadjuvant hormonal therapy sensitivity

doi: 10.1016/j.neo.2022.100875

Figure Lengend Snippet: Clinicopathological analysis for CREB5 by immunohistochemistry in 85 PCa patients.

Article Snippet: Briefly, sections were incubated overnight with primary antibody against CREB5 (Abnova, 8A5, USA.

Techniques: Immunohistochemistry, Expressing, Significance Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: A trio of tumor suppressor miRNA downregulates CREB5 dependent transcription to modulate neoadjuvant hormonal therapy sensitivity

doi: 10.1016/j.neo.2022.100875

Figure Lengend Snippet: CREB5 is highly expressed in NHT-R PCa tissues. (A-D) CREB5 expression measured by IHC assay in Qilu cohort. Representative IHC images of CREB5 expression in PCa biopsy tissues with NHT-S (A1) or NHT-R (A2) in Qilu cohort. Quantitative analysis of cases with CREB5 positive was shown in A3. ROC curve was constructed to validate the sensitivity and specificity of CREB5 in predicting NHT-R (B). Representative IHC images of CREB5 expression in cases with Gleason score <8 (C1), and Gleason score ≥8 (C2-C3). ROC curve was constructed to validate the sensitivity and specificity of CREB5 in predicting GS (D). (E-G) The relevance between CREB5 and the three miRNAs expression in Qilu cohort. (H-K) Analysis of CREB5 expression in TCGA dataset. (H) CREB5 levels in PCa subgroups with different Gleason scores; (I) CREB5 levels in PCa subgroups with different clinical stages; (J) CREB5 levels in PCa subgroups with different pathological stages; (K) CREB5 levels in PCa subgroups with lymph node metastasis. (L-M) Analysis of CREB5 expression in MSKCC dataset. (L)The expression of CREB5 in primary and metastatic PCa tissues compared with the matched normal prostate samples in MSKCC dataset. (M) The correlation between CREB5 expression and biochemical recurrence-free survival in MSKCC cohort (Kaplan–Meier survival, Log-rank test). (N) The correlation between CREB5 expression and biochemical recurrence-free survival in GSE116918 data (Kaplan–Meier survival, Log-rank test). TCGA The Cancer Genome Atlas. GS, Gleason score. cT, clinical stage. pT, pathological stage. LNM, lymph node metastasis. BCR, biochemical recurrence. Error bars represent means ± SD of three independent experiments. Original magnification of IHC images, × 200. * P < 0.05.

Article Snippet: Briefly, sections were incubated overnight with primary antibody against CREB5 (Abnova, 8A5, USA.

Techniques: Expressing, Construct

Journal: Oncology Letters

Article Title: CREB5 promotes tumor cell invasion and correlates with poor prognosis in epithelial ovarian cancer

doi: 10.3892/ol.2017.7234

Figure Lengend Snippet: cAMP response element-binding protein 5 (CREB5) is negatively associated with ovarian cancer prognosis. (A) CREB5 expression profiling from GSE41 2 datasets (n=46). (B) Overall survival of patients with low vs. high CREB5 expression in the GSE4122 dataset (n=580). (C) Relapse-free survival of patients with low vs. high CREB5 expression in the GSE4122 dataset (n=484).

Article Snippet: After blocking, the membranes were incubated with anti-CREB5 monoclonal antibodies (1:1,000; GTX44660; BD Biosciences, Franklin Lakes, NJ, USA) overnight at 4°C.

Techniques: Binding Assay, Expressing

Journal: Oncology Letters

Article Title: CREB5 promotes tumor cell invasion and correlates with poor prognosis in epithelial ovarian cancer

doi: 10.3892/ol.2017.7234

Figure Lengend Snippet: (A and B) qPCR analysis of CREB5 expression in (B) 10 ovarian cancer cell lines and (A) 10 ovarian cancer samples compared to human ovarian surface epithelial cells (HOSEpiC) and IOSE80. Transcript levels were normalized to GAPDH expression. Bars represent the mean ± standard deviation of three independent experiments. *P<0.05. (C) Western blot analysis of CREB5 protein expression in normal ovarian epithelial cells (HOSEpiC and IOSE80) and primary ovarian tumors from 10 patients with ovarian cancer (T1-T10). GAPDH was used as the loading control. (D) Western blot assay of CREB5 protein expression in normal ovarian epithelial cells and ovarian cancer cell lines. IOSE80 cells were used as the negative control.

Article Snippet: After blocking, the membranes were incubated with anti-CREB5 monoclonal antibodies (1:1,000; GTX44660; BD Biosciences, Franklin Lakes, NJ, USA) overnight at 4°C.

Techniques: Expressing, Standard Deviation, Western Blot, Negative Control

Journal: Oncology Letters

Article Title: CREB5 promotes tumor cell invasion and correlates with poor prognosis in epithelial ovarian cancer

doi: 10.3892/ol.2017.7234

Figure Lengend Snippet: (A) Immunohistochemical staining of CREB5 in human ovarian epithelial cancer tissues. *P<0.05. (B) Overall survival of patients with low vs. high expression of CREB5 in human ovarian surface epithelial cells (P<0.01). (C) Relapse-free survival (RFS) of patients with low vs. high CREB5 expression in ovarian cancer cells (P<0.01).

Article Snippet: After blocking, the membranes were incubated with anti-CREB5 monoclonal antibodies (1:1,000; GTX44660; BD Biosciences, Franklin Lakes, NJ, USA) overnight at 4°C.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Oncology Letters

Article Title: CREB5 promotes tumor cell invasion and correlates with poor prognosis in epithelial ovarian cancer

doi: 10.3892/ol.2017.7234

Figure Lengend Snippet: The relationship between CREB5 and clinical pathological characteristics in 125 patients with ovarian cancer.

Article Snippet: After blocking, the membranes were incubated with anti-CREB5 monoclonal antibodies (1:1,000; GTX44660; BD Biosciences, Franklin Lakes, NJ, USA) overnight at 4°C.

Techniques: Expressing

Journal: Oncology Letters

Article Title: CREB5 promotes tumor cell invasion and correlates with poor prognosis in epithelial ovarian cancer

doi: 10.3892/ol.2017.7234

Figure Lengend Snippet: Univariate and multivariate analysis of factors associated with overall survival in 125 ovarian cancer patients.

Article Snippet: After blocking, the membranes were incubated with anti-CREB5 monoclonal antibodies (1:1,000; GTX44660; BD Biosciences, Franklin Lakes, NJ, USA) overnight at 4°C.

Techniques: