**4.6. Evaluation of autophagy by Western blot analysis**

One of the most widely used method for the examination of autophagy activity is by elucidating the protein expression of the autophagy markers through immunoblotting. The fluctuation in the expression can help in showing the effect of different interventions such as gene silencing or inhibition on autophagy activity. The following protocol for determination of autophagy marker expressions is adapted from the general protocol for Western blotting (Bio-Rad).

#### *4.6.1. Materials*

MTT or any real-time luminescence-based assays can be carried out for this purpose. After that, the user should run the silencing experiments using serial-diluted siRNA followed by Western blot to determine what is the minimum effective siRNA concentration for the best gene-silencing results. These steps should be repeated for every cell line that is going to be

Immunofluorescence is a common method of immunostaining. This technique uses the specificity of antibodies to their antigen, a specific biomolecule target within or around a cell for the visualization of the distribution of the target molecules in the cells. Through this technique, researchers can visualize the location of the desired targets as well as qualitatively analyze protein concentration in the cells. The following protocols are carried out in tissue-culture flat-bottom 96-well plate.

Fragment (Alexa Fluor® 488 conjugated)—Cell Signaling

tested using the siRNA because there will be some differences between cell lines.

**4.5. Evaluation of autophagy by immunofluorescence**

**1.** LC3B (D11) XP® Rabbit mAb—Cell Signaling Technology, #3868

*4.5.1. Materials*

84 Cell Culture

*4.5.2. Methods*

to fixation.

15 minutes.

temperature.

X-100 while the cells are blocked.

**2.** Anti-rabbit IgG (H + L), F(ab')2

**3.** DAPI—Cell Signaling Technology, #4083

**4.** Phosphate buffered salts (PBS) tablets—Takara, #T900

**8.** Triton X-100 (for molecular biology)—Sigma, #T8787

**5.** Tissue culture-treated 96-well flat-bottom plate—TPP, #92096

**6.** Albumin, bovine serum, fraction V, low heavy metals—Merck, #12659-100GM **7.** Methanol, methyl alcohol, Grade AR—Riendemann Chmidt, #M2097-1-2500

**1.** Cells were seeded into 96-well plates and grown to a confluency of ~50%.

**2.** Treatments such as starvation and autophagy inhibition were carried out in the well prior

**3.** The spent media were removed and the cells were washed with 1X PBS once, before 200 uL ice-cold 100% methanol was added into each well, followed by incubation in −20°C for

**4.** The methanol was removed from each well, and the wells were washed for three times

**5.** 5% bovine serum albumin (BSA) dissolved in 1X PBS with 0.3% Triton X-100 was prepared, and 50 uL is added into each well for blocking. The cells were incubated for 1 hour at room

**6.** Antibody diluent buffer was prepared by dissolving 1% BSA in 1X PBS with 0.3% Triton

with 200 uL 1X PBS incubated for 5 minutes between each wash on the bench.

Technology, #4412

	- **a.** HT-29 (ATCC HTB-38)
	- **b.** HCT 116 (ATCC CCL-247)
	- **c.** CCD-112CoN (ATCC CRL-1541)

*4.6.2. Methods*

mixing.

were kept.

media with the inhibitor.

protease inhibitor cocktail.

the well of a gradient gel.

TBST at 1:2000 dilution.

loading buffer and 2 μL of 2-mercaptoethanol.

using FlashBlot transfer buffer at 55 V for 1 hour.

prevent non-specific binding of antibody.

and 5 minutes for another 2 times.

**a.** Anti-rabbit secondary antibody: LC3B

**b.** Anti-mouse secondary antibody: β-Actin

proteins were viewed by using ImageQuant LAS 500.

**1.**In a 24-well plate, the cells were seeded at 50% confluency and left to incubate overnight. **2.**The media were then changed accordingly, with normal media, serum-free media, and

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**4.**The cells were then lysed with lysis cocktail consisting of 1X RIPA lysis buffer and 1X

**5.**The lysates were vortexed briefly every 5 minutes for 30 minutes and kept in ice in between

**6.**The lysates were then centrifuged at 16000×g for 15 minutes at 4°C, and the supernatants

**7.**The lysates were then quantified by using BCA protein assay and immediately diluted with deionized water to obtain 13 μL of 20 μg lysate and then added to 5 μL of 4X SDS

**8.**The samples were boiled at 90°C for 10 minutes, and 10 μL of the samples were loaded into

**9.**The lysates were separated by SDS electrophoresis by using Tris/Glycine/SDS running buffer at 150 V for approximately 45 minutes and transferred to a PVDF membrane by

**10.**The membrane was then blocked by using 5% Blotting Grade Blocker in TBST for 1 hour to

**11.**The membrane was then incubated with primary antibody diluted with the Blocker in

**13.**Any unbound antibodies were then removed by washing with TBST for 10 minutes once

**14.**Secondary antibodies conjugated with horseradish peroxidase (HRP) were then prepared

**16.**The membrane was incubated for 1 hour at room temperature, and unbound antibodies are washed away with TBST once for 10 minutes and for 5 minutes for another 4 times. **17.**The WesternBright Sirius HRP substrate was then dropped on the membrane, and the

**18.**The blot images were then analyzed using ImageJ software for densitometry analysis.

**12.**The incubation was done overnight at 4°C or 1 hour at room temperature.

by diluting the antibody in the Blocker in TBST at 1:3000 dilution.

**15.**The membrane was then incubated in the secondary antibody:

**3.**After 24-hour incubation, the cells were collected and rinsed with PBS.

**Figure 4.** Immunofluorescence analysis of autophagy marker LC3B expression in colon cells after treatment. Immunofluorescence analysis of starvation-induced and PI3K-inhibited 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 fixation and staining. The DAPI-stained nuclei is in blue, while the LC3B expression is in green (Alexa Fluor 488 conjugated antibody).

	- **a.** β-Actin—#3700
	- **b.** LC3B—#4599
	- **a.** Anti-mouse IgG, HRP-linked antibody—#7076S
	- **b.** Anti-rabbit IgG, HRP-linked antibody—#7074S

### *4.6.2. Methods*

**3.**Protease inhibitor cocktail, 100X—Cell Signaling Technology, #5871

**7.**4–20% Mini-PROTEAN® TGX™ Precast Protein Gels, 15-well—Bio-Rad #4561096

**Figure 4.** Immunofluorescence analysis of autophagy marker LC3B expression in colon cells after treatment. Immunofluorescence analysis of starvation-induced and PI3K-inhibited 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 fixation and staining. The DAPI-stained nuclei is in blue, while

**4.**Pierce BCA protein assay kit—ThermoFisher Scientific, #23227

**6.**Tris/Glycine/SDS running buffer, 10X—Bio-Rad, #1610772

**8.**FlashBlot transfer buffer—Advansta, 150,421–95

**10.**Blotting Grade Blocker—Bio-Rad, #170–6404 **11.**Primary antibody (Cell Signaling Technology)

**12.**Secondary antibody (Cell Signaling Technology)

**a.** Anti-mouse IgG, HRP-linked antibody—#7076S **b.** Anti-rabbit IgG, HRP-linked antibody—#7074S

**13.**WesternBright Sirius HRP substrate—Advansta, 170,501–39

**a.** β-Actin—#3700 **b.** LC3B—#4599

86 Cell Culture

**9.**Immun-Blot PVDF membrane—Bio-Rad, #1620177

the LC3B expression is in green (Alexa Fluor 488 conjugated antibody).

**5.**2-mercaptoethanol—Merck, 60–24-2

	- **a.** Anti-rabbit secondary antibody: LC3B
	- **b.** Anti-mouse secondary antibody: β-Actin

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 the treatment.

**5. Notes and limitations**

unexplored cellular pathways.

**6. Conclusions**

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

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The phenomenon of autophagy has been hinted since decades ago and has been a hot area of research ever since the 1990s. Extensive studies have been done on the characterization, mechanism, function, and its association with a multitude of diseases including cancer. While a simple search of autophagy in Google may yield an insurmountable amount of information, its role, mechanism, and function in relation to cancer are still not fully understood and present multiple contradictions between and within different cancer types. The simplified literature review and the protocols enclosed in this book chapter will hopefully help researchers in further understanding the roles and mechanisms of autophagy in different cancer cell types,

**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.
