Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: Soft agar assay analysis of HMEC TERT/ST/ER-RasV12 cells transduced with retroviral vectors encoding control or TP53 shRNAs. Cells were plated in soft agar in medium containing growth supplements (EGF, insulin, hydrocortisone) and 4-OHT (to activate Ras V12 ). Results (left panel) shown are the average colony number ± SD in biological triplicates. Representative MTT-stained colonies are shown in the right panel. (ns: not significant, student’s t-test) DOI: http://dx.doi.org/10.7554/eLife.07101.004

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: Soft Agar Assay, Transduction, Retroviral, Control, Staining

Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: Western blot analysis corresponding to to assess the knockdown efficiency of TP53 shRNA. β-ACTIN serves as the loading control. DOI: http://dx.doi.org/10.7554/eLife.07101.005

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: Western Blot, Knockdown, shRNA, Control

Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: ( A ) HMEC TERT/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 Ras V12 ). 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. HMEC TERT/ST/ER-RasV12 cells were generated by overexpressing TERT, SV40 small T antigen and ER-HRAS V12 . 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 HMEC TERT/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. β-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 HMEC TERT/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 ( TGM2 R cDNA) 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 ). β-ACTIN serves as the loading control. (EV, Empty Vector) ( H ) Soft agar assay analysis of BJ TERT/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 3T3 ER-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

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: Transduction, Retroviral, Control, Staining, shRNA, Expressing, Mutagenesis, Cloning, Generated, Knockdown, Western Blot, Plasmid Preparation, Soft Agar Assay

Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: Western blot analysis corresponding to to assess the knockdown efficiency of TP53 shRNA. β-ACTIN serves as the loading control. DOI: http://dx.doi.org/10.7554/eLife.07101.006

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: Western Blot, Knockdown, shRNA, Control

Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: By utilizing a publicly available breast cancer dataset (GSE3494), a chi-square test was used to identify genes with an asymmetric distribution in breast tumors with ‘low’ expression (lowest = 0, highest = 1) in 205 TP53 wild-type samples and 46 TP53 -mutant samples. The cut-off level was determined at the average minus one standard deviation cut-off (horizontal axis). A total of 122 genes with lower expression in a significant number of TP53 wild-type samples compared to TP53 mutant samples were selected based on their χ2 p values (p<0.01). MINA was selected as one of the genes in the candidate list and its normalized mRNA signal is shown as an example. DOI: http://dx.doi.org/10.7554/eLife.07101.007

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: Expressing, Mutagenesis, Standard Deviation

Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: Known TP53 downstream target genes, BAX , and transcriptional cofactor of TP53, BRD7 , were also in the candidate list and downregulated in a subset of TP53 wild-type tumors relative to TP53 mutant tumors by chi-square analysis (p<0.01). DOI: http://dx.doi.org/10.7554/eLife.07101.008

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: Mutagenesis

Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: TGM2 expression analysis is shown to illustrate lower expression in a subset of TP53 wild-type tumors compared to TP53 mutant tumors by chi-square analysis (p<0.01) DOI: http://dx.doi.org/10.7554/eLife.07101.009

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: Expressing, Mutagenesis

Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: Western blot of TGM2, CDKN1A and TP53 corresponding to . β-ACTIN serves as the loading control. DOI: http://dx.doi.org/10.7554/eLife.07101.011

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: Western Blot, Control

Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: Western blot of TGM2 and TP53 corresponding to . β-ACTIN serves as the loading control. DOI: http://dx.doi.org/10.7554/eLife.07101.013

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: Western Blot, Control

Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: ( A ) qRT-PCR analysis of the effect of control or TGM2 shRNAs (denoted respectively as control and TGM2 #1) on mRNA expression of either TP53, CDKN1A, MDM2, or TGM2 in HMEC TERT/ST/ER-RasV12 cells. The levels of mRNA were normalized to TBP expression and to control cells. The data indicate the average ± SD of biological triplicates. (**p<0.01, student’s t -test to control cells) ( B ) Western blot analysis of HMEC TERT/ST/ER-RasV12 cells stably transduced with retroviruses expressing control or TP53 shRNAs. β-ACTIN serves as the loading control. ( C ) qRT-PCR analysis of the cells in ( B ). The levels of TGM2 mRNA were normalized to TBP expression and to control cells. The data indicate the average ± SD of biological triplicates. (**p<0.01, student’s t -test to control cells) ( D ) Western blot analysis of HMEC TERT/ST/ER-RasV12 cells stably transduced with a retrovirus expressing mCherry with either control or TP53 shRNAs. The populations were verified to have more than 70% mCherry positive cells, and then retrovirally-transduced and selected to express an empty vector or a 6x His-tag TP53 shRNA-resistant overexpression vector (His-tag TP53 R cDNA). β-ACTIN serves as the loading control. (EV, Empty Vector) ( E ) qRT-PCR analysis of cells in ( D ) for TGM2 mRNA normalized to TBP expression and to control cells. The data indicate the average ± SD of biological triplicates. (*p<0.05; **p<0.01, student’s t -test) ( F ) qRT-PCR analysis of BJ TERT/ST/ER-RasV12/shp16 cells stably transduced with retroviruses expressing control or TP53 shRNAs. The levels of mRNA were normalized to TBP expression and to control cells. The data indicate the average ± SD of biological triplicates. (**p<0.01, student’s t -test to control cells) ( G ) qRT-PCR analysis of wild-type and Tp53 -/- MEFs. The levels of mRNA were normalized to Gapdh expression and to control cells. The data indicate the average ± SD of biological triplicates. (**p<0.01, student’s t -test to control cells). DOI: http://dx.doi.org/10.7554/eLife.07101.016

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: Quantitative RT-PCR, Control, Expressing, Western Blot, Stable Transfection, Transduction, Plasmid Preparation, shRNA, Over Expression

Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: ( A ) qRT-PCR analysis of HMEC TERT/ST/ER-RasV12 cells stably transduced with retroviruses expressing control or TP53 shRNAs. Cells were treated with Nutlin-3a (5 µM or 10 µM) for 2 days. The levels of mRNA were normalized to TBP expression and to control cells. CDKN1A is used as the positive control to see TP53 activation. The data indicate the average ± SD of biological triplicates. ( B ) qRT-PCR analysis of BJ TERT/ST/ER-RasV12/shp16 cells stably transduced with retroviruses expressing control or TP53 shRNAs. Cells were treated with Nutlin-3a (10 µM) for 2 days. The levels of mRNA were normalized to TBP expression and to control cells. CDKN1A is used as the positive control to see TP53 activation. The data indicate the average ± SD of biological triplicates. ( C and D ) Luciferase reporter assays using a series of promoter deletion mutants of the TGM2 gene. The number (bp) indicates the position relative to the translational start site (ATG). Reporter plasmids containing the indicated deletion constructs were transfected into H1299 cells with control or TP53 plasmid, and luciferase activity was monitored. The average value of the luciferase activity from the cells transfected with CMV-TP53 and the reporter plasmid containing TGM2 (-1530 to -78) promoter fragment is set at 1, and the relative activity is shown. The data indicate the average ± SD of biological triplicates. ( E ) TP53 binds to the TGM2 promoter. ChIP assay was performed with an antibody detecting endogenous TP53, or IgG (negative control) using HMEC TERT/ST/ER-RasV12 cells. The potential TP53 response element in the TGM2 promoter identified in ( D ) was analyzed by PCR. CDKN1A and GAPDH are served as the positive and negative control respectively. DOI: http://dx.doi.org/10.7554/eLife.07101.017

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: Quantitative RT-PCR, Stable Transfection, Transduction, Expressing, Control, Positive Control, Activation Assay, Luciferase, Construct, Transfection, Plasmid Preparation, Activity Assay, Negative Control

Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: Luciferase reporter assays using a series of promoter deletion mutants of the TGM2 gene. The number (bp) indicates the position relative to the translational start site (ATG). Reporter plasmids containing indicated deletion constructs were transfected into H1299 cells with control or TP53 plasmid, and luciferase activity was monitored. The average value of the luciferase activity from the cells transfected with CMV-TP53 and the reporter plasmid containing TGM2 (-159 to -78) promoter fragment is set at 1, and the relative activity is shown. The data indicate the average ± SD of biological triplicates. DOI: http://dx.doi.org/10.7554/eLife.07101.018

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: Luciferase, Construct, Transfection, Control, Plasmid Preparation, Activity Assay

Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: The genomic sequence and location of 82 bp fragment required and sufficient for TP53-mediated transactivation is shown. Chromosome coordinates are given according to GRCh38/hg38 Human Genome Reference. The sequence and location of the primers used for ChIP analysis are also shown (the position is indicated by the arrow). Genbank accession number (NM_004613.2) of TGM2 mRNA is shown. The TGM2 gene structure, TP53 responsive element, and primers were drawn proportionally to their sizes. The location of the primers for ChIP analysis were chosen to avoid the difficulty of amplifying the highly GC-rich region on the TGM2 promoter to increase the sensitivity of the ChIP analysis. DOI: http://dx.doi.org/10.7554/eLife.07101.019

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: Sequencing

Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: ( A ) Formation of red punctae following autophagy induction in the absence of growth supplements. HMEC TERT/ST/ER-RasV12 cells ( TP53 +/+ ) or TP53 CRISPR knockout HMEC TERT/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) HMEC TERT/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 HMEC TERT/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. β-ACTIN serves as the loading control. ( F ) Western blot analysis of HMEC TERT/ST/ER-RasV12 cells and two independent TP53 CRISPR knockout HMEC TERT/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. β-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 HMEC TERT/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

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: CRISPR, Knock-Out, Transfection, Plasmid Preparation, Incubation, Microscopy, Control, Knockdown, shRNA, Western Blot, Clone Assay, Quantitative RT-PCR, Expressing

Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: DNA sequencing result of TP53 -/- clones showing frameshift mutations in exon 4 of TP53 gene for CRISPR TP53 knockout HMEC TERT/ST/ER-RasV12 ( TP53 -/- ) clone 1 and clone 2. DOI: http://dx.doi.org/10.7554/eLife.07101.021

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: DNA Sequencing, Clone Assay, CRISPR, Knock-Out

Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: ( A ) Soft agar assay analysis of HMEC TERT/ST/ER-RasV12 cells transduced with a retrovirus expressing mCherry with either control, CDKN1A or TP53 shRNAs. The populations were verified to have more than 70% mCherry positive cells. The cells were then additionally transduced with the indicated shRNA constructs, selected with 4 µg/ml of blasticidin, and evaluated by soft agar assay analysis. The results shown are the average colony number ± SD from biological triplicates. (*p<0.05, ns: not significant, student’s t -test) ( B ) Formation of tumors in NOD/SCID mice. 500,000 HMEC TERT/ST/RasV12 cells transduced with the indicated vectors were injected subcutaneously in mice (n=6). (Top) The number of tumors observed after 4- and 6-weeks from the time of injection. (Bottom) Pictures of tumors excised 4 weeks after injection. ( C and D ) HMEC TERT/ST/ER-RasV12 cells were transduced with a retrovirus expressing mCherry with either control or CDKN1A shRNAs. The populations were verified to have more than 70% mCherry positive cells. The cells were then additionally transduced with the indicated shRNA constructs, selected with 4 µg/ml of blasticidin, and evaluated by soft agar assay analysis. The results shown are the average colony number ± SD from biological triplicates. (*p<0.05, **p<0.01, ns: not significant, student’s t -test) ( E and F ) HMEC TERT/ST/ER-RasV12 cells were transduced with a retrovirus expressing mCherry with either control or TGM2 (TGM2 #1) shRNAs. The populations were verified to have more than 70% mCherry positive cells. The cells were then additionally transduced with the indicated shRNA constructs, selected with 4 µg/ml of blasticidin, and evaluated by soft agar assay analysis. The results shown are the average colony number ± SD from biological triplicates. (*p<0.05, ns: not significant, student’s t -test). ( G ) Western blot analysis of HMEC TERT/ST/ER-RasV12 cells treated with TGM2 inhibitors. The cells were incubated in medium without EGF, insulin, and hydrocortisone in the presence of Z-DON (50 µM) or LDN 27219 (10 µM) for 24 hr. β-ACTIN serves as the loading control. ( H ) Soft agar assay analysis of HMEC TERT/ST/ER-RasV12 cells transduced with a retrovirus expressing mCherry with either control or TGM2 (TGM2 #1) shRNAs. The populations were verified to have more than 70% mCherry positive cells, and additionally transduced and selected with 4 µg/ml of blasticidin to express an empty vector (EV) or a TGM2 cDNA resistant to TGM2 shRNA wild-type (WT), C277S, or R580A mutants (denoted as TGM2 WT R , TGM2 C277S R , or TGM2 R580A R cDNA) by retroviruses. Quantification shows average colony number ± SD in biological triplicates. (**p<0.01, *p<0.05, ns: not significant, student’s t -test). DOI: http://dx.doi.org/10.7554/eLife.07101.023

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: Soft Agar Assay, Transduction, Expressing, Control, shRNA, Construct, Injection, Western Blot, Incubation, Plasmid Preparation

Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: Western blot analysis of TGM2, TP53, and CDKN1A corresponding to are shown. β-ACTIN serves as the loading control. DOI: http://dx.doi.org/10.7554/eLife.07101.025

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: Western Blot, Control

Journal: eLife

Article Title: Transglutaminase 2 contributes to a TP53-induced autophagy program to prevent oncogenic transformation

doi: 10.7554/eLife.07101

Figure Lengend Snippet: Stress, in this case the depletion of growth supplements, induces autophagy and TP53-dependent expression of TGM2 . TGM2 facilitates autophagic flux by promoting autophagic protein degradation and autolysosome clearance. Loss of TGM2 expression synergizes with loss of CDKN1A expression to promote malignant transformation of HMECs. Therefore, TGM2-mediated autophagy and CDKN1A-mediated cell cycle arrest are potentially two critical barriers in the TP53 pathway that prevent oncogenic transformation of HMECs. DOI: http://dx.doi.org/10.7554/eLife.07101.037

Article Snippet: The protein lysates were then sonicated and immunoprecipitated with mouse anti-p53 antibody (Santa Cruz, DO-1) or mouse IgG as the negative control.

Techniques: Expressing, Transformation Assay