Fig 2: NF2 and RB1 score as sensitizing candidates with vinorelbine. (A) Unique gene‐trap insertions in NF2 and RB1 in untreated (n = 4, left panel), docetaxel‐treated (n = 2, panel in the center), and vinorelbine‐treated (n = 2, right panel) conditions. A representative example of each screening condition is shown including the P‐value comparing the depicted drug‐treated replicate to the depicted untreated replicate, determined by Fisher's exact t‐tests. Y‐axis represents the sense to antisense integration ratio, while log10 of sense/total number of insertions is plotted on the x‐axis. Loss of RB1 scored at times with a ratio > 0.5 in untreated and docetaxel‐treated screens, indicating that disruptive sense integrations caused a survival benefit. In vinorelbine screens, a ratio < 0.5 was observed for both RB1 and NF2. Noteworthy, loss of NF2 was of slight disadvantage for survival even under untreated conditions, represented by a ratio < 0.5. (B) Confirmation of functional inactivation of NF2 and RB1 by western blotting with the respective antibodies (n = 2). (C) Validation of loss of NF2 and RB1 as sensitizing factor for vinorelbine with two independent knockout clones each. Equal numbers of cells were exposed to half, full, or twice the drug concentrations which have been used in the screens. Bar plots show mean quantification of three biological replicates with SEM; **P = 0.001–0.01; *P = 0.01–0.05.

Fig 3: Peroxynitrite selectively down-regulates mitochondrial metabolism in human MD-Schwann cells. A, representative IR Western blots of nitrated and total Hsp90 in homogenates of vestibular schwannomas from three NF2 patients (VS 1 to 3), and human (HSC) and mouse (MSC) WT- and MD-Schwann cells (n = 3–4); and B, in homogenates (Hom) and mitochondrial (Mit) and cytosolic (Cyt) fractions of human and mouse WT- and MD-Schwann cells subjected to subcellular fractionation (n = 3). +C: positive control, nitrated recombinant Hsp90. C, the mitochondrial membrane potential of human WT- and MD-Schwan cells after 48 h incubation in the absence or presence of urate (100 µm) was determined after incubation of the cells with the probe JC-1 (5 µm) for 30 min and expressed as the percentage of the ratio of red over green JC-1 fluorescence (n = 3 with 8 replicates). *, p < 0.05 versus untreated WT; **, versus untreated MD by Kruskal-Wallis test. D, the OCR of human WT- and MD-Schwann cells after 48 h of incubation in the absence and presence of urate (100 µm) was measured by extracellular flux analysis in basal conditions and after the sequential addition of oligomycin (1 µm, oligo), FCCP (2 µm), and antimycin A (10 µm, AA) (mean ± S.E. of 5 independent experiments with 5 replicates). E, analysis of the different OCR parameters after sequential addition of the inhibitors, expressed as percentage of WT-Schwann cells (n = 5 with 5 replicates). *, p < 0.001 versus untreated WT-Schwann cells; **, versus untreated MD-Schwann cells by one-way ANOVA followed by Bonferroni post test.

Fig 4: LUZP1 inhibits protein phosphatase 1c ß/d (PP1c ß/d) in a microtubule (MT)-facilitated manner No difference in Shroom3 localization between Venus-LUZP1-expressing LUZP1 knockout (REV) and LUZP1 knockout (LUZP1 KO) Eph4 cells, shown by representative confocal micrographs of co-cultures of WT and LUZP1 KO cells. Scale bar, 10 µm.Pull-down assays between Merlin and LUZP1, showing that Merlin did not bind LUZP1. IB, immunoblotting.No difference in MT organization between wild-type (WT) and LUZP1 KO Eph4 cells, shown by representative confocal micrographs of co-cultures of WT and LUZP1 KO Eph4 cells. Scale bar, 10 µm.No difference in a-tubulin intensities within circumferential rings (CRs) between WT and LUZP1 KO cells, shown by bar plots with dot density plots. (25.97 ± 4.69 arbitrary units [a.u.] [WT] vs. 27.03 ± 6.20 a.u. [KO]). n = 3. P = 0.42 (unpaired t-test). Bars and error bars represent the mean ± standard deviation (SD).Representative confocal micrographs of co-culture of Venus-LUZP1-expressing LUZP1 knockout (REV) and LUZP1 KO Eph4 cells treated with nocodazole in the apical and basal planes. Scale bar, 10 µm. Nocodazole treatment reversed the apical constriction of REV Eph4 cells. Scale bar, 10 µm.No difference in the degree of co-localization between LUZP1 and di-phosphorylated myosin light chain (ppMLC) after nocodazole treatment, shown by bar plots with dot density plots quantifying co-localization using Pearson's correlation coefficients (r) (r = 0.48 ± 0.12 [control]; r = 0.47 ± 0.04 [nocodazole]; r = 0.49 ± 0.12 [washout]). n = 3. P = 0.22 (Kruskal–Wallis test). Bars and error bars represent the mean ± SD. In vitro Merlin phosphorylation assay using 1 µg MTs in addition to 100 ng GST-Merlin, 2 pg p21-activated kinase 1 (PAK1), 1 mM ATP, 1 µg GST- PP1c ß/d, and 0–5 µg GST-LUZP1, showing that MTs facilitated LUZP1-mediated inhibition of PP1c ß/d (1.00 [1st lane, control] vs. 1.73 ± 0.79 [2nd lane] vs. 3.03 ± 1.17 [3rd lane] vs. 6.06 ± 1.89 [4th lane] vs.2.69 ± 1.58 [5th-lane] vs. 4.45 ± 2.28 [6th lane] vs. 5.46 ± 2.68 [7th lane] vs. 8.32 ± 3.76 [8th lane]). n = 5. *P < 0.05 (Kruskal–Wallis test followed by Steel test [compared with 1st lane]). Bars and error bars represent the mean ± SD. Source data are available online for this figure.

Fig 5: LUZP1 inhibits the activity of protein phosphatase 1c ß/d (PP1c ß/d), the catalytic subunit of myosin phosphatase A schematic drawing of myosin phosphatase. Myosin phosphatase consists of PP1c ß/d, myosin phosphatase targeting subunit 1 (MYPT1), and a small 20-kDa regulatory subunit (M20). PP1c ß/d represents a catalytic subunit responsible for dephosphorylating myosin light chain (MLC), whereas MYPT1 targets myosin phosphatase to MLC by binding both PP1c ß/d and MLC.Representative confocal micrographs of co-cultures of wild-type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 µm.Bar plots with dot density plots showing that PP1c mean intensities within CRs are similar between WT and LUZP1 KO cells (28.68 ± 9.60 arbitrary units [a.u.] [WT] vs. 25.04 ± 9.47 a.u. [LUZP1 KO]). P = 0.09 [Mann–Whitney U test]. n = 3. Bars and error bars represent the mean ± standard deviation (SD).Co-immunoprecipitation of HA-PP1c ß/d and GFP-LUZP1. LUZP1 binds to PP1c ß/d. IB, immunoblotting. In vitro MLC phosphorylation assay using 1 µg GST-PP1c ß/d in addition to 25 ng GST-MLC, 4 ng GST-ROCK1 catalytic domain, 1 mM ATP, and 0–5 µg GST-LUZP1. Quantification of the di-phosphorylated MLC (ppMLC)/MLC ratio relative to the control showed that LUZP1 upregulated ppMLC/MLC levels in a dose-dependent manner (1.00 [1st lane, control] vs. 1.27 ± 0.33 [2nd lane] vs. 1.76 ± 0.68 [3rd lane] vs. 2.53 ± 1.65 [4th lane] vs. 2.93 ± 2.45 [5th lane]). n = 3 or 6. **P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 1st lane]). Bars and error bars represent the mean ± SD. In vitro Merlin phosphorylation assay using 1 µg GST-PP1c ß/d, 100 ng GST-Merlin, 2 pg p21-activated kinase 1 (PAK1), and 5 µg GST-LUZP1. Quantification of the phosphorylated Merlin (pMerlin)/Merlin ratio relative to the control showed that LUZP1 upregulated pMerlin/Merlin levels (0.23 ± 0.15 [1st lane] vs. 1.00 [2nd lane, control] vs. 0.32 ± 0.17 [3rd lane] vs. 0.97 ± 0.42 [4th lane] vs. 1.25 ± 0.39 [5th lane]). n = 4 or 9. *P < 0.05, **P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 3rd lane]). Bars and error bars represent the mean ± SD.A schematic drawing of the relationships among ppMLC, LUZP1, and myosin phosphatase at tight junction (TJ)-associated CRs to promote apical constriction. Source data are available online for this figure.