myc tag sequence  (Addgene inc)

Journal: bioRxiv

Article Title: An extra-glycolytic function for hexokinase 2 as an RNA-binding protein regulating SOX10 mRNA translation in melanoma

doi: 10.1101/2024.08.13.607712

Figure Lengend Snippet: (A) RT-qPCR quantification representing the % of SOX10 mRNA (left panel) or HPRT mRNA (right panel) in fractions (horizontal axes) obtained by sucrose-gradient (10-50%) ultracentrifugation of lysates from A375 cells transfected with siRNAs targeting HK2 (siHK2, light red) or control (siCTRL, grey) (n = 2 biological replicates). (B) Western blot analysis of the SOX10 protein level in distinct melanoma cell lines transfected with siRNAs targeting HK2 (siHK2, light red), GAPDH (siGAPDH, light blue) or control (siCTRL, grey). The SOX10 protein quantification is normalized to VCL expression. p -values were calculated by two-tailed unpaired t -test (SD, n = 3 biological replicates) and only significant comparisons are shown (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). (C) RT-qPCR quantification of the SOX10 mRNA in different melanoma cell lines transfected with siRNAs targeting HK2 (siHK2, light red), GAPDH (siGAPDH, light blue) or control (siCTRL, grey). p-values were calculated by ordinary one-way ANOVA (SD, n = 3 biological replicates).

Article Snippet: To establish the stable overexpression of SOX10 (Myc-DDK tagged; OriGene, #RC203545L3V) in A375 and A2058 cell lines, cells were transduced with pLenti-C-Myc-DDK-P2A-Puro vector expressing SOX10 (NM_006941) ORF nucleotide sequence (OriGene, #RC203545L3V) according to manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Transfection, Control, Western Blot, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: An extra-glycolytic function for hexokinase 2 as an RNA-binding protein regulating SOX10 mRNA translation in melanoma

doi: 10.1101/2024.08.13.607712

Figure Lengend Snippet: (A) RIP experiment performed on A375 and A2058 melanoma cell lines (SD, n = 3 biological replicates). SOX10 mRNA was analysed in IgG and HK2 immunoprecipitated samples and expressed as percentage of the mRNA present in the input. GAPDH , TBP and ACT mRNAs were used as negative controls. p -values were calculated by ordinary two-way ANOVA with Dunnett’s multiple comparisons test (SD, n = 3 biological replicates) (**** p ≤ 0.0001). (B) In silico prediction of HK2- SOX10 mRNA interaction propensities using the CatRAPID algorithm. The highest interaction score observed between HK2 and the SOX10 mRNA is at the SOX10 5’UTR. Upper panel: HK2- SOX10 mRNA interaction profile. Lower panel: schematic representation of the SOX10 mRNA. (C) RIP experiment performed on A375 cells transfected with luciferase reporters containing the SOX10 5’UTR and 3’UTR sequences upstream of the Renilla reporter gene. An Empty reporter was used as control. Renilla mRNA was analyzed in IgG and HK2 immunoprecipitated samples and expressed as percentage of the mRNA present in the input. p -values were calculated by ordinary two-way ANOVA with Dunnett’s multiple comparisons test (SEM, n = 6 biological replicates) (* p ≤ 0.05). (D) Representative fluorescence images and quantification of RNA-proximity ligation assays (PLA) of HK2 protein and SOX10 mRNA in A375 cells transfected with siRNAs targeting SOX10 (siSOX10, orange) or control (siCTRL, grey) (n = 3 biological replicates). After 48h, cells were fixed, permeabilized, and incubated with anti-sense SOX10 5’UTR oligonucleotide probes and anti-HK2 antibody. Scale bar, 10 μm. HK2- SOX10 mRNA PLA signal (yellow), Phalloidin staining (green), and DAPI staining (blue). The significance for PLA values was derived from the Mann-Whitney statistical test (SD, ** p ≤ 0.01).

Article Snippet: To establish the stable overexpression of SOX10 (Myc-DDK tagged; OriGene, #RC203545L3V) in A375 and A2058 cell lines, cells were transduced with pLenti-C-Myc-DDK-P2A-Puro vector expressing SOX10 (NM_006941) ORF nucleotide sequence (OriGene, #RC203545L3V) according to manufacturer’s instructions.

Techniques: Immunoprecipitation, In Silico, Transfection, Luciferase, Control, Fluorescence, Ligation, Incubation, Staining, Derivative Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: An extra-glycolytic function for hexokinase 2 as an RNA-binding protein regulating SOX10 mRNA translation in melanoma

doi: 10.1101/2024.08.13.607712

Figure Lengend Snippet: (A) Schematic of full-length (FL) and mutated SOX10 5’UTR (Δ1-6) reporters cloned upstream of the Renilla reporter. The deleted regions of ∼50nts are represented as red dashed lines. (B) Luciferase assay performed in A375 cells co-transfected with a Firefly reporter and the Renilla reporters in (A). An empty Renilla vector and the SOX10 3’UTR Renilla reporter were used as controls. The activity of the Firefly and Renilla reporters was measured 48h after transfection. The Renilla activity was normalized by the Firefly activity, and the data shown is relative to the Empty vector. p -values were calculated by Mann-Whitney statistical test (SEM, n = 4 biological replicates) (∗ p < 0.05). (C) RT-qPCR quantification of the Renilla mRNA from the same transfected cells in (B). The Renilla expression was normalized by the Firefly expression, and the data shown is relative to the expression of the Empty vector. p -values were calculated by Mann-Whitney statistical test (SEM, n = 4 biological replicates). (D) RIP experiment performed on A375 cells transfected with Renilla luciferase reporters containing the SOX10 5’UTR FL and Δ4, Δ5 and Δ6. Renilla mRNA was analyzed in IgG and HK2 immunoprecipitated samples and expressed as percentage of the mRNA present in the input. p -values were calculated by ordinary two-way ANOVA with Dunnett’s multiple comparisons test (SEM, n = 3 biological replicates) (* p ≤ 0.05).

Article Snippet: To establish the stable overexpression of SOX10 (Myc-DDK tagged; OriGene, #RC203545L3V) in A375 and A2058 cell lines, cells were transduced with pLenti-C-Myc-DDK-P2A-Puro vector expressing SOX10 (NM_006941) ORF nucleotide sequence (OriGene, #RC203545L3V) according to manufacturer’s instructions.

Techniques: Clone Assay, Luciferase, Transfection, Plasmid Preparation, Activity Assay, MANN-WHITNEY, Quantitative RT-PCR, Expressing, Immunoprecipitation

Journal: bioRxiv

Article Title: An extra-glycolytic function for hexokinase 2 as an RNA-binding protein regulating SOX10 mRNA translation in melanoma

doi: 10.1101/2024.08.13.607712

Figure Lengend Snippet: (A) Western blot analysis of the ectopic SOX10 Myc-DDK expression in A375 and A2058 melanoma cells. VCL was used as loading control (n = 3 biological replicates). (B) A375 and A2058 cells from (A) were plated 24h after transfection with siRNAs targeting HK2 (siHK2, light red) or control (siCTRL, grey). The colonies were stained with crystal violet after 10 days, and the clonogenic cell growth was measured. Upper panel: percentage of area covered by crystal violet stained cell colonies. Lower panel: representative images of three independent experiments. p -values were calculated by ordinary two-way ANOVA with Turkey’s multiple comparison test (SD, n = 3 biological replicates), and only significant differences within the same cell line are shown (* p ≤ 0.05, **** p ≤ 0.0001). (C) Cell proliferation assay performed in A375 and A2058 cells from (A). Cells were plated 24h after transfection with siRNAs targeting HK2 (Empty, light blue; SOX10, light orange) or control (Empty, dark blue; SOX10 dark orange). p -values were calculated by ordinary two-way ANOVA with Turkey’s multiple comparison test (SD, n = 3 biological replicates), and only significant differences within the same cell line are shown (**** p ≤ 0.0001).

Article Snippet: To establish the stable overexpression of SOX10 (Myc-DDK tagged; OriGene, #RC203545L3V) in A375 and A2058 cell lines, cells were transduced with pLenti-C-Myc-DDK-P2A-Puro vector expressing SOX10 (NM_006941) ORF nucleotide sequence (OriGene, #RC203545L3V) according to manufacturer’s instructions.

Techniques: Western Blot, Expressing, Control, Transfection, Staining, Comparison, Proliferation Assay