Journal: Molecular Biomedicine

Article Title: Lipid nanoparticle-mediated transcription factor EB mRNA delivery for the treatment of metabolic dysfunction-associated steatotic liver disease

doi: 10.1186/s43556-026-00615-0

Figure Lengend Snippet: LNP-TFEB and S211A enhanced autophagy and promoted lipid metabolism in the mouse liver. a Representative Western blot images and b corresponding quantitative analysis of TFEB (TFEB and FLAG-tag detection confirmed LNP-mediated expression of TFEB and S211A), autophagy-related proteins (LC3, P62), lipid metabolism-related proteins (PPARα, ATGL, CPT1A) and lipid synthesis-related proteins (FASN, ACC, SREBP1) in mouse liver ( n = 4 or 6 per group). c Relative hepatic mRNA expression levels of autophagy-related genes, lipid catabolism-related genes and lipid synthesis and transport-related genes, as determined by qPCR ( n = 5 or 6 per group). d Representative hematoxylin and eosin (H&E) staining images of liver sections and the corresponding NAFLD activity score (NAS) ( n = 6 per group). e Representative images of Sirius Red staining and quantification of the relative fibrotic area ( n = 6 per group). f Representative BODIPY fluorescence staining images of neutral lipids in liver sections and quantification of fluorescence intensity. cv: Central vein ( n = 6 per group). g Representative F4/80 immunofluorescence staining images showing macrophage infiltration and the corresponding quantitative analysis ( n = 5 per group). Scale bar = 100 μm. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The membranes were blocked with 5% skim milk for 2 h and then incubated overnight at 4 °C with the following primary antibodies: anti-TFEB (1:1,000, Bethyl, A303-673A), anti-LC3 (1:1,000, Abclonal, A15591), anti-p62 (1:1,000, Abclonal, A7758), anti-phospho-eIF4EBP1 (1:1,000, Abclonal, AP1363), anti-eIF4EBP1 (1:1,000, Abclonal, A24691), anti-FGF21 (1:1,000, Abclonal, A23463), anti-FASN (1:1,000, Epyzime, R013857), anti-SREBP1 (1:1,000, Epyzime, P010134), anti-ACC (1:1,000, Epyzime, P105115), anti-CPT1A (1:1,000, Epyzime, P106431), anti-PPARα (1:1,000, Epyzime, P010554), anti-ATGL (1:1,000, Epyzime, R011456), and anti-β-actin (1:1,000, Proteintech, 20,536–1-AP).

Techniques: Western Blot, Analysis, FLAG-tag, Expressing, Staining, Activity Assay, Fluorescence, Immunofluorescence

Journal: Advanced Science

Article Title: Phosphorylated STYK1 Promotes Gemcitabine Resistance via Autophagy‐Mediated Cancer Stemness in Pancreatic Cancer

doi: 10.1002/advs.202521488

Figure Lengend Snippet: Gemcitabine induced AMPK to amplify STYK1‐mediated autophagy activation. (A–D) Western blotting analysis and quantification of LC3, p62, STYK1, and p‐AMPK T172 protein expression levels in PANC‐1/GEM cells with wildtype and stable STYK1 knockdown, treated with gemcitabine at different concentrations and for different durations (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 according to Student's t ‐test. (E) KEGG enrichment analysis of differentially expressed genes between gemcitabine‐resistant pancreatic cancer cells and pancreatic cancer cells ( GSE80617 ). Differentially expressed genes were identified using the criteria of |log2FC| > 1 and p < 0.05. The dot size represents the number of genes enriched in each pathway, and the color represents the enrichment significance based on −log10( p value). (F, G) Western blotting analysis and quantification of LC3, p62, and p‐AMPK T172 protein expression levels in PANC‐1/GEM cells with wildtype and stable STYK1 knockdown treated with or without the AMPK inhibitor Compound C (10 µM, 24 h) or gemcitabine (5 µM, 24 h) (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 according to Student's t ‐test. (H, I) Western blotting analysis and quantification of LC3 and p‐AMPK T172 protein expression levels in AMPK‐deficient PANC‐1 cells transfected with Flag‐STYK1 and treated with or without gemcitabine (1 µM, 24 h) (n = 3). ** p < 0.01; *** p < 0.001; **** p < 0.0001 according to Student's t ‐test. (J, K) Co‐immunoprecipitation analysis of the interaction between STYK1 and VPS34 with Beclin1 or ATG14L in HEK293T cells co‐transfected with STYK1 and Flag‐Beclin1 or Flag‐ATG14L, treated with or without treatment AMPK activator AICAR (1 mM, 4 h). Cell lysates were used for IP and Western blotting with the indicated antibodies. Data were expressed as mean ± SD, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 according to Student's t ‐test.

Article Snippet: Tissue sections were blocked with 5% normal goat serum with 0.1% Triton X‐100 and 3% H 2 O 2 in PBS for 60 min at room temperature and then incubated with anti‐ki67 ( GB111499 ‐100, Servicebio), anti‐CK19 (GB11197, Servicebio), anti‐CD44 (15675‐1‐AP, Proteintech), anti‐CD133 (18470‐1‐AP, Proteintech), anti‐LC3 (14600‐1‐AP, Proteintech), anti‐p62 (66184‐1‐Ig, Proteintech) overnight, which were then detected by HRP conjugated DAB.

Techniques: Activation Assay, Western Blot, Analysis, Expressing, Knockdown, Transfection, Co-Immunoprecipitation Assay

Journal: Advanced Science

Article Title: Phosphorylated STYK1 Promotes Gemcitabine Resistance via Autophagy‐Mediated Cancer Stemness in Pancreatic Cancer

doi: 10.1002/advs.202521488

Figure Lengend Snippet: Peptide disrupting STYK1‐AMPK interaction inhibits autophagy and pancreatic cancer stemness. (A) Co‐immunoprecipitation analysis of the interaction between HA‐AMPK and GFP‐tagged STYK‐driving peptide or Flag‐STYK1 in HEK293T cells. Cell lysates were used for IP and Western blotting with the indicated antibodies. (B) SPR analysis of the binding kinetics between the peptide CP‐SAIDP5 and AMPK. (C) Co‐immunoprecipitation analysis of endogenous STYK1 and AMPK in PANC‐1/GEM cells after treatment with STYK1‐driven peptide CP‐SAIDP5 (5 µM, 24 h). Cell lysates were used for IP and Western blotting with the indicated antibodies. (D) Western blotting analysis of LC3, p62, and p‐AMPK T172 expression in PANC‐1/GEM cells after treatment with peptide CP‐SAIDP5 (5 µM, 24 h) or gemcitabine (5 µM, 24 h) alone or in combination. (E, F) Representative confocal images of red and yellow spots in PANC‐1/GEM cells after treatment with peptide CP‐SAIDP5 (5 µM, 24 h) or gemcitabine (5 µM, 24 h) alone or in combination. Quantitative measurement of the number of autolysosomes (red dots) and autophagosomes (yellow dots) (n = 3). Scale bar: 10 µm. * p < 0.05; ** p < 0.01; *** p < 0.001 according to Student's t ‐test. (G) Co‐immunoprecipitation analysis of the interaction between Beclin1 and ATG14L and VPS34 after treatment of PANC‐1/GEM cells with peptide CP‐SAIDP5 (5 µM, 24 h) or gemcitabine (5 µM, 24 h) alone or in combination. Cell lysates were used for IP and Western blotting with the indicated antibodies. (H) Western blotting analysis of CD44 and CD133 expression levels after treatment of PANC‐1/GEM cells with peptide CP‐SAIDP5 (5 µM, 24 h) or gemcitabine (5 µM, 24 h) alone or in combination. (I) RT‐qPCR analysis of CD44 , CD133 , and stemness‐associated transcription factors OCT4 , NANOG , and SOX2 after treatment of PANC‐1/GEM cells with peptide CP‐SAIDP5 (5 µM, 24 h) or gemcitabine (5 µM, 24 h) alone or in combination (n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 according to Student's t ‐test. (J, K) Flow cytometric analysis and quantification of CD44 + CD133 + cells in PANC‐1/GEM cells treated with peptide CP‐SAIDP5 (5 µM, 24 h) or gemcitabine (5 µM, 24 h) alone or in combination. Numbers in the graphs indicate the percentage of this subpopulation (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001 according to Student's t ‐test. (L, M) Representative images of AsPC‐1/GEM cells treated with peptide CP‐SAIDP5 (5 µM, 24 h) or gemcitabine (5 µM, 24 h) alone or in combination, cultured in serum‐free medium for 14 days. The number of spheroids >70 µm in diameter was statistically analyzed (n = 3). Scale bar: 100 µm. * p < 0.05; ** p < 0.01 according to Student's t ‐test. Data were expressed as mean ± SD.

Article Snippet: Tissue sections were blocked with 5% normal goat serum with 0.1% Triton X‐100 and 3% H 2 O 2 in PBS for 60 min at room temperature and then incubated with anti‐ki67 ( GB111499 ‐100, Servicebio), anti‐CK19 (GB11197, Servicebio), anti‐CD44 (15675‐1‐AP, Proteintech), anti‐CD133 (18470‐1‐AP, Proteintech), anti‐LC3 (14600‐1‐AP, Proteintech), anti‐p62 (66184‐1‐Ig, Proteintech) overnight, which were then detected by HRP conjugated DAB.

Techniques: Co-Immunoprecipitation Assay, Analysis, Western Blot, Binding Assay, Expressing, Confocal, Quantitative RT-PCR, Cell Culture, Spheroids

Journal: Advanced Science

Article Title: Phosphorylated STYK1 Promotes Gemcitabine Resistance via Autophagy‐Mediated Cancer Stemness in Pancreatic Cancer

doi: 10.1002/advs.202521488

Figure Lengend Snippet: STYK1‐driving peptides inhibit gemcitabine‐resistant pancreatic cancer development. (A, B) Representative images and quantitative analysis of Transwell assays with and without matrigel of PANC‐1/GEM and AsPC‐1/GEM cells treated with peptide CP‐SAIDP5 (5 µM, 24 h) or gemcitabine (5 µM for PANC‐1/GEM cells, or 40 µµ for AsPC‐1/GEM cells, 24 h) alone or in combination (n = 3). Scale bar: 100 µm. * p < 0.05; ** p < 0.01; *** p < 0.001 according to Student's t ‐test. (C) Representative images of excised tumors from gemcitabine‐resistant PDX tumor‐bearing mice treated with CP‐SAIDP5 peptide (25 mg/kg), gemcitabine (25 mg/kg), or their combination (n = 4). (D) Tumor weights of excised PDX tumors from the indicated treatment groups (n = 4). ** p < 0.01; *** p < 0.001; **** p < 0.0001 according to Student's t ‐test. (E) Tumor growth curves of PDX‐bearing mice in the indicated treatment groups (n = 4). ** p < 0.01; **** p < 0.0001 according to Student's t ‐test. (F) Body weight changes in gemcitabine‐resistant PDX tumor‐bearing mice following treatment with CP‐SAIDP5 peptide (25 mg/kg), gemcitabine (25 mg/kg), or combination therapy. (n = 4). ns (not significant) according to Student's t ‐test. (G) Representative H&E staining images and immunohistochemistry images of Ki67, CD44, CD133, LC3, and p62 expressed in resected tumor tissues (n = 4). Scale bar: 50 µm. (H) Representative histological images and H&E staining images of pancreatic tissues after peptide CP‐SAIDP5 (25 mg/kg) or gemcitabine (25 mg/kg) alone or co‐treated KPC mice (n = 3). Scale bar: 40 µm. I–K) Quantification of H&E staining, Sirius Red or Alcian blue staining in the indicated groups (n = 3). *** p < 0.001; **** p < 0.0001 according to Student's t ‐test. (L–Q) Concentrations of WBC, RBC, PLT, BUN, AST, ALT in the serum of peptide CP‐SAIDP5 or gemcitabine alone or co‐treated KPC mice (n = 3). ns (not significant) according to Student's t ‐test. Data were expressed as mean ± SD.

Article Snippet: Tissue sections were blocked with 5% normal goat serum with 0.1% Triton X‐100 and 3% H 2 O 2 in PBS for 60 min at room temperature and then incubated with anti‐ki67 ( GB111499 ‐100, Servicebio), anti‐CK19 (GB11197, Servicebio), anti‐CD44 (15675‐1‐AP, Proteintech), anti‐CD133 (18470‐1‐AP, Proteintech), anti‐LC3 (14600‐1‐AP, Proteintech), anti‐p62 (66184‐1‐Ig, Proteintech) overnight, which were then detected by HRP conjugated DAB.

Techniques: Analysis, Staining, Immunohistochemistry