Caco2 response to methotrexate, lactoferrin, and methotrexate-lactoferrin co-exposure

bulk sequencing outputs of Caco2 cells after exposure to permeability modifying and permeability rescuing agents. To identify the molecular drivers for methotrexate-induced barrier dysfunction, we conducted RNA sequencing on Caco2 spheroids treated with methotrexate and lactoferrin. Given our observation that barrier function was compromised as early as 4-6 hours after exposure to methotrexate, we isolated RNA from spheroids 4-hour post-treatment to capture the transcriptional events responsible for initiating the processes. Overall design: Caco-2 spheroids were exposed to methotrexate, lactoferrin, and lactoferrin with methotrexate. RNA was extracted using RNeasy Plus Micro kit (Qiagen, Hilden, Germany) after 4 hour exposure as per protocol. The cDNA library preparation and paired-end sequencing was performed by Novogene Corporation Inc. (Sacramento, CA). Analysis was performed using the DEseq2 package in R-Suite (Warsaw, Poland). Differentially expressed genes were identified as genes that had >2-fold change in expression with adjusted p-value less than 0.01. Gene ontology analysis on the top 500 upregulated genes was performed using the PGSEA package in R.