AMPK Knockout Impairs the Formation of Three-Dimensional Spheroids

Park, Yea-In; Park, Rackhyun; Lee, Siyun; Lee, Chunghyeon; Yoo, Inkyu; Ka, Hakhyun; Huh, Yang Hoon; Hong, Jongkwang; Park, Junsoo

doi:10.3390/life15040525

Open AccessArticle

AMPK Knockout Impairs the Formation of Three-Dimensional Spheroids

by

Yea-In Park

^1,†,

Rackhyun Park

^2,†

,

Siyun Lee

^1,†

,

Chunghyeon Lee

¹,

Inkyu Yoo

¹,

Hakhyun Ka

¹,

Yang Hoon Huh

³,

Jongkwang Hong

¹

and

Junsoo Park

^1,*

¹

Division of Biological Science and Technology, Yonsei University, Wonju 26493, Republic of Korea

²

Department of Life Science, Yong-In University, Yongin 17092, Republic of Korea

³

Electron Microscopy Research Center, Korea Basic Science Institute (KBSI), Cheongju 28119, Republic of Korea

^*

Author to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Life 2025, 15(4), 525; https://doi.org/10.3390/life15040525

Submission received: 25 February 2025 / Revised: 19 March 2025 / Accepted: 20 March 2025 / Published: 22 March 2025

(This article belongs to the Section Physiology and Pathology)

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Abstract

AMP-activated protein kinase (AMPK) is an important regulator of cellular energy homeostasis, and AMPK contributes to cell growth, apoptosis, and autophagy. Although most cell studies have been performed using two-dimensional (2D) cell culture, recent studies have demonstrated that the three-dimensional (3D) spheroid technique is helpful in various cell research fields, such as tumor biology, due to its resemblance to the 3D tissue structure. However, the role of AMPK in 3D spheroid formation has not been characterized clearly. This study used the AMPK knockout cell line to examine the role of AMPK in 3D spheroid formation and is the first report describing the generation of 3D spheroids using AMPK knockout cells. While control cells produced round spheroids with a similar length-to-width ratio, AMPK knockout produced an oval shape with a more significant length-to-width ratio. We demonstrate that AMPK knockout spheroids contain significantly more prominent lysosomes in each cell, indicating that autophagic flux is impaired in 3D spheroids. Finally, flow cytometry analysis showed that AMPK knockout spheroids contain more apoptotic cells than control cells. These results indicate that AMPK is required for efficient 3D spheroid formation.

Keywords: AMPK; knockout; 3D culture; spheroid; lysosome

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MDPI and ACS Style

Park, Y.-I.; Park, R.; Lee, S.; Lee, C.; Yoo, I.; Ka, H.; Huh, Y.H.; Hong, J.; Park, J. AMPK Knockout Impairs the Formation of Three-Dimensional Spheroids. Life 2025, 15, 525. https://doi.org/10.3390/life15040525

AMA Style

Park Y-I, Park R, Lee S, Lee C, Yoo I, Ka H, Huh YH, Hong J, Park J. AMPK Knockout Impairs the Formation of Three-Dimensional Spheroids. Life. 2025; 15(4):525. https://doi.org/10.3390/life15040525

Chicago/Turabian Style

Park, Yea-In, Rackhyun Park, Siyun Lee, Chunghyeon Lee, Inkyu Yoo, Hakhyun Ka, Yang Hoon Huh, Jongkwang Hong, and Junsoo Park. 2025. "AMPK Knockout Impairs the Formation of Three-Dimensional Spheroids" Life 15, no. 4: 525. https://doi.org/10.3390/life15040525

APA Style

Park, Y.-I., Park, R., Lee, S., Lee, C., Yoo, I., Ka, H., Huh, Y. H., Hong, J., & Park, J. (2025). AMPK Knockout Impairs the Formation of Three-Dimensional Spheroids. Life, 15(4), 525. https://doi.org/10.3390/life15040525

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

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AMPK Knockout Impairs the Formation of Three-Dimensional Spheroids

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