sj-xlsx-1-cix-10.1177_11769351221127875 – Supplemental material for SurviveAI: Long Term Survival Prediction of Cancer Patients Based on Somatic RNA-Seq Expression

Supplemental material, sj-xlsx-1-cix-10.1177_11769351221127875 for SurviveAI: Long Term Survival Prediction of Cancer Patients Based on Somatic RNA-Seq Expression by Omri Nayshool, Nitzan Kol, Elisheva Javaski, Ninette Amariglio and Gideon Rechavi in Cancer Informatics</p

Up-regulation of MDM2 is the major inhibitor mechanism of p53 apoptotic transcription in hypoxia.

<p>(A) Total mRNAs were reverse transcribed from RKO and A549 cell treated with cobalt or 2% O₂ for 16 h for PCR analyses of MDM2 gene expression. GAPDH was used as internal control. (B) 293 cells were co-transfected with p53AIP1-luc reporter and HIPK2-Flag or K1182R-Flag (MDM2 degradation-resistant mutant) expression vectors and 24 h later treated with CoCl₂ for 16 h, before luciferase activity was assayed. RLU: relative luciferase unit. <i>Columns</i>, mean of three independent experiments performed in duplicate; <i>bars</i>, S.D. * <i>P</i><0.01. (C) Cells were treated as in (B) and after treatment equal amounts of total cell extracts were subjected to Western immunoblotting using the indicated antibodies: anti-Flag (to detect ectopic HIPK2-Flag expression), anti-Ser46 and anti-p53 antibodies. (D) RKO cells were transfected with siMDM2 and 36 h later equal amount of total cell extracts were analyzed by Western immunoblotting with specific anti-MDM2 antibody. (E) RKO cells were transfected with siMDM2 and 24 h later were treated with CoCl₂ and ADR for 16 h. Equal amount of total cell extracts were analyzed by Western immunoblotting with specific antibodies detecting p53Ser46 phosphorylation and PARP cleavage (arrows show cleaved and uncleaved forms); total p53 is also shown. Anti-tubulin was used as protein loading control. (F) RKO cells, stably transfected with p53AIP1-luc reporter, were transfected with siMDM2 and 24 h later treated with CoCl₂ and ADR for 16 h before luciferase activity was assayed. RLU: relative luciferase unit. <i>Columns</i>, mean of three independent experiments performed in duplicate; <i>bars</i>, S.D.</p

Cobalt downregulates HIPK2 protein levels and abrogates HIPK2 recruitment onto HIF-1α promoter.

<p>(A) RKO and A549 cells were treated with cobalt for 16 h in the presence or absence of proteasome inhibitor MG132 (40 µmol/L for 6 h) and the vehicle DMSO. Equal amount of total cell extracts were analyzed by Western immunoblotting with specific antibody detecting endogenous HIPK2 protein levels; anti-Hsp70 was used as protein loading control. (B) Total mRNAs were reverse transcribed from RKO and A549 cell treated with cobalt for 16 h for PCR analyses of HIPK2 gene expression. GAPDH was used as internal control. (C) Chromatin immunoprecipitation (ChIP) analysis performed with anti-HIPK2 and anti-HDAC1 antibodies on RKO cells treated with cobalt for 8 and 16 h. PCR analyses were performed on the immunoprecipitated DNA samples using specific primers for the human <i>HIF-1α</i> promoter. Amplification of <i>GAPDH</i> promoter (right panel) was used as control of HIPK2 binding specificity to the <i>HIF-1α</i> promoter. A sample representing linear amplification of the total input chromatin (Input) was included as control. Additional controls included immunoprecipitation performed with non-specific immunoglobulins (No Ab).</p

Effect of zinc on reversing hypoxia-induced HIPK2 dysfunction and protecting against hypoxia pathway.

<p>(A, left panel) Subconfluent RKO cells were exposed to CoCl₂ and ZnCl₂ alone or in combination for 16 h, and equal amount of total cell extracts analyzed by Western immunoblotting with specific antibody showing endogenous HIPK2 level. Anti-tubulin was used as protein loading control (right panel). Equal amount of nuclear extracts of RKO cells treated as above, were analyzed by Western immunoblotting with specific antibodies detecting HIF-1α levels. Anti-Hsp70 was used as protein loading control. (B) RKO cells treated as in (A) were lysed for nuclear and cytoplasmic fractionation and analyzed by Western immunoblotting using anti-HIPK2 antibody. Anti-tubulin and anti-NFYB antibodies were used for protein loading control of cytoplasmic and nuclear fractions, respectively. (C) RKO and A549 cells were treated with 2% O₂ in the presence or absence of zinc for 16 h, and equal amount of nuclear cell extracts analyzed by Western immunoblotting with specific antibodies detecting HIF-1α and HIPK2 nuclear levels. Anti-Hsp70 was used as protein loading control. (D) Kinase assay of ectopic HIPK2 in 293 cells transfected with Flag empty or HIPK2-Flag expression vector left untreated or treated with cobalt or zinc for 16 h. Equal amount of total cell extracts were immunoprecipitated using anti-Flag antibody and assayed for kinase activity using MBP as substrate. Equal expression of MBP protein was confirmed by comassie-staining of kinase assay. (E) Chromatin immunoprecipitation (ChIP) analysis performed with anti-HIPK2 antibody on RKO cells treated as in (A). PCR analyses were performed on the immunoprecipitated DNA samples using specific primers for the human <i>HIF-1α</i> promoter. Amplification of <i>GAPDH</i> promoter (right panel) was used as control of HIPK2 binding specificity to the <i>HIF-1α</i> promoter A sample representing linear amplification of the total input chromatin (Input) was included as control. Additional controls included immunoprecipitation performed with non-specific immunoglobulins (No Ab). (F, upper panel), Total mRNAs were reverse transcribed from RKO cells treated with ZnCl₂ and CoCl₂ for 24 h and ADR for 16 h respectively, for PCR analyses of HIF-1 target genes <i>Bcl2</i>, <i>MDR1</i>, and <i>VEGF</i>. Ratio: expression ratio to GAPDH. (F, lower level) densitometric analysis of gene expression plotted as the expression ratio to GAPDH, used as internal control. Student's <i>t</i> test was used for statistical analysis of comparison between the values of cobalt and cobalt plus zinc, and of CoCl₂/ADR and CoCl₂/ADR/ZnCl₂ as shown. * <i>P</i><0.01. (G) RKO cells depleted of HIPK2 function by siHIPK2 were treated with ZnCl₂ and CoCl₂ for 24 h and ADR for 16 h, respectively and total mRNAs reverse transcribed for PCR analyses as in (F).</p

Microarray bioinformatical analysis of the RA effects on hMSC cultured for 5 days without serum.

<p>A: Venn diagram showing the number of differentially expressed transcripts in DMEM and RA treated hMSC when compared to 10%FBS treated cells. B: The genes regulated specifically by RA (i.e. blue or yellow in the diagram depicted in A) were clustered by Ingenuity software. This software contains a database with different transcripts arranged in networks according to their known biological interactions. According to the number of transcripts regulated in each of these networks, the program scores them. The best scored network is depicted here. Direct interactions are represented by continuous arrows and direct by dotted ones. Increase in expression is represented by red color and decrease by green. The number under each transcript name is the logarithmic change in comparison to 10%FBS control cells RNA. The type of interaction is indicated by a label and the type of molecule is indicated by the shape of the box, being both detailed at the right of the figure.</p

Primers used for QRT-PCR in this study.

<p>Primers used for QRT-PCR in this study.</p

Periostin can rescue the effect of RA on hMSC osteogenesis.

<p>A: hMSC were treated for 5 days with either DMEM, 0.5 µM RA in DMEM (RA), 20 ng/ml EGF+ 5 ng/ml bFGF (EGF+FGF), 20 ng/ml EGF +5 ng/ml bFGF +0.5 µM RA (EGF+FGF+RA), 10%FBS (FBS), or 0.5 µM RA in 10%FBS (FBS+RA). The RNAs were purified and the cDNAs were synthesized using reverse transcriptase. Periostin transcript was quantified in each sample. The error bars represent relative expression normalized to RNF10 expression and referred to the relative expression on DMEM as mean ± s.e.m. All the treatments were performed in triplicates in two independent experiments with cDNA from different donors and the significance of the results was assessed using one way ANOVA and Tukey's multiple comparison test. B–G: hMSC were cultured in osteoblasts differentiation medium supplemented with 0.5 µM RA (B–D), or in 10%FBS +0.5 µM RA (negative control) (E–G), for 21 days over uncoated plastic (B and E), 2.5 µg laminin/well coated plastic (C and F), or 2.5 µg periostin/well coated plastic (D and G). Osteoblasts were stained with alizarin red. Size bars = 100 µm.</p

Therapeutic efficacy of zinc combined with chemotherapy.

<p>(A) RKO cells were implanted into nude mice by i.m. injection and allowed to develop into palpable ∼300 mm³ tumor nodules at the injection sites and the randomized to four treatment groups: (<i>i</i>) vehicle (PBS) alone, (<i>ii</i>) ADR alone (10 mg/kg body weight) delivered once intraperitoneally (i.p.) at day seven (arrow), (<i>iii</i>), ZnCl₂ alone (10 mg zinc/kg body weight) administrated once daily by oral administration, and (<i>iv</i>) ZnCl₂ combined with chemotherapy: tumors were pre-treated at day 7 with ZnCl₂ for 8 h before delivering ADR and thereafter, ZnCl₂ was administered once daily for the next 2 weeks. Each experiment was conducted two independent times, each time with eight mice per group. Tumor volumes were measured every other day following the establishment of xenografts in mude mice. <i>Y axis</i>, tumor volume; <i>X axis</i>, calibration time (days) after cell injection (p.i.: post-injection). Student's <i>t</i> test was used for statistical analysis of comparison between the values of ADR and ADR in combination with zinc treatments, as shown. * <i>P</i><0.01. (B) Picture of explanted tumors showing tumor volumes at day 18. (C) RNA samples from explanted tumors, at day 18 after cell injection, treated with ADR and zinc alone or in combination were used for reverse-transcription (RT)-PCR. The mRNA levels were normalized to GAPDH expression. (D) Densitometric analysis of gene expression plotted as expression ratio to GAPDH. Average of two different tumors for each treatment group. Student's <i>t</i> test was used for statistical analysis of comparison between the values of ADR and ADR in combination with zinc treatments, as shown. * <i>P</i><0.01.</p

RA and EGF+bFGF effects on Erk phosphorylation and on cell cycle progression.

<p>A: Western blot analysis of hMSC that were cultured for 5 days in either DMEM alone (DMEM) or in the presence of 0.5 µM RA (RA) or in the presence of 20 ng/ml EGF+ 5 ng/ml bFGF (EGF+bFGF), with or without the addition of 0.5 µM RA (EGF+bFGF+RA). As control, cells were cultured with 10%FBS (FBS), or with 0.5 µM RA in 10%FBS (FBS+RA). Whole-cell protein extracts from these differently treated cells were fractionated on a denaturating 12% polyacrylamide gel, transferred to nitrocellulose and detected with anti phosphorylated Erk antibody (pErk1/2). The membrane was stripped twice, one for detection with anti total Erk antibody (Erk1) and the second for β-actin antibody detection used as loading control. B: Densitometry analysis of A. The bars represent relative expression normalized to β-actin expression and referred to this ratio in DMEM. C–J: Cell cycle progression by FACS of hMSC that were cultured for 5 days with DMEM (C), 0.5 µM RA in DMEM (D), 20 ng/ml EGF (E), 5 ng/ml bFGF (F), 5 ng/ml bFGF +20 ng/ml EGF (G), or 5 ng/ml bFGF +20 ng/ml EGF +0.5 µM RA (H). In addition, hMSC were cultured for 2 days in DMEM (I) or in the presence of 0.5 µM RA (J) before replacement of the medium with 20 ng/ml EGF +5 ng/ml bFGF in DMEM for further 2 days. At the end of the experiment the cells were harvested by trypsinization, permeabilized and stained with propidium iodide to measure the DNA content by FACS.</p

Antibodies used for Western blot in this study.

<p>Antibodies used for Western blot in this study.</p