Journal: iScience

Article Title: Targeting host lipogenesis with a diarylamide inhibitor disrupts SARS-CoV-2 replication

doi: 10.1016/j.isci.2026.117111

Figure Lengend Snippet: NCOA1 is a host factor targeted by compound 10 to regulate lipid metabolic programs (A) Validation of DARTS assay by western blot in Huh-7 cell lysates. Compd. 10 (50 μM) treatment increases the protease resistance of NCOA1 protein. (B and C) CETSA analysis reveals that compd. 10 (50 μM) induced thermal stabilization of NCOA1 is abolished by D276A and R281A mutations. Experiments were performed in HEK293T cells. (D and E) Western blot analysis shows a dose-dependent reduction of NCOA1 protein levels by compd. 10 . Data were analyzed by one-way ANOVA followed by Dunnett’s test (∗∗ p < 0.01). (F and G) CHX chase assay demonstrates accelerated degradation of NCOA1 in the presence of compd. 10 (8 μM). Student’s t test was used for statistical analysis (∗ p < 0.05, ∗∗ p < 0.01). (H and I) CHX chase analysis shows reduced stability of PPARγ protein upon compd. 10 (8 μM) treatment. Student’s t test was used for statistical analysis (∗∗ p < 0.01). (J and K) Western blot analysis shows that autophagy-lysosome inhibitors (3-MA, BafA1), but not the proteasome inhibitor MG132, rescue NCOA1 protein levels reduced by compd. 10 (8 μM), indicating lysosome-dependent degradation. Data were analyzed by two-way ANOVA followed by Šídák’s test (∗∗ p < 0.01, ∗∗∗ p < 0.001). Data are presented as mean ± SD from at least three independent experiments.

Article Snippet: Bafilomycin A1 (BafA1) , MCE , Cat# HY-100558.

Techniques: Biomarker Discovery, Western Blot

Journal: Advanced Science

Article Title: CDK4/MERCs/PINK1 Axis Drives PFOA/HFPO‐TA‐Induced Cardiac Senescence via Mitophagy Defect and cGAS‐STING Activation: In Vitro Amelioration by Cycloastragenol

doi: 10.1002/advs.77280

Figure Lengend Snippet: PFOA/HFPO‐TA impairs PINK1 mitochondrial localization and autophagic flux in HL‐1 cells. (A) Microscopic observation was made after culture. (B) MMP and ROS levels. (C) Representative IF images of PINK1. HL‐1 cells were transfected with Mito‐DsRed. (D) Western blot analysis of p62 and LC3‐II/I protein expression under Baf A1 treatment. (E) Western blot analysis of p21 and p16 protein expression under Baf A1 treatment. (F‐G) Quantitative results of proteins. (H) SA‐β‐gal staining under Baf A1 treatment. (I) Schematic diagram illustrating impaired PINK1 mitochondrial localization and mitophagic flux caused by MERCs disruption. Data are presented as the mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For cellular stimulation, HL‐1 cells were treated with different reagents, including 20 nM Baf A1 and 1 μM H‐151 (MCE, USA).

Techniques: Transfection, Western Blot, Expressing, Staining, Disruption

Journal: bioRxiv

Article Title: SH3KBP1/CIN85, a new actor of ER-phagy in muscle

doi: 10.64898/2026.07.15.737746

Figure Lengend Snippet: SH3KBP1 downregulation affects basal autophagy in muscle cells. ( A ) Representative confocal images of C2C12 myoblasts stably expressing control shRNA (shCTRL) or a SH3KBP1-targeting shRNA (shSH3KBP1) and transfected with the tandem fluorescent reporter mRFP-GFP-LC3. The cells were treated with DMSO (vehicle) or BafA1. GFP + RFP + vesicles (yellow) correspond to APs, whereas RFP + -only vesicles (red) correspond to ALs. Scale bar: 30 µm (overview images); 10μm (insets). B ) Quantification of autophagic activity expressed as the ratio of GFP + RFP + vesicles on total RFP + vesicles in shCTRL and shSH3KBP1 C2C12 cells; n=3 independent experiments. ( C and D ) Representative western blot analysis showing the kinetics of LC3B-II accumulation in shCTRL and shSH3KBP1 C2C12 myoblasts ( C ) or in 5-days differentiated myotubes ( D ) treated with 100 nM BafA1 for the indicated periods of time and the corresponding quantification of LC3B-II levels normalized to ACTB levels; n= 3 independent experiments. ( E and F ) Rescue experiment. Representative western blot analysis of the kinetics of LC3B-II accumulation in shCTRL and shSH3KBP1 C2C12 myoblasts transfected with an empty vector (pCVM-GFP) or shSH3KBP1 myoblasts re-expressing a full-length flag-tagged SH3KBP1 (Flag-SH3KBP1_FL) and then treated with 100 nM BafA1 for the indicated times ( E ) and its quantification as a ratio between LC3B-II levels and ACTB levels ( F ); n=3 independent experiments. Data are represented as mean +/-SD. Statistical significances between shCTRL and shSH3KBP1 C2C12 cell lines were assessed using Mann-Whitney tests.

Article Snippet: BafA1 (MedChemExpress, HY-100558; 100 nM,) was used to block the autophagic flux.

Techniques: Stable Transfection, Expressing, Control, shRNA, Transfection, Activity Assay, Western Blot, Plasmid Preparation, MANN-WHITNEY

Journal: bioRxiv

Article Title: SH3KBP1/CIN85, a new actor of ER-phagy in muscle

doi: 10.64898/2026.07.15.737746

Figure Lengend Snippet: SH3KBP1 associates to ER and autophagosomes. ( A and B ) Representative confocal images of C2C12 myoblasts ( A ) and 5 days-differentiated myotubes ( B ) co-immunostained for SH3KBP1 (red) and CKAP4 (green). Nuclei were stained with DAPI (blue). Scale bar: 30 µm (overviews images); 10μm (insets in A and B). n=4 independent experiments ( C ) Representative western blot of co-immunoprecipitation experiments performed using GFP-Trap magnetic particles in C2C12 myoblasts transiently transfected with either CMV-GFP vector or the expression vector for full-length GFP-tagged-SH3KBP1 (SH3KBP1_FL). The day after, cells were either left untreated (DMSO) or submitted to autophagy induction by EBSS combined to BafA1 (100 nM) or by LOP (15 µM) combined to BafA1 for 4 hours. Input and immunoprecipitation (IP) fractions were analyzed with GFP, CKAP4 and LC3 antibodies. ( D ) Quantification of CKAP4-SH3KBP1 complexes assessed by measuring CKAP4 levels in the IP-fraction, normalized to immunoprecipitated GFP-SH3KBP1 (IP-fraction). Data are presented as mean +/- SD; n=5 independent experiments. ( E ) Quantification of LC3B-II-SH3KBP1 complexes as in (D); n=4 independent experiments. ( F ) Representative western blots of GFP-Trap experiments performed in C2C12 cells transfected with pCMV-GFP or expression vectors of GFP-tagged full-length SH3KBP1 (SH3KBP1_FL), GFP-tagged N-ter part of SH3KBP1 (SH3KBP1_Nter) or GFP-tagged C-ter part of SH3KBP1 (SH3KBP1_Cter). GFP and LC3B were revealed in the IP and input fractions. ( G ) Quantification of SH3KBP1-LC3B-II complexes as shown in (D). Data are presented as mean +/- SD; n=5 independent experiments. Statistical significances were assessed using Welch’s ANOVA tests, followed by Games-Howell post hoc tests.

Article Snippet: BafA1 (MedChemExpress, HY-100558; 100 nM,) was used to block the autophagic flux.

Techniques: Staining, Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: SH3KBP1/CIN85, a new actor of ER-phagy in muscle

doi: 10.64898/2026.07.15.737746

Figure Lengend Snippet: SH3KBP1 is degraded by ER-phagy. ( A, B, C and D ) Representative confocal images of SH3KBP1 (red) co-immunostained with LC3B (green, A ) or LAMP1 (green, B ) in C2C12 myoblasts ( A and C ) and 5 days-differentiated myotubes ( B and D ). Cells were left untreated (DMSO, vehicle) or treated with BafA1 (100 nM, 6 h) or EBSS+BafA1 (6 h) ( A and B ) or with EBSS (4h) or LOP (15 µM, 6 h) ( C and D ). Nuclei were stained with DAPI (blue). Scale bar: 30 µm (overviews images); 10μm (insets). Quantifications show the number of SH3KBP1-LC3B ( A and B ) or SH3KBP1-LAMP1 ( C and D ) colocalization events per cell. Data are presented as mean +/- SD; n=3. Statistical significance was assessed using Kruskal-Wallis test followed by Dunn’s multiple comparisons test. ( E and F ) Representative western blot showing SH3KBP1, LC3B and ACTB (loading control) in C2C12 cells treated with LOP (15 µM, 0 to 6 h) ( E ), and the corresponding quantification of SH3KBP1 levels normalized to ACTB levels ( F ). Data are presented as mean +/- SD; n=3 independent experiments. Statistical analysis was performed using Welch’s ANOVA followed by Games-Howell multiple comparisons test. ( G and H ) Representative western blot showing SH3KBP1, LC3B and ACTB (loading control) in C2C12 cells treated with BafA1 (100nM, 0 to 6h) ( G ), and the corresponding quantification of SH3KBP1 levels normalized to ACTB levels ( H ). Data are presented as mean +/- SD; n=5 independent experiments. Statistical analysis was performed using Kruskal-Wallis followed by Dunn’s multiple comparisons test.

Article Snippet: BafA1 (MedChemExpress, HY-100558; 100 nM,) was used to block the autophagic flux.

Techniques: Staining, Western Blot, Control