**5. Notes and limitations**

As shown in **Figure 5**, the level of the LC3B-II proteins is generally lower than LC3B-I in cells grown at normal condition. However, following the inhibition with LY294002, the conversion of the LC3B-I to LC3B-II increases dramatically especially for the cancer cell lines. This shows an increase in the formation of the autophagophore in response to the inhibition. This is similar to the findings of Luo et al. where the inactivation of the PI3K/Akt pathway results in an increase in expression of LC3B-II proteins [62]. Meanwhile, following starvation, the cell lines also showed a marked increase in conversion of LC3B-I to LC3B-II indicating increase in autophagy activity. The increase in formation of autophagosome in cells undergoing starvation and inhibition by LY294002 is due to the effect of the PI3K pathway on the mammalian target of rapamycin complex 1 (mTORC1). In nutrient-deprived cells, IκB kinase (IKK) expression has been shown to be upregulated, while p85 regulatory subunit of PI3K has been shown to be a substrate of IKK. During starvation, the increase in IKK expression leads to the increase in phosphorylation of the p85 subunit of PI3K leading to inactivation of PI3K pathway [63]. The inactivation of the PI3K pathway inactivates mTORC1, which has an inhibitory effect on ULK1–Atg13–FIP200 complex, an autophagy initiation complex [64, 65]. So the inactivation of the PI3K pathway in both starvation and LY294002 treatment leads to the activation of autophagy by ULK-1 complex. However, the monitoring of only one protein marker is not enough to conclusively indicate the effect of

**Figure 5.** Western blot analysis of LCB-I and LCB-II. Top panel: Western blot analysis of starvation-induced and PI3Kinhibited colorectal cancer cell lines: HT29, HCT116, and CCD112. Cells were seeded at 70,000 per well and incubated for 24 hours prior to treatment. Each cell line was subjected to two different treatments: Starvation in serum-free media and inhibition of PI3K in media containing 50 μM of LY294002. Cells were incubated for 24 hours prior to harvesting. Cell lysates were run on 4–20% gradient gels under reducing conditions, and proteins were immunodetected on a PVDF membrane with rabbit anti-LC3B Mab (#3868) and mouse anti-β-actin Mab (#3700) from cell signaling technology. Both antibodies were diluted to 1:2000 with 5% milk in TBST. The bands were subsequently visualized with HRP-labeled anti-rabbit IgG antibodies (#7074) for LC3B and anti-mouse IgG antibodies (#7076) for β-actin (cell signaling technology) diluted at 1:3000 with 5% milk in TBST. Bottom panel: Densitometry analysis of protein bands. The analysis was done by using image J, and the relative protein levels were calculated by dividing absolute protein level of LC3B with β-actin.

the treatment.

88 Cell Culture

The enclosed protocols in this chapter focus on evaluating autophagy using in vitro cancer cell lines but not limited to them. It should be noted that this fundamental cellular mechanism can be detected and studied in other cell types such as leucocytes, fibroblasts, stem cells, and so on. Autophagy is also extensively studied in fixed and live tissues (which are not discussed here) with regard to cancers and other diseases. We have only included Western blot and immunofluorescence protocols because of their simplicity and cost-effectiveness. Due to availability, colorectal cancer cell lines were used as study models for autophagy in this chapter. It must be noted that the expression pattern of studied proteins may vary among cell lines and across different cell types. Hence, the enclosed data should only be used as a reference. Researchers are advised to perform their own optimization experiments and baseline studies based on the given protocols. There are numerous varying parameters that may contribute to varying outcomes including brand and manufacturer of reagents and consumables, ambient conditions, personnel, instrumentation, and so on. Here, we have only targeted one of the autophagy effectors, LC3B for demonstration. It should be noted that there is a list of autophagy-related proteins/mRNA/DNA (described in Section 3) that can be studied according to the researchers' target of interest with respect to the nature of the research project. In addition, a plethora of autophagy-associated inducer or inhibitor (described in Section 4) can be chosen to study a specific protein/mRNA or pathway in autophagy. Last but not least, to further understand how autophagy functions and its association with a disease or disorder, it is always more favorable to study two or more autophagy-related targets concurrently to maximize the gained output and cost-effectiveness. Our enhanced understanding on autophagy and the development of technology allowed the study of autophagy to be made easier through panel assays such as Autophagy Regulators Panel (Millipore), CYTO-ID Autophagy detection kit (Enzo Life Sciences), Autophagy Antibody Sampler Kit (Cell Signaling Technology), and Autophagy Detection Kit (Abcam). Newly engineered study models such as ATG, p62, and ULK-1 knockout cell lines and animals have also been generated for in-depth study of autophagy pathway. In addition, the advancement in bioinformatics also helps in data organization and analysis as well as deciphering the potential interaction of autophagy with other unexplored cellular pathways.
