Fig 2: TEX19 is predominantly localized to the cytoplasm in normal testis and cancer tissues: a Staining of a human testicular seminiferous tubule indicates that TEX19 (red – bottom left panel) is predominantly located in the basal layer of cells and is mostly cytoplasmic. Co-staining with an anti-vimentin antibody (green – top right), which marks Sertoli cells, reveals that TEX19 staining is adjacent to, but not overlapping with vimentin. Bar = 50 μm. b Nuclear foci are apparent when testis are stained with anti-TEX19 (red; blue = DAPI) antibody. White arrows in the merged image show the localization of the nuclear staining with anti-TEX19 antibodies (red; DAPI = blue). c Co-staining with anti-MAGE-A1 antibodies (spermatogonial cells; green) and anti-TEX19 antibodies (red; blue = DAPI) indicate that TEX19 nuclear foci are associated with spermatagonia. White arrows indicate anti-TEX19 stained nuclear foci. Bar = 10 μm. d Staining of clear margin morphologically normal colon tissue taken from a cancer patient indicates that there is no staining with anti-TEX19 antibody (red; right hand panel; blue = DAPI, left hand panel). Bar = 100 μm. e Staining of matched (to tissue shown in D) colon tumour material shows regions of intense staining with anti-TEX19 antibodies (red; blue = DAPI). Bar = 100 μm. f Enlargement of the colon cancer region of intense anti-TEX19 signal (white box; scale bar is 30 μm) shows that anti-TEX19 staining (red) is mostly cytoplasmic

Fig 3: Analysis of TEX19 depleted SW480 cells: a TEX19 depleted SW480 cells do not have cleaved caspase-3 indicting there is no measurable apoptosis. b TEX19 depleted SW480 cells (lower image) do not form full spheres and exhibit no staining with ß-galactosidase indicating that they are not undergoing lysosome-dependent senescence (upper image shows SW480 spheres in the absence of TEX19 siRNA staining positive for ß-galactosidase due to natural senescence of internal cells; bar = 250 µm). c FACS of TEX19 siRNA treated SW480 cell populations (a minimum of 10,000 cells were analyzed in each case). d TEX19 siRNA treated cells have a lower proportion of the population in G2 and a higher proportion in S phase. e Western blot of whole cell extracts of SW480 cells depleted for TEX19 have lower levels of phosphor-histone H3 indicating fewer cells in mitosis. f Western blot and RT-qPCR analysis demonstrating TEX19 depletion (for data shown in a and c-e)

Fig 4: TEX19 is required for cancer cell proliferation and self-renewal: a Left hand line plot: siRNA-mediated (siRNA A) TEX19 mRNA depletion results in inhibition of SW480 cancer cell proliferation (* ≤ 0.05; unpaired t-test); right hand bar chart: RT-qPCR analysis of TEX19 mRNA levels for the cell cultures shown in the adjacent cell count plot (** ≤ 0.01; unpaired t-test; siRNA treatment occurred every 24 h and RNA extraction for RT-qPCR was taken 24 h following final siRNA treatment). b Extreme limiting dilution assay shows that TEX19 siRNA treatment (siRNA A) results in loss of self-renewal capacity in SW480 cancer cells (*** ≤ 0.001; chi square test). Sphere formation was monitored following 10 days incubation. c Left hand line plot: shRNA-mediated (+dox) TEX19 mRNA depletion results in reduction in proliferative potential of SW480 cancer cells (* ≤ 0.05; unpaired t-test; RNA extraction for RT-qPCR was taken 24 h following final Dox treatment); right hand bar chart: RT-qPCR analysis of TEX19 mRNA levels for cell cultures shown in the adjacent plot (* ≤ 0.05; unpaired t-test). d Left hand line plot: SW480 cell carrying a dox-inducible shRNA cassette generate tumours with reduced volume when the mice are fed with dox (red line) compared with no dox (grey line). P value is derived from a Wilcoxon matched pairs test (2-tailed); right hand bar chart: RT-qPCR analysis of TEX19 mRNA levels within final tumours extracted from mice used in the experimental set shown in the adjacent line plot (* ≤ 0.05; unpaired t-test: RNA was extracted for RT-qPCR analysis immediately following termination and tumour extraction for both +/- Dox)

Fig 5: TEX19 has nuclear and cytoplasmic localization in cancer cells: a Sub-confluent SW480 cells show TEX19 (red) localized to both nucleus and cytoplasm. Distinct cells show distinct proportions of cellular TEX19 within the nucleus. DNA is stained with DAPI (blue); bar = 40 μm. b Western blot analysis of nuclear and cytoplasmic extracts of sub-confluent SW480 cells indicates the majority of TEX19 is cytoplasmic, but a clear nuclear fraction is detected. Lamin (nucleus) and tubulin (cytoplasm) are used as controls (controls show clean faction signals, indicating no inter-fraction contamination). c Sub-confluent SW480 cells treated with the nuclear export blocking agent LMB demonstrates that TEX19 (red) can accumulate in the nucleus. DNA is stained by DAPI (blue); bar = 20 μm. d Western blot analysis of chromatin associated TEX19 demonstrates that TEX19 does not have a tight association with chromatin, even when cells are blocked in M phase with colcemid. Histone H3 is a control for tight chromatin association (only dissociated with 1.0 M NaCl); tubulin is mostly cytoplasmic (see b) and does not associate with chromatin during the chromatin preparation. e TEX19 is predominantly cytoplasmic at higher cell densities. Staining of sub-confluent HCT116 cells (top panels) indicates that TEX19 (red; blue = DAPI) is nuclear and cytoplasmic at this cell density, as for SW480 cells. When cells reach confluence (lower panels) the majority of TEX19 is cytoplasmic. Bar = 20 μm. f When SW480 cells are densely associated in spheres TEX19 (red; DAPI = blue) is mostly cytoplasmic. Bar = 100 μm. NTERA2 cells show strong nuclear staining with anti-TEX19 antibodies (green) irrespective of level of confluence. Red = anti-tubulin; Blue = DAPI; bar = 20 μm. g NTERA2 cells exhibit predominantly nuclear TEX19. Green = anti-TEX19; Blue = DAPI; Red = anti-tubulin; bar = 20 μm