Journal: bioRxiv

Article Title: Fasting disrupts the InsP₆–HDAC3 axis to drive ER stress–mediated clearance of DNA-damaged cells and enforce tissue quality control

doi: 10.64898/2026.05.24.727426

Figure Lengend Snippet: (A) Workflow for proteomic analysis of FACS-purified IECs from Ileum. Schematic created in BioRender (CHATTERJEE, S., 2026; https://BioRender.com/mtrovit ). (B) Volcano plot showing differentially expressed proteins in starved versus untreated mouse crypts. (C) Pathway enrichment analysis of upregulated proteins. (D) Heatmap of selected proteins associated with ER stress (red), integrated cellular stress (blue), and starvation responses (green, positive control), highlighting CDK5RAP3. (E) Network visualization of enriched biological processes and pathways related to cellular stress responses, including ER stress, unfolded protein response, and proteotoxic stress. (F) Immunoblot analysis of CDK5RAP3, CHOP, IRE1, and calreticulin in untreated, starved, and refeed conditions. The tests were conducted under two different exposures, 3 sec blue and 45 sec pink, and were validated using antibodies from Proteintech yellow and Bethyl green. (G) Volcano plot of bulk RNA sequencing from purified crypts of untreated and starved mice, highlighting CDK5RAP3 among differentially expressed transcripts during fasting. (H) Heat map of CUT&RUN analysis of H3K27ac signal in untreated and starved samples (highlighted blue). (I) Overlap of RNA-seq, and CUT&RUN datasets. (J) Genome browser tracks showing H3K27ac enrichment at the CDK5RAP3 transcription start sites (highlighted blue). (K) Immunoprecipitation of pan-acetylated proteins from 400 μg protein lysate followed by immunoblotting for CDK5RAP3 from untreated and starved samples. Data represent n = 3 independent biological replicates unless otherwise indicated; mean ± SD where applicable.

Article Snippet: Eluted DNA from CUT&RUN was used for library construction with the CUTANATM CUT&RUN Library Prep Kit (EpiCypher, Cat. #14-1001 and #14-1002), according to the manufacturer’s instructions.

Techniques: Purification, Positive Control, Western Blot, RNA Sequencing, Immunoprecipitation

Journal: bioRxiv

Article Title: Fasting disrupts the InsP₆–HDAC3 axis to drive ER stress–mediated clearance of DNA-damaged cells and enforce tissue quality control

doi: 10.64898/2026.05.24.727426

Figure Lengend Snippet: (A) Experimental conditions including untreated (UT), heat shock (HS), cold shock (CS), osmotic stress (OS), oxidative stress (OXS), UV-induced DNA damage (UV), amino acid deprivation (A.A), and growth factor deprivation (GFD) followed by western blot. Created in BioRender. CHATTERJEE, S. (2026) https://BioRender.com/mtrovit . (B) Immunoblot analysis of pan-histone acetylation (PanAc) and total H3 in HEK, HCT116, and MEF cells under the indicated conditions. (C) Quantification of PanAc normalized to H3 in HEK, HCT116, and MEF cells (n = 3; mean ± SD). (D) Heat map of CUT&RUN analysis showing H3K27ac signal distribution in UT and GFD conditions in HCT116 cells. (n = 2). (E) Genome browser tracks showing H3K27ac enrichment at the CDK5RAP3 locus in untreated and GFD HCT116 cells. (n = 2). (F) Heatmap and volcano plot of gene expression changes under GFD in HCT116 cells highlighting CDK5RAP3. (G) Overlap of CDK5RAP3-associated datasets from HCT116 and mouse samples. (H) Immunoprecipitation of acetylated proteins (from 400 μg lysate) followed by immunoblotting for CDK5RAP3 from HDAC1 and HDAC3 knockout HCT116 cells under UT and GFD conditions. (n=3). (I) Immunoblot analysis of CDK5RAP3, H3K27ac, H4K16ac, and histone controls following treatment with histone acetyltransferase inhibitors under GFD conditions. MG149 (35µM) Tip60 inhibitor, A485 (10µM) P300 inhibitor treated in GFD for 12 hours.(n=3) (J) Immunoblot analysis of CDK5RAP3, RPL26 UFMylation, CHOP, and IRE1 in UT and GFD HCT116 cells.(n=3) (K) Schematic of HDAC3 PROTAC-mediated degradation. Created in BioRender (CHATTERJEE, S., 2026; https://BioRender.com ). Western blot analysis in HCT116 cells assessed the time-dependent degradation of HDAC3 after PROTAC treatment and the effect of compound washout. (L) Immunoblot and quantification of IPMK and IPPK protein levels in HCT116 cells (n = 3 to 13; mean ± SD). (M) ChIP–qPCR analysis of CDK5RAP3 under UT, GFD, and GFD+CP-InsP₆ treated HCT116 cells (n = 3; mean ± SD). (N) Immunoprecipitation of CDK5RAP3 from 400 μg of protein lysate from HCT116 cells followed by immunoblotting for acetylation under indicated conditions. (n = 3) All dots in bar plots represent individual biological replicates.

Article Snippet: Eluted DNA from CUT&RUN was used for library construction with the CUTANATM CUT&RUN Library Prep Kit (EpiCypher, Cat. #14-1001 and #14-1002), according to the manufacturer’s instructions.

Techniques: Western Blot, Gene Expression, Immunoprecipitation, Knock-Out, ChIP-qPCR