**4.4. siRNA knockdown for autophagy**

siRNAs have been developed by companies that specifically targets different genes that produce important proteins that play roles in the activation of autophagy. Some examples of currently available siRNAs are those that target Atg3, Atg5, Atg7, Atg10, Atg12, Atg13, Atg14, and Atg101. When researchers first receive the siRNA, the user should run an optimization experiment to determine a few characteristics of the siRNA. The toxicity of such siRNA should first be tested, and the IC50 should be determined to better understand what concentration range might work the best and have the least impact on cell viability. Cytotoxicity assays such as


**Table 1.** Autophagy inhibitors that can be used in vitro.

**Figure 3.** Spheroid formation of colorectal cancer (CRC) cell lines. 1 × 10<sup>5</sup>

until spheroids were formed. The images above were taken at 72 hours after seeding. HCT116 and HT29 both formed a round spheroid in the well, while SW480 formed an irregular spheroid. No spheroid was formed 72 hours after seeding.

low-attachment 96-well plate and incubated at 37°C at 5% CO2

**4. In vitro studies of cellular autophagy**

82 Cell Culture

**4.1. 2D and 3D establishment using CRC cell lines**

CRC cell lines such as HT29, SW48, HCT116, and SW480 are adherent cell lines. By default, they will grow in normal tissue-cultured flat-bottom plates or flask in a monolayer, which is a 2D structure. However, monolayer morphology is not the natural appearance of all the established cell lines. Therefore, scientists have developed 3D models to bridge the difference between in vitro and in vivo experiments. For 3D model establishment, the cells were seeded into a round-bottom ultra-low-attachment 96-well plate and allowed to settle down and grow. The cells were observed every day until clear spheroids can be seen. It is important to capture images every day until clear spheroids can be seen. Some cell lines such as SW480 might not form clear rounded spheroids. Once the spheroids are formed, it is important to not disrupt spheroid formation when handling it in situations such as changing media. This is because the cells were seeded in ultra-low-attachment plate, which means they no longer attach to the bottom of the well. Careless handling of the spheroids might cause disruption to it or worse, the loss of spheroids. Although the process of 3D formation is straight forward, not all cell lines are able to form spheroids. Such example is the SW48, which when seeded into the well, remained in multiple clusters instead of forming spheroids as shown in **Figure 3**. Through the formation of 3D models, scientists can better study the gene expressions and cell behaviors [52], as well as carry out drug response experiments on a model that better mimics the natural

cells were seeded into round-bottom ultra-

with 95% humidity. The cells were observed every day

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

**7.** Primary antibody was diluted in antibody diluent buffer at the recommended concentra-

Cell-Based Assays for Evaluation of Autophagy in Cancers

http://dx.doi.org/10.5772/intechopen.80088

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**8.** After blocking, the blocking buffer was removed, and 50 uL of the diluted antibody was

**9.** The primary antibody was removed the next day, and the wells were washed as mentioned

**10.**Fluorophore-conjugate secondary antibody targeting the primary antibody is diluted in antibody diluent buffer (Step 6) at the recommended concentration by the manufacturer. For this experiment, Alexa Flour 488-conjugated anti-Rabbit IgG antibody was diluted 1:1000. 50 uL of diluted secondary antibody was added into each well and incubated at

**11.**The Secondary Antibody was removed and the wash step in Step 4 was repeated 5 times.

**12.**DAPI was diluted to a concentration of 0.5 ug/mL in 1X PBS, and 50 uL was added into

**13.**The wells were washed 1 time according to Step 4 and viewed using a fluorescence micro-

Cells were treated with LY294002, which is a cell-permeable inhibitor for phosphoinositide 3-kinase (PI3K) that acts on the enzyme ATP binding site (**Table 1**). As PI3K is strictly required for autophagy, PI3K inhibition sequentially leads to autophagy inhibition. As shown in **Figure 4**, both CCD112 and HT29 showed an increase in LC3B signal after starvation and LY294002 treatment. The signal is correlated to the increase in autophagosome formation after the treatment. However, such result was not obvious for HCT116, which has a high autophagosome formation in the untreated cell lines. This may be a result of continuous activation of

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).

each well in the dark. The wells were incubated for 5 minutes at RT in the dark.

tion by the manufacturer. LC3B was diluted 1:200 for this experiment.

added into each well. The plate was incubated at 4°C overnight.

room temperature for 2 hours in the dark.

autophagy pathway due to KRAS mutation.

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

in Step 4.

scope in the dark.

*4.6.1. Materials*

**1.** Cell lines (ATCC):

**a.** HT-29 (ATCC HTB-38)

**b.** HCT 116 (ATCC CCL-247)

**c.** CCD-112CoN (ATCC CRL-1541)

**2.** RIPA lysis buffer, 10X—Cell Signaling Technology, #9806S

#### **4.5. Evaluation of autophagy by immunofluorescence**

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.
