Fig 3. Modulation of EGR1 impacts on TFEB-driven tumorigenesis.

(A) Representative image of colony formation capacity of HeLa WT (upper) and HeLa-FLCN KO cells (lower) stably infected with shRNAs against luciferase (shLUC) and EGR1 (shEGR1). (B) Bar plots showing the quantification of colonies’ number and size (%Area) in shLUC and shEGR1 engineered cells. Mean ± SD values are shown. ANOVA was used; *p < 0.05, **p < 0.01, ***p < 0.001. (C) (Left) Bar plots showing TFEB and EGR1 mRNAs quantification as measured by qPCR in HeLa-FLCN KO cells treated with shLUC and shEGR1. Mean ± SD values are shown. ANOVA was used; *p < 0.05, **p < 0.01, ***p < 0.001. (Right) Representative image of immunoblot analysis of EGR1 and TFEB levels in HeLa-FLCN KO cells stably infected with shLUC and shEGR1. GAPDH was used as a loading control. (D) Bright-field representative images of 3D spheroids generated from HeLa-FLCN KO cells stably infected with shLUC and shEGR1 over a 9-day course. (E) Line plot showing High-Content Imaging-based quantification of 3D spheroids size (Area µm²). Mean ± SD values relative to 9 and 7 replicates for shLUC and shEGR1 spheroids, respectively. ANOVA was used; *p < 0.05, **p < 0.01, ***p < 0.001. (F) Bright-field representative images of 3D spheroids generated from shLUC and shEGR1 HeLa-FLCN KO cells infected with a doxycycline-inducible TFEB overexpressing construct. Images are relative to 3D spheroids at 2 and 8 days of culture, in the absence (CTRL) or presence (TFEB) of doxycycline. (G) (Left) Line plot showing High-Content Imaging-based quantification of 3D spheroids size (Area µm²) over 8 days of culture, in the absence (CTRL) or presence (TFEB) of doxycycline. Mean ± SD values relative to 5 replicates for each condition. (Right) Results from the ANOVA followed by Tukey’s multiple comparisons significance test; *p < 0.05, **p < 0.01, ***p < 0.001. Individual quantitative observations that underlie the data summarized here can be located under the Supporting information file as S1 Data. Uncropped images can be found in the Supporting information file as S1 Raw Images. EGR1, early growth response 1; TFEB, transcription factor EB; WT, wild-type.