Journal: bioRxiv

Article Title: Internal Translation of p53 Oncoproteins During Integrated Stress Response Confers Survival Advantage on Cancer Cells

doi: 10.1101/2023.03.03.531004

Figure Lengend Snippet: a , Sequence alignment of regions surrounding internal translation initiation sites (TIS) in p53 from 37 different mammalian species using MAFFT server within Jalview. TIS are shown in red and were labelled with codon numbers corresponding to the human p53 sequence: ATG40, ATG133 and ATG160. b-e , Western blot analyses (WB) of A549, HT1080, NIH3T3 and LN229 cells endogenously expressing p53 and cultured under normal conditions (-) or integrated stress response (ISR) conditions through treatment with thapsigargin (Th, 16h), tunicamycin (Tu, 16h) or etoposide (Eto, 21h). f-h , WB of p53-null H1299 cells expressing no p53 or Δ133p53 wild-type (Δ133) ( f, h ) or tagged full-length p53 (Flag-p53-HA) ( g ) or Δ133p53 mutated at translation initiation starts AUG160 (M160A Δ133) or AUG133 (M133A Δ133) ( h ), and subjected or not to ISR (Th 16h or Eto 36h), as indicated. DO12, monoclonal anti-p53 (aa 256-267) antibody. MAP4, monoclonal anti-Δ133p53 specific antibody. Bp53.10, monoclonal anti-p53 (aa 374-378) antibody. CM1, polyclonal anti-p53 antibody. P-eIF2α, phosphorylated eIF2α (ISR marker). Shown are representative data of at least three independent experiments. The numbers in parenthesis under the WBs specify the amounts of protein for the indicated bands relative to bands showing numbers in bold, according to WB quantifications and normalization against loading control (α-tubulin or vinculin).

Article Snippet: Primary antibodies used for WB were CM-1, Bp53.10, DO12 and MAP4 for p53 isoforms, anti-α-tubulin (Calbiochem DM1A), anti-Flag (Sigma M2), anti-Flag (BioLegend L5), anti-HA (Roche 3F10), anti-Lamin B1 (Santa Cruz Biotechnology A-11), anti-vinculin (Santa Cruz Biotechnology H-10), anti-Phospho-eIF2α (Ser51) (Cell Signaling Technology).

Techniques: Sequencing, Western Blot, Expressing, Cell Culture, Marker, Control

Journal: bioRxiv

Article Title: Internal Translation of p53 Oncoproteins During Integrated Stress Response Confers Survival Advantage on Cancer Cells

doi: 10.1101/2023.03.03.531004

Figure Lengend Snippet: a , Western blot analyses (WB) of H1299 cells expressing Δ133p53 and treated with DMSO (-) or with ISR activator thapsigargin (Th, 16h) and proteasome inhibitor (MG132, 4h) or translation inhibitor (cycloheximide, CHX, 4h). b , Likelihood of translation initiation from start codons homologous to human TIS (hTIS) 1, 40, 133 and 160 in 6 different mammalian species, as predicted by NetStart 1.0 (top) and ATGpr (bottom). c, d , WB of H1299 cells expressing 5’cap-translation–blocking bicistronic mRNA containing Δ160p53 and its 5’UTR (b-5’Δ160, represented in ( c ), top panel) and treated with DMSO (-) or Th (16h) as indicated. A549 cells endogenously expressing p53 were also analysed for comparison ( c ). P-eIF2α, phosphorylated eIF2α (ISR marker). e , WB of H1299 cells expressing bicistronic Δ133p53 mRNA (b-Δ133) and treated with DMSO (-, 36h), Th (16h) or DNA damaging drug etoposide (Eto, 36h). f , WB of H1299 cells expressing tagged and mutated p53, as indicated, and treated with Th (16h). fs indicates frameshift mutations of one nucleotide at the indicated codon (157 or 130). g-j , Luminescence readings (% increase of Firefly Luciferase / Renilla Luciferase ratio over empty control) of A549, H1299, HCT116 or HeLa cells expressing empty bicistronic dual-luciferase mRNA (b-Luc, represented in ( g )) or b-Luc mRNAs containing the first 432 or 258 nucleotides of Δ160p53 (b-Δ160(432) Luc or b-Δ160(258) Luc, respectively) or 78 nucleotides of the 5’-UTR of Δ160p53 (b-Δ160’s 5’UTR(78) Luc) or the positive control c-myc IRES (b-c-mycIRES Luc), as indicated, and treated or not with Th (16h). Shown are averages ± s.d. of n experiments as indicated or representative data of at least three independent experiments (* P < 0.05, **P < 0.01 and ***P < 0.005 compared to negative control). CM1, polyclonal anti-p53 antibody. The numbers in parenthesis under the WBs specify the amounts of protein for the indicated bands relative to bands showing numbers in bold, according to WB quantifications and normalization against α-tubulin.

Article Snippet: Primary antibodies used for WB were CM-1, Bp53.10, DO12 and MAP4 for p53 isoforms, anti-α-tubulin (Calbiochem DM1A), anti-Flag (Sigma M2), anti-Flag (BioLegend L5), anti-HA (Roche 3F10), anti-Lamin B1 (Santa Cruz Biotechnology A-11), anti-vinculin (Santa Cruz Biotechnology H-10), anti-Phospho-eIF2α (Ser51) (Cell Signaling Technology).

Techniques: Western Blot, Expressing, Blocking Assay, Comparison, Marker, Luciferase, Control, Positive Control, Negative Control

Journal: bioRxiv

Article Title: Internal Translation of p53 Oncoproteins During Integrated Stress Response Confers Survival Advantage on Cancer Cells

doi: 10.1101/2023.03.03.531004

Figure Lengend Snippet: a , Luminescence readings for renilla luciferase (RLuc) and firefly luciferase (FLuc) in HeLa cells not expressing any luciferase (pcDNA3.1 vector and non-transfected (NT)); or transfected with bicistronic dual-luciferase mRNAs containing p53 IRES(160) with 432 nucleotides (SV40 | b-Δ160 (432) Luc), p53 IRES(160) with 510 nucleotides (SV40 | b-Δ160 (510) Luc) or positive control for cryptic promoter activity, MLH1’s 5’UTR (SV40 | b-MLH1 Luc); or transfected with similar bicistronic constructs but lacking the SV40 promoter (labelled “–” instead of “SV40”). b , Schematic of the general bicistronic dual-luciferase mRNA (where “x” is replaced with the sequence of study), showing target locations for siRNA used in (c) and PCR primers used in (d). c , Luminescence readings for renilla luciferase (RLuc) and firefly luciferase (FLuc) in HeLa cells expressing empty bicistronic dual-luciferase mRNA (b-Luc) or b-Luc mRNAs containing p53 IRES(160) with 432 nucleotides (b-Δ160(432) Luc), p53 IRES(160) with 510 nucleotides (b-Δ160 (510) Luc) or EMCV IRES (b-EMCV IRES Luc), and treated with siRNA control (GFP) or siRNA targeting RLuc, as indicated. d , RT-PCR of total RNA extracted from HeLa cells expressing the indicated bicistronic dual-luciferase mRNAs using primer sets I (top panel) or II (lower panel) shown in (c). PCR without RT (PCR) served as negative control and PCR of plasmid DNA (pPCR) as positive control. Shown are averages ± s.d. or representative data of at least three independent experiments (# P > 0.05, * P < 0.05, and ***P < 0.005 as indicated).

Article Snippet: Primary antibodies used for WB were CM-1, Bp53.10, DO12 and MAP4 for p53 isoforms, anti-α-tubulin (Calbiochem DM1A), anti-Flag (Sigma M2), anti-Flag (BioLegend L5), anti-HA (Roche 3F10), anti-Lamin B1 (Santa Cruz Biotechnology A-11), anti-vinculin (Santa Cruz Biotechnology H-10), anti-Phospho-eIF2α (Ser51) (Cell Signaling Technology).

Techniques: Luciferase, Expressing, Plasmid Preparation, Transfection, Positive Control, Activity Assay, Construct, Sequencing, Control, Reverse Transcription Polymerase Chain Reaction, Negative Control

Journal: bioRxiv

Article Title: Internal Translation of p53 Oncoproteins During Integrated Stress Response Confers Survival Advantage on Cancer Cells

doi: 10.1101/2023.03.03.531004

Figure Lengend Snippet: a, b , Immunoprecipitation (IP) of full-length (FL) FLag-R273Hp53-HA protein with Flag antibody (a ) or Δ160p53-HA or Δ133p53-HA together with Δ160p53-HA with HA antibody ( b ) followed by Western blot analyses (WB) with polyclonal anti-p53 antibody CM1. WCL, whole cell lysate before IP. Supernatant, supernatant of samples after IP and before washes. IgG, control samples incubated with purified rabbit IgG instead of Flag or HA antibody. The numbers in squares under the WB in (b) specify the relative percentage amount of FLp53 protein compared to the amount of IPed Δ160p53-HA or Δ133p53-HA in the same lane, without any adjustments (†) or considering expression levels of Δ160p53-HA protein from Δ133p53-HA mRNA and equal binding capacity as in the Δ160p53-HA IP sample (§). c , Fluorescent imaging of H1299 cells expressing bicistronic Δ160p53-mCherry mRNA and treated with DMSO (-) or ISR-inducing drug Thapsigargin for 16h. d, e , RT-qPCR quantifications of indicated endogenous mRNAs from H1299 cells expressing or not different p53 proteins, as specified. Shown are averages ± s.d. of n experiments as indicated or representative data of at least three independent experiments (# P > 0.05, * P < 0.05, **P < 0.01 and ***P < 0.005 compared to p53 alone (d) or no p53 (e)). CM1, polyclonal anti-p53 antibody.

Article Snippet: Primary antibodies used for WB were CM-1, Bp53.10, DO12 and MAP4 for p53 isoforms, anti-α-tubulin (Calbiochem DM1A), anti-Flag (Sigma M2), anti-Flag (BioLegend L5), anti-HA (Roche 3F10), anti-Lamin B1 (Santa Cruz Biotechnology A-11), anti-vinculin (Santa Cruz Biotechnology H-10), anti-Phospho-eIF2α (Ser51) (Cell Signaling Technology).

Techniques: Immunoprecipitation, Western Blot, Control, Incubation, Purification, Expressing, Binding Assay, Imaging, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Internal Translation of p53 Oncoproteins During Integrated Stress Response Confers Survival Advantage on Cancer Cells

doi: 10.1101/2023.03.03.531004

Figure Lengend Snippet: a , Western blot analyses (WB) of H1299 cells expressing mutant R248Q p53 and treated with morpholino (MO) against p53 IRES(160) (MO-i) or control MO (MO-ctl). b, c , WB of LN299 ( b ) or A549 ( c ) cells expressing endogenous p53 and treated or not with thapsigargin (Th, 16h) or DNA-damaging agent etoposide (Eto, 21h) and morpholino against p53 IRES(160) (MO-i) or control MO (MO-ctl) or siRNA against exons 2 and 3 of p53 mRNA (si-e2-3) or control siRNA (si-ctl) or Endo-Porter alone (endop., MO delivery reagent). * indicates knock-down (KD) of Δ133p53 and Δ160p53; + indicates FLp53 and Δ40p53 KD by siRNA. d-f , Fluorescence-activated cell sorting (FACS) analyses of cell-cycle phase and apoptosis in A549 cells expressing endogenous p53 mRNA with ( d, e ) or without ( f ) co-expression of bicistronic Δ160p53 and treated or not with Th (16h) and control morpholino (MO-ctl) or morpholino against p53 IRES(160) (MO-i), as indicated. Shown are averages ± s.d. of n experiments as indicated or representative data of at least three independent experiments (*P < 0.05 compared to control MO). Bp53.10, monoclonal anti-p53 (aa 374-378) antibody. Numbers in parenthesis under the WB specify amounts of Δ160p53 protein for indicated bands relative to band showing number in bold, according to WB quantifications and normalization against α-tubulin.

Article Snippet: Primary antibodies used for WB were CM-1, Bp53.10, DO12 and MAP4 for p53 isoforms, anti-α-tubulin (Calbiochem DM1A), anti-Flag (Sigma M2), anti-Flag (BioLegend L5), anti-HA (Roche 3F10), anti-Lamin B1 (Santa Cruz Biotechnology A-11), anti-vinculin (Santa Cruz Biotechnology H-10), anti-Phospho-eIF2α (Ser51) (Cell Signaling Technology).

Techniques: Western Blot, Expressing, Mutagenesis, Control, Knockdown, Fluorescence, FACS

Journal: American journal of hematology

Article Title: Expression of p53 protein isoforms predicts survival in patients with multiple myeloma.

doi: 10.1002/ajh.26507

Figure Lengend Snippet: FIGURE 1 Schematic representation of human p53 protein isoforms, and the region containing the epitope for the p53 antibodies used in this study. (A) The main domains of p53 protein isoforms and their locations are represented by colors and amino acid (aa) numbering, respectively. The C-terminal sequences specific to the β (DQTSFQKENC) and γ (MLLDLRWCYFLINSS) variants are also shown. The molecular weight of each p53 isoform protein is indicated. The α, β, TA, long, and short protein isoforms are specifically recognized by A300-249A, KJC8, DO-1, and DO-11, respectively. (B) Number of MM patients with and without expression of each p53 isoform. (C) Number of MM patients with and without expression of TA isoforms, differentiating the two bands at 55–57 and 60–63 kDa that correspond to the TAp53α and TAp53β/γ isoforms, respectively. BR, basic region, aa 364–393 (yellow); DBD, DNA-binding domain, aa 101–292 (green); NLS, nuclear localization signal, aa 305–322 (orange); OD, oligomerization domain, aa 326–356 (red); PRD, proline-rich domain, aa 64–92 (blue); TAD1, transactivation domain 1, aa 1–42 (purple); TAD2: transactivation domain 2, aa 43–63 (violet) [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense machine (ProteinSimple, San Jose, CA) in accordance with the manufacturer's protocols and as previously described.20,21 The primary antibodies used in the study were: mouse monoclonal DO-11 (BioRad; MCA1704, aa 181–190), whose epitope is present in the common region DBD and allows detection of all p53 protein isoforms; mouse monoclonal DO-1 (Santa Cruz Biotechnology; sc-126, aa 11–25), whose epitope is situated in the transactivation domain 1 that is present only in the TAp53α, TAp53β, and TAp53γ protein isoforms; rabbit polyclonal anti-p53 A300-249A-T (Bethyl Laboratories, Inc.; aa 375–393), which is specific to the α isoforms (TAp53α, Δ40p53α, Δ133p53α, and Δ160p53α); and anti-GAPDH (Cell Signaling; rabbit mAb #2118), which was used as the endogenous control.

Techniques: Molecular Weight, Expressing, Binding Assay

Journal: American journal of hematology

Article Title: Expression of p53 protein isoforms predicts survival in patients with multiple myeloma.

doi: 10.1002/ajh.26507

Figure Lengend Snippet: FIGURE 2 Association of p53 protein isoforms with deletion of 17p and with standard and high cytogenetic risk. Distribution of the expression of total p53 protein, long, TAp53β/γ and short isoforms, based on the presence or absence of the 17p deletion. Expression levels of the TAp53β/γ in MM patients with standard and high cytogenetic risk. The statistically significant differences between groups were determined by the Mann–Whitney U test (p values indicated) [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense machine (ProteinSimple, San Jose, CA) in accordance with the manufacturer's protocols and as previously described.20,21 The primary antibodies used in the study were: mouse monoclonal DO-11 (BioRad; MCA1704, aa 181–190), whose epitope is present in the common region DBD and allows detection of all p53 protein isoforms; mouse monoclonal DO-1 (Santa Cruz Biotechnology; sc-126, aa 11–25), whose epitope is situated in the transactivation domain 1 that is present only in the TAp53α, TAp53β, and TAp53γ protein isoforms; rabbit polyclonal anti-p53 A300-249A-T (Bethyl Laboratories, Inc.; aa 375–393), which is specific to the α isoforms (TAp53α, Δ40p53α, Δ133p53α, and Δ160p53α); and anti-GAPDH (Cell Signaling; rabbit mAb #2118), which was used as the endogenous control.

Techniques: Expressing, MANN-WHITNEY

Journal: American journal of hematology

Article Title: Expression of p53 protein isoforms predicts survival in patients with multiple myeloma.

doi: 10.1002/ajh.26507

Figure Lengend Snippet: FIGURE 3 Probability of survival without progression and of overall survival of MM patients by level of p53 protein isoforms. (A) TTP and OS probabilities in patients with high levels of short isoforms. (B) TTP and OS probability according to the expression of TAp53β/γ isoforms. The log- rank (Mantel–Cox) test p values are shown. MM, multiple myeloma; OS, overall survival; TA, transactivation domain; TTP, survival without progression [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense machine (ProteinSimple, San Jose, CA) in accordance with the manufacturer's protocols and as previously described.20,21 The primary antibodies used in the study were: mouse monoclonal DO-11 (BioRad; MCA1704, aa 181–190), whose epitope is present in the common region DBD and allows detection of all p53 protein isoforms; mouse monoclonal DO-1 (Santa Cruz Biotechnology; sc-126, aa 11–25), whose epitope is situated in the transactivation domain 1 that is present only in the TAp53α, TAp53β, and TAp53γ protein isoforms; rabbit polyclonal anti-p53 A300-249A-T (Bethyl Laboratories, Inc.; aa 375–393), which is specific to the α isoforms (TAp53α, Δ40p53α, Δ133p53α, and Δ160p53α); and anti-GAPDH (Cell Signaling; rabbit mAb #2118), which was used as the endogenous control.

Techniques: Expressing

Journal: American journal of hematology

Article Title: Expression of p53 protein isoforms predicts survival in patients with multiple myeloma.

doi: 10.1002/ajh.26507

Figure Lengend Snippet: FIGURE 4 Probability of survival without progression and of overall survival of MM patients by cytogenetic risk and level of p53 protein isoforms, simultaneously. Analysis of MM patients with standard and high cytogenetic risk in combination with high and low expression levels of short (A) and TAp53β/γ (B) protein isoforms. The log-rank (Mantel–Cox) test p values are shown. (C) Forest plot of multivariate models with probabilities for each factor associated with TTP and OS of patients, based on the expression level of the studied p53 protein isoforms and age at diagnosis (years), ISS III versus I/II, high-level LDH, plasmacytoma and high versus standard cytogenetic risk (N = 145). 95% Confidence intervals are indicated in parentheses. MM samples with missing values were excluded from the model. ISS, International Staging System; LDH, lactate dehydrogenase; MM, multiple myeloma; TA, transactivation domain; TTP, survival without progression [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense machine (ProteinSimple, San Jose, CA) in accordance with the manufacturer's protocols and as previously described.20,21 The primary antibodies used in the study were: mouse monoclonal DO-11 (BioRad; MCA1704, aa 181–190), whose epitope is present in the common region DBD and allows detection of all p53 protein isoforms; mouse monoclonal DO-1 (Santa Cruz Biotechnology; sc-126, aa 11–25), whose epitope is situated in the transactivation domain 1 that is present only in the TAp53α, TAp53β, and TAp53γ protein isoforms; rabbit polyclonal anti-p53 A300-249A-T (Bethyl Laboratories, Inc.; aa 375–393), which is specific to the α isoforms (TAp53α, Δ40p53α, Δ133p53α, and Δ160p53α); and anti-GAPDH (Cell Signaling; rabbit mAb #2118), which was used as the endogenous control.

Techniques: Expressing, Biomarker Discovery