Extended Data Figure 5. Merlin expression correlates with sensitivity to ferroptosis in mesothelioma cell lines.

a. Expression of Lats1/2 was tested in the indicated mesothelioma cell lines by Western blot.

b. Spheroids were treated with 10 μM erastin for 24 h before SYTOX Green staining.

c. Western blot confirming knockdown efficiency of Merlin in MSTO-211H cells.

d. Confluent MSTO-211H cells expressing shNT or shMerlin as indicated were treated with 1 µM RSL3, with or without 2 µM Fer-1. Cell death (left, 24 h after treatment) and lipid ROS production (right, 16 h) were measured. Data are plotted as mean ± s.d.; n = 3 biological replicates. P-values were acquired by one-way ANOVA, **** p<0.0001.

e. Merlin-mutant Meso33 cells were reconstituted with wild type Merlin. Expression of Merlin was confirmed by Western blot.

f. Localisation of YAP (green) under sparse or confluent conditions in Meso33 cells expressing wild type Merlin was determined by immunofluorescence.

g. Meso33 cells expressing wild type Merlin were cultured under sparse or confluent conditions and stimulated with cystine-free media. Cell death was measured by SYTOX Green staining coupled with flow cytometry after 24 h of treatment. Data are plotted as mean ± s.d.; n = 3 biological replicates. P-values were acquired by two-tailed t-test, n.s., p=0.1874, * p=0.0104.

h. Meso33 cells expressing wild type Merlin were cultured as described in (g). Lipid ROS was measured after 16 h of cystine starvation. Data are plotted as mean ± s.d.; n = 3 biological replicates. P-values were acquired by two-tailed t-test, n.s., p=0.4860, * p=0.0201.

i. Meso33 spheroids harbouring Dox-inducible Merlin expression were grown in the presence or absence of Dox for 72 h, at which point 10 µM erastin was added. After 24 h, spheroids were stained with SYTOX Green for cell death.