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. 2021 Oct 26;13(21):5373. doi: 10.3390/cancers13215373

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© 2021 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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Requirement of TSPAN5 for HCC proliferation, migration, invasion and spheroid formation. (A) HuH7 cells transiently transfected with negative control (sictrl) and TSPAN5, CDH2 or VCAN siRNA (siTSPAN5, siCDH2 and siVCAN) were counted daily for 5 days. Values are means ± SD (n = 3); *, p < 0.05. (B) HepG2 DLC1 wt (HepG2 wt) cells transiently transfected with negative control (sictrl) as well as HepG2 DLC1 KO cells transiently transfected with negative control (sictrl) and TSPAN5 siRNA (siTSPAN5) were counted daily for 5 days. Values are means ± SD (n = 3); *** p < 0.001. Lysates of HepG2 DLC1 wt (wt ctrl) and HepG2 DLC1 KO (DLC1 KO) cells were immunoblotted with anti-DLC1 and anti-HSP90 antibodies to demonstrate the DLC1 KO. Representative blots of three independent experiments are shown. TSPAN5 knockdown efficiency was determined by qRT-PCR with gene-specific TSPAN5 and 18S rRNA primers. Values are means ± SD (n = 3); *, p < 0.05. (C) HuH7 cells expressing scrambled (sictr) and TSPAN5 siRNA (siTSPAN5) were transiently transfected with MRTF-A-N100 and counted daily for 5 days. Values are means ± SD (n = 3). (D) HuH7 cells were transiently transfected with scrambled siRNA (sictrl) and TSPAN5 siRNA (siTSPAN5). TSPAN5 knockdown efficiency was determined by qRT-PCR with gene-specific TSPAN5 and 18S rRNA primers. Transfected HuH7 cells were subjected to Matrigel^® invasion assay chambers and after 48 h invaded cells were stained by crystal violet and counted. Statistical analysis was performed using the unpaired t-test. Values are means ± SD (n = 3); *** p < 0.001. (E) HuH7 transfected with negative control siRNA (sictrl), CDH2 siRNA (siCDH2), VCAN siRNA (siVCAN) and TSPAN5 siRNA (siTSPAN5) were transduced to a special spheroid plate and spheroid formation was monitored for 11 days. The spheroid average of day 2 and day 11 were compared (bottom panel). Values are means ± SD (n = 3); *** p < 0.001. The whole Wester Blots are available at Figure S10.