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. Author manuscript; available in PMC: 2020 Nov 1.
Published in final edited form as: Nat Cancer. 2020 Apr 20;1(5):533–545. doi: 10.1038/s43018-020-0056-0

Extended Data Fig. 6. ISR pathway activation enhances PD-L1 translation, and regulation of the PD-L1 5′ UTR reporter by UROD depletion.

Extended Data Fig. 6.

a, qRT-PCR analysis of mean Pd-l1 mRNA, normalized to Actin, in S/S or A/A MEFs expressing either control or Urod shRNA with 3 technical replicates per sample. Experiment was performed two independent times with similar results. Data from a representative experiment are shown. b,c, qRT-PCR analysis of PD-L1 mRNA in 6 individual ribosomal fractions, with 3 technical replicates per sample for two independent primer pairs. Quantification of combined fractions (<3 and >3) from (b) are shown in main Fig. 5c. d,e, qRT-PCR analysis of PD-L1 mRNA in 6 individual ribosomal fractions, with 3 technical replicates per sample for two independent primer pairs. Quantification of combined fractions (<3 and >3) from (d) are shown in main Fig. 5d. f,g, qRT-PCR analysis of ATF4 mRNA in ribosomal fractions from main Fig. 5a (f) or main Fig. 5b (g). ATF4 mRNA abundance was calculated as described in main Fig. 5c and d. Error bars in f and g represent SDs from the mean from three independent fractions (<3 or >3 ribosomes). A student’s two-tailed t-test was performed to determine statistical significance. ***p=0.00062 (f); ***p=0.0008 (g). h,i, qRT-PCR analysis of ATF4 mRNA in 6 individual ribosomal fractions. j, qRT-PCR analysis of mean Luciferase mRNA, normalized to Actin, in MEF cells transfected with reporter constructs shown in main Fig. 5e, with 3 technical replicates, shown as individual data points. Experiment was performed two independent times, with similar results. Data from a representative experiment are shown. k, qRT-PCR analysis of mean Luciferase mRNA normalized to ACTIN in H358 cells from main Fig. 5g across n=3 technical replicates, shown as individual data points. l, qRT-PCR analysis of mean Luciferase mRNA normalized to Actin in MEF cells from main Fig. 5h across n=3 technical replicates, shown as individual data points. Experiment in (k) was performed in an independent cell line, shown in (l).