Fig. 1: PANX1 5′ UTR expression correlates with its protein abundance. | Oncogenesis

Fig. 1: PANX1 5′ UTR expression correlates with its protein abundance.

From: Quercetin induces pannexin 1 expression via an alternative transcript with a translationally active 5′ leader in rhabdomyosarcoma

Fig. 1

A RNA-seq reads mapped to the 5′ UTR and exon 1 regions of PANX1 from representative RNA libraries prepared from three biological replicates of Rh30 (aRMS) cells and as compared to representative RNA libraries from six normal human skeletal muscle tissue samples retrieved from Ryan et al., JCI Insight 2018. The Y-axis shows RNA-seq read coverage. B Ratio of normalized counts of RNA-seq reads mapped to the 5′ UTR region over the CDS region of PANX1. Note that there is no read mapped to the 5′ UTR region of PANX1 in Rh30 RNA samples. All normal skeletal muscle RNA-seq reads were retrieved from Ryan et al., JCI Insight 2018. Statistical analysis was performed using a two-way ANOVA with Tukey’s post-hoc test. **P < 0.01 between normal skeletal muscle and Rh30. C RT-qPCR of PANX1 from Rh18 (eRMS) (n = 3) and Rh30 cells (n = 3) using primers specific to its 5′ UTR (−276 to +1 relative to the PANX1 ATG start codon) and exon-exon junctions. Undifferentiated (n = 3) and differentiated (n = 3) human skeletal muscle myoblasts (HSMM) were used as controls. Results from each amplified region were normalized independently to their differentiated (Diff. HSMM) HSMM controls in the respective PANX1 mRNA regions. **P < 0.01 and ***P < 0.001 compared to Undiff. HSMM; #P < 0.05,##P < 0.01 and ###P < 0.001 compared to Rh18; &&P < 0.01 and &&&P < 0.001 compared to Rh30 (n = 3). D Representative Western blots and their quantification E showing levels of myosin heavy chain (MHC), a marker for terminal myogenic differentiation of HSMM, and PANX1 (n = 3). GAPDH was used as loading control. Arrowhead indicates a non-specific immunoreactive band (n.s.), which was further confirmed by (F) Western blotting analysis (n = 3) of Rh30 cells 72 h post-siRNA-mediated knockdown of PANX1. Both wildtype Rh30 cells (WT) and Rh30 cells transfected with a scrambled siRNA sequence (Control siRNA) were used as controls. GAPDH was used as loading control. Arrowhead highlights the non-specific immunoreactive band (n.s.) also seen in (D). ***P < 0.001 compared to Undiff. HSMM, Rh18, and Rh30. Results are expressed as mean ± s.d. Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc tests in (C) and (E).

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