Fig 1: EGFR up-regulated p-JNK1/2/JNK1/2 ratio and NF-κB p65 expression and partly offset the suppressive effect of shGPNMB on the JNK/NF-κB signaling pathway while shEGFR worked inversely. (a and b) Relative βIII tubulin and CNPase expressions were measured by Western blot assay. (c and d) Expressions of JNK/NF-κB signaling pathway-related indicators were measured by Western blot assay. GAPDH was employed as the internal reference. ^ P < 0.05 or ^^^ P < 0.001 vs NC + shNC; ### P < 0.001 vs GPNMB + shNC; && P < 0.01, &&& P < 0.001 vs NC + shEGFR; ε P < 0.05 or εεε P < 0.001 vs shNC + NC; Ω P < 0.05 or ΩΩΩ P < 0.001 vs shGPNMB + NC; ΔΔΔ P < 0.001 vs shNC + EGFR. All results represent means ± SD of triplicate determinations. JNK 1/2: c‑Jun NH2‑terminal kinase 1/2; p-JNK 1/2: phosphorylated JNK1/2; NF-κB p65: nuclear factor κB p65.
Fig 2: GPNMB promoted cell viability and neuronal differentiation while shGPNMB performed differently. (a) Relative βIII tubulin expression was measured by qRT-PCR and Western blot assay. Upregulation of βIII tubulin expression suggested the successful differentiation of the neural stem cells into neurons. (b and c) Relative GPNMB expression was measured by qRT-PCR and Western blot assay. (d and e) Transfection efficiency was validated by qRT-PCR and Western blot assay. (f) Cell viability was measured via CCK-8. GPNMB promoted while shGPNMB inhibited cell viability. (g) Immunofluorescence assay was adopted to assess the status of neuronal differentiation (magnification 200×, scale bar 100 µm). Red part referred to the targeted proteins, and blue meant the nuclei as stained by DAPI. (h) Relative neural differentiation markers’ (βIII tubulin and CNPase) expressions were measured by Western blot assay. GAPDH was set as the internal reference. ** P < 0.01 or *** P < 0.001 vs Control; + P < 0.05 or +++ P < 0.001 vs NC; ## P < 0.01, ### P < 0.001 vs shNC. All results represent means ± SD of triplicate determinations. qRT-PCR: quantitative reverse-transcription polymerase chain reaction; GPNMB: glycoprotein non-metastatic melanoma B; shNC: short-hairpin-negative control; CCK-8: cell counting kit-8; CNPase: 2′,3′-cyclic nucleotide 3′ phosphodiesterase.
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