Journal: Foods

Article Title: Lipid-Lowering Effects of Inonotus obliquus Polysaccharide In Vivo and In Vitro

doi: 10.3390/foods10123085

Figure Lengend Snippet: Preliminary characterization of IOP. ( a ) UV–Vis analysis of IOP. ( b ) Molecular weight of IOP. ( c ) The standard monosaccharide. ( d ) The monosaccharide composition of IOP. 1. D—mannose, 2. D—ribose, 3. L—rhamnose, 4. D—glucuronic acid, 6. D—galacturonic acid, 7. D—glucose, 8. D—galactose, 10. D—xylose, 11. L—Arabinose, 12. L—fucose. ( e ) FTIR analysis of IOP. ( f ) The SEM images of IOP observed at magnifications of 500×, 1000×, and 1500×.

Article Snippet: At 15 V, the morphologies of IOP under 500×, 1000×, and 1500× magnification were observed using SEM (TM-3000 scanning electron microscope, Hitachi, Ltd., Tokyo, Japan).

Techniques: Molecular Weight

Journal: International Journal of Molecular Sciences

Article Title: Exosomal CD40, CD25, and Serum CA19-9 as Combinatory Novel Liquid Biopsy Biomarker for the Diagnosis and Prognosis of Patients with Pancreatic Ductal Adenocarcinoma

doi: 10.3390/ijms26041500

Figure Lengend Snippet: Experimental overview of exosome enrichment and characterization. ( A ) Schematic representation of exosome isolation from peripheral blood and MACSPlex exosome assay. ( B ) Transmission Electron Microscopy (TEM) images showing enriched plasma exosomes and exosome marker CD9 from two PDAC patients. ( C , D ) Measurement of nanoparticle concentration (N/mL plasma) by the nanoparticle tracking analysis (NTA) in controls (n = 36), pancreatitis (n = 16), and PDAC groups (n = 32). ( E ) Exosomal protein concentration, measured by the BCA assay, in controls (n = 43), pancreatitis (n = 20), and PDAC groups (n = 35). ( F ) Measurement of protein concentration by Western blot. Data are mean ± s.d.

Article Snippet: Proteins were separated by SDS-PAGE (SDS polyacrylamide gel electrophoresis), transferred to nitrocellulose membranes, blocked in 5% non-fat milk in PBS with 0.5% Tween-20, and immunostained overnight at 4 °C using the following primary antibodies in a 1/1000 dilution in TBST: CD9 (BD555370), CD63 (BD556019), CD40 (Cat. #66965-1-Ig, Proteintech, Rosemont, IL 60018, USA).

Techniques: Isolation, Transmission Assay, Electron Microscopy, Clinical Proteomics, Marker, Concentration Assay, Protein Concentration, BIA-KA, Western Blot

Journal: NPJ Science of Learning

Article Title: Training on multiple days results in better learning in embedded eyeblink conditioning in young human adults

doi: 10.1038/s41539-025-00347-w

Figure Lengend Snippet: a A CS-US paired trial using an embedded protocol. b 3-day acquisition protocol. c The experimental setup consists of custom-made glasses, attached with magnetic sensor and airpuff nozzle. The magnet is glued to the participant’s left eyelid. The CS, US, and GMR chip readings are recorded and controlled via Spike2 v9.10 and CED Micro1401 AD converter. Created in BioRender.com .

Article Snippet: The GMR sensor data was sampled at 1000 Hz and transferred to a computer via a Micro1401 AD converter (Cambridge Electronic Design).

Techniques:

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: P. gingivalis (P. g) Infection Causes a Significant Increase in the Protein HSp27, Accompanied by Large Spatial Accumulation of Hsp27 with the Bacteria in a Temporal Manner in Primary GECs. ( A ) Representative confocal microscopy images of P. g -infected human primary GECs at an MOI 100, at 6 h and 24 h after infection. GECs were then stained for P. g (rabbit anti-P. g; Alexa 488; green) or HSp27 (mouse anti-HSp27; Alexa 568; red). GECs were then imaged via the Leica DM6 CS Stellaris 5 Confocal/Multiphoton System at 63x. ( Ai ) Imaris Software was used to create a zoomed image of infected GECs and was used to calculate the amount of co-localization between P. g and HSp27. HSp27 was found to readily colocalize with P. g , having an average Pearson correlation coefficient of 0.87 via the Imaris software. Scale bar is 30 µm for 63x and Zoomed Magnification. (B ) P. g was added at MOI 100 to GECs, which were incubated 6 or 12h. Cell lysates were then analyzed via western blot. ( Bi ) Quantitative ImageJ analyses of western blot results. Data is represented as Mean±SD, where n=3 and p<0.05 was considered as significant via Two-Tailed Student T-test. *p<0.05.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Infection, Bacteria, Confocal Microscopy, Staining, Software, Incubation, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: The Integrity of P. gingivalis (P. g) -Specific Autophagosomes is Highly Dependent on HSp27 Presence. Human primary GECs were treated with HSP27siRNA (100nM) for 48 h prior to incubation with P. g ( MOI 100) for 6 h. Autophagosomes were then isolated and analyzed via Confocal Microscopy. ( A ) Schematic autophagosomal isolation method of infected GECs. ( B ) Confocal microscopy images of autophagosomes (ThiolTracker Violet; blue) were obtained via Super Resolution Zeiss Airyscan LSM 880 at 20x. ( C ) Autophagosomes were stained for P. g (rabbit anti- P. g ; Alexa 488; green) and reduced GSH (ThiolTracker Violet; blue). Confocal microscopy images of autophagosomes were obtained via Super Resolution Zeiss Airyscan LSM 880 at 20x. ( Ci ) Quantitative ImageJ analysis of Confocal microscopy results was then performed. Data is represented as Mean±SD, where n=25 and p<0.05 was considered as statistically significant (Student two-tailed T-test). **p<.005

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Incubation, Isolation, Confocal Microscopy, Infection, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: The Magnetic-Labelling Process Does Not Impact P. gingivalis (P. g) Infection in GECs. P. g was labeled with lipobiotin (5 μM) and were then incubated in MagCellect Streptavidin Ferrofluid. Human Primary GECs were then infected for 24 h. Representative confocal microscopy images of P. g- infected GECs at an MOI 100, were taken at 24 h after infection using via Zeiss LSM 880 (63x). P. g (rabbit anti-P . gingivalis ; Alexa 488; green) was detected in the GECs. Actin (red) was stained utilizing Rho-Phallodin.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Infection, Labeling, Incubation, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Intracellular P. gingivalis ( P. g ) Significantly Induces and Co-Localizes with LC3C, an Isomer of LC3, and this Specific Event is Highly Dependent on HSp27 for Successful Autophagic Survival. Human primary GECs were treated with HSp27 siRNA (100nM) for 48 h. P. g was added at MOI 100 to GECs, which were incubated for 6 or 24 h. ( A ) GECs were targeted for P. g (rabbit anti- P. g ; goat anti-rabbit Ultra Small Gold Antibody) and labeled P. g was found to be readily ensconced within double-membraned autophagosomes in GECs. Following HSp27 depletion, P. g appeared to readily start to degrade. Representative transmission electron microscopy images of P. g -infected GECs were also taken at 80 kV and 100000x magnification. Scale bar is 800 nm. ( B ) 6 h and 24h P. g-infected GECs were also stained for P. g (rabbit anti-P . g ; Alexa 488; green) and LC3C (mouse anti-LC3C; Alexa 568; red) to examine whether LC3C characterizes P. g -specific autophagosomes. These cells were then imaged via confocal microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 63x. The range of z-stacks was kept consistent and representative images were selected from the mid-ranged sections. ( Bi ) The Imaris software was utilized to obtain a xoomed 63x Orthogonal Image of 24 h P. g infection and found heightened co-localization between P. g and LC3C. LC3C was found to readily colocalize with P. g, having an average Pearson correlation coefficient of 0.96 via the Imaris post-processing software. ( C ) Lysates of infected and HSp27-depleted GECs were also analyzed via western blotting. Non-target controls were performed and not shown. (Ci) Quantitative ImageJ analysis was performed for the western blot results. Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via Student two-tailed T-test. **p<.005.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Incubation, Labeling, Transmission Assay, Electron Microscopy, Infection, Staining, Confocal Microscopy, Software, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 Presence Permits the Prolonged Existence of LC3C-characterized, P. gingivalis Specific Autophagosomes by Hampering Canonical Autolysosomal Fusion in Primary GECs. ( A ) Human primary GECs were transfected with mCherry-eGFP-LC3C for 48 h. Select GECs were also treated with 1 uM of the autophagolysosomal fusion inhibitor Bafilomycin A1, 1 uM Pepstatin A, or 5 mM 3-MA. Others were treated with Hsp27 siRNA (100nM) for 24 h. P. g was added at MOI 100 to GECs, which were incubated for 24 h. ( A ) GECs were then stained for P. g (mouse anti- P.g; Alexa 405; blue) and were mounted. GECs were then imaged via confocal microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 63x. ( Ai ) Imaris was used to obtain a zoomed 63x orthogonal image of 24 h P. g infection and measure the high co-localization levels between P. g and the LC3C Reporter System. P. g localized readily to the LC3C construct, with a Pearson correlation coefficient of 0.82. (B) Separately, GECs were additionally stained for P. g (rabbit anti-P . gingivalis ; Alexa 488; green) and LAMP-1 (mouse anti-LAMP-1; Alexa 568; red) and were imaged. The range of all z-stacks was kept consistent and representative images were selected from the mid-ranged sections. Scale bar is 40 µm for 63x Magnification. ( Bi ) Imaris was used to obtain a zoomed 63x orthogonal image of 24 h P. g infection and measure the co-localization levels between P. g and LAMP-1 in infected and treated GECs. While P. g infected GECs did not exhibit high co-localization with LAMP-1 (Pearson correlation coefficient of .25), their HSp27-depleted counterparts did, with an average Pearson correlation coefficient of 0.83. The Scale bar is 20 µm for 63x Magnification. ( C ) Finally, GECs treated with autophagic inhibitors were targeted for P. g (rabbit anti- P. g ; goat anti-rabbit Ultra Small Gold Antibody) and labeled P. g was found to be readily ensconced within double-membraned autophagosomes in GECs. Representative transmission electron microscopy images of P. g -infected GECs were taken at 80 kV and 30000x or 100000x magnification. Following HSp27 depletion, P. g appeared to readily start to degrade, however treatment with late-stage autophagic inhibitors Bafilomycin A1 or Pepstatin A appeared to rescue P. g from degradation. Representative transmission electron microscopy images of P. g -infected GECs were taken at 80 kV and 30000x or 100000x magnification. Scale bar is 800 nm.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Transfection, Incubation, Staining, Confocal Microscopy, Infection, Construct, Labeling, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Depletion of LC3C via siRNA Collapses P. gingivalis (P. g) -Induced Non-Canonical Autophagosomal Integrity. Human primary GECs were treated with LC3C siRNA (100nM) for 48h. P. g was added at MOI 100 to GECs for 6, 12, or 24 h. ( A ) Intracellular P. g survival after LC3C siRNA depletion was determined using a standard antibiotic protection assay. In brief, any extracellular bacteria were killed via 1h gentamicin (300 μg/mL) and metronidazole (200 μg/mL) treatment. cDNAs were synthesized for qPCR using P. g -specific 16S rRNA primers to quantify intracellular levels of live P. g . Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via Student two-tailed T-test. *p<.05 **p<.005. ( B ) P. g -specific autophagosomes were also selectively isolated. Autophagosomes were stained for P. g (rabbit anti- P. g ; Alexa 488; green) and reduced GSH (ThiolTracker Violet; blue). Confocal images of P. g -specific autophagosomes at 6 h post-infection (63x) were taken utilizing the Super Resolution Zeiss Airyscan LSM 880.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Bacteria, Synthesized, Two Tailed Test, Isolation, Staining, Infection

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Δ ndk-P. gingivalis (P. g) Undergoes Canonical Degradative Autophagosomal Trafficking in Human Primary GECs. Primary GECs were infected with WT P. gingivalis (P. g) versus Δ ndk - P. g at MOI 100 for 3, 12, or 24h. ( A ) TEM analysis used Immunogold labeling for P. g was performed on WT P. g versus Δ ndk - P. g at 3 or 24h post-infection. Images were acquired at 40000x magnification utilizing a Hitachi H-7000 TEM (Hitachi High Technologies America, Inc.) affixed to a Veleta camera with iTEM. ( B ) Separately, GECs were additionally stained for P. g (rabbit anti-P . gingivalis ; Alexa 488; green) and LAMP-1 (mouse anti-LAMP-1; Alexa 568; red) and were imaged. The range of all z-stacks was kept consistent and representative images were selected from the mid-ranged sections. Co-localization analysis of P. g and LAMP-1 was additionally carried out using the Zeiss LSM 880 Confocal Software, determining that the WT P. g had a Pearsons value of .137 while the Δ ndk - P. g had a Pearsons value of .704.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Infection, Labeling, Staining, Software

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: The Depletion of HSp27 Abrogates the Inhibition of Oxidative Stress and Severely Impacts the Intracellular Survival of P. gingivalis (P. g) Studied in Human Primary Organotypic Cultures of Gingiva. To create the organotypic culture systems, human primary GECs and Fibroblasts Cells (FBCs) were co-cultured together upon a collagen raft. Select rafts were then treated with HSP27 siRNA (100nM) for 48 h. Select rafts were also treated with N-acetyl Cysteine (NAC) (50 uM) for 1 h. P. g was added at MOI 100 to rafts, which were incubated for 24 h. Rafts were then collected and sectioned so that immunofluorescence could be performed. ( A ) Representative images of H&E stained raft culture systems at 20x magnification, which clearly mimic the oral gingival crevice. Scale bar is 50 µm. E: Multilayer Undifferentiated Epithelial Cells, C: Collagen Matrix; F: Fibroblasts. ( B ) Rafts were stained for P. g (rabbit anti- P. g; Alexa 488; green) or HpS27 (mouse anti-HSp27; Alexa 568; red). Rafts were then imaged via confocal microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 20x and 63x. Scale bar is 50 um for 20x and 20 um for 63x and Zoomed View. The range of z-stacks was kept consistent. HSp27 was once again found to readily co-localize with P.g with a Pearson coefficient of .83 as determined via the Imaris Software. ( Bi ) A Zoomed (4x) version of the 63x magnification was created using the Imaris Software, highlighting the intracellular nature of individual P. g. ( C ) GECs were additionally treated with HSp27 siRNA (100nM) for 48 h. Select GECs were then treated with N-acetyl Cysteine (NAC) (50 uM) for 1 h and/or eATP (3mM) for 30 min. P. g was added at MOI 100 to GECs, which were incubated 6 h. If any extracellular bacteria were present, they were killed by gentamicin (300 μg/mL) and metronidazole (200 μg/mL) treatment for 1 h. cDNAs were synthesized for qPCR using P. g -specific 16S rRNA primers to quantify intracellular level of live P. g . Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via One-Way Anova Test. ** p<0.005.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Inhibition, Cell Culture, Incubation, Immunofluorescence, Staining, Confocal Microscopy, Software, Bacteria, Synthesized

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Cross-Sectional Human in Situ Sample and Expression Analyses Support High Levels and Increased Co-localization of P. gingivalis (P. g) , HSp27, and LC3C in Periodontitis-Afflicted Oral Tissues. Publicly available mRNA expression data (GEO accession: GSE79705) was obtained from previously collected and examined periodontitis-afflicted and healthy gingival tissues. This microarray expression data was then analyzed via GEO2R and the relative levels of ( A ) HSp27 and ( B ) LC3C were obtained and compared. Data is represented as Mean±SD, where n=12 and p<0.05 was considered as statistically significant via One-Way Anova. *p<0.05. Representative confocal images of gingival biopsy specimens from healthy individuals and periodontitis-afflicted patients were also taken and examined. DAPI staining was utilized to visualize cellular DNA. ( C ) P. g (mouse anti-P . gingivalis ; Alexa 488; green) and LC3C (rabbit anti-LC3C; Alexa 594; red) were detected via dual staining. ( D ) HSp27 (mouse anti-HSp27; Alexa 488; green) and LC3C detection (rabbit anti-LC3C; Alexa 594; red) were also detected. Images were then captured using super resolution confocal laser scanning microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 10x and 63x magnification with oil immersion. Zoomed (2x) 3D versions of the 63x magnifications were obtained via the Imaris software. The range of z-stacks was kept consistent. SC: Stratum corneum, SL: Stratum lucidum, SG: Stratum granulosum, SB: Stratum basale, LT: Lamina propria. Scale bars = 200µm for 10x and 20 µm for 63x. Quantification of mean fluorescence intensity provided in Supplement. LC3C and HSp27 both were found to exhibit high levels of co-localization with each other and with P. g, as the Pearson coefficient was calculated to be .93 for LC3C and P. g and .85 for LC3C and HSp27 via the Imaris Software at the most severe state of disease.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: In Situ, Expressing, Microarray, Staining, Confocal Laser Scanning Microscopy, Software, Fluorescence

Journal: Oncotarget

Article Title: Dihydroartemisinin induces autophagy-dependent death in human tongue squamous cell carcinoma cells through DNA double-strand break-mediated oxidative stress

doi: 10.18632/oncotarget.17520

Figure Lengend Snippet: (A) Autophagosomes were checked by transmission electron microscopy. Cal-27 cells were treated with or without 24.5μM DHA for 24h, then fixed, embedded, sectioned, mounted, and stained as described in Materials and Methods. The autophagosome was indicated by white arrow. Scale bar 500 μm. (B) The quantitative data for autophagosomes were obtained from 3 sets of experiments. A total of 20 cells were counted for each variable. * P <0.05 vs. NC group. (C) DHA-induced autophagosomes were detected in Cal-27 cells by immunofluorencent staining of LC3B (green) (1000×). Cells were treated as described above. F-actin (red) was stained with Phalloidine. Nuclei (blue) were counter-stained with DAPI. (D) Quantitative analysis of autophagosomes with green-fluorescent puncta. Fifty cells were counted for each variable. * P <0.05 vs. NC group. (E) The autophagy-associated proteins LC3B-I/II were detected by Western blotting. Cal-27 cells were treated with indicated concentrations of DHA for 24 h, and then harvested for examining the expression levels. 40μM Etopside was used as DNA double-strand break positive control. GAPDH was used as a loading control. All experiments were performed in triplicates.

Article Snippet: The primary antibodies were rabbit anti-LC3B antibody (#2775, CST, diluted at 1:400), rabbit anti-Beclin-1 antibody (#3495, CST, diluted at 1:400), rabbit anti-Phospho-STAT3 (Tyr705) antibody (#3495, CST, diluted at 1:1500), rabbit anti-STAT3 antibody (#8232, CST, diluted at 1:1000), rabbit anti-H2b antibody (BS1657, Bioworld, diluted 1:500) and rabbit anti-GAPDH polyclonal antibody (#2118, CST, diluted at 1:1000).

Techniques: Transmission Assay, Electron Microscopy, Staining, Western Blot, Expressing, Positive Control, Control

Journal: Oncotarget

Article Title: Dihydroartemisinin induces autophagy-dependent death in human tongue squamous cell carcinoma cells through DNA double-strand break-mediated oxidative stress

doi: 10.18632/oncotarget.17520

Figure Lengend Snippet: H2b and GAPDH served as the quality control for the nuclear fraction and the cytoplasmic fraction, respectively. Cyt: cytoplasmic proteins; Nuc: nuclear proteins.

Article Snippet: The primary antibodies were rabbit anti-LC3B antibody (#2775, CST, diluted at 1:400), rabbit anti-Beclin-1 antibody (#3495, CST, diluted at 1:400), rabbit anti-Phospho-STAT3 (Tyr705) antibody (#3495, CST, diluted at 1:1500), rabbit anti-STAT3 antibody (#8232, CST, diluted at 1:1000), rabbit anti-H2b antibody (BS1657, Bioworld, diluted 1:500) and rabbit anti-GAPDH polyclonal antibody (#2118, CST, diluted at 1:1000).

Techniques: Control

Journal: Advanced Science

Article Title: Epigenetic Regulation of DAPK1 and Netrin‐1 Drives Diabetic Encephalopathy

doi: 10.1002/advs.202502535

Figure Lengend Snippet: DAPK1 levels were increased in the brain of diabetic mice with diabetic encephalopathy. A) Schematic overview of diabetic mouse modeling and cognitive‐related behavior assessments. B,C) Representative swimming paths on day 7, B) latency to reach the platform from days 1–7, C) swimming paths on day 9 and number of crossing the platform region on day 9 in the Morris water maze task for diabetic groups (6 month old STZ‐treated mice and db/db mice) and control groups (6 month old SCB‐treated mice and db/m mice). n = 10–12 per group. D) Western blot analysis and E) quantification of DAPK1 protein expression relative to β‐actin in the hippocampus of diabetic and control groups. n = 6 per group. F) Representative images and G) quantitative analysis of immunohistochemical staining for DAPK1 in hippocampal subregions (CA1, DG, and CA3) of diabetic and control mice. n = 6 slices from 3 mice per group. H) Distribution of DAPK1 (red) in excitatory pyramidal neurons (CaMKII, green) and its absence in inhibitory neurons (GAD1, green). Nuclei were stained with DAPI (blue). Higher‐magnification images of the regions marked with white squares in the CA1 area are shown on the right. I) Colocalization analysis of DAPK1 with CaMKII (up) and GAD1 (down), showing overlay levels and Pearson's correlation coefficient. J) Representative images and K) qualification of microglia (IBA1, red) and astrocytic (GFAP, green) activation in the hippocampus of diabetic and control groups. n = 6 slices from three mice per group. L) Representative images and M) quantification of apoptotic cells in the hippocampus of diabetic and control groups, as indicated by TUNEL‐positive staining (green). Nuclei were stained with DAPI (blue). The regions marked with white squares in the CA1 area are shown at higher magnification on the right. The percentage of apoptotic cells was calculated with the number of TUNEL‐positive cells divided by the total number of DAPI‐labeled cells in the same microscopic field. n = 4 slices from three mice per group. N,O) Electrophysiological recordings of N) field excitatory postsynaptic potential (fEPSP) slope to evaluate the LTP at the CA3‐CA1 synapse. n = 9 cells from three mice per group. Data are presented as mean ± S.E.M., and statistical analysis was performed using an unpaired Student's t ‐test or one‐way or two‐way analysis of variance (ANOVA), unless otherwise specified. ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking with 5% nonfat milk for 30 min, the membranes were incubated with primary antibodies overnight at 4 °C to DAPK1 (rabbit, 1:1000, No.25136‐1‐AP, Proteintech), DAPK1 (mouse, 1:1000, No.67815‐1‐Ig, Proteintech), β‐actin (mouse, 1:800, No.66009‐1‐Ig, Proteintech), HNF1A polyclonal antibody (rabbit, 1:1000, No. 22426‐1‐AP, Proteintech), Phospho‐HNF1A (Ser247) Ab (rabbit, 1:1000, AF7045, Affinity), or Netrin‐1 (rabbit, 1:500, DF8579, Affinity), followed by washes with Tris‐buffered saline (PBS)‐Tween 20.

Techniques: Control, Western Blot, Expressing, Immunohistochemical staining, Staining, Activation Assay, TUNEL Assay, Labeling

Journal: Advanced Science

Article Title: Epigenetic Regulation of DAPK1 and Netrin‐1 Drives Diabetic Encephalopathy

doi: 10.1002/advs.202502535

Figure Lengend Snippet: Knockdown hippocampus DAPK1 in excitatory neurons rescued diabetic encephalopathy in diabetes. A) Schematic of the strategy for conditional DAPK1 kinase domain knockout in hippocampal excitatory neurons using CaMKII‐CreERT and tamoxifen (TAM) induction in diabetic mice. Western blot analysis of DAPK1 and β‐actin protein expression in the hippocampus of STZ/KD f/f and STZ/KD −/− groups. n = 2 per group. B) Representative path traces to the hidden platform on day 7 and latency over 7 days in different groups. n = 10 per group. C) Swimming paths on day 9 and number of crossing the platform region on day 9 in the MWM. n = 10 per group. D) Immunofluorescence staining of IBA1 (red) and GFAP (green) in the hippocampus, showing changes in microglial and astrocytic activation across groups. n = 6 slices from three mice per group. E) TUNEL staining of hippocampal sections for apoptosis detection, with DAPI counterstaining; apoptotic cells appear green in images. F) Quantification of TUNEL‐positive cells in the hippocampus, showing reduced apoptosis in DAPK1 −/− mice. n = 6 slices from three mice per group. G) Electrophysiological recordings illustrating normalized field excitatory postsynaptic potential (fEPSP) slopes in CA3–CA1 synapses, with reduced long‐term potentiation (LTP) impairment in DAPK1 −/− mice. H) Summary of fEPSP slope changes among groups, highlighting improved synaptic function in DAPK1 −/− mice. n = 9 cells from three mice per group. I) Representative traces (up) and quantification (down) of paired‐pulse facilitation (PPF) recorded from hippocampal slices. n = 10 cells from three mice per group. J) Representative traces (up) and quantification (down) of AMPA/NMDA current ratio measured in CA1 neurons. n = 10 cells from three mice per group. K) Representative transmission electron microscopy (TEM) images showing hippocampal synapses in the stratum radiatum. L) Quantification of postsynaptic density (PSD) length from TEM images. n = 130–150 synapses from 3 to 5 mice per group. Data are shown as mean ± S.E.M., and statistical analysis was performed using one‐way or two‐way analysis of variance (ANOVA). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking with 5% nonfat milk for 30 min, the membranes were incubated with primary antibodies overnight at 4 °C to DAPK1 (rabbit, 1:1000, No.25136‐1‐AP, Proteintech), DAPK1 (mouse, 1:1000, No.67815‐1‐Ig, Proteintech), β‐actin (mouse, 1:800, No.66009‐1‐Ig, Proteintech), HNF1A polyclonal antibody (rabbit, 1:1000, No. 22426‐1‐AP, Proteintech), Phospho‐HNF1A (Ser247) Ab (rabbit, 1:1000, AF7045, Affinity), or Netrin‐1 (rabbit, 1:500, DF8579, Affinity), followed by washes with Tris‐buffered saline (PBS)‐Tween 20.

Techniques: Knockdown, Knock-Out, Western Blot, Expressing, Immunofluorescence, Staining, Activation Assay, TUNEL Assay, Transmission Assay, Electron Microscopy

Journal: Advanced Science

Article Title: Epigenetic Regulation of DAPK1 and Netrin‐1 Drives Diabetic Encephalopathy

doi: 10.1002/advs.202502535

Figure Lengend Snippet: The elevation of DAPK1 is driven by suppression of miR‐216a‐5p. A) Relative mRNA levels of DAPK1 in hippocampus of control (SCB and db/m) and diabetic (STZ and db/db) mice, showing no significant differences across groups. n = 5. B) Heatmap of miRNA expression profiles in different groups. C) Diagram to display the conserved binding site in DAPK1 3′UTR to the miR‐216a‐5p. The mutant sequence in 3′UTR of DAPK1 for luciferase analysis was provided at the bottom. D) Luciferase reporter assay of wild‐type (WT) and mutant (MUT) DAPK1 3′UTR constructs in cells transfected with miR‐216a‐5p mimic or scramble control. n = 6 per group. E,F) N2a cells were transfected with miR‐216a‐5p agomir or antagomir. E) The cell lysates were collected, and the protein levels of DAPK1 were then detected by western blot (WB) after 48 h, F) with quantification. n = 4 per group. G,H) Immunofluorescent staining showing DAPK1 (green) and miR‐216a‐5p (red) in the hippocampus of db/m and db/db mice, with DAPI counterstaining (blue). Correlation analysis between miR‐216a‐5p and DAPK1 levels, showing a significant negative relationship ( R 2 = 0.7465, p < 0.001). n = 15 slices per group from five mice. Data are presented as mean ± S.E.M., and statistical analysis was performed using an unpaired Student's t ‐test or linear regression. * p < 0.05, ** p < 0.01, and **** p <0.0001.

Article Snippet: After blocking with 5% nonfat milk for 30 min, the membranes were incubated with primary antibodies overnight at 4 °C to DAPK1 (rabbit, 1:1000, No.25136‐1‐AP, Proteintech), DAPK1 (mouse, 1:1000, No.67815‐1‐Ig, Proteintech), β‐actin (mouse, 1:800, No.66009‐1‐Ig, Proteintech), HNF1A polyclonal antibody (rabbit, 1:1000, No. 22426‐1‐AP, Proteintech), Phospho‐HNF1A (Ser247) Ab (rabbit, 1:1000, AF7045, Affinity), or Netrin‐1 (rabbit, 1:500, DF8579, Affinity), followed by washes with Tris‐buffered saline (PBS)‐Tween 20.

Techniques: Control, Expressing, Binding Assay, Mutagenesis, Sequencing, Luciferase, Reporter Assay, Construct, Transfection, Western Blot, Staining

Journal: Advanced Science

Article Title: Epigenetic Regulation of DAPK1 and Netrin‐1 Drives Diabetic Encephalopathy

doi: 10.1002/advs.202502535

Figure Lengend Snippet: Knockdown of DAPK1 in excitatory neurons restores Netrin‐1 (Ntn1) expression. A) Heatmap showing the relative mRNA levels of various neurotrophic factors, including Ntn1, in db/db mice compared to controls. n = 3 per group. B) Western blot analysis of Ntn1 protein levels in the hippocampus of db/m and db/db mice, with quantification showing a significant reduction of Ntn1 in db/db mice. n = 5 per group. C) Western blot analysis of Ntn1 in DAPK1‐KD +/− and DAPK1‐KD −/− diabetic mice treated with tamoxifen (TAM), with quantification indicating restoration of Ntn1 levels following DAPK1 knockdown. n = 5 per group. D) Immunofluorescent staining for DAPK1 (green) and Ntn1 (red) in the hippocampus of different groups, with merged images showing colocalization. E) Quantitative analysis of Ntn1 levels in different group mice, demonstrating increased Ntn1 expression following DAPK1 kinase domain knockdown. n = 6 slices per group from three mice. F) Correlation analysis between the relative protein levels of DAPK1 and Ntn1 ( R 2 = 0.4808, p < 0.001). n = 6 per group. G) Schematic of the dual‐luciferase reporter assay construct used to assess Ntn1 promoter activity in response to DAPK1 kinase domain knockdown. H) Relative luciferase activity in cells after cotransfection with the reporter vector and the control or effector vector for 48 h. I) Western blot analysis of HNF1A and Ntn1 levels in control and DAPK1‐overexpressing (oe‐DAPK1) cells, with J) quantification showing a decrease in HNF1A and Ntn1 levels upon DAPK1 overexpression. n = 3 per group. K) Chromatin immunoprecipitation (ChIP) assay was performed to assess the binding of HNF1A to the Ntn1 promoter in HEK293T cells transfected with either DAPK1 vector, wild‐type HNF1A, or phosphorylation‐deficient mutant HNF1A‐S249A. Immunoprecipitated DNA was quantified using qPCR targeting the Ntn1 promoter region. n = 3 per group. L) Luciferase reporter assay showing the transcriptional activity of wild‐type HNF1A and its phosphorylation mutants (S249A and S249D) on the Ntn1 promoter. HEK293T cells were co‐transfected with the Ntn1 promoter‐luciferase construct and the indicated HNF1A variants. Luciferase activity was measured 24 post‐transfection and normalized to Renilla luciferase. n = 5 per group. Data are presented as mean ± S.E.M., and statistical analysis was performed using an unpaired Student's t ‐test or one‐way or two‐way analysis of variance (ANOVA), unless otherwise specified. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking with 5% nonfat milk for 30 min, the membranes were incubated with primary antibodies overnight at 4 °C to DAPK1 (rabbit, 1:1000, No.25136‐1‐AP, Proteintech), DAPK1 (mouse, 1:1000, No.67815‐1‐Ig, Proteintech), β‐actin (mouse, 1:800, No.66009‐1‐Ig, Proteintech), HNF1A polyclonal antibody (rabbit, 1:1000, No. 22426‐1‐AP, Proteintech), Phospho‐HNF1A (Ser247) Ab (rabbit, 1:1000, AF7045, Affinity), or Netrin‐1 (rabbit, 1:500, DF8579, Affinity), followed by washes with Tris‐buffered saline (PBS)‐Tween 20.

Techniques: Knockdown, Expressing, Western Blot, Staining, Luciferase, Reporter Assay, Construct, Activity Assay, Cotransfection, Plasmid Preparation, Control, Over Expression, Chromatin Immunoprecipitation, Binding Assay, Transfection, Phospho-proteomics, Mutagenesis, Immunoprecipitation

Journal: PLOS ONE

Article Title: Use of extracellular vesicle microRNA profiles in patients with acute myeloid leukemia for the identification of novel biomarkers

doi: 10.1371/journal.pone.0306962

Figure Lengend Snippet: (A) The size and concentration trend of vesicles by fractions are presented. The eluted fractions (11 and 12; 0.5 mL each) were used for vesicle isolation. Samples were diluted 10-fold. (B) The size distribution of the isolated vesicles was determined using nanoparticle tracking analysis (NTA). The average size of BM aspirate serum or plasma-derived vesicles was 115.5 ± 2.7 nm and 101.5 ± 4.4 nm, respectively; these sizes were within the size range of typical EVs. Samples were diluted 10-fold. (C) In transmission electron microscopy (TEM) images, the size of isolated vesicles was <200 nm, and they were visualized as cup-shaped vesicles under high magnification. (D) Western blotting showed that the isolated vesicles were positive for the markers of EVs (CD63 and CD81). EVs, extracellular vesicles; BM, bone marrow; AML, acute myelogenous leukemia.

Article Snippet: After blocking with 5% skim milk (w/v) in 0.1% TBST for 2 h, the membranes were probed overnight at 4°C with 1:1000 dilutions of rabbit anti-CD63 polyclonal antibody and rabbit anti-CD81 polyclonal antibodies (all from Bioss Antibodies, MA, USA).

Techniques: Concentration Assay, Isolation, Derivative Assay, Transmission Assay, Electron Microscopy, Western Blot