Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: a , Tau puncta formation (left) and degradation of preformed tau puncta (right) were quantified in HEK293 cells expressing 0N4R P301S Tau-Venus in the presence or absence of ULK1 activator Rac-(BL)-918. n = a total of 50 randomly selected fields per group from 3 biological replicates. b , c , HEK293 P301S tau-Venus cells were transfected with siRNA-ULK1 ( b ), siRNA-ULK2 ( c ) or siRNA-scramble ( b , c ), and transfected cells were incubated with or without 5-μM Rac-(BL)-918 in the presence of 10-nM tau seeds. Graphs in a – c show normalized fluorescence intensity relative to vehicle control ( a ) or in the absence of Rac-(BL)-918 ( b , c ), as indicated. n = a total of 54 randomly selected fields per group from 3 biological replicates. d , Rac-(BL)-918 inhibited the formation of tau puncta dependent on ULK1, FUNDC1, PINK1, Ambra1 and Beclin1. HEK293 P301S tau-Venus cells were transfected with siRNA-scramble or a siRNA candidate, followed by treatment with 10-nM tau seeds and 5-μM Rac-(BL)-918 simultaneously. n = a total of 36–40 randomly selected fields per group from 3 biological replicates. e , Rac-(BL)-918 increased the degradation of preformed tau puncta dependent on ULK1 and Beclin1. HEK293 P301S tau-Venus cells were transfected with siRNA-scramble or a siRNA candidate, followed by subsequent treatment with 10-nM tau seeds and 5-μM Rac-(BL)-918. n = a total of 36–40 randomly selected fields per group from 3 biological replicates. f , Associative memory tests were administered to adult day-2 transgenic C. elegans expressing hTau[P301L] n -sid-1 OV in the presence of siRNA targeting unc-51/ULK1 or control siRNA in the presence of Rac-(BL)-918, where indicated. Data are shown in the format of % CI, where a lower score corresponds to greater response to odorant and better memory function. n = 3 biological sets. g , Associative memory tests were administered to adult day-2 transgenic C. elegans expressing hTau[P301L] n -sid-1 OV in the absence or presence of siRNA to pink-1 or fndc-1/FUNDC1 . Data are shown in the format of % CI, where a lower score corresponds to greater response to odorant and better memory function. n = 5 biological sets. In the box plots, the center line denotes the median, box range indicates the 25th–75th percentile and whiskers denote minimum and maximum values. Unless specified elsewhere, data are mean ± s.e.m. Results represent pooled data from three to five biological replicates. Violin and box plots are centered around the median with interquartile ranges and all data points shown; the shape of the plot reflects the distribution of data. Statistical significance was determined using the Kruskal–Wallis test (non-normal data distribution) or one-way ANOVA (normal data distribution) ( a – e ) or two-way ANOVA followed by Tukey’s multiple comparisons test ( f , g ).

Article Snippet: The siRNA reagents used including siRNA targeting ULK1 (catalog no. SR322391, OriGene), PINK1 (catalog no. SR324912, OriGene), Parkin (catalog no. SR321228, OriGene), FUNDC1 (catalog no. SR315322, OriGene), Ambra1 (catalog no. SR310808, OriGene), BNIP3 (catalog no. SR300461, OriGene), BNIP3L/NIX (catalog no. SR300462, OriGene), GSK3-beta (catalog no. SR301979, OriGene), ULK2 (catalog no. SC-44183, Santa Cruz Biotechnology), Atg5 (catalog no. SR322789, OriGene), Beclin1 (catalog no. SR322490, OriGene), Sirt1 (catalog no. SR323581, OriGene) or scramble control siRNA Oligo Duplexes at 100 nM.

Techniques: Expressing, Transfection, Incubation, Fluorescence, Control, Transgenic Assay

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: ( a ) Effects of ULK1 activators (2, 4 µM of LYN-1604 dihydrochloride; 0.5, 5 µM of (Rac)-BL-918) and inhibitors (5, 10 µM of SBI-0206965; 2.5, 5 µM of XST-14) on Tau seed-induced formation of Tau puncta in HEK293 cells expressing 0N4R P301S Tau-Venus. Data are pooled from 3 biological replicates. (b) Effects of ULK1 activators (2, 4 µM of LYN-1604 dihydrochloride; 0.5, 5 µM of (Rac)-BL-918) and inhibitors (5, 10 µM of SBI-0206965; 2.5, 5 µM of XST-14) on degradation of preformed Tau puncta in the HEK293 cells expression 0N4R P301S Tau-Venus. Data are pooled from 3 biological replicates. ( c ) mt-Keima and YPH-Parkin expressing HeLa cells were used to evaluate the effects on mitophagy induction by treating a positive control CCCP. Representative images are shown. ( d-f ) Quantification of mitophagy events by treating ULK1 activators, Rac-BL-918 (0.5, 5 µM) and LYN-1604 (2, 4 µM), as well as ULK1 inhibitors SBI-0206965 (5, 10 µM) and XST-14 (2.5, 5 µM). One dot represents the average value of one image. Data were pooled from 3 biological replicates. ( g-h ) Representative images ( g ) and quantification ( h ) of mitophagy induction from primary cortical neurons by treating ULK1 activators, Rac-BL-918 (5 µM) and LYN-1604 (4 µM), as well as ULK1 inhibitors SBI-0206965 (5 µM) and XST-14 (5 µM). Mitophagy was detected using a mitophagy detection dye kit. Nuclei were stained with DAPI. Scale bar = 20 μm. ( i ) Representative blots of target proteins (ULK1, ULK2, PINK1, GSK3β, Parkin, Atg5, FUNDC1, AMBRA1, BNIP3, Nix, Beclin1) and loading control (GAPDH or β-tubulin) in HEK293 cells expressing 0N4R P301S Tau-Venus transfected with siRNAs (100 nM, 48 h) or scrambled siRNA. ( j ) Associative memory tests were administered to adult day 2 transgenic C. elegans expressing hTau[P301L]n-sid-1 OV in the presence of ULK1 inhibitors (10, 100 µM of SBI-0206965; 5, 50 µM of XST-14). Attraction to odorant is quantified as Chemotaxis Index (% CI), where lower score corresponds to greater response to odorant/better memory function. Unless specified elsewhere, data are mean ± S.E.M. Statistical analyses performed were one-way ANOVA followed by Dunnett’s multiple comparisons test ( a , b , d-f , h ); two-way ANOVA followed by Tukey’s multiple-comparisons test ( j ).

Article Snippet: The siRNA reagents used including siRNA targeting ULK1 (catalog no. SR322391, OriGene), PINK1 (catalog no. SR324912, OriGene), Parkin (catalog no. SR321228, OriGene), FUNDC1 (catalog no. SR315322, OriGene), Ambra1 (catalog no. SR310808, OriGene), BNIP3 (catalog no. SR300461, OriGene), BNIP3L/NIX (catalog no. SR300462, OriGene), GSK3-beta (catalog no. SR301979, OriGene), ULK2 (catalog no. SC-44183, Santa Cruz Biotechnology), Atg5 (catalog no. SR322789, OriGene), Beclin1 (catalog no. SR322490, OriGene), Sirt1 (catalog no. SR323581, OriGene) or scramble control siRNA Oligo Duplexes at 100 nM.

Techniques: Expressing, Positive Control, Staining, Control, Transfection, Transgenic Assay, Chemotaxis Assay

Journal: Frontiers in Pharmacology

Article Title: AP39 alleviates HHCY-induced myocardial remodeling by regulating FUNDC1-mediated mitochondrial dynamics via S-sulfhydration of NEDD8/CUL4B

doi: 10.3389/fphar.2026.1729145

Figure Lengend Snippet: (A) GO analysis revealed that cardiomyocytes under HHCY conditions were mainly enriched in mitochondrial processes, mitochondrial function, and inner mitochondrial membrane signaling pathways. (B) GSEA of AP39-intervened samples that showed significant enrichment and upregulation of the mitophagy pathway. (C) Cystoscope analysis the top 5 ranked genes were SQSTM1, PINK1, HIF1α, FUNDC1, and TBK1. Expression of FUNDC1, LC3A/B in SD rats induced by HHCY (D) and H9c2 cardiomyocytes (E) in each group. (*p < 0.05 vs. Control; **p < 0.01 vs. Control; #p < 0.05 vs. HHCY; ##p < 0.01 vs. HHCY; $p < 0.05 vs. HHCY + AP39, $$$p < 0.005 vs. HHCY + AP39); (F) Expression of P53,P16 in H9c2 cardiomyocytes in each group after siRNA interference. (*p < 0.05 vs. Control; #p < 0.05 vs. HHCY; $$$p < 0.005 vs. HHCY + AP39); (G) Observation of changes in Mitophagy formation in each group under transmission electron microscopy. Expression of MFN1, MFN2, Drp1 in SD rats induced by HHCY (H) and H9c2 cardiomyocytes (I) in each group. (*p < 0.05 vs. Control; **p < 0.01 vs. Control; ***p < 0.005 vs. Control; #p < 0.05 vs. HHCY; ##p < 0.01 vs. HHCY; ###p < 0.005 vs. HHCY; $p < 0.05 vs. HHCY + AP39, $$p < 0.01 vs. HHCY + AP39, $$$p < 0.005 vs. HHCY + AP39); (J) Co-localization of FUNDC1 and LC3B in cardiomyocytes in each group under fluorescence microscopy. (K) Mitochondrial membrane potential changes after siRNA interference. (Red fluorescence indicates high mitochondrial membrane potential; green fluorescence indicates low mitochondrial membrane potential). Gene Ontology: GO; high homocysteine: HHCY; Gene Set Enrichment Analysis: GSEA.

Article Snippet: All primary antibodies used for Western blotting (WB) and immunofluorescence (IF) were purchased from the corresponding commercial suppliers, and their detailed information is listed as follows: GAPDH (Proteintech, USA; 10494-1-AP; WB 1:8000), α-smooth muscle actin (α-SMA) (Proteintech, USA; 14395-1-AP; WB 1:4000), Collagen type III (Proteintech, USA; 22734-1-AP; WB 1:800), FUNDC1 (Proteintech, China; AWA51295 ; IF 1:100, WB 1:1000), LC3B (Proteintech, USA; 14600-1-AP; IF 1:500, WB 1:4000), MFN1 (Proteintech, USA; 13798-1-AP; WB 1:5000), MFN2 (Proteintech, USA; 12186-1-AP; WB 1:20000), DRP1 (Proteintech, USA; 12957-1-AP; WB 1:5000), P53 (Proteintech, USA; 10442-1-AP; WB 1:20000), P16 (Proteintech, USA; 10883-1-AP; WB 1:3000), NEDD8 (Abways, China; AWA11003 ; WB 1:1000), and CUL4B (Abways, China; AWA48229 ; WB 1:1000).

Techniques: Membrane, Protein-Protein interactions, Expressing, Control, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy

Journal: Frontiers in Pharmacology

Article Title: AP39 alleviates HHCY-induced myocardial remodeling by regulating FUNDC1-mediated mitochondrial dynamics via S-sulfhydration of NEDD8/CUL4B

doi: 10.3389/fphar.2026.1729145

Figure Lengend Snippet: (A) Co-Immunoprecipitation (Co-IP) analysis showing the binding of FUNDC1 to DRP1 in cardiomyocytes of each group; (B) Expression of P53, P16 in H9c2 cardiomyocytes in each group after siRNA interference. (*p < 0.05 vs. Control; **p < 0.01 vs. Control; #p < 0.05 vs. HHCY; ##p < 0.01 vs. HHCY; $p < 0.05 vs. HHCY + AP39); (C) Detection of cellular senescence in heart tissues of each group (upper panel) and in cardiomyocytes of each group (lower panel) by β-galactosidase assay.

Article Snippet: All primary antibodies used for Western blotting (WB) and immunofluorescence (IF) were purchased from the corresponding commercial suppliers, and their detailed information is listed as follows: GAPDH (Proteintech, USA; 10494-1-AP; WB 1:8000), α-smooth muscle actin (α-SMA) (Proteintech, USA; 14395-1-AP; WB 1:4000), Collagen type III (Proteintech, USA; 22734-1-AP; WB 1:800), FUNDC1 (Proteintech, China; AWA51295 ; IF 1:100, WB 1:1000), LC3B (Proteintech, USA; 14600-1-AP; IF 1:500, WB 1:4000), MFN1 (Proteintech, USA; 13798-1-AP; WB 1:5000), MFN2 (Proteintech, USA; 12186-1-AP; WB 1:20000), DRP1 (Proteintech, USA; 12957-1-AP; WB 1:5000), P53 (Proteintech, USA; 10442-1-AP; WB 1:20000), P16 (Proteintech, USA; 10883-1-AP; WB 1:3000), NEDD8 (Abways, China; AWA11003 ; WB 1:1000), and CUL4B (Abways, China; AWA48229 ; WB 1:1000).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: AP39 alleviates HHCY-induced myocardial remodeling by regulating FUNDC1-mediated mitochondrial dynamics via S-sulfhydration of NEDD8/CUL4B

doi: 10.3389/fphar.2026.1729145

Figure Lengend Snippet: (A) KEGG analysis revealed that cardiomyocytes under HHCY conditions were mainly enriched in ubiquitination pathway. (B) Heatmap showing differentially expressed CULLIN family genes in HHCY hearts. P value is shown. (C) Expression of NEDD8, Cul4b in SD rats induced by HHCY in each group. (*p < 0.05 vs. Control; #p < 0.05 vs. HHCY); (D) Co-Immunoprecipitation (Co-IP) analysis showing the binding of FUNDC1 to Cul4b in cardiomyocytes of each group; (E–G) Expression of Cul4b, FUNDC1, P53, P16 in H9c2 cardiomyocytes in each group. (*p < 0.05 vs. Control; #p < 0.05 vs. HHCY; ##p < 0.01 vs. HHCY); (H) Co-immunoprecipitation (Co - IP) and ubiquitination assays were performed to assess FUNDC1 ubiquitination in Control, HHCY, and HHCY + AP39 groups. (I,J) Expression of FUNDC1, NEDD8, CUL48in H9c2 cardiomyocytes in each group. (K) Expression of NEDD8, CUL48, S-NEDD8, S-CUL4B in H9c2 cardiomyocytes in each group. (*p < 0.05 vs. Control; #p < 0.05 vs. HHCY; $p < 0.05 vs. HHCY + AP39).

Article Snippet: All primary antibodies used for Western blotting (WB) and immunofluorescence (IF) were purchased from the corresponding commercial suppliers, and their detailed information is listed as follows: GAPDH (Proteintech, USA; 10494-1-AP; WB 1:8000), α-smooth muscle actin (α-SMA) (Proteintech, USA; 14395-1-AP; WB 1:4000), Collagen type III (Proteintech, USA; 22734-1-AP; WB 1:800), FUNDC1 (Proteintech, China; AWA51295 ; IF 1:100, WB 1:1000), LC3B (Proteintech, USA; 14600-1-AP; IF 1:500, WB 1:4000), MFN1 (Proteintech, USA; 13798-1-AP; WB 1:5000), MFN2 (Proteintech, USA; 12186-1-AP; WB 1:20000), DRP1 (Proteintech, USA; 12957-1-AP; WB 1:5000), P53 (Proteintech, USA; 10442-1-AP; WB 1:20000), P16 (Proteintech, USA; 10883-1-AP; WB 1:3000), NEDD8 (Abways, China; AWA11003 ; WB 1:1000), and CUL4B (Abways, China; AWA48229 ; WB 1:1000).

Techniques: Ubiquitin Proteomics, Expressing, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Fibroblast growth factor-inducible 14 accelerates pulmonary fibrosis by inducing fibroblast senescence in mice

doi: 10.1007/s00018-026-06161-w

Figure Lengend Snippet: Activation of Fn14 inhibits mitophagy in fibroblasts. A , primary fibroblasts were treated with rTWEAK (100 ng/mL) for 48 h. Cell samples were harvested after 48 h. Volcano plot showing the changes of fibroblast genes (fold change ≥ 2). B , KEGG enrichment analysis for differentially expressed genes in Control vs. rTWEAK-treated primary fibroblasts. C , Mitochondrial membrane potential was measured by JC-1 staining. D , the expression of Mfn1 , Opa1 , and Dnm1 mRNA in primary fibroblasts was detected by Real-time PCR ( n = 3). E-J , The protein levels of L-OPA1, MFN1, FUNDC1, PINK1, and LC3 were determined by Western blot analysis ( n = 3). K , Immunofluorescence and confocal microscopy show the colocalization of Mito Tracker (green) and LAMP1 (red) in rTWEAK-treated primary fibroblasts, scale bar = 50 μm. * P < 0.05 and ** P < 0.01

Article Snippet: Anti-FUNDC1 monoclonal antibody , CST , 49,240 , 1: 2000.

Techniques: Activation Assay, Control, Membrane, Staining, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Confocal Microscopy