Journal: bioRxiv
Article Title: NF1 Loss Remodels Tumor Niches for Immune Evasion
doi: 10.64898/2026.01.11.698818
Figure Lengend Snippet: (A) GSEA Hallmark pathway analysis showing EMT, TGFβ, angiogenesis, and hypoxia are upregulated as a result of NF1 knockdown (B). Heatmap showing differentially expressed genes between shNF1 and shSCR expressing cells. (C) Western blots showing NF1 and EGFR expression before and after DOX-induced NF1 expression (rNF1) compared to control (rControl) in NF1 Mut STC-08-175 and Mewo melanoma cells. HSP90 served as a loading control. (D) GSEA Hallmark pathway analysis showing IFNα and IFNɣ responses are upregulated, and EMT, hypoxia and inflammatory response are downregulated as a result of NF1 ectopic expression. (E) Heatmap showing significant differential expressed genes comparing NF1 ectopic expression to control. (F) Growth curves of NF1 Mut STC-08-175 melanoma cells ectopically expressing rNF1 compared to control. P -values were calculated with a two-sided t-test at the experimental endpoint. Data points represent mean ± SD. (n=12). (G) Western blots of NF1 co-immunoprecipitation showing EGFR levels after pulling down with NF1 or IgG control antibodies after DOX-induced NF1 expression (rpbNF1) or control (rbpControl) in STC-08-175 or Mewo NF1 Mut melanoma cells. (H) Western blots comparing NF1 and EGFR, expression in shNf1 compared to shSCR RIM-3 murine melanoma cells. HSP90 served as a loading control. (I) RT-PCR data showing Nf1 and Egfr expression changes in shNf1compared to shSCR expressing RIM3 melanoma cells. Bar graphs indicate mean ± SD (n=3). P -values were calculated with a two-sided t-test. (J) Growth curves of RIM-3 melanoma cells expressing shNf1 compared to shSCR (control). P -values were calculated with a two-sided t-test at the experimental endpoint. Data points represent mean ± SD. (n=12). (K) Heatmap showing differentially expressed antigen presentation genes in RIM-3-shNF1 melanoma cells after EGFR knockdown. (L) GSEA Hallmark Gene Sets analysis showing enriched pathways in shEGFR RIM-3-shNF1 cell lines. (M) Heatmap showing differentially expressed HLA genes in shEGFR and shSCR (control) expressing NF1 Mut STC-07-127 melanoma cells. (N) Western blots comparing HLA and EGFR expression with and without afatinib treatment in three human NF1 Mut melanoma cell lines. HSP90 served as a loading control. (O) Flow cytometry analysis showing MHC class I antigen presentation difference between IFNɣ-stimulated or non-stimulated NF1 Mut STC-08-175 melanoma cells with and without afatinib treatment. Bar graphs indicate mean ± SD (n=3). P -values were calculated with a two-sided t-test.
Article Snippet: Tumor volumes were calculated with the formula: (v (π/6*l*w2), where l = length in mm, w = width in mm, v=mm3). shNF1 RIM-3 tumors were allowed to reach ∼100-200 mm 3 for growth curves or ∼500 mm 3 for flow analysis before being randomly assigned to either afatinib treatment (Medchem Express, BIBW 2992, Cat# HY-10261), PD-1(CD279) anti-mouse Mab (Bioxcell, Cat# BE0146), a combination of both treatments or the appropriate vehicle control.
Techniques: Knockdown, Expressing, Western Blot, Control, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Immunopeptidomics, Flow Cytometry
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