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. 2021 Oct 7;220(12):e202103087. doi: 10.1083/jcb.202103087

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© 2021 Herbst et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure S3. — Verification of nuclear export inhibitor LMB and S6K inhibitor Ly2584702 and quantification of cytoplasmic PDCD4. Related to Figs. 3 and 4. (A) Violin plots of normalized nuclear PDCD4 ICC intensity. Basal, n = 117 cells; TTX, n = 118 cells; Bic, n = 109 cells; CHX-basal, n = 123 cells; CHX-TTX, n = 120 cells; and CHX-Bic, n = 104 cells from 3 sets of cultures. Basal median = 1.00; TTX median = 0.9119; Bic median = 0.4924; CHX-basal median = 0.8890; CHX-TTX median = 1.033; CHX-Bic median = 0.5375. Basal versus Bic, P < 0.0001; TTX versus Bic, P < 0.0001; CHX-basal versus CHX-Bic, P < 0.0001; CHX-TTX versus CHX-Bic, P < 0.0001. (B) Violin plots of normalized cytoplasmic PDCD4 ICC intensity in the same cells as in A. Basal median = 1.00; TTX median = 0.9501; Bic median = 0.7138; CHX-basal median = 1.112; CHX-TTX median = 1.191; CHX-Bic median = 0.8626. Basal versus Bic, P < 0.0001; TTX versus Bic, P < 0.0001; CHX-basal versus CHX-Bic, P < 0.0001; CHX-TTX versus CHX-Bic, P < 0.0001. (C) Violin plots of normalized nuclear PDCD4 ICC intensity. Control, n = 137 cells; and LMB, n = 122 cells from three sets of cultures. Control median = 1.00; LMB median = 1.513. Control versus LMB, P < 0.0001. (D) Violin plots of normalized cytoplasmic PDCD4 ICC intensity in the same cells as in Fig. 3 A. Basal median = 1.00; TTX median = 1.158; Bic median = 0.9127; LMB-basal median = 1.031; LMB-TTX median = 1.005; LMB-Bic median = 0.7122. Basal versus Bic, P = 0.034; TTX versus Bic, P < 0.0001; LMB-basal versus LMB-Bic, P < 0.0001; LMB-TTX versus LMB-Bic, P < 0.0001. (E) Violin plots of normalized cytoplasmic PDCD4 ICC intensity in the same cells as in Fig. 3 B. Basal median = 1.00; TTX median = 1.174; Bic median = 0.8439; Epox-basal median = 0.8789; Epox-TTX median = 0.9596; Epox-Bic median = 0.9077. Basal versus Bic, P = 0.001; TTX versus Bic, P < 0.0001; Epox-basal versus Epox-Bic, P = 1; Epox-TTX versus Epox-Bic, P = 0.8258. (F) Violin plots of normalized cytoplasmic PDCD4 ICC intensity in the same cells as in Fig. 3 C. Basal median = 1.00; TTX median = 1.093; Bic median = 0.9226; Bort-basal median = 0.9229; Bort-TTX median = 0.8904; Bort-Bic median = 0.9472. Basal versus Bic, P = 0.0156; TTX versus Bic, P < 0.0001; Bort-basal versus Bort-Bic, P = 1; Bort-TTX versus Bort-Bic, P = 0.3544. (G) Violin plots of normalized cytoplasmic PDCD4 ICC intensity in the same cells as in Fig. 3 D. Basal median = 1.00; TTX median = 1.173; Bic median = 0.8955; MLN-basal median = 0.8940; MLN-TTX median = 0.9603; MLN-Bic median = 0.8633. Basal versus Bic, P = 0.0324; TTX versus Bic, P < 0.0001; MLN-basal versus MLN-Bic, P = 0.6294; MLN-TTX versus MLN-Bic, P = 0.11. (H) Violin plots of normalized cytoplasmic HA ICC intensity in the same cells as in Fig. 4 C. WT-basal median = 1.00; WT-TTX median = 1.161; WT-Bic median = 0.8621; S71A-basal median = 0.9676; S71A-TTX median = 1.209; S71A-Bic median = 1.063. WT-basal versus WT-Bic, P = 0.0012; WT-TTX versus WT-Bic, P < 0.0001; S71A-basal versus S71A-Bic, P = 0.6704; S71A-TTX versus S71A-Bic, P = 0.2012. (I) Violin plots of normalized cytoplasmic PDCD4 ICC intensity in the same cells as in Fig. 4 E. Basal median = 1.00; TTX median = 1.14; Bic median = 0.95; LY-basal median = 1.05; LY-TTX median = 1.04; LY-Bic median = 0.76. Basal versus Bic, P = 0.4742; TTX versus Bic, P = 0.0088; LY-basal versus LY-Bic, P < 0.0001; LY-TTX versus LY-Bic, P < 0.0001. Note that, in these experiments, the basal versus Bic-induced decrease in cytoplasmic PDCD4 is not statistically significant for the untreated (no inhibitor) cells. The Bic-induced cytoplasmic decrease is smaller and less consistent than the nuclear decrease, with statistical significance in the control cells of Fig. S3, E–G, but not Fig. S3, I and K. Nonetheless, there is a significant Bic-induced decrease of cytoplasmic PDCD4 in the treated (LY2584702) cells, demonstrating that S6K is not required for the Bic-induced decrease of PDCD4. (J) Western blot of protein lysates from neurons treated with or without LY2584702. Western blot was stained with antibodies for phospho-S6 (Ser235/236) and total S6 to confirm that LY2584702 inhibits S6K activity. (K) Violin plots of normalized cytoplasmic PDCD4 ICC intensity in the same cells as in Fig. 4 F. Basal median = 1.00; TTX median = 1.114; Bic median = 0.9326; Ro-31-8425 (Ro)-basal median = 1.059; Ro-TTX median = 1.172; Ro-Bic median = 1.195. Basal versus Bic, P = 0.2592; TTX versus Bic, P = 0.0008; Ro-basal versus Ro-Bic, P = 0.0118; Ro-TTX versus Ro-Bic, P = 0.9892. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001; Mann-Whitney U test with Bonferroni correction.