Figure 4: Ferritinophagy is induced in response to MEKi treatment.

A. Schematic showing lysosome purification using affinity-based capture from KP4 cells stably expressing T192-mRFP-3xHA and treated with DMSO or MEKi.

B. Volcano plot of lysosome proteomics data from KP4 cells expressing T192-mRFP-3xHA treated with DMSO or MEKi. Data are plotted as log2 fold change (MEKi/DMSO) versus −log10 of the p-value (see Table S5). Ferritin light chain (FTL) and Ferritin heavy chain (FTH1) are indicated in red.

C. Average peptide counts for the indicated proteins from n=3 biological replicates per condition.

D. Immunoblot for the indicated proteins in input and lysosomal fractions isolated from KP4 cells expressing T192-mRFP-3xHA and treated with DMSO or MEKi. Note the enrichment of FTH, FTL, NCOA4, and LC3B in the MEKi lysosome fraction. NPC1 and LAMP1 serve as loading controls while the absence of AIF, RCAS1 and PDI confirms organelle purity. FTH, FTL, NCOA4, and LC3B are also increased in the input indicating increased overall expression in MEKi-treated cells.

E. Immuno-fluorescence staining of LAMP2 (red) and FTL (green) in KP4 cells following DMSO or MEKi treatment in the presence or absence of acute BafA1 treatment (500nM for 4h) to suppress lysosome degradation. Arrowheads in insets show increased colocalization of FTL and LAMP2 in cells co-treated with MEKi+BafA1. Scale, 20μm.

F. Quantification of percentage colocalization between FTL and LAMP2 in cells in E. n=10–15 fields per condition.

G. Live cell imaging of KP4 cells incubated with FerroOrange iron dye and treated with the indicated agents.

H. Quantification of fluorescence intensity of images in G from n = 7–10 fields per condition.

I. Immunoblot for total FTH1, FTL and NCOA4 in KP4 cells treated with the indicated concentrations of MEKi for 48h.

J. Immunoblot for total FTH1 and FTL in KP4 cells treated with 1μM ERKi for 48h.

K. Schematic (left) and immunoblot for the indicated proteins (right) of KP4 xenografts isolated from nude mice treated daily for 11 days with vehicle or MEKi (Trametinib 2mg/kg).

L. Immunoblot for total Transferrin receptor (TfR1) and Ferroportin (FPN) in KP4 cells treated with DMSO or MEKi.

M. Flow cytometry-based measurement of cell surface Transferrin receptor in KP4 cells treated with DMSO or MEKi.

N. Uptake of fluorescently labeled Transferrin in KP4 cells treated with DMSO or MEKi.

Data are the mean ± s.d. and P values were determined using an unpaired two-tailed Student’s t-test (F, M, N) and one-way ANOVA (H). MEKi treatment was 100nM for 48h unless otherwise indicated.