Supplementary Figure legends
Fig. S1. p53 is eliminated by Snail/K-Ras
(A) Ubiquitinylated p53 was not induced by oncogenic K-Ras. HCT116 p53- cells were transfected with HA-Ub and/or p53 and K-ras. After transfection, cell lysate was IPed with p53 Ab and checked Ubiquitination with the HA-Ub Ab. (B) To examine the location of disappeared p53,we performed the immuno-staining with p53Abafter transfection with indicating vectors for 24 hrs into HCT116 p53 deficient cells. From this experiment, we obtained the very interesting result that p53 were found in non-cellular vacuoles (white arrows; vacuoles are DAPI negative), when oncogenic K-Ras or Snail was transfected. Moreover, in co-transfected cells, the vacuoles obviously increased. In these cases, we did not observe the increase of apoptotic bodies or nuclear condensation. (C) Reduced p53 by oncogenic K-Ras or Snail was detected in media. After transfection with indicating vectors to HCT116, we divided the cell into 4 fractions and the media into 2 fractions through centrifugation to precipitated cell debris and apoptotic body. In cellular fractions, we could observe the reduction of p53 by oncogenic K-Ras and Snail. In addition, we could observe the similar expression pattern in centrifugedprecipitated fraction (centrifuged ppt). However, in media, we could find the p53 and Snail in oncogenic K-Ras or Snail transfected samples. (D) To address the engagement of autophagic mechanism for p53 degradation, we blocked the beclin through si-RNA. However, differentially from si-Snail, si-beclin could not increase p53 expression in A549, suggesting that the reduction of p53 would not be achieved by autophagic mechanism.
Fig. S2. p53 is secreted as vesicle-like structure
(A) In capan-1 cells, we could observe the vesicle-like extra-nuclear p53 (arrows), where Snail is co-localized. However, DN-Ras eliminated vesicular architecture (right panel). Capan-1 cells were fixed with 100 % Me-OH after transfection with EV or DN-Ras for 24 hr, and stained with p53 (red) and Snail (green). More interesting feature was that p53 containing vesicles were negative to DAPI, a marker of DNA. (B) Secreted p53 is induced by oncogenic K-Ras and suppressed by si-Snail and DN-Ras. Panc-1 was transfected with indicating vectors for 24 hr. To concentrate p53, we performed the GST-pull down using Snail-GST protein. WCL was used for control. (C) To measure the quantity of p53, we compared the p53 expression of media without concentration or GST-pull down in MCF-7 cells. In this cell, transfection of K-Ras or Snail can reduce intracellular p53 expression, whereas it could induce median p53. Considering loading portion, reduced p53 in intracellular portion would be secreted into medium. (D) vesicle-like p53 and Snail are coated by nuclear membrane emerin. (E) Extra-nuclear emerin (p53 containing vesicle; arrows) is eliminated by si-Snail and DN-Ras. HCT116 p53-/- cells were transfected with indicating vectors for 24 hr and stained with p53 (red) and Emerin (green). (F) in vitro binding assay between Lamin A and Snail. Recombinant Snail proteins were incubated with 293 cell lysate. After washing with PBS and RIPA, Snail-associated Lamin A was detected by SDS-PAGE and WB analysis. C-terminal region of Snail is responsible for binding with Lamin A.
Fig. S3.Secreted p53 is located in cytoplasmic membrane.
(A) Immunostaining of p53 and Snail in pre-fixed cells with PFA. HCT116 p53- cells were transfected with indicating vectors for 24 hr, and fixed with PFA without washing and permeablization. After washing, cells were incubated with p53 DO-1 and Snail Ab for 4 hr. (B) Reduced p53 and snail was recovered in media. HCT116 p53- cells were transfected with indicating vectors for 24hr. Cell media were collected and cell debris were eliminated by centrifugation. After precipitation by 100% Et-OH, precipitated proteins were loaded. To confirm the median p53, we also performed the WB analysis with HA-Ab.
Fig. S4. p53 is secreted by vesicle transport.
(A) In PC3, we transfected Snail with p53 and examined the expression of p53. In control, p53 was strongly expressed in nucleus. However, in snail transfected cells, p53 was reduced and localized in cytosol as small vesicle. When cells were treated with Noc, p53, in Snail transfected cells, was stocked in cytosol and co-localized with Snail. (B) To monitor real-time movement of p53-secretory vesicle, we transfected GFP-p53 into PC3 and monitored the expression of GFP. Since our condition has limitation, we can observe the cells for 30 min. however, we can find out that Snail/K-ras promotes p53 exporting as vesicle. When Aph/Noc treated, p53 vesicles are stocked in cytoplasm.
Fig. S5. Different destination of p53 and Snail after secretion.
(A) When recombinant p53-His (DNA binding domain that is also responsible for Snail binding) was incubated with cell or conditioned media, it is disappeared except for HCT116 whole cell lysate. in contrast, Lamin A (His) is recovered from media. This result suggests that p53-His is easily digested by secreted protease and resorbed by HCT116. Recombinant proteins (2 g/ml) were incubated with fresh media (media), A549-conditioned media, PC3 cells (addition of cell culture condition) or HCT116 for 2 hr. And then, culture media and WCL after washing with PBS were collected and applied to SDS-PAGE/WB analysis. (B) Snail fragments are recovered from media. Each fragment of Snail is incubated with A549 and MKN 45 for 2 hr. Media and WCL were prepared as the same methods of (A), and subjected into SDS-PAGE and WB analysis. (C) To examine the K-ras specific uptake, we incubated His-p53 and lamin A with 293 cells after transfection with H-, N-, and K-ras vectors for 24 hr. After 1 hr incubation, we collected media and analyzed remaining proteins. Comparing to Lamin A, p53-His is eliminated in K-ras transfected cell’s media. Instead, His-p53 is recovered in WCL. (D) expression of caveolin-1 is related with K-Ras status. Several kinds of cell lines were used for measuring of caveolin-1 expression. K-Ras mutated cell lines (+) show elevated expression of caveolin-1. (E) p53 endocytosis is suppressed by si-caveolin-1. A549 cells were transfected with si-caveolin-1 for 24 hr and incubated with recombinant p53-His for indicating time. After harvest, His-p53 in cell was determined by WB analysis.
Fig. S6. His-p53 is resorbed by K-ras mutated cells.
(A) A549 cells were incubated with propidium iodine (50 g/ml) and/or His-p53 (2 g/ml) for 4 hr. When PI was treated alone, this chemical was not accumulated in cells. However, when PI was co-treated with His-p53, it was obviously detected inner cells. Endocytosis inhibitor could block the accumulation of PI, suggested that PI might be endocytosed with His-p53. (B) However, we did not observe the PI accumulation in MKN 45 despite of same experimental condition.
Fig. S7. Cancer tissues secretes p53 and Snail
(A) WB analysis using tissue cultured media. We obtained 10 pair of cholangioma and 4 HCC samples. These tissues were dissected them into small pieces and incubated in RPMI-1640 medium. After collection of cultured media, we analyzed p53 and Snail expression. Actin was used for negative control and Pouseu S for loading control. (B) Non-cancer bile duct tissue did not show the expression of p53 and Snail in tissue cultured media. (C) ELISA of p53. Tissue cultured media was applied for p53 ELISA. (D) Presence of anti-p53 Ab in cultured media. To measure the p53 Ab, we incubated tissue media with GST-p53-bead. After washing with RIPA and PBS, we checked the presence of human Ab. (E) Presence of Snail Ab. To measure the Snail Ab, we incubated tissue media with bead conjugated GST-Snail-N-terminus. After collection of GST-Snail, we examined the co-precipitated human Ab.
Fig. S8. Anti-Snail Ab is detected in lung cancer blood serum. Using blood serum of lung cancer, we examined the presence of snail Ab. Among 30 tested samples, about half of them showed the positive signal. Clinical information of them was filed in Table S2. Interestingly, one of 9 normal patients showed the positive for this analysis. Since normal populations of this analysis were obtained from non-cancer patients, we are now following the person.
Fig. S9. Summarized Diagram.
When cells gain oncogenic K-Ras, p53 and Snail are secreted. In extracellular space, p53 would be digested by extracellular proteases and eliminated by endocytosis by neighboring K-Ras mutated cells. In contrast, secreted Snail would be exposed to immune system. Thus, anti-Snail Ab may be produced.
Supplementary Table
Table S1 summary and clinical information of our tissues used in Fig. S7
Table S2 clinical information of lung cancer patients, used in Fig. S8.
1