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. 2021 Jun 6;23(6):594–606. doi: 10.1016/j.neo.2021.05.014

Fig. 2.

Fig. 2

Repressing LH2 expression blunts HNSCC cell migration and invasion.

(A) Evaluation of the CRISPRi system for inducible silencing of LH2 expression. To validate 5 sgRNAs targeting the PLOD2 gene promoter region, LH2-high UM-SCC-74A cells stably expressing doxycycline-inducible dCas9-KRAB were transduced with each sgRNA, cells were treated +/- 1 µg/mL doxycycline for 7 d, and quantitative real-time PCR was used to assess LH2 mRNA levels. Representative data of independent biologic replicates are presented as the mean ± SD (4 technical replicates per biologic replicate; Student's t test, *P < 0.05, ****P < 0.0001). Also see Supplementary Figure 2A.

(B) Left, UM-SCC-74A_TRE3GdCas9-KRABU6sgRNA cells were treated +/- 1 µg/mL doxycycline for 7 d, total protein extracted, and western blot run to validate repression at the protein level. This blot is representative of independent biological replicates. Right, quantification of LH2 protein levels. Band intensities were normalized to β-actin and are shown as the fold change relative to the no doxycycline condition.

(C) Wound healing (scratch) assay performed with the stable UM-SCC-74A_TRE3GdCas9-KRABU6sgRNA4 cells treated +/- 1 µg/mL doxycycline for 7 d. Images were captured every hour for 24 h to quantify rates of wound closure. Representative data of independent biologic replicates are presented as the mean ± SD (3 independent FOV were captured and quantified per biologic replicate). Also see Supplementary Figure 2B.

(D) Left, transwell invasion assays were performed for UM-SCC-74A_TRE3GdCas9-KRABU6sgRNA4 cells using Boyden chambers precoated with Matrigel. After 24 h, cells were stained with 0.05% crystal violet, imaged, and cell invasion quantified. Right, quantification of invasion presented as either the number of cells invaded per field of view (FOV), or by extracting the crystal violet and analyzing absorbance at 570 nm. Representative data of independent biologic replicates are presented as the mean ± SD (>5 independent FOV were quantified per biologic replicate).