Journal: Aging Cell

Article Title: Down‐Regulation of TFEB With Defective Autophagy in the Susceptibility of Aging Kidneys to Septic Acute Kidney Injury

doi: 10.1111/acel.70644

Figure Lengend Snippet: Modulation of autophagy in senescent BUMPT cells bidirectionally regulates their susceptibility to LPS‐induced injury. (A–F) Treatment of LPS‐treated senescent BUMPT cells with Tat‐Beclin 1 peptide (30 μM) and chloroquine (20 μM), respectively. (A) Representative immunoblots of LC3‐I/II, c‐CAS3 and GAPDH expression of BUMPT cells treated with Tat‐Beclin 1 peptide, and (B) corresponding densitometric quantification. (C) Representative immunoblots of LC3‐I/II, c‐CAS3 and GAPDH expression of BUMPT cells treated with chloroquine and (D) corresponding densitometric quantification. (E) Representative images of TUNEL assay (red). Hochest was used to stain the nucleus (blue); scale bar = 50 μm. (F) Relative mRNA expression levels of the genes encoding Il‐6, Mcp‐1 and Tnfα , assessed by RT‐qPCR. All quantitative data are expressed as mean ± SEM. * p < 0.05.

Article Snippet: For LPS treatment, cells were incubated with DMEM medium containing 0.2% FBS and 10 μg/mL LPS for 24 h. For Tat‐Beclin 1 peptide (HY‐P2260, Medchemexpress) treatment, cells were incubated with DMEM medium containing 0.2% FBS and 30 μM Tat‐Beclin 1 peptide for 8 h. For chloroquine (C6628, Sigma‐aldrich) treatment, cells were incubated with DMEM medium containing 0.2% FBS and 20 μM for an additional 8 h. For detection of autophagic flux in cells, normal BUMPT cells and senescence‐induced BUMPT cells were transiently transfected with mRFP‐GFP‐LC3 (ptfLC3, 21,074, Addgene).

Techniques: Western Blot, Expressing, TUNEL Assay, Staining, Quantitative RT-PCR

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: BMC Molecular and Cell Biology

Article Title: Alcohol exposure induces ferroptosis-dominated programmed cell death in esophageal epithelial cells

doi: 10.1186/s12860-026-00589-5

Figure Lengend Snippet: A short-term exposure to 5.0% ethanol triggers autophagy that supports cell growth. A . An equal number of HEEC cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 5 min, 30 min, or 2, 6, and 12 h. Cells were fixed in 4% paraformaldehyde and stained for LC3B. An FITC-conjugated secondary antibody was used to develop the signal. Cell nuclei were counterstained with DAPI. Scale bar = 5 μm. B . An equal number of HEEC cells was seeded in 96-well plates and incubated with complete medium (control), medium containing 5.0% ethanol, or medium containing 5.0% ethanol plus 10 µM Chloroquine (CQ) for 5 min, 30 min, 2 h, 6 h, or 12 h. Cell viability was assessed using CCK-8 kits. * indicates a significant change between EtOH and EtOH/CQ. C . An equal number of Het1A cells was seeded in 96-well plates and incubated with the complete medium containing DMSO (control), the medium containing 5.0% ethanol, or the medium containing 5.0% ethanol plus 10 µM Chloroquine (CQ) for 5 min, 30 min, 2 h, 6 h, or 12 h. Cell viability was assessed using CCK-8 kits. * indicates a significant change between EtOH and EtOH/CQ. D . An equal number of HEEC or Het1A cells was seeded on the coverslips and incubated with the complete medium containing DMSO (control), the medium containing 5.0% ethanol, or the medium containing 5.0% ethanol plus 10 µM Chloroquine (CQ) for 30 min. Cells were fixed in 4% paraformaldehyde and stained for PCNA. An FITC-conjugated secondary antibody was used to develop the signal. Cell nuclei were counterstained with DAPI. Scale bar = 5 μm. E . An equal number of HEEC or Het1A cells was seeded in 6-well plates and incubated with complete medium containing DMSO (control), medium containing 5.0% ethanol, or medium containing 5.0% ethanol plus 10 µM Chloroquine (CQ) for 30 min. Protein was extracted and analyzed by Western blotting. The membranes were probed for LC3B, PCNA, Beclin-1, or GAPDH. F . Quantitative analyses of LC3B, PCNA, and Beclin-1 expression against GAPDH in HEEC cells. * indicates a significant change compared to the control (DMSO). G . Quantitative analyses of LC3B, PCNA, and Beclin-1 expression against GAPDH in Het1A cells. * indicates a significant change compared to the control (DMSO)

Article Snippet: The following primary antibodies were used: Beclin-1 (Origene, #TA502643), PCNA, CASP1 (Abcam, #ab179515), CASP3 (Santa Cruz Biotechnology, #sc-271759), CASP8 (Origene, #TA374288), CASP9 (Origene, #TA4227045), endoG, GPX4 (Origene, #TA423164M), GSDMD, IL-1β (Santa Cruz Biotechnology, #sc-12742), MLKL, phosphor-MLKL (Abcam, #ab196436), SLC7A11 (Origene, #TA423232), LC3B, BAX, GAPDH (OriGene, #TA800894), and β-actin (Santa Cruz Biotechnology, Inc. #sc-69879).

Techniques: Incubation, Control, Staining, CCK-8 Assay, Western Blot, Expressing