Journal: iScience

Article Title: BMAL1 upregulates STX17 levels to promote autophagosome-lysosome fusion in hippocampal neurons to ameliorate Alzheimer's disease

doi: 10.1016/j.isci.2024.111413

Figure Lengend Snippet: Figure 5. The AD model showed circadian rhythm disturbance and decreased Bmal1 expression (A) The protocol of synchronize in vivo and in vitro. (B) APP/PS1 mice displayed circadian rhythm disorders, increased daytime activity, and prolonged free-running cycles.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-LC3B antibody Abcam Cat #ab192890 BMAL1 (D2L7G) Rabbit mAb Cell Signaling Technology Cat #14020S STX17 Polyclonal antibody Proteintech Cat #17815-1-AP VAMP8 Polyclonal antibody Proteintech Cat #15546-1-AP SNAP29 Polyclonal antibody Proteintech Cat #12704-1-AP Anti-SQSTM1 / p62 antibody Abcam Cat #ab109012 Anti-Amyloid Precursor Protein antibody Abcam Cat #ab32136 Bacterial and virus strains Lentivirus-overexpressing STX17 Shanghai Genechem N/A Lentivirus-overexpressing BMAL1 Shanghai Genechem N/A STX17-siRNA Hanbio Biotechnology N/A Chemicals, peptides, and recombinant proteins RNAiso Plus TaKaRa Cat #9109 RIPA buffer Boster Cat #AR0102 Glutaraldehyde,2.5%(EM Grade) Solarbio Cat #P1126 Critical commercial assays PrimeScript RT Master Mix TaKaRa Cat #RR036A SYBR Premix Ex TaqTMII TaKaRa Cat #DRR041A Experimental models: Cell lines HT22 Chinese Academy of Sciences Cell Bank CSTR:19375.09.3101MOUGNM47 Experimental models: Organisms/strains SPF mice Model Animal Research Center APP/PS1 transgenic mice Software and algorithms ImageJ ImageJ https://imagej.nih.gov/ij/ GraphPad Prism GraphPad Prism https://www.graphpad.com/ Other Code of GSE21779 GEO https://www.ncbi.nlm.nih.gov/gds/ Code for data analysis and visualization bioinformatics https://www.bioinformatics.com.cn Code for revealed the binding sites JASPAR http://jaspar.genereg.net/ Data: Western Blot and Microscopy data Lead contact 163.wangxh@163.com

Techniques: Expressing, In Vivo, In Vitro, Activity Assay

Journal: iScience

Article Title: BMAL1 upregulates STX17 levels to promote autophagosome-lysosome fusion in hippocampal neurons to ameliorate Alzheimer's disease

doi: 10.1016/j.isci.2024.111413

Figure Lengend Snippet: Figure 6. BMAL1 regulates STX17 to affect autophagy and amyloid deposition (A) JASPAR analysis revealed the recognition sites of BMAL1 on the promoter sequence of STX17. (B) Detection of luciferase activity after the STX17 promoter sequence plasmid and BMAL1 plasmid were transfected into HT22 cells. (C) Autophagic flow was partially restored in APP-overexpressed HT22 after BMAL1 overexpression. (D) Amyloid deposition decreases after BMAL1 overexpression. n = 6; scale bar, 50 mm. *p < 0.05 vs. the Lv-OE-APP+Lv-NC group; **p < 0.01 vs. the Lv-OE- APP+Lv-NC group. Data are represented as mean ± SD.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-LC3B antibody Abcam Cat #ab192890 BMAL1 (D2L7G) Rabbit mAb Cell Signaling Technology Cat #14020S STX17 Polyclonal antibody Proteintech Cat #17815-1-AP VAMP8 Polyclonal antibody Proteintech Cat #15546-1-AP SNAP29 Polyclonal antibody Proteintech Cat #12704-1-AP Anti-SQSTM1 / p62 antibody Abcam Cat #ab109012 Anti-Amyloid Precursor Protein antibody Abcam Cat #ab32136 Bacterial and virus strains Lentivirus-overexpressing STX17 Shanghai Genechem N/A Lentivirus-overexpressing BMAL1 Shanghai Genechem N/A STX17-siRNA Hanbio Biotechnology N/A Chemicals, peptides, and recombinant proteins RNAiso Plus TaKaRa Cat #9109 RIPA buffer Boster Cat #AR0102 Glutaraldehyde,2.5%(EM Grade) Solarbio Cat #P1126 Critical commercial assays PrimeScript RT Master Mix TaKaRa Cat #RR036A SYBR Premix Ex TaqTMII TaKaRa Cat #DRR041A Experimental models: Cell lines HT22 Chinese Academy of Sciences Cell Bank CSTR:19375.09.3101MOUGNM47 Experimental models: Organisms/strains SPF mice Model Animal Research Center APP/PS1 transgenic mice Software and algorithms ImageJ ImageJ https://imagej.nih.gov/ij/ GraphPad Prism GraphPad Prism https://www.graphpad.com/ Other Code of GSE21779 GEO https://www.ncbi.nlm.nih.gov/gds/ Code for data analysis and visualization bioinformatics https://www.bioinformatics.com.cn Code for revealed the binding sites JASPAR http://jaspar.genereg.net/ Data: Western Blot and Microscopy data Lead contact 163.wangxh@163.com

Techniques: Sequencing, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Over Expression

Journal: Journal of Functional Foods

Article Title: Egg white ovomucin hydrolysate inhibits intestinal integrity damage in LPS-treated Caco-2 cells

doi: 10.1016/j.jff.2021.104822

Figure Lengend Snippet: Fig. 4. Abundance changes of tight junction proteins in differentiated Caco-2 cells treated with ovomucin-protex 26L hydrolysate (1.0 mg/mL) for 24 h and LPS (20 μg/mL) for 2 h. Whole cell lysates were used for Western blot analysis of claudin-2 (A), claudin-3 (B), occludin (C), and ZO-1 (D), respectively. Band densities were quantified using Image Studio software (LI-COR Biosciences). The data are expressed as mean ± SEM of 4 independent experiments. Con means control group. *p < .05, **p < .01, significantly different from control group. #p < .05, significantly different from the LPS group.

Article Snippet: Rabbit monoclonal primary antibodies against claudin-2, claudin-3, occludin, ZO-1, phospho-NF-κB p65 (Ser536), NF-κB p65, phospho-p44/42 MAPK (ERK1/2) (Thr202/Try204), and p44/42 MAPK (ERK1/2) were obtained from Cell Signaling Technology (Whitby, ON, Canada).

Techniques: Western Blot, Software, Control

Journal: Journal of Functional Foods

Article Title: Egg white ovomucin hydrolysate inhibits intestinal integrity damage in LPS-treated Caco-2 cells

doi: 10.1016/j.jff.2021.104822

Figure Lengend Snippet: Fig. 5. Cellular distribution of tight junction proteins. Caco-2 cells were cultured on the coverslips for 21 d to undergo differentiation and treated with ovomucin- protex 26L hydrolysate for 24 h followed by LPS (20 μg/mL) for 2 h. After treatment, cells were fixed and stained with primary anti-claudin-2/3, anti-occludin, and anti-ZO-1 antibodies at 4 ℃ overnight. Anti-rabbit CFTM 488A antibody (Sigma) was used to visualize the localization of tight junction proteins by a CSU10 spinning disk confocal microscope (Quorum Technologies, Canada). Con means control group.

Article Snippet: Rabbit monoclonal primary antibodies against claudin-2, claudin-3, occludin, ZO-1, phospho-NF-κB p65 (Ser536), NF-κB p65, phospho-p44/42 MAPK (ERK1/2) (Thr202/Try204), and p44/42 MAPK (ERK1/2) were obtained from Cell Signaling Technology (Whitby, ON, Canada).

Techniques: Cell Culture, Staining, Microscopy, Control

Journal: Journal of Functional Foods

Article Title: The dermato-protective effects of lucidone from Lindera erythrocarpa through the induction of Nrf2-mediated antioxidant genes in UVA-irradiated human skin keratinocytes

doi: 10.1016/j.jff.2014.10.019

Figure Lengend Snippet: Fig. 4 – Lucidone protects HaCaT cells from UVA-induced apoptosis. HaCaT cells were pre-treated with lucidone (1–4 µM) for 24 h and then irradiated or not with 15 J/cm2 UVA. (A) DNA fragmentation was determined by the TUNEL assay. The photographs show TUNEL-positive cells under fluorescence microscopy (magnification ×200) from three separate samples. Each photograph is representative of one of the three independent assays. (B) Immunoblotting was performed to monitor the levels of the apoptotic regulatory proteins Bcl-2 and Bax. An equal amount (60 µg) of total lysate from each sample was resolved by SDS-PAGE (8–15% polyacrylamide gel) with β-actin as a control. The relative changes in the intensity of the protein bands were measured by commercially available quantitative software as described in the Materials and Methods. (C) Lucidone alleviates UVA-induced mitochondrial dysfunction. The mitochondrial membrane potential was determined by the uptake of MitoTracker Green (CMXRos) by the mitochondria. HaCaT cells were pretreated with lucidone (4 µM) for 24 h, and then exposed to UVA (15 J/cm2). After UVA exposure, cells were incubated with MitoTracker (1 µM) for 30 min at 37 °C. The cell nucleus was stained with DAPI (1 µg/mL) for 5 min and examined by fluorescence microscopy (magnification × 200) as described in the Materials and Methods. The results are the mean ± SD of three assays. **p < 0.01; ***p < 0.001 compared with untreated control cells. #p < 0.05; ##p < 0.01; compared with UVA-irradiated cells.

Article Snippet: Antibodies against Bax, Bcl-2, Nrf2, Keap-1, NQO-1, and β-actin were obtained from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Irradiation, TUNEL Assay, Microscopy, Western Blot, SDS Page, Control, Software, Membrane, Incubation, Staining

Journal: Journal of Functional Foods

Article Title: The dermato-protective effects of lucidone from Lindera erythrocarpa through the induction of Nrf2-mediated antioxidant genes in UVA-irradiated human skin keratinocytes

doi: 10.1016/j.jff.2014.10.019

Figure Lengend Snippet: Fig. 5 – Lucidone up-regulates the antioxidant markers Nrf2, HO-1, NQO-1, and γ-GCLC, and it down-regulates their endogenous inhibitor Keap-1 in UVA-irradiated HaCaT cells. Cells were pre-treated with lucidone (1–4 µM for 24 h) and then irradiated with 15 J/cm2 UVA. (A, B) Western blot analysis results show the effects of lucidone on the total protein levels of Nrf2, Keap-1, HO-1, NQO-1, and γ-GCLC in whole cells (A) and on the protein level of Nrf2 in the cytosolic and nuclear fractions (B). An equal amount (50 µg) of total lysate from each sample was resolved by 8–15% SDS-PAGE with β-actin as a control. The relative changes in the intensities of the protein bands were measured by commercially available quantitative software. (C) Immunofluorescence staining shows the changes of Nrf2 in lucidone-treated HaCaT cells. Cells were stained with DAPI (1 µg/mL) for 5 min and examined by fluorescence microscopy (magnification ×200). (D) Lucidone stimulates Nrf2- mediated ARE activity. Cells were co-transfected with luciferase reporters and pGL3-ARE; then, the luciferase activity was determined and normalized to β-gal activity and is shown as relative luciferase activity. (E) The amount of intracellular GSH was measured using a commercially available ELISA kit, as described in the Materials and Methods. The results are the mean ± SD of three assays. *p < 0.05; **p < 0.01; compared with untreated control cells. #p < 0.05; ##p < 0.01; compared with UVA-irradiated cells.

Article Snippet: Antibodies against Bax, Bcl-2, Nrf2, Keap-1, NQO-1, and β-actin were obtained from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Irradiation, Western Blot, SDS Page, Control, Software, Staining, Microscopy, Activity Assay, Transfection, Luciferase, Enzyme-linked Immunosorbent Assay