Supplemental Figure and Movie legends
Supplemental figure legends
Figure S1Microarray analysis of isogenic RT4P and RT4v6 cells to understand the key signaling pathways altered. RT4P and RT4v6 cells were subjected to microarray analysis for mRNA expression. The mRNAs with maximum contrast were grouped into glucose metabolism (a) and DNA damage response (b). Color code: Red-More expression; Green-less expression. Note the RT4v6 have downregulated expression of mRNAs connected to glucose metabolism and DNA damage response. (c) Transmission electron microscopy revealing the ultrastructure of blebbishields. Note the two distinct type of electron dense nucleoli. The fenestrated active nucleoli (aNu) is usually bound within nuclear membrane (NM). The cytoplasmic nucleoli (cNu) are usually found in the vicinity of broken nuclear membranes (black arrows). Note that the cytoplasmic nucleoli are perfectly round in structure. Note the stalk is made of thick cytoplasmic material. Consistent with apoptotic Golgi fragmentation, the apoptotic Golgi clusters are detected (AGC). Cytoplasm (C), nucleus (N), mitochondria (Mt), rough endoplasmic reticulum (RER).
Figure S2(a) RT4P blebbishield formation in the presence of Hoechst-33342 stain showing the main body is composed of nuclear materials. RT4P cells were subjected to blebbishield ejection using BE medium with 1M cisplatin and LiCl and the Hoechst-33342 (10 nM) was added at 23rd hour of BE medium treatment and the blebbishield formation was imaged by time-lapse microscopy. Note that the main body harbors DNA. (Also see main figure-1C for main body). (b and c) Determination of Oct4 expression in RT4P cells and blebbishields by Western blotting. Panel b is normal exposure with NTERA-2 cl.D1 [NT-2/D1] cells as positive control. Panel c is a comparison of RT4P cells with various bladder cancer cells (Oct4 is detected in trace quantities after overnight exposure and hence RT4P cells are not negative for Oct4 expression).
Figure S3Blebbishield derived colonies are capable of undergoing second round of blebbishield formation (Blebbishield serial ejection-2; BSE-2) from low Hoechst-33342 positive cells. (a) RT4P blebbishields were allowed to grow in normal MEM for 30 days and the blebbishield derived colonies were subjected to second round of blebbishield ejection using BE medium with 1M cisplatin and imaged using time-lapse microscopy. The apoptotic phase (3 hour panel) and bleb-fusion stages (4 hour panel) were shown enlarged. (b) RT4v6 blebbishields were allowed to grow in normal MEM for 30 days and the blebbishield derived colonies were subjected to second round of blebbishield ejection using BE medium with 1M cisplatin in the presence of 10 nM Hoechst-33342 and imaged to show the low Hoechst-33342 positive cells in the middle of the colony (upper panel). The same colony from panel-A was imaged using time-lapse microscopy to show the blebbishield formation from the low Hoechst-33342 positive cells. Note the high Hoechst-33342 positive cells in the periphery undergo pyknosis before the cells from the interior of the colony undergo blebbishield formation. The apoptotic phase (Panel 12:20 hours) and bleb-fusion stages (Panel 13:55 hours) were shown enlarged.
Figure S4(a) Tumor incidence pattern of RT4P and RT4P-BSE2 cells in nude mice sub-cutaneous injection model. Note that the tumor incidence is almost similar. (b) Minor differences in tumor weight of Orthotopic tumors induced by unsorted RT4v6 (n=10) and VEGFR2High RT4v6 sorted (n=9; 1 mice with unknown tumor status due to cannibalism among 10 mice) cells. (c) Minor lethal phenotype observed in VEGFR2High RT4v6 sorted cells injected mice (Orthotopic). Note: the endpoint of experiments were as per IACUC guidelines and hence the mice were not followed-up for death, instead euthanized on day 23 post orthotopic injection.
Supplemental Movie legends
Movie S1Blebbishield formation of RT4P cells in response to combined inhibition of p70S6K, GSK-3 and induction of DNA damage. RT4P cells at high density (8x107 cells/T-75 flask) were treated with 15ml BE medium (with 1mM cisplatin and indicated components in materials and methods) at 24 hours post seeding and subjected to time-lapse microscopy in DIC bright field settings. Frame rate for imaging: 30 seconds/frame; Note the cell undergo apoptotic blebbing (apoptotic phase) and the blebs fuse together to form blebbishields (fusion phase). Also note that, the blebbishields are anchored to the substratum by a thin stalk. (For time labeling and stalk labeling see main figure-1c and supplemental figure S1c).
Movie S2RT4P Blebbishields form spheres by fusion. RT4P blebbishields were isolated as described in experimental procedures section. These blebbishields were plated in BE medium with 1M cisplatin and imaged by time-lapse microscopy under bright field settings. Frame rate: 4 minutes/frame; Frame size: 467x371m. Note: The blebbishield which undergo fusion to form stem cell sphere is highlighted by a red circle.
Movie S3The spheres formed from RT4P blebbishields are capable of morphological differentiation under normal culture conditions. RT4P blebbishields were isolated and plated in BE medium with 1M cisplatin to form spheres for 24 hours. The BE medium was replaced with normal MEM and imaged by time-lapse microscopy under bright field settings to study the morphological differentiation potential of spheres. Frame rate: 3 minutes/frame; Frame size: 467x371m. Note: The colonies undergo differentiation and acquire the original morphology of RT4P cells in the end of the video. The cells pinch off their parts as a part of their differentiation, a possible process to expel the materials ingested from previous BE medium with cisplatin treatment.
Movie S4The spheres formed from RT4P blebbishields exhibit vesicle pinching in response to BE medium with 10M cisplatin. RT4P blebbishields were isolated and plated in BE medium with 1M cisplatin to form spheres for 24 hours. This medium was replaced with BE medium with 10M cisplatin and imaged by time-lapse microscopy under bright field settings to study the drug resistance property of spheres. Frame rate: 1 minute/frame; Frame size: 467x371m. Note: The spheres constantly produce long blebs that are often pinched off from the colony (Arrows).
Movie S5The spheres formed from RT4P blebbishields exhibit vesicle burst in response to Hoechst-33342. RT4P blebbishields were isolated and plated in normal MEM for 24 hours to form spheres. This medium was replaced by normal MEM with Hoechst-33342 stain and imaged by time-lapse microscopy to study the dye export property of spheres. See main text for Hoechst-33342 concentration. Frame rate: 30 seconds/frame; Frame size: 236x190m. Note: The spheres immediately produce blebs that lack DNA but burst out rapidly.