Journal: bioRxiv
Article Title: Pasta, an age-shift transcriptomic clock, maps the chemical and genetic determinants of aging and rejuvenation
doi: 10.1101/2025.06.04.657785
Figure Lengend Snippet: ( a ) Representative images from Senescence Associated Beta Galactosidase assay performed on A-375 cells exposed to Pralatrexate (Prltx). Doxorubicin (Doxo) was used as positive control for senescence induction. Scale Bar: 10 μm. ( b ) qPCR analysis of CDKN1A and IL6 mRNA levels in A-375 cells in response to Prtlx and Doxo.; N=3 biological replicates; *** p< 0.001 (one-way ANOVA). ( c ) Cell proliferation assay by Edu incorporation performed in A-375 cells exposed to Prltx and Doxo (one representative experiment with n=3 replicates/condition). *** p< 0.001 (one-way ANOVA, calculated on Edu + cells). ( d ) Immunoblotting showing LMNB1, CDKN1A and GAPDH protein levels in A-375 cells exposed to Prltx and Doxo (N=3 biological replicates). ( e ) Representative images from Senescence Associated Beta Galactosidase assay performed on MDA-231 cells exposed to Pralatrexate (Prltx). Doxorubicin (Doxo) was used as positive control for senescence induction. Scale Bar: 10 μm. ( f ) qPCR analysis of CDKN1A and IL6 mRNA levels in MDA-231 cells in response to Prltx and Doxo.; N=3 biological replicates; *p< 0.05 (one-way ANOVA). ( g ) Cell proliferation assay by Edu incorporation performed in MDA-231 cells exposed to Prltx and Doxo (one representative experiment with n=3 replicates/condition). ** p< 0.01; *** p< 0.001 (one-way ANOVA, calculated on Edu + cells). ( h ) Immunoblotting showing LMNB1, CDKN1A and GAPDH protein levels in A-375 cells exposed to Prltx and Doxo (N=3 biological replicates). (i) qPCR analysis of Oct4, Sox2 and Nanog levels in PC3 cells exposed to Piperlongumine (Piplng). * p< 0.05 Student’s t test. (j) Immunoblotting showing OCT3/4 and alpha-tubulin protein levels in PC3 cells incubated with Piplng..
Article Snippet: Non-specific binding sites were saturated by incubating membranes for 1□ h in 0.05% Tween 20 (#P9416, Sigma Aldrich) v-v in Tris-buffered saline (TBS) supplemented with 5% BSA (Sigma-Aldrich, #10735078001, w:v in TBS), followed by an overnight incubation with primary antibodies specific for Waf1/Cip1/CDKN1A p21 (Santa Cruz Biotechnology, #sc-6246), LaminB1 (12987-1-AP, Proteintech) and Oct3/4 (sc-5279, Santa Cruz Biotechnology).
Techniques: β-Gal Assay, Positive Control, Proliferation Assay, Western Blot, Incubation
Santa Cruz Biotechnology is a verified supplier 
