HRP Anti-GAPDH antibody [mAbcam 9484] - Loading Control

• WB

Lab

Western blot - HRP Anti-GAPDH antibody [mAbcam 9484] - Loading Control (AB9482)

All lanes:

Western blot - HRP Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9482) at 1/1000 dilution

Lane 1:

HeLa Whole Cell Lysate at 10 µg

Lane 2:

Hek293 Whole Cell Lysate at 10 µg

Lane 3:

NIH 3T3 Whole Cel lLysate at 10 µg

Lane 4:

PC12 Whole Cell Lysate at 10 µg

Predicted band size: 36 kDa

Observed band size: 37 kDa

true

Exposure time: 4min

• WB

CiteAb

Western blot - HRP Anti-GAPDH antibody [mAbcam 9484] - Loading Control (AB9482)

Western Blotting using HRP Anti-GAPDH antibody [mAbcam 9484] - Loading Control, ab9482. Publication image from Swanton, C. et al., 2016, Genome Biol, 27634334. Legend direct from paper.

HER2 expression and PTEN contribute to APOBEC3 activity. aAPOBEC3B mRNA expression following silencing of HER2 expression in BT474 cells by RNAi. HER2 levels were depleted by RNAi and, after 72 h, cells were harvested and mRNA extracted. Following cDNA synthesis, APOBEC3B mRNA levels were determined by quantitative PCR; *p < 0.01 (t-test). siNT non-targeting control siRNA. b BT474 cells were treated as in a and, following lysis, western blots were probed with the indicated antibodies. c BT474 and MDA-MB-361 cells were treated as in a and, following lysis, samples were subjected to cytidine deamination assay to determine levels of APOBEC3 activity. d BT474 cells were treated with 10 nM afatinib or 30 nM lapatinib for 24 h followed by mRNA isolation and quantitative PCR to determine APOBEC3B mRNA expression levels; *p < 0.01, ***p < 0.005 (t-test). e BT474 cells were treated as in d and, following lysis, western blots were probed with the indicated antibodies. f BT474 cells were treated as in d and, following lysis, samples were subjected to cytidine-based deamination assay to determine levels of APOBEC3 activity. g MCF10A cells were treated with or without 2 mM hydroxyurea (HU) and exposed to the indicated drugs for 48 h followed by APOBEC3 cytidine deamination assay. h MDA-MB-453 cells were treated with the indicated drugs for 48 h followed by APOBEC3 cytidine deamination assay. i PTEN levels were depleted from MCF7 cells growing on glass coverslips by RNAi. Cells were fixed and stained with 53BP1 and cyclin A antibodies. The fraction of cyclin A-negative cells displaying more than five 53BP1 nuclear foci were scored; *p < 0.05 (t-test). j PTEN levels were depleted from MCF7 cells by RNAi. After 72 h cells were harvested and samples were subjected to cytidine deamination assay to determine APOBEC3 activity. k APOBEC3 activity in response to RAS induction and hydroxyurea (HU) treatment. MCF10A-ER : HRAS V12 cells were induced with tamoxifen (4-hydroxytamoxifen; 4-OHT) in either the presence or absence of hydroxyurea for 48 h, followed by mRNA isolation, cDNA synthesis and quantitative PCR to determine APOBEC3B expression levels. l MCF10A-ER : HRAS V12 cells were treated as in k. Cells were subsequently lysed and subjected to APOBEC3 cytidine deamination assay. LY LY294002, MK MK2206, NT non-targeting, RAPA rapamycin, UNT untreated

false

• WB

CiteAb

Western blot - HRP Anti-GAPDH antibody [mAbcam 9484] - Loading Control (AB9482)

Western Blotting using HRP Anti-GAPDH antibody [mAbcam 9484] - Loading Control, ab9482. Publication image from Swanton, C. et al., 2016, Genome Biol, 27634334. Legend direct from paper.

HER2 expression and PTEN contribute to APOBEC3 activity. aAPOBEC3B mRNA expression following silencing of HER2 expression in BT474 cells by RNAi. HER2 levels were depleted by RNAi and, after 72 h, cells were harvested and mRNA extracted. Following cDNA synthesis, APOBEC3B mRNA levels were determined by quantitative PCR; *p < 0.01 (t-test). siNT non-targeting control siRNA. b BT474 cells were treated as in a and, following lysis, western blots were probed with the indicated antibodies. c BT474 and MDA-MB-361 cells were treated as in a and, following lysis, samples were subjected to cytidine deamination assay to determine levels of APOBEC3 activity. d BT474 cells were treated with 10 nM afatinib or 30 nM lapatinib for 24 h followed by mRNA isolation and quantitative PCR to determine APOBEC3B mRNA expression levels; *p < 0.01, ***p < 0.005 (t-test). e BT474 cells were treated as in d and, following lysis, western blots were probed with the indicated antibodies. f BT474 cells were treated as in d and, following lysis, samples were subjected to cytidine-based deamination assay to determine levels of APOBEC3 activity. g MCF10A cells were treated with or without 2 mM hydroxyurea (HU) and exposed to the indicated drugs for 48 h followed by APOBEC3 cytidine deamination assay. h MDA-MB-453 cells were treated with the indicated drugs for 48 h followed by APOBEC3 cytidine deamination assay. i PTEN levels were depleted from MCF7 cells growing on glass coverslips by RNAi. Cells were fixed and stained with 53BP1 and cyclin A antibodies. The fraction of cyclin A-negative cells displaying more than five 53BP1 nuclear foci were scored; *p < 0.05 (t-test). j PTEN levels were depleted from MCF7 cells by RNAi. After 72 h cells were harvested and samples were subjected to cytidine deamination assay to determine APOBEC3 activity. k APOBEC3 activity in response to RAS induction and hydroxyurea (HU) treatment. MCF10A-ER : HRAS V12 cells were induced with tamoxifen (4-hydroxytamoxifen; 4-OHT) in either the presence or absence of hydroxyurea for 48 h, followed by mRNA isolation, cDNA synthesis and quantitative PCR to determine APOBEC3B expression levels. l MCF10A-ER : HRAS V12 cells were treated as in k. Cells were subsequently lysed and subjected to APOBEC3 cytidine deamination assay. LY LY294002, MK MK2206, NT non-targeting, RAPA rapamycin, UNT untreated

false

• WB

CiteAb

Western blot - HRP Anti-GAPDH antibody [mAbcam 9484] - Loading Control (AB9482)

Western Blotting using HRP Anti-GAPDH antibody [mAbcam 9484] - Loading Control, ab9482. Publication image from Rorbach, J. et al., 2016, Nat Commun, 27356879. Legend direct from paper.

Identification of pathogenic compound heterozygous mutations in NSun3.(a) OXPHOS complex activity in unfrozen patient muscle homogenate (red line). Grey bars represent the normal range. Note the different scale for complex I–II as compared with complex III–V. (b) Segregation analysis and electropherogram corresponding to the genomic DNA mutations identified in the family of a patient carrying NSun3 mutations. The patient's parental allele carries a c.295C>T transition in exon 3, while the maternal allele has a 3,314 nt deletion spanning exon 3 (c.123-615_466+2155del). (c) Mutation analysis of NSun3 mRNA level in human dermal fibroblasts of wild-type (wt/wt), patient (mut/mut) and patient cells rescued with a NSUN3 construct (mut/mut+NSUN3). Gel electrophoresis of DNA fragments obtained in a reverse transcriptase–PCR using total RNA from the indicated samples. (d) Sanger sequencing of the bands from the mut/mut sample excised from the gel presented in c showing a stop mutation in the full-length NSUN3 mRNA and the lack of exon 3 on the shorter band. (e) Schematic overview of the NSUN3 gene, summarizing the mutations in the patient cells (red) on both genomic DNA (gDNA) and mRNA level. (f) Immunofluorescence labelling of a Flag-tagged NSun3 construct (red) in HeLa cells. Cells were counterstained for the mitochondrial import receptor subunit TOM20 (green) and DAPI (blue). Scale bar, 10 µm. (g) Sub-cellular localization of NSun3 analysed by western blotting with antibodies against NSun3, TOM22 (mitochondrial outer membrane), mtSSB1 (mitochondrial matrix), GAPDH (cytosol), Histone H3 (nucleus). HEK293T cells were fractionated into debris (‘D', lane 2), cytosol (‘C', lane 3) and mitochondria (‘M', lanes 4–6) ‘T' indicates the total cell lysate. ‘fl' indicates full-length TOM22, ‘tr' stands for truncated TOM22.

false

• WB

CiteAb

Western blot - HRP Anti-GAPDH antibody [mAbcam 9484] - Loading Control (AB9482)

Western Blotting using HRP Anti-GAPDH antibody [mAbcam 9484] - Loading Control, ab9482. Publication image from Swanton, C. et al., 2016, Genome Biol, 27634334. Legend direct from paper.

APOBEC3 activity and replication stress in breast cancer cell lines. aAPOBEC3B (black), APOBEC3G (grey) and APOBEC3A (white) mRNA expression in 15 breast cancer cell lines as determined by quantitative PCR. HER2+ cell lines (red), basal cell lines (black), luminal cell lines (green). SKBR3 cells have a null mutation for APOBEC3B. Error bars represent standard deviation. b APOBEC3 activity in the 15 breast cancer cell lines used in a. Cells were lysed and subjected to oligonucleotide-based cytidine deamination assay followed by electrophoresis on 15 % TBE-urea gels. c Cells were grown for two population doublings on glass coverslips followed by fixation and staining with 53BP1 and cyclin A antibodies. The fractions of cyclin A-negative cells displaying more than five 53BP1 nuclear foci were scored. APOBEC3B mRNA expression was determined by quantitative PCR from parallel cell lysates. A Spearman’s rank correlation test was performed to correlate the fraction of 53BP1 nuclear bodies in cell lines with the level of APOBEC3B (r = 0.62, p = 0.0284). Error bars represent standard deviation. d BT474 cells were treated with 12.5–300 µM nucleosides for 72 h prior to lysis. Western blots were probed with the indicated antibodies. e BT474 cells were treated as in d followed by lysis and an APOBEC3 cytidine deamination assay

false

• WB

CiteAb

Western blot - HRP Anti-GAPDH antibody [mAbcam 9484] - Loading Control (AB9482)

Western Blotting using HRP Anti-GAPDH antibody [mAbcam 9484] - Loading Control, ab9482. Publication image from Swanton, C. et al., 2016, Genome Biol, 27634334. Legend direct from paper.

Induction of replication stress and APOBEC3 activity in breast cancer cell lines. a MCF10A cells were treated with the indicated drugs for 48 h followed by mRNA extraction, cDNA synthesis and quantitative PCR for APOBEC3B and APOBEC3G expression levels. b MCF10A cells were treated as in a followed by western blotting with the indicated antibodies. c MCF10A cells were treated as in a prior to lysis and a cytidine deamination assay for APOBEC3 activity using probe 2. d MCF10A cells were treated as in a followed by fixation and immunofluorescence for Ser139 γH2AX and S4/8 replication protein A phosphorylation (pRPA). Red asterisks indicate treatments inducing APOBEC3B mRNA, protein expression, activity levels and S4/8 RPA phosphorylation. e MCF10A cells were pre-treated with 300 µM exogenous nucleosides followed by incubation with the indicated drugs for an additional 24 h. Following lysis, APOBEC3 activity was measured by a cytidine deamination assay. f Ribonucleotide reductase subunits RRM1, RRM2 and RRM2B were depleted from MCF10A cells by RNA interference and, after 72 h, cells were lysed and subjected to an APOBEC3 cytidine deamination assay. 5FU 5-fluorouracil, MMS methyl methanesulfonate, siNT non-targeting control siRNA

false