Western Blot Analysis of Microtubule-Associated Protein Light Chain 3 to Monitor Autophagosomal Dynamics in...

Autophagy plays a complex role in pancreatic ductal adenocarcinoma (PDAC), contributing to tumor progression, stress adaptation, and therapy resistance. Accurate assessment of autophagosomal dynamics is therefore essential for studies of cancer biology. Among the available methods, immunoblotting of microtubule-associated protein light chain 3 (LC3) is widely used to monitor autophagosomal dynamics by distinguishing between the cytosolic (LC3-I) and lipidated, autophagosome-associated (LC3-II) forms. However, the low molecular weight of LC3 and the minimal difference in electrophoretic mobility between these isoforms present technical challenges that require careful optimization. Presented here is a reproducible protocol for the semi-quantitative analysis of LC3-II levels by Western blot in KPC-derived murine pancreatic cancer cells. The method incorporates optimized conditions for cell lysis, electrophoresis, protein transfer, and antibody-based detection to ensure reliable separation and detection of LC3 isoforms. In addition, a standardized workflow for densitometric analysis of LC3-II bands using Fiji (ImageJ) is provided. The sensitivity of the method is demonstrated through the detection of increased LC3-II levels under conditions of autophagy induction (gemcitabine treatment) and impaired autophagic flux (VMP1 knockdown). Critical technical parameters that influence data interpretation, including lysis buffer composition and antibody specificity, are also examined, together with common experimental pitfalls. Although LC3 immunoblotting alone is insufficient to fully define autophagic flux, when combined with complementary assays it provides a robust and accessible approach for monitoring autophagic activity. The protocol can be adapted to other experimental contexts, facilitating mechanistic studies of autophagy in cancer models.