Supplementary Material: Peroxisomes protect lymphoma cells from HDAC inhibitor-mediated apoptosis

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1 Supplementary Material: Peroxisomes protect lymphoma cells from HDAC inhibitor-mediated apoptosis Michael S Dahabieh 1,2, ZongYi Ha 1,5, Erminia Di Pietro 3,5, Jessica N Nichol 1, Alicia M Bolt 1,4, Christophe Goncalves 1, Daphné Dupéré-Richer 1, Filippa Pettersson 1, Koren K Mann 1,2,4, Nancy E Braverman 3, Sonia V del Rincón *,1, Wilson H Miller Jr. *,1,2 Affiliations: 1 Lady Davis Institute, McGill University 2 Department of Experimental Medicine, McGill University 3 Department of Medical Genetics and Pediatrics, McGill University Health Centre 4 Department of Oncology, McGill University 5 These authors contributed equally to the work. * To whom correspondence should be addressed: wilson.miller@mcgill.ca, sonia.delrincon@mcgill.ca

2 Supplementary materials and methods: Mined cdna analyses: Gene set expression profiles corresponding to vehicle and HDACi-treated U937 (GEO: GSE58421), MM231 (GEO: GSE72688), NB4, HL-60, and K562 (GEO: GSE32240) were analyzed with respect to the GO term peroxisome organization. Heat maps of expressed genes only were generated via hierarchical clustering of rows with Euclidian distance as a metric and average linkage method using Morpheus (Broad institute, Cambridge, MA, USA). Phase contrast images: Cells were transfected per the methods section in main text, treated with 2 um Vor for 18h, viewed a DM IL LED (Leica, Wetzlar, Germany) microscope and images were captured via an Infinity3 (Lumenera, Sarasota, FL, USA) camera. Supplementary Figure Captions: Fig. S1. HDACi induce upregulation of peroxisomes across multiple cancer cell lines. (A) Microarray heatmap of cdnas corresponding to GO term: peroxisome organization (GO: ) in Vor-treated (2 µm, 12h) U937 cells (GEO: GSE58421), performed in biological duplicate (N=2). (B) Mined, microarray expression profile of SAHA-treated (5 µm, 24h) MDA- MB-231 cells from N=3 (GEO accession: GSE72688). Gray bars indicate transcripts with undetectable expression. (C) Microarray expression profile (GEO accession: GSE32240) in NB4, K562 and HL-60 leukemia cell lines upon treatment with (1mM, 48h) valproic acid (VPA). Note: values found on GEO correspond to Log 2 of the ratio of VPA vs vehicle treated. (D)

3 Immunoblots of PEX3 and PMP70 (β-actin loading control) in vehicle (DMSO) and Vor-treated (2 µm) HL-60 and (5 µm) MM-231 cells. (E) Total PlsEtn levels (sn-1, PlsEtn), (F) PlsEtn 16:0, (G) 18:0 and (H) 18:1 after vehicle and 6,12, and 18h Vor (2 µm) treatment of U937 cells. Fig. S2. PEX3 knockdown potentiates Vor-induced apoptosis in lymphoma model systems. (A) PMP70 immunofluorescence staining (DAPI nuclear stain) of siscr and sipex3-2 vehicle (DMSO) and Vor-treated (2 µm, 12h) U937 cells. Vor was added 72h post-transfection. Graph represents means ± SEM (one way analysis of variance, Tukey) of N=3 with 5 images taken per condition and ~4 cells per image. Scale bar represents 10 µm. * P 0.05 and ***P (B) Immunoblots comparing siscr vs sipex3-2 knockdown in U937 cells following DMSO and Vor (2 µm, 12h) treatment. Shown are additional peroxisome biogenesis and assembly components PEX16 and PEX19, as well as apoptosis marker CL-CASP3. (C) Flow cytometry scatter plots of AnnexinV-FITC (horizontal axis) and propidium iodide (PI, vertical axis), of siscr and sipex3-2 upon vehicle and Vor treatment (2 µm, 24h) of U937 cells and (D) corresponding to bar graphs of apoptotic cells representing the sum of Annexin-V (+)/PI(+) and Annexin-V(+)/PI(-). Graph represents means ± SEM (one way analysis of variance, Tukey) of N=3 (technical triplicate). *** P (E) Phase contrast cell images corresponding to treatments upon PEX3 knockdown in U937 cells. Scale bar represents 10 µm. (F) Relative PEX3, and CL-CASP3 protein levels upon of sipex3-1 and sipex3-2 transfection and Vor treatment (4 µm, 16h) in OCI-LY8 cells, and (G) Flow cytometry scatter plots of AnnexinV- FITC (horizontal axis) and propidium iodide (PI, vertical axis), of siscr and sipex3-1, sipex3-2 upon vehicle and Vor treatment (4 µm, 24h) of OCI-LY8 cells and (H) corresponding bar graphs of apoptotic cells representing the sum of Annexin-V (+)/PI(+) and Annexin-V(+)/PI(-).

4 Graph represents means ± SEM (one way analysis of variance, Tukey) of N=3 (technical triplicate). *** P Fig. S3. PEX19 knockdown potentiates Vor-induced apoptosis in lymphoma model systems. (A) Immunoblot of PEX19 levels upon siscr and sipex19 (2 sequences) transfection following vehicle (DMSO) and Vor (2 µm, 12h) treatment in U937 cells, and (B) OCI-LY8 cells (4 µm, 12h). (C) Flow cytometry scatter plots of AnnexinV-FITC (horizontal axis) and propidium iodide (PI, vertical axis), of siscr and sipex3 (sequences 1 and 2) upon vehicle and Vor treatment (2 µm, 24h) of U937 cells and (D) OCI-LY8 cells (4 µm Vor, 24h). (E) and (F) correspond to bar graphs of apoptotic cells from (C) and (D), respectively, representing the sum of Annexin-V (+)/PI(+) and Annexin-V(+)/PI(-). Graph represents means ± SEM (one way analysis of variance, Tukey) of N=3 (technical triplicate). ** P 0.01, *** P Fig. S4. B8 cells possess elevated plasmalogen levels and undergo apoptosis upon PEX3 knockdown. Analyses of (A) PlsEtn 18:0, and (B) 16:0 upon vehicle (DMSO) and 12h Vor (2 µm) treatment in U937 cells and B8 cells (2 µm Vor). Graphs represent means ± SEM (one way analysis of variance, Tukey) of N=3 (technical triplicate). ** P 0.01, *** P (C) PMP70 immunofluorescence staining (DAPI nuclear stain) of siscr and sipex3-2 vehicle (DMSO) and Vor-maintained B8 cells. Graph represents means ± SEM (unpaired t-test) of N=3 (technical triplicate). ***P Scale bar represents 10 µm. (D) Immunoblots of PEX3, PEX16, PEX19, and β-actin (loading control), 96h post transfection with siscr and sipex3-2 in B8 cells (2 µm Vor). (E) Flow cytometry scatter plots of AnnexinV-FITC (horizontal axis) and

5 propidium iodide (PI, vertical axis), of siscr and sipex3-1 and (F) siscr vs sipex3-2 in Vorcultured B8 cells. Graph represents means ± SEM (unpaired t-test) of N=3 from. ** P = Fig. S5. Catalase protects U937 and B8 cells from H 2 O 2 -mediated apoptosis. (A) quantitation of catalase puncta in vehicle (DMSO) and Vor-treated (2 µm, 12h) U937 cells and B8 cells chronically maintained in 2 µm Vor. Graph represents means ± SEM of N=6 with 5 images taken per condition with ~ 6 cells per image. One-way ANOVA analysis. *** P-value (B) Representative immunoblots of catalase, γh2ax, CL-PARP and b-actin (loading control) upon H 2 O 2 (0.25 mm, 12h) treatment of siscr and sicat-1 transfected B8 cells. (C) Flow cytometry scatter plots of AnnexinV-FITC (horizontal axis) and propidium iodide (PI, vertical axis), of siscr and sicat (sequences 1 and 2) upon vehicle and 0.25 mm H 2 O 2 treatment (24h) of U937 cells and (D) B8 cells in 0.50 mm H 2 O 2 (24h). Cells were treated with H 2 O 2 48h posttransfection. (E) and (F) correspond to bar graphs of apoptotic/necrotic cells from (C) and (D), respectively, representing the sum of Annexin-V (-)/PI(+), Annexin-V (+)/PI(+) and Annexin- V(+)/PI(-). Graph represents means ± SEM (one way analysis of variance, Tukey) of N=3 (technical triplicate). ** P 0.01, *** P Fig. S6. Knockdown of catalase sensitizes B8 cells to HDACi. (A) Immunoblots of catalase, γh2ax, HO-1, CL-CASP3 and b-actin (loading control), upon siscr and sicat-2 treatment. Samples were harvested 96h post-transfection. (B) Flow cytometry scatter plots of AnnexinV- FITC (horizontal axis) and propidium iodide (PI, vertical axis) of siscr and sicat-2 treated cells, 96h post-transfection, and (C) corresponding % apoptotic cells graphs of PI/AnnexinV-

6 FITC double positive and AnnexinV-FITC positive cells. Graph represents means ± SEM (unpaired t-test) of N=3. * P = Fig. S7. HDACi-mediated apoptosis is potentiated via catalase knockdown in U937 and OCI- LY8 cells. (A) Immunoblots of catalase and β-actin (loading control) 48h post-transfection with siscr and sicat-1 and sicat-2 sequences in U937, and (B) OCI-LY8 cells. (C) Flow cytometry scatter plots of AnnexinV-FITC (horizontal axis) and propidium iodide (PI, vertical axis), of siscr and sicat (sequences 1 and 2) upon vehicle and Vor treatment (2 µm, 24h) of U937 cells and (D) OCI-LY8 cells (4 µm Vor, 24h). (E) and (F) correspond to bar graphs of apoptotic cells from (C) and (D), respectively, representing the sum of Annexin-V (+)/PI(+) and Annexin-V(+)/PI(-). Graph represents means ± SEM (one way analysis of variance, Tukey) of N=3 (technical triplicate). *** P Fig. S8. Panobinostat increases peroxisomal protein abundance and potentiates apoptosis upon PEX3 knockdown. (A) Flow cytometry scatter plots of AnnexinV-FITC (horizontal axis) and propidium iodide (PI, vertical axis), of siscr and sipex3 (sequences 1 and 2) upon vehicle and pano treatment (40 nm, 24h) of U937 cells and (B) corresponding bar graphs of % apoptotic cells representing the sum of Annexin-V (+)/PI(+) and Annexin-V(+)/PI(-). Graph represents means ± SEM (one way analysis of variance, Tukey) of N=3 (technical triplicate). * P 0.01, *** P (C) Immunoblots of catalase, PEX3, PEX19, PEX11B and b-actin (loading control) upon treatment 6 and 12h panobinostat (pano) treatment (40 nm) in U937 cells, and constant maintenance in B8 cells (40 nm, 1 week). (D) Flow cytometry scatter plots of AnnexinV-FITC (horizontal axis) and propidium iodide (PI, vertical axis) corresponding to

7 vehicle (DMSO)-treated U937 and B8 cells, alongside U937 cells treated with 40 nm pano for 48h and B8 cells maintained in 40 nm pano for 1 week. (E) Bar graphs of apoptotic/necrotic cells from (D), representing the sum of Annexin-V (-)/PI(+), Annexin-V (+)/PI(+) and Annexin- V(+)/PI(-). Graph represents means ± SEM (one way analysis of variance, Tukey) of N=3 (technical triplicate). *** P Supplementary Tables: Table S1: KEGG pathways enriched in panobinostat-treated DLBCL responders versus nonresponders. See methods section in manuscript (GSEA) for analyses details. Table S2: MEF2B mutation status does not correlate with enhanced peroxisome gene expression. Analyses were performed from 4 patients with MEF2B mutant status and no correlation was found between elevated expression of KEGG peroxisome transcripts. See methods section in manuscript (GSEA) for analyses details.

Confocal immunofluorescence microscopy

Confocal immunofluorescence microscopy HL-6 and cells were cultured and cytospun onto glass slides. The cells were double immunofluorescence stained for Mt NPM1 and fibrillarin (nucleolar marker). Briefly,