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: Scientific reports

Article Title: Functional roles of pantothenic acid, riboflavin, thiamine, and choline in adipocyte browning in chemically induced human brown adipocytes.

doi: 10.1038/s41598-024-69364-w

Figure Lengend Snippet: Figure 3. Concentration-dependent effects of PA on UCP1 expression in ciBAs. (A) The expression of UCP1, CIDEA, and FABP4 was measured by qRT-PCR analysis in ciBAs treated with Ch at concentrations from 0.25 to 16 μg/mL throughout the experiments. (B) The expression was measured in ciBAs treated with PA at concentrations from 0.25 to 16 μg/mL. (C) UCP1 and β-Actin proteins were detected by immunoblotting analysis in ciBAs treated with PA at various concentrations. (D) Representative images of Lipi-Red staining (red), UCP1 expression (green), and DAPI (blue) in the ciBAs treated with PA at various concentrations throughout the experiments. The area of the staining for Lipi-Red and UCP1 was quantified by ImageJ software. (E–G) Glycerol secretion, triglyceride accumulation, and glycerol-3-phosphate dehydrogenase 1 (GPDH) activity were measured in ciBAs treated with PA at various concentrations. (H) The phosphorylation of CREB and HSL proteins was quantified by immunoblotting analysis in ciBAs treated with PA at 0.5 μg/mL and 16 μg/ mL throughout the experiments. The band intensities were quantified by densitometry using ImageJ software. (I) Cellular mitochondria contents were evaluated by MitoTracker staining in ciBAs treated with PA at various concentrations. Data represent mean ± SD (n = 3). One-way ANOVA with Tukey’s multiple comparison tests: * p < 0.05, ** p < 0.01, *** p < 0.001, N.S.; not significant.

Article Snippet: The membranes were blocked with 3% skim milk followed by incubation with antibodies against UCP1 (MAB6158, R&D Systems, MN, USA), ATGL (#2138, Cell Signaling Technology, MA, USA), CEBPA (#8178, Cell Signaling Technology), β-Actin (A5316, Sigma-Aldrich), P-CREB (#9198, Cell Signaling Technology), CREB (#9197, Cell Signaling Technology), P-HSL (#18381, Cell Signaling Technology), and HSL (#4107, Cell Signaling Technology) at 4 °C overnight.

Techniques: Concentration Assay, Expressing, Quantitative RT-PCR, Western Blot, Staining, Software, Activity Assay, Phospho-proteomics, Comparison

Journal: The Tohoku journal of experimental medicine

Article Title: Low-intensity aerobic exercise mitigates exercise-induced bronchoconstriction by improving the function of adrenal medullary chromaffin cells in asthmatic rats.

doi: 10.1620/tjem.234.99

Figure Lengend Snippet: Fig. 5. Low- or moderate-intensity aerobic exercise inhibits the ERK/CREB signaling and downstream gene expression in rat adrenal medulla. The relative levels of phosphorylated ERK and CREB were characterized by Western blot, and the relative levels of c-FOS, EGFR1, c-JUN, JUNB, and FOSB mRNAs to the control β-actin in the adrenal medullary tissues of individual rats were characterized by qRT-PCR. Data are representative images or expressed as the mean ± s.d. of individual groups of rats (n = 4 per group) from three separate experiments. (A) The relative levels of phosphorylated ERK and CREB. (B) The relative levels of c-FOS, EGFR1, c-JUN, JUNB, and FOSB mRNAs. Control: Control rats; Low: The rats with low-intensity aerobic exercise; Mod: The rats with moderate-intensity aerobic exercise; OVA: The rats were sensitized and challenged with OVA; OVA + Low: The rats were sensitized and challenged with OVA and subjected to low-intensity aerobic exercise; OVA + Mod: The rats were sensitized and challenged with OVA and subjected to moder- ate-intensity aerobic exercise. *P < 0.05 vs. the control group; ▲P < 0.05 vs. the OVA group; #P < 0.05 vs. the OVA + Low group.

Article Snippet: The animals were sacrificed and studied on day 56. incubated with anti-peripherin (1:20,000), anti-neurofilament-68 (NF68, 1:5,000), anti-chromogranin A (CgA, 1:10,000), anti-PNMT (1:1,000), anti-ERK1/2 (extracellular signal-regulated kinase, 1:1,000), anti-phosphorylated ERK1/2 (anti-p-ERK1/2, 1:500, Abcam, Cambridge, UK), anti-CREB (cAMP responsive element binding protein, 1:1,000), anti-phosphorylated CREB (anti-p-CREB, 1:1,000), or anti-β-actin (1:1,000, Cell Signaling Technology, Beverly, USA) overnight at 4°C.

Techniques: Gene Expression, Western Blot, Control, Quantitative RT-PCR

Journal: Oncology reports

Article Title: The pancreatic cancer secreted REG4 promotes macrophage polarization to M2 through EGFR/AKT/CREB pathway.

doi: 10.3892/or.2015.4357

Figure Lengend Snippet: Figure 3. Involvement of EGFR/AKT/CREB pathway in REG4-mediated macrophage polarization to M2. (A) Representative western blots of phosphorylated EGFR, AKT and CREB in macrophage cultures treated with REG4 (10 nM) for different time intervals indicated. (B) Quantitative analysis of the immunoblots of p-EGFR, p-AKT and p-CREB normalized with EGFR, AKT and CREB, respectively. Data are mean ± SEM from four independent experiments. *p<0.05, **P<0.01 compared to untreated cells. (C) Representative western blotting images showing the phosphorylation of EGFR, AKT and CREB in macrophages cultures treated with or without REG4 (10 nM) for 30 min in the absence or presence of LY294002. (D) Quantitative analysis of the immunoblots of p-EGFR, p-AKT and p-CREB normalized with EGFR, AKT and CREB, respectively, in macrophages cultures treated with or without REG4 (10 nM) for 30 min in the absence or presence of LY294002. Data are mean ± SEM from four independent experiments. *p<0.05 compared to untreated cells; #P<0.05 compared with REG4 treatment alone. (E) Representative western blots of CD163, CD68, and CREB in REG4-treated macrophages which were infected with control shRNA (shCON) or shCREB lentiviral particles. (F) Quantitative analysis of the immunoblots of CD163, CD68 and CREB in REG4-treated macrophages which were infected with control shRNA (shCON) or shCREB lentiviral particles. Data are mean ± SEM from three independent donors. *p<0.05 compared with control.

Article Snippet: The lentiviral CREB shRNA, targeting human CREB (NM_004379) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, uSA).

Techniques: Western Blot, Phospho-proteomics, Infection, Control, shRNA

Journal: Oncology reports

Article Title: The pancreatic cancer secreted REG4 promotes macrophage polarization to M2 through EGFR/AKT/CREB pathway.

doi: 10.3892/or.2015.4357

Figure Lengend Snippet: Figure 4. The conditioned medium of Panc1 cells induces macrophage polarization to M2. (A) Western blots of REG4 in the culture medium or cell lysate of Panc1, AsPC1 and BxPC3 cells. Shown are representatives of three independent experiments with similar results. (B) Representative western blots of CD206 and CD163 in macrophage cultures treated with or without the conditioned medium of Panc1, AsPC1 and BxPC3 cell cultures, respectively. (C) Quantitative analysis of the immunoblots of CD163 and CD206 in macrophage cultures treated with or without the conditioned medium of Panc1 (CMPanc1), AsPC1 (CMAsPC1) and BxPC3 (CMBxPC3) cell cultures, respectively. Data are shown by mean ± SEM from four independent experiments. *p<0.05 compared to untreated cells; #P<0.05 compared with CMPanc1 treatment. (D) Western blots of REG4 in the culture medium and cell lysate of Panc1 cells infected with or without lentiviral REG4 shRNA. Shown are representatives of three independent experiments with similar results. (E) Representative western blots of CD206 and CD163 in macrophage cultures treated with the conditioned medium Panc1 cells infected with lentiviral control shRNA (CMPanc1/shCon) or REG4 shRNA (CMPanc1/shREG4). (F) Quantitative analysis of the immunoblots of CD163 and CD206 in macrophage cultures treated with CMPanc1/shCon or CMPanc1/shREG4. *p<0.05 compared with control.

Article Snippet: The lentiviral CREB shRNA, targeting human CREB (NM_004379) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, uSA).

Techniques: Western Blot, Infection, shRNA, Control