Fig 2: Suppression of nsPEF-induced biotin-cadaverine conjugation by TG2 RNAi. (A) HeLa S3 cells were transfected with either siRNA targeting TG2 (TG) or control siRNA (ctl). At 48 h after transfection, the cells were loaded with biotin-cadaverine, exposed to 0 or 35 shots of 20 kV·cm-1 nsPEFs, and further incubated at 37 °C for 1 h. Western blotting was performed to analyze biotinylated proteins, TG2, and Ku70 (loading control). (B) Signal intensities of biotinylated proteins and TG2 in the western blot were quantified by densitometry and normalized relative to the signal of Ku70. The protein level in the control cells (transfected with control siRNA, no nsPEF exposure) was arbitrarily set to 1. Data represent the means ± standard deviations) from three independent experiments. Double asterisk (**) indicates P < 0.01, and a single asterisk (*) refers to P < 0.05.

Fig 3: TSP1 secretion is associated with type I collagen when C7 is absent or when TSP1 is ectopically expressed.(A) Left: PLA showing proximity between TSP1 and C1 in primary NHF and RDEBF with or without recombinant C7 expression. Scale bar: 20 µm. Right: Graph shows mean ± SEM signal intensity, 30 cells were counted from each replicate experiment (n=3), ****: p<0.0001. (B) Left: Immunoblot of C1, and TSP1 from total cell lysates (IC) and conditioned media (EC) from primary RDEBF treated with scramble siRNA (siMock) or C1 siRNA (siC1). Right: Graph shows TSP1 secretion levels measured by immunoblot quantification of TSP1 (cell media)/TSP1 (cell lysate), with mean ± SEM from replicate experiments (n=3). ***: p<0.001. (C) Left: Immunoblot of C1 and C7 from total cell lysate as well as TSP1 from total cell lysates (IC) and conditioned media (EC) isolated from NHF treated with siMock, siC1 or C7 siRNA (siC7). Right: Graph shows TSP1 secretion levels measured by immunoblot quantification of TSP1 (cell media)/TSP1 (cell lysate), with mean ± SEM from replicate experiments (n=2). ***: p<0.001, ****: p<0.0001. (D) Left: Immunoblot of C1 from conditioned media and TANGO1 from total cell lysate as well as TSP1 from total cell lysates (IC) and conditioned media (EC) isolated from NHF treated with siMock (first column), siC1, TANGO1 siRNA (siTANGO1) or combination of both siC1 and siTANGO1. Right: Graph shows TSP1 secretion levels measured by immunoblot quantification of TSP1 (cell media)/TSP1 (cell lysate), with mean ± SEM from replicate experiments (n=3). *: p<0.05, **: p<0.01, ****: p<0.0001. (E) Left: PLA showing proximity between TGM2 and C1 in primary NHF, RDEBF and RDEBF with recombinant C7 expression. Scale bar: 20 µm. Right: Graph shows mean ± SEM signal intensity, 30 cells were counted from each replicate experiment (n=3), ****: p<0.0001. (F) Left: PLA showing proximity between TSP1 and C1 in primary NHF transiently transfected with an empty vector (NHF+Mock) or CMV-driven TSP1 vector (NHF+TSP1). Scale bar: 20 µm. Graph shows mean ± SEM PLA signal intensity, 30 cells were counted from each replicate experiment (n=3), ****: p<0.0001. Right: Immunostaining with anti-TSP1 antibody confirms transfection of TSP1. Scale bar: 20 µm. (G) Left: PLA between TSP1 and TANGO1 in NHF transiently transfected by empty vector (NHF+Mock) or CMV-driven TSP1 vector (NHF+TSP1). Scale bar: 20 µm. Right: Graph shows mean ± SEM PLA signal intensity, 30 cells were counted from each replicate experiment (n=3), ns: not significant. (H). Left: Immunoblot of TSP1, C7, C12 and C1 from both IC and EC, pSMAD3 and SMAD3 from IC after being transiently transfected with empty plasmid (pUC19) or TSP1 containing pUC19 vector (TSP1). GAPDH serves as loading control. Right: Graphs show mean ± SEM of TGFß activation by quantifying the ratio of pSMAD3/SMAD3, and the secretion levels of TSP1, C7, C12 and C1 calculated by IC/EC ratios from replicate experiments (n=3). *: p<0.05; ns: not significant.

Fig 4: Absence of growth supplements induces TP53-dependent autophagy.(A) Formation of red punctae following autophagy induction in the absence of growth supplements. HMECTERT/ST/ER-RasV12 cells (TP53+/+) or TP53 CRISPR knockout HMECTERT/ST/ER-RasV12 cells (TP53-/-) cells were transfected with the plasmid RFP-GFP-LC3. Cells were incubated in medium without EGF, insulin, and hydrocortisone for 24 hr before visualization on a confocal microscope. Scale bar: 5 µm (B) Quantification of the fraction of red punctate cells within the total population of transfected cells shown in (A). Red punctate cells were counted as cells containing only RFP signal without visible overlap of GFP signal in the cytoplasm; transfected cells were counted as cells containing either RFP signals or a mix of RFP and GFP signals in the cytoplasm (>250 cells were counted). (**p<0.01, student’s t-test) (C) Control or TP53 knockdown (TP53 shRNA) HMECTERT/ST/ER-RasV12 cells were seeded, treated and visualized as in (A). (D) Quantification of the fraction of red punctate cells within the total population of transfected cells shown in (C), treated as in (B). (**p<0.01, student’s t-test) (E) Western blot analysis of TP53+/+ cells, TP53-/- CRISPR knockout HMECTERT/ST/ER-RasV12 clones, as well as control and TP53 shRNA cells treated with or without chloroquine (CQ, 50 µM, 2 hr) after incubation in medium without EGF, insulin, and hydrocortisone for 24 hr. ß-ACTIN serves as the loading control. (F) Western blot analysis of HMECTERT/ST/ER-RasV12 cells and two independent TP53 CRISPR knockout HMECTERT/ST/ER-RasV12 clones. Cells were incubated in medium in the presence or absence of EGF, insulin, and hydrocortisone (denoted as EIH) for 48 hr. ß-ACTIN serves as the loading control. (G) qRT-PCR analysis of cells in (F). TGM2 mRNA expression was normalized to TBP mRNA expression. The mean value of TGM2 mRNA expression in TP53+/+ cells with presence of EIH is set at 1, and relative expression is shown. (**p<0.01, ns: not significant, student’s t-test) (H) Western blot analysis of HMECTERT/ST/ER-RasV12 cells expressing either control or TP53 shRNA. Cells were treated the same as in (F). (I) qRT-PCR analysis of cells in (H). Data are shown as in (G).DOI: http://dx.doi.org/10.7554/eLife.07101.020

Fig 5: TGM2 suppresses transformation of primary human mammary epithelial cells in soft agar.(A) HMECTERT/ST/ER-RasV12 cells were transduced with retroviral vectors encoding control or TP53 shRNAs and plated in soft agar in medium with 4-OHT (to activate RasV12). Growth supplements (EGF, insulin, hydrocortisone) were withheld for the first 3 days. Results (left panel) shown are the average colony number ± SD in biological triplicates. Representative MTT-stained colonies are shown in the right panel. (**p<0.01 compared to control cells, student’s t-test) (B) Flow diagram for the shRNA screen. The candidate gene list of 122 genes is selected by comparing genes with lower expression in a significant number of TP53 wild-type tumor samples versus TP53 mutant tumor samples using expression array (GSE3494) consisting of 251 breast cancer samples with TP53 mutation status, followed by designing and cloning of shRNAs against the genes. These genes could be under selective pressure to lose expression only in TP53 wild-type tumors, thus could be potential members of the TP53 pathway. HMECTERT/ST/ER-RasV12 cells were generated by overexpressing TERT, SV40 small T antigen and ER-HRASV12. The shRNAs against the candidate genes were introduced into the cells and observed for colony formation in soft agar in the primary and secondary screen. (C) Soft agar analysis for HMECTERT/ST/ER-RasV12 cells expressing control, TGM2- (denoted as TGM2#1 or TGM2#2), TP53-, or CDKN1A- shRNAs using the conditions described in (B). Quantification shows average colony number ± SD in biological triplicates. (*p<0.05; **p<0.01 compared to control cells, student’s t-test) (D) Knockdown efficiency of TGM2 with two independent shRNAs. TGM2 protein expression was analyzed by Western blotting. ß-ACTIN serves as the loading control. (E) TGM2 mRNA expression was quantified by qPCR, normalized to TBP expression and to control vector in biological triplicates, and represented as the average fold change ± SD. (**p<0.01 compared to control cells, student’s t-test) (F) Soft agar assay analysis of HMECTERT/ST/ER-RasV12 cells transduced with a retrovirus expressing mCherry with either control or TGM2#1 shRNAs. The populations were verified to have more than 70% mCherry positive cells, and additionally transduced and selected to express an empty vector (EV) or a shRNA-resistant TGM2 cDNA (TGM2RcDNA) by retroviruses. Quantification shows average colony number ± SD in biological triplicates. (**p<0.01, student’s t-test) (G) Western blot analysis of TGM2 protein expression for (F). ß-ACTIN serves as the loading control. (EV, Empty Vector) (H) Soft agar assay analysis of BJTERT/ST/ER-RasV12/shp16 cells transduced with a retrovirus expressing control, TGM2-, or TP53- shRNA. Quantification shows average colony number ± SD in biological triplicates. (*p<0.05; **p<0.01, compared to control cells, student’s t-test) (I) Soft agar assay analysis of NIH 3T3ER-RasV12 cells transduced with a retrovirus expressing control or Tgm2-shRNAs. Quantification shows average colony number ± SD in biological triplicates. (**p<0.01 compared to control cells, student’s t-test).DOI: http://dx.doi.org/10.7554/eLife.07101.003