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Figure 5. Abrogation of Nrg-ErbB-PI3K-PI5K-mTorc2 signaling promotes Mitf nuclear translocation (A) Diagram of activating (green) and inhibiting (red) phosphorylation sites (P) of the Mit/Tfe family. The kinases that regulate each of those phosphorylation sites and the pharmacological inhibitors used in primary culture to target each of these kinases are indicated. (B) Diagram of SC primary culture with inhibitors. (C–N0) Primary SC culture derived from dissociated sciatic nerves of P4 WT mice, stained for Mitf (green) and Hoechst (blue). (C–C0) Primary SCs were cultured with DMSO (control), (D-D0) trametinib (MEK), (E and E0) SCH772984 (ERK1/2), (F and F0) enzastaurin (PKC), (G and G0) BIO (GSK3), (H and H0) MK-2206 (AKT), (I and I0) Torin (mTorc1/2), (J and J0) YM201636 (Pikfyve), (K and K0) VPS34-IN1 (Vps34), (L and L0) <t>canertinib</t> (ErbB), (M and M0) hNrg1, and (N and N0) rapamycin (mTorc1). Scale bar, 30 mm. (O) Examples of Mitf subcellular localization (top) and quantification of Mitf enrichment in the cytoplasm (pink), uniformly distributed (purple) or enriched in the nucleus (blue). n = 3 independent experiments for each inhibitor, n > 100 cells per experiment. The data are represented as the mean + SEM. (P and Q) Models of Mitf cytoplasmic retention and translocation into the SC nucleus.
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Figure 5. Abrogation of Nrg-ErbB-PI3K-PI5K-mTorc2 signaling promotes Mitf nuclear translocation (A) Diagram of activating (green) and inhibiting (red) phosphorylation sites (P) of the Mit/Tfe family. The kinases that regulate each of those phosphorylation sites and the pharmacological inhibitors used in primary culture to target each of these kinases are indicated. (B) Diagram of SC primary culture with inhibitors. (C–N0) Primary SC culture derived from dissociated sciatic nerves of P4 WT mice, stained for Mitf (green) and Hoechst (blue). (C–C0) Primary SCs were cultured with DMSO (control), (D-D0) trametinib (MEK), (E and E0) SCH772984 (ERK1/2), (F and F0) enzastaurin (PKC), (G and G0) BIO (GSK3), (H and H0) MK-2206 (AKT), (I and I0) Torin (mTorc1/2), (J and J0) YM201636 (Pikfyve), (K and K0) VPS34-IN1 (Vps34), (L and L0) canertinib (ErbB), (M and M0) hNrg1, and (N and N0) rapamycin (mTorc1). Scale bar, 30 mm. (O) Examples of Mitf subcellular localization (top) and quantification of Mitf enrichment in the cytoplasm (pink), uniformly distributed (purple) or enriched in the nucleus (blue). n = 3 independent experiments for each inhibitor, n > 100 cells per experiment. The data are represented as the mean + SEM. (P and Q) Models of Mitf cytoplasmic retention and translocation into the SC nucleus.

Journal: Cell reports

Article Title: Mitf is a Schwann cell sensor of axonal integrity that drives nerve repair.

doi: 10.1016/j.celrep.2023.113282

Figure Lengend Snippet: Figure 5. Abrogation of Nrg-ErbB-PI3K-PI5K-mTorc2 signaling promotes Mitf nuclear translocation (A) Diagram of activating (green) and inhibiting (red) phosphorylation sites (P) of the Mit/Tfe family. The kinases that regulate each of those phosphorylation sites and the pharmacological inhibitors used in primary culture to target each of these kinases are indicated. (B) Diagram of SC primary culture with inhibitors. (C–N0) Primary SC culture derived from dissociated sciatic nerves of P4 WT mice, stained for Mitf (green) and Hoechst (blue). (C–C0) Primary SCs were cultured with DMSO (control), (D-D0) trametinib (MEK), (E and E0) SCH772984 (ERK1/2), (F and F0) enzastaurin (PKC), (G and G0) BIO (GSK3), (H and H0) MK-2206 (AKT), (I and I0) Torin (mTorc1/2), (J and J0) YM201636 (Pikfyve), (K and K0) VPS34-IN1 (Vps34), (L and L0) canertinib (ErbB), (M and M0) hNrg1, and (N and N0) rapamycin (mTorc1). Scale bar, 30 mm. (O) Examples of Mitf subcellular localization (top) and quantification of Mitf enrichment in the cytoplasm (pink), uniformly distributed (purple) or enriched in the nucleus (blue). n = 3 independent experiments for each inhibitor, n > 100 cells per experiment. The data are represented as the mean + SEM. (P and Q) Models of Mitf cytoplasmic retention and translocation into the SC nucleus.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Chicken Polyclonal Anti-Glial Fibrillary Acidic Protein (GFAP) Aviva Systems Biology Cat# OAPC00115; RRID: N/A Donkey anti-Goat IgG (H + L) CrossAdsorbed Secondary Antibody; Alexa FluorTM 555 Thermo Fisher Scientific A-21432; RRID:AB_2535853 Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody; Alexa FluorTM 555 Thermo Fisher Scientific Cat# A-31570; RRID:AB_2536180 Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody; Alexa FluorTM 647 Thermo Fisher Scientific Cat# A-31573; RRID:AB_2536183 Goat anti-Chicken IgY (H + L) Secondary Antibody; Alexa FluorTM 555 Thermo Fisher Scientific A-21437; RRID:AB_2535858 Goat Anti-Human Sox10 Polyclonal antibody R and D Systems Cat# AF2864; RRID:AB_442208 Goat anti-Mouse IgG (H + L) Highly CrossAdsorbed Secondary Antibody; Alexa FluorTM Plus 680 Thermo Fisher Scientific Cat# A32729; RRID:AB_2633278 Goat anti-Mouse IgG (H + L) Secondary Antibody; DyLightTM 800 4X PEG Thermo Fisher Scientific Cat# SA5-35521; RRID:AB_2556774 Goat anti-Rabbit IgG (H + L) Highly CrossAdsorbed Secondary Antibody; Alexa FluorTM 680 Thermo Fisher Scientific Cat# A-21109; RRID:AB_2535758 Goat anti-Rabbit IgG (H + L) Secondary Antibody; DyLightTM 800 4X PEG Thermo Fisher Scientific Cat# SA5-35571; RRID:AB_2556775 Goat Polyclonal Anti-GFAP Antibody Aviva Systems Biology Cat# OAEB01041; RRID:AB_10885969 Mouse Anti-Paxillin Monoclonal Antibody; Unconjugated; Clone 349 BD Biosciences Cat# 610051; RRID:AB_397463 Mouse Anti-Rabbit Glyceraldehyde-3phosphate dehydrogenase (GAPDH) Monoclonal Antibody; Clone 6C5 Fitzgerald Industries International Cat# 10R-G109a; RRID:AB_1285808 Rabbit Polyclonal Anti-MiTF antibody Abcam Cat# ab122982; RRID:AB_10902226 Rabbit Polyclonal anti-MITF C terminus (CGTMPESSPAYSIPRKMGSNLEDILMD) This paper # 7416; RRID: N/A Rabbit Polyclonal anti-MITF N terminus (DLVNRIIKQEPVLENCSQE) This paper # 7414; RRID: N/A Rabbit Polyclonal anti-S100 Abcam Cat# ab868; RRID:AB_306716 Biological samples Mouse: Germplasm; Mtmr2tm1a(KOMP)Mbp KOMP/IKMC Project # 30275 Chemicals, peptides, and recombinant proteins Alexa FluorTM 488 Phalloidin Thermo Fisher Scientific Cat# A12379 BIO Sigma-Aldrich Cat# B1686; CAS 667463-62-9 Canertinib (CI-1033) Selleck Chemicals LLC Cat# S1019; CAS 267243-28-7 Enzastaurin (LY317615) Selleck Chemicals LLC Cat# S1055; CAS 170364-57-5 (Continued on next page) Cell Reports 42, 113282, November 28, 2023 19

Techniques: Translocation Assay, Phospho-proteomics, Derivative Assay, Staining, Cell Culture, Control