Fig 2: Universal high APOBEC3C expression in PDAC denotes shorter OS.The Mantel–Cox test was used to compare Kaplan–Meier survival curves. A, B Representative IHC images showing relatively high expression (A patient ID: 470506) and relatively low expression (B patient ID: 372428) of APOBEC3C in PDAC tissues. Sixty PDAC specimens (FUSCC cohort B1) underwent IHC staining with an APOBEC3C antibody to corroborate the qRT–PCR results. Positive APOBEC3C staining was located in the nucleus and cytoplasm of pancreatic tumour cells. C A cutoff value of 0.011802837 (qRT–PCR result) stratified the patients in FUSCC cohort B into the high APOBEC3C expression subgroup (n = 109) and low APOBEC3C expression subgroup (n = 15) with a difference in OS. The mRNA expression level was determined by performing qRT–PCR with cDNAs in triplicate, and the normalised mean value was used for analysis. D A cutoff value of 353.34 TPM stratified TCGA cohort into the high APOBEC3C expression subgroup (n = 69) and the low APOBEC3C expression subgroup (n = 94) with a trend towards a difference in OS. E A cutoff value of 372.77 TPM stratified the QCMG cohort into the high APOBEC3C expression subgroup (n = 62) and the low APOBEC3C expression subgroup (n = 8) with a difference in OS. PDAC, pancreatic ductal adenocarcinoma; A3C, APOBEC3C; HR, hazard ratio.

Fig 3: APOBEC3C is expressed at the highest level among APOBECs in PDAC.A, B The mRNA expression levels of APOBECs in TCGA (n = 163) and FUSCC cohort A (n = 124). C, D The Pearson correlation coefficients were calculated to examine the correlations between APOBEC expression in TCGA cohort (C) and FUSCC cohort A (D). *p < 0.05, **p < 0.01. Detailed information was shown in Table S5. E The height of the bar represents the median mRNA expression level of a specific APOBEC. GEPIA2 normalised data by the maximum median expression value across all blocks and then compared data between pancreatic tumour tissues (TCGA database, n = 163) and normal tissues (TCGA and GTEx databases, n = 171) using one-way ANOVA, *p-value < 0.01. Corresponding box plots are displayed in Fig. S3. F Expression of APOBEC enzymes in PDAC cell lines. The mRNA expression level was determined by performing qRT–PCR on validated cDNAs in triplicate. G, H Representative images (patient ID.408371, FUSCC cohort B1) of IHC staining for APOBEC3 in PDAC (G) and normal pancreatic tissues (H). Normal pancreatic tissues from six patients with PDAC were stained, and the results are presented in Fig. S4. PDAC, pancreatic ductal adenocarcinoma; TPM: transcripts per million mapped reads.

Fig 4: HIV-1 replication phenotypes following A3C disruption and variant complementation in non-permissive CEM2n cells. a Immunoblots of endogenous A3C in SupT11, H9, and CEM2n cells. Tubulin was used as a loading control. b Immunoblots of endogenous A3C in CEM2n clones following targeted disruption of A3C exon 3 by Cas9/gRNA complexes. Tubulin was used as a loading control. c A3C exon 3 sequences from parental CEM2n and an A3C-disrupted clone (CEM2n ?A3C). d Flow cytometry plots for CEM2n ?A3C cell pools 72 h post-transduction with GFP-reporter complementation vectors. The percentage of GFP+ cells is indicated for each population. e Immunoblots of A3C in the parental CEM2n line, CEM2n ?A3C, and complemented CEM2n ?A3C derivatives. Tubulin was used as a loading control. f, g Spreading infection kinetics of Vif-proficient and Vif-deficient HIV-1 (initial MOI = 0.02) in CEM2n, CEM2n ?A3C, and the indicated complemented conditions. SupT11 cells are included as a control permissive cell type. Virus infectivity was determined by infection of CEM-GFP with culture supernatants followed 48 h later by quantification with flow cytometry. Each spreading infection experiment was performed 3 times and representative data are shown

Fig 5: The chromosomal distribution of kataegis regions with enriched C > X substitutions is correlated with APOBEC3C expression.The MeiChuncai/KataegisPortal R package was used to detect kataegis in FUSCC cohort A (n = 124) and calculate the C > X mutation proportion in each kataegis region. The Ramtools R package was used to obtain trinucleotide motifs of mutations. Each PDAC sample harboured 125 to 173 kataegis regions. Detailed information about kataegis in FUSCC cohort A is recorded in Tables S7-10. A Representative rainfall plot showing a PDAC sample (Patient ID: 442043) harbouring 159 kataegis regions. The x-axis represents the SNV position, and the y-axis represents the intermutational distance. As the density of the mutation distribution may influence the observation of kataegis, we separately display kataegis regions on chromosomes 1, 7, 9, 11, and 17 to show the kataegis regions with enriched C > X conversions. Kataegis were pointed out by arrows, and kataegis with enriched C > X mutations were labelled by solid arrows. B Nonparametric Spearman correlation test to determine whether the count of kataegis regions with enriched C > X substitutions or the count of total kataegis regions correlated with APOBEC3C expression. C Heatmap showing the chromosomal distribution of kataegis regions with enriched C > X substitutions (more than 5 C > X substitutions per 1000 bp). The colour ladder was based on the counts of kataegis regions. The y-axis represents the hospital ID of each sample in FUSCC. D Genes with enriched C > X kataegis regions. Genes with frequencies lower than 20% were omitted from this figure. The complete list is recorded in Table S9. SNV, single-nucleotide variant; PDAC, pancreatic ductal adenocarcinoma; chr, chromosome; r, correlation. E, F, G Comparison of total kataegis count (E), C > X mutation-enriched kataegis count (F) and proportion of C > X mutation-enriched kataegis in total kataegis (G) between APOBEC3C highest expressed samples (n = 25) and APOBEC3C lowest expressed samples (n = 25) from FUSCC cohort A.