Journal: bioRxiv

Article Title: Adaptive regulation of dNTP homeostasis confers osimertinib resistance in EGFR mutant non-small cell lung carcinoma

doi: 10.64898/2025.12.24.696437

Figure Lengend Snippet: (A) A549, PC-9, and HCC827 cells were treated with 5 nM osimertinib (Osi). Protein levels of phosphorylated CHK1 (p-CHK1), phosphorylated CHK2 (p-CHK2), total CHK1, and CHK2 were analyzed by western blotting. GAPDH served as a loading control. Data are representative of three independent experiments. (B) Cells were treated as in panel A, with MG132 added 2 hours before harvest. Protein levels of p-CHK1, p-CHK2, CHK1, and CHK2 were assessed by western blotting. ACTB was used as a loading control. Data represent three independent experiments. (C) Proliferation assays in HCC827 and PC-9 cells treated with increasing concentrations of osimertinib alone or in combination with PV1019 (CHK2 inhibitor, concentration specified). (D) PC-9 cells were treated with DMSO, 5 nM Osi, 10 µM PV1019, or the combination for the indicated times. Nuclear fractions were analyzed by western blotting for RRM1, RRM2, RRM2B, POLD1, POLH, and TNNT3. ACTB and Histone H3 were used as loading controls. (E) PC-9 cells were treated with DMSO, 5 nM Osi, 10 nM LY2606368 (CHK1/CHK2 inhibitor), or the combination for the indicated times. Nuclear extracts were analyzed as in panel D. Data are representative of three independent experiments.

Article Snippet: RRM1 (#10526-1-AP), RRM2 (11661-1-AP), RRM2B (#18005-1-AP), CHK1 (#25887-1-AP), CHK2 (#13954-1-AP), EGFR (#66455-1-Ig), beta Actin (#20536-1-AP), HA tag (#51064-2-AP), Histone H3 (#17168-1-AP), GAPDH (#60004-1-Ig), POLD1(#15646-1-AP), POLH (#28133-1-AP), MYBL2 (#18896-1-AP) and TNNT3 (#19729-1-AP) were purchased from Proteintech.

Techniques: Western Blot, Control, Concentration Assay

Journal: Biomolecules

Article Title: Mitochondrial Dysfunction and Protein Homeostasis in Aging: Insights from a Premature-Aging Mouse Model

doi: 10.3390/biom14020162

Figure Lengend Snippet: Ubiquitin–proteasome system activity in the liver: ( A – C ) caspase- (β1), trypsin- (β2), and chymotrypsin-like (β5) activities of the 20S proteasome in liver lysates from 45 ± 2-week-old wild-type mice (WT), >120-week-old wild-type mice (old-WT), and 45 ± 2-week-old mtDNA-mutator mice (Mut). Mitochondrial dysfunction and aging both significantly affected chymotrypsin-like activity. The activities for WT vs. old-WT vs. Mut were caspase-like: 2820 ± 114 vs. 3080 ± 56 vs. 2560 ± 146 FU/h; trypsin-like: 2650 ± 206 vs. 4800 ± 137 vs. 2350 ± 198 FU/h; and chymotrypsin-like: 4370 ± 372 vs. 2890 ± 197 vs. 2940 ± 91.37 FU/h (one-way ANOVA and Tukey’s post hoc analysis). ( D , E ) Level of 20S proteasome catalytic subunits β1 (PSMB1), β2 (PSMB2), and β5 (PSMB5) were assessed by western blot. No differences in protein expression were found between the three groups. One representative western blot and the quantification of the protein levels calculated by normalizing the band intensity with the corresponding actin in six mice are shown. ( F , G ) Accumulation of poly-ubiquitinated proteins was assessed by western blot. One representative western blot and the quantification of ubiquitinated protein levels in six mice are shown (one-way ANOVA and Tukey’s post hoc analysis). Data shown are mean ± S.E.M with p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.0001 (****); ns = non-significant; FU = fluorescence unit, AU = arbitrary unit. Original figures can be found in .

Article Snippet: For fluorescence analysis of mitochondria, fibroblasts were grown on round cover slips and transfected with a mitochondrial marker plasmid (mitoKaede expressing plasmid #28133, Addgene, Cambridge, MA, USA) [ ].

Techniques: Ubiquitin Proteomics, Activity Assay, Western Blot, Expressing, Fluorescence

Journal: Biomolecules

Article Title: Mitochondrial Dysfunction and Protein Homeostasis in Aging: Insights from a Premature-Aging Mouse Model

doi: 10.3390/biom14020162

Figure Lengend Snippet: Summary of alterations of proteasomal and lysosomal activity in liver and cerebellum caused by mitochondrial dysfunction and aging.

Article Snippet: For fluorescence analysis of mitochondria, fibroblasts were grown on round cover slips and transfected with a mitochondrial marker plasmid (mitoKaede expressing plasmid #28133, Addgene, Cambridge, MA, USA) [ ].

Techniques: Activity Assay