Fig 2: Nrf2 upregulation modulates microRNA miR-200c-3p and Notch1 and Dll4 mRNA in the migrating front. (A–C) Live single cell gene expression measurements with the dsLNA probes in RT4 cells. From left to right: fluorescence images of control and SFN (7.5 µM, 24 h) cases, tracing of relative fluorescence intensity per tissue depth measured as number of cell layers, and heatmap of representative cells at the leading edge measuring (A) microRNA miR-200c-3p, (B) Notch1 mRNA, and (C) Dll4 mRNA levels in a cell migration assay. (D–F) FISH assay in fixed RT4 cells. From left to right: fluorescence images of control and SFN cases, tracing of relative fluorescence intensity per tissue depth measured as number of cell layers, and heatmap of representative cells at the leading edge measuring (D) microRNA miR-200c-3p, (E) Notch1 mRNA, and (F) Dll4 mRNA levels in a scratch cell migration assay. Scale bars, 50 µm. Each cell at the leading edge is indicated by “Cell #” where cell #1 refers to the first measured cell from top to bottom. Data represent mean ± S.E.M. The two-way ANOVA with the post-hoc Tukey test was performed to compare across groups. At least 500 cells were analyzed for each condition (miRNA-200c-3p, Notch1 mRNA, and Dll4 mRNA). Results are from three experiments performed across different days.

Fig 3: Leader cell formation at the leading edge. (A) Representative bright-field images of leader cells after a 24 h cell migration assay for CRISPR/Cas9 NFE2L2-KO Pool RT4 cells (KO), RT4 cells (Control) and sulforaphane treated (7.5 μM, 24 h) RT4 cells (SFN), respectively. (B) Plot showing leader cells per millimeter for CRISPR/Cas9 NFE2L2-KO Pool RT4 cells (KO), RT4 cells (Control) and sulforaphane treated RT4 cells (SFN), respectively. One-way ANOVA with post-hoc Tukey’s test was used to compare across groups and the ROUT method was used to identify outliers (KO n = 46, Control n = 100, SNF n = 86 leading edges). (C–H) Representative immunocytochemistry images of leader cells in the control case (RT4 cells) characterizing gene expression for (C) E-cadherin, (D) Jagged1, (E) ZEB1, (F) Notch1, (G) Nrf2, (H) Dll4. (I,J) Representative overlay images using the dsLNA biosensors to measure mRNA levels of (I) Dll4 and (J) Notch1, respectively. (K,L) Mean intensity of leader vs. follower cells for levels of (K) Dll4 mRNA (n = 6 follower cells) and (L) Notch1 mRNA (n = 6 follower cells). (K,L) represent the quantification of one leader cell and its follower cells. Immunocytochemistry data are from four experiments, and dsLNA biosensor data are from three experiments. Scale bars, 20 μm.

Fig 4: The effects of Ailanthone and sulforaphane on leader and follower cells. (A) Representative image of a sprout protruding from a 3D microtumor formed by HeLa cells. (B) Unlike the number of sprouts, the morphology (e.g., width and length) of the sprouts was not significantly affected by Alia and SFN treatment. One-way ANOVA with post-hoc Tukey’s test was used to compare across groups (Aila n = 29 branches, Control n = 49 branches, SNF = 27 branches). The experiment was performed three times across different days. (C) Overlay images measuring mRNA levels of Dll4, Notch1, Jagged1, and miR-200c in leader and follower cells. Scale bars, 50 µm. (D) Relative expressions of Dll4 mRNA, Notch1 mRNA, Jagged1 mRNA, and miR-200c between leader and follower cells in invading sprouts. Leader cells in the control case generally expressed high Dll4 and Jag1 (mean values of leader/follower expression >1) while follower cells expressed higher Notch1 and miR200c across cases (mean values of leader/follower expression <1). The D’Agostino and Pearson test was used to determine data that are not normally distributed (Dll4: Alia p-value = 0.4740, Control p-value<0.0001, SFN p-value = 0.2233. Notch1: Alia p-value = 0.2618, Control p-value<0.0001, SFN p-value = 0.0259. Jag1: Alia p-value <0.0001, Control p-value = 0.0169, SFN n is too small. mi200c: Alia p-value <0.0001, Control p-value p-value<0.0001, SFN n is too small). For Dll4, Notch1, Jag1, and miR200c respectively: Aila n = 9, 13, 8, 12 branches, Control n = 22, 37, 18, 14 branches, SNF = 12, 13, 7, seven branches. Results are from three experiments performed across different days.

Fig 5: Nrf2 regulates the Notch signaling pathway in the migrating front. (A–C) Immunocytochemistry of RT4 bladder cancer cells measuring Jagged1 (left column), Notch1 (center column), and Dll4 (right column) protein levels in a cell migration assay for (A) CRISPR/Cas9 NFE2L2-KO Pool RT4 cells (KO), (B) RT4 cells (Control), and (C) sulforaphane treated (7.5 µM, 24 h) RT4 cells (SFN), respectively. Scale bars, 50 µm. (D–F) Quantification of immunocytochemistry data. (D) Average intensity of each cell in the whole monolayer. One-Way ANOVA with post-hoc Tukey’s test was used to compare across groups (Jagged1: KO n = 307 cells, Control n = 526 cells, SFN n = 423 cells. Notch1: KO n = 307 cells, Control n = 370 cells, SFN n = 423 cells. Dll4: KO n = 460 cells, Control n = 1135 cells, SFN n = 570 cells). (E) Tracing of relative fluorescence intensity per tissue depth measured as number of cell layers. Data represent mean ± S.E.M. (F) Heatmap of representative cells at the leading edge for Jagged1, Notch1, and Dll4, respectively. Each cell at the leading edge is indicated by “Cell #” where cell #1 refers to the first measured cell from top to bottom. Results are from four experiments performed across different days.