The adsorbent SiPy displayed good adsorption capacity of 90.25, 75.38, 55.23, and 35.12 mg g−1 for Cu(ii), Zn(ii), Cd(ii), and Pb(ii), respectively, at 25 min and pH = 6. The adsorption behaviors of metal ions onto the SiPy adsorbent fitted well with the pseudo-second-order kinetic mode and the isotherm was better described by the Langmuir isotherm. The thermodynamic studies disclose spontaneous and endothermic adsorption process. Furthermore, the SiPy adsorbent retained good selectivity and regeneration properties after five adsorption–desorption cycles of Cu(ii). A computational investigation of the adsorption mechanism indicates that the N-pyridine, O-hydroxyl, and ether O-atoms play a predominant role during the capture of Cu(ii), Zn(ii), Cd(ii), and Pb(ii). This study proposes the SiPy adsorbent as an attractive material for the selective removal of Cu(ii) from real river water and real industrial wastewater.The removal of heavy metals is attracting considerable attention due to their undesirable effects on the environment. In this investigation, a new adsorbent based on silica functionalized with pyridin-2-ylmethanol (SiPy) was successfully synthesized to yield to a hybrid material. FTIR, SEM, TGA, and specific surface area analysis were used to characterize the structure and morphology of the SiPy hybrid material. Various heavy metal ions such as Cu( The removal of heavy metals is attracting considerable attention due to their undesirable effects on the environment. However, their use cannot be avoided because of their necessity in tanneries, batteries, paints, coatings, dyeing, textile mill, and other growing industries.5 These industries release heavy metal ions into the environment and polluted water is used for irrigation and consumption.6 It has become essential to search for new methodologies to extract them from environmental aqueous solutions.With intensive industrial activity and urbanization, aquatic environment contamination by heavy metal ions has become one of the major environmental pollution sources of groundwater and surface waters7 reverse osmosis,8 chemical precipitation,9 membrane filtration,10 photocatalysis methods,11,12 filtration,13 electrochemical technology,14 solvent extraction,15 and flocculation.16 However, these techniques are limited in their practical use due to the generation of hazardous by-products.17 Adsorption is however considered a promising and effective purification technique for heavy metal ions due to its simplicity, low cost, and high efficiency.18In recent years, various techniques and technologies were used for the removal of heavy metal ions such as ion exchange,19 zeolites,20 chitosan,21 and cellulose22 have gained attention due to their promising potential in environmental domains. Mesoporous silica, however, offers a number of assets such as a large specific surface area, high thermal and mechanical stabilities, as well as regular porosity and flexibility toward surface chemical modification.23,24 In this respect, mesoporous silica gel can be easily functionalized to significantly increase the sorption of heavy metals ions from wastewater.25–32 The efficiency and selectivity of these adsorbents partly rely on the donor atoms such as oxygen, nitrogen, and sulfur onto the surface of the material that are responsible for forming a complex with heavy metals.33–37 In this context, pyridine has attracted attention because of its structure and well-known coordination chemistry.38,39 Pyridine is thus an excellent grafting group for functionalizing silica and can be widely applied in environmental cleanup via the chelating reaction.40–42Conventionally, adsorbents such as carbon nanotubes,43In this work, a silica gel impregnated with a ligand-based functionalized pyridine was synthesized and characterized for the extraction of heavy metal from aqueous solutions. On the other hand, density functional theory (DFT), noncovalent interaction (NCI), quantum theory of atoms in molecules (QTAIM) approaches, and the localized orbital locator (LOL) were employed to gain a better understanding of the metal ion's adsorption mechanism and selectivity of the ligand structure.2.2.1.2 g−1, pore diameter of 52–73 Å, and pore volume of 0.7–0.85 cm3 g−1 was activated before use by heating at 120 °C for 24 h. The silylating agent 3-glycidoxypropyltrimethoxysilane was used without purification. All metal ion quantities were determined by atomic absorption spectroscopy (AAS) using a Spectra Varian A.A. 400 spectrophotometer. The pH value was controlled using a pH 2006, J. P. Selecta; elemental analyses were performed by the Microanalysis Centre Service (CNRS). FT-IR spectra were obtained using a PerkinElmer System 2000. SEM images were obtained on a FEI-Quanta 200. Mass loss determinations were performed in a 90 : 10 O2(g)/N2(g) atmosphere on a PerkinElmer Diamond TG/DTA at a heating rate of 10 °C min−1. The specific surface area of modified silica was determined using the BET equation. The nitrogen adsorption–desorption was obtained by means of a Thermoquest Sorptomatic 1990 analyzer after the material had been purged in a stream of dry nitrogen.All solvents and chemicals  were of analytical grade and used without further purification. Silica gel (Sigma-Aldrich) with particle size in the range of 70–230 mesh, surface area of 470–530 m2.2.4  and dry THF (90 mL), ethyl picolinate was added  at 0 °C and the reaction was stirred at reflux for 4 h. After the reaction was completed, the mixture was cooled to 0 °C, and a solution of NaOH  and distilled water (17 mL) were added successively. The resulting product was purified by column chromatography  to afford compound L1 in 86% yield. Brown liquid; Rf = 50% ; IR : ν(OH) = 3411; ν  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>N) = 1553; ν (CC) = 1452; 1H NMR (DMSO-d6): δ 4.53 ; 5.38 ; 7.21 ; 7.44 ; 7.76 ; 8.44 ; 13C NMR (DMSO-d6): δ 64.52 ; 120.60 ; 122.35 ; 137.02 ; 148.89 ; 162.31 ; MS: m/z, 110.05 (M + H)+.To a mixture of LiAlH2.3.44 Briefly, 10 g of activated silica was dissolved in toluene (40 mL), in which were added triethylamine (240 μL). Then, 3-glycidoxypropyltrimethoxysilane (13.3 mL) was added to the reaction and stirred at solvent reflux temperature for a period of 24 h under nitrogen atmosphere. The final product was filtered and extracted via Soxhlet extraction with ethanol and dichloromethane (1 : 1) for 12 h. The crude product was vacuum dried at 70 °C over 24 h.The surface modification of mesoporous silica material by 3-glycidoxypropyltrimethoxysilane (SiEp) was carried out according to our published method.2.4.1) (After converting the hydroxyl-pyridine (L1)  to its a2.5.ii)] = 10 to 300 mg L−1), time (5 to 35 min), pH (1 to 7), temperature (25 to 45 °C), and selectivity  parameters on the adsorption of Cu(ii), Zn(ii), Cd(ii), and Pb(ii) were studied in the batch method. Briefly, 10 mg of the SiPy adsorbent was dispersed to 10 mL of aqueous solutions. The pH values were adjusted with diluted HCl and NaOH solutions, the mixture was shaken at 25 °C for 25 min. After extraction, the metal ion concentrations were monitored by AAS and the adsorption capacity was calculated using 45qe (mg g−1) denotes the adsorption capacity, C0 (mg L−1) and Ce (mg L−1) are the initial and equilibrium concentrations respectively, V (mL) refers to the volume of the solution, and W (mg) refers to the mass of the adsorbent.The effect of concentration ,51 combined with the 6-311G+ basis set,52 for M(ii) complexes to the basis LANL2DZ level.53DFT,N09 ref.  and Mult3.3.1.1, which was converted to its pyridine alcoholate using metallic sodium in THF, which was then immobilized on the SiEp surface in DMF at reflux.45The synthesis of the new material SiPy is presented in 3.2.3.2.1.The result of the elemental analysis, determined for SiPy, showed high percentages for carbon (5.97%) and nitrogen (1.08%), which are not present on the starting silica. This result indicates that the organic matter (pyridine unit) has been immobilized on the silica network, thus supporting a successful functionalization. This immobilization of the organic matter on the inorganic silica network was also confirmed by the analyses below.3.2.2.−1, 1087 cm−1, and 798 cm−1 related to the silica backbone were identified.54 The SiPy material was analyzed using FTIR spectra and compared with pure silica SiG and SiEp showed a new peak at 1462 cm−1, which was attributed to the stretching vibration of CN. This spectrum thus shows that the material was successfully modified by the pyridine derivative.The specific peaks observed at 3440 cm and SiEp. After f3.2.3.As we can see from SEM imaging , the sur3.2.4.55 The surface area for free silica SiG is 305.21 m2 g−1 with a pore volume of 0.770 cm3 g−1. The BET surface area of SiEpand SiPy decreased to 277.08 and 261.33 m2 g−1, respectively, due to pore blocking by the chemical bonding of free silica with organic functional groups. It means that part of the pores were blocked by organic groups, which led to a decrease in the size of the pores. The total pore volume of free silica was 0.770 cm3 g−1 and it decreased to 0.68 and 0.66 cm3 g−1 for SiEp and SiPy, respectively.While a porous structure was observed for SiPy, it was of interest to determine its eventual mesoporous character by nitrogen adsorption/desorption . Such a 3.2.5.56,57SiEp also exhibited two decomposition stages at 1.34% from 25–110 °C and 10.8% from 200 to 800 °C due to the desorption of water and the degradation of organic moieties grafted to silica. Therefore, the two distinct weight loss steps of SiPy represent a loss of 4.13% in the 25–100 °C range and 12.4% in the interval of 270–800 °C, which were attributed to the vaporization of water and degradation of organic compounds.The thermal stability of SiG, SiEp, and SiPy was examined by TGA analysis . SiG sho3.3.3.3.1.3O+ in the suspension, which are available to protonate the pyridine unit and oxygen atoms and renders the surface of SiPy positively charged, which had a repulsive force that hampered the removal of metal ions. At pH ≥8, SiPy adsorption efficiency remains almost constant, indicating the formation of metal hydroxide precipitates for Cu(ii), Zn(ii), Cd(ii), and Pb(ii) ions.The adsorption of heavy metal ions depends on the pH of the solution, which is a dominant parameter affecting the adsorption. In this investigation, the effect of pH on the adsorption experiments was performed on SiPy over the range of 1.0–7.0 with optimal initial concentration . Herein,3.3.2.ii), Cd(ii), Cd(ii), and Pb(ii) was varied from 5 to 35 min. From The study of the impact of the contact time is very important for the investigation of metal ions removal. For this purpose, the time of adsorption of Cu(ii), Zn(ii), Cd(ii), and Pb(ii) by SiPy were studied using first, second-order, and intra-particle diffusion models. The linear form of the three models can be calculated as 58,59The potential mechanisms controlling the sorption of Cu(qe (mg g−1) and qt (mg g−1) are the amounts of adsorbate at equilibrium and at time t (s), respectively, and K1 (min−1) is the rate constant of the first-order adsorption.Pseudo-first-order model:K2 (g (mg min−1)−1) is the second order rate constant of adsorption.Pseudo-second-order model:kpi (mg g−1 min−1/2) is the intra-particle diffusion rate constant, and constant Ci indicates the thickness of the boundary layer.Intra-particle diffusion model:ii), Zn(ii), Cd(ii), and Pb(ii) experimental adsorption rate data were fitted to the three kinetic models. All the kinetic data of adsorption onto SiPy, calculated from the related linear fitting curves, are shown in Fig. S1 and S2.R estimated from the pseudo-second-order kinetic model were significantly higher than those obtained from the pseudo-first-order kinetic model. The results suggested a better fitting of the pseudo-second-order, assuming that the adsorption was a chemisorption process.The Cu, Zn(ii), Cd(ii), and Pb(ii) ions is higher at the beginning of the process due to the availability of a large number of active sites on the adsorbent surface. The lower slopes of the second parts are due to the decrease in the concentration gradient, which makes the diffusion of Cu(ii), Zn(ii), Cd(ii), and Pb(ii) ions into the mesopores of the adsorbent slower, thus leading to a low removal rate. As the data showed, intra-particle diffusion was the real rate-controlling process of the adsorption process.Then, it is important to further study the intra-particle diffusion model, which has been widely applied to provide the rate-controlling steps affecting the adsorption mechanism.3.3.3.ii), Zn(ii), Cd(ii), and Pb(ii). These very high adsorption capacities can be justified by the available adsorption sites on the surface of the adsorbents, which are easily occupied by the metal ions. But the number of active sites on the SiPy surface is limited when they are fully occupied. Thus, saturated adsorption would be achieved at that time.62 The equilibrium adsorption of the metal ions on SiPy was in the order Cu(ii) > Zn(ii) > Cd(ii) > Pb(ii)  .63,64 which are recalled below:The adsorption mechanism of metal ions and the SiPy adsorbent was investigated by Langmuir, Freundlich, Dubinin–Radushkevich (D–R), and Temkin isotherm models,qe (mg g−1) and q (mg g−1) represent the equilibrium and the saturated adsorption capacity, respectively. Ce (mg g−1) is the concentration of metal ions in solution at equilibrium, qm (mg g−1) is the theoretical saturation adsorption capacity, and KL (L mg−1) is the equilibrium Langmuir constant.Langmuir model:KF is the Freundlich constant and n is the value used to suggest the heterogeneity of the interface.Freundlich model:β (mol J−1)2 is a constant related to the mean free energy E of adsorption (E = (2β)−0.5), and ε (J mol−1) is the Polanyi potential related to the equilibrium concentration (ε = RT ln(1 + 1/Ce)).Dubinin–Radushkevich (D–R) model:bt (J mol−1) and At (L mg−1) are the Temkin isotherm constant, T defines the Kelvin temperature (K), and R (8.314 J mol−1 K−1) defines the universal gas constant.Temkin model:ii) Zn(ii), Cd(ii), and Pb(ii) onto the surface of SiPy are presented in Fig. S4–S7.The plots of the linear fitting curves of the Langmuir, Freundlich, D–R, and Temkin isotherm mod-els for the adsorption of Cu with 95.60 mg g−1, a high value that shows that the SiPy adsorbent could be used potentially for the adsorption of Cu(ii) from industrial wastewater and river waters.It is obvious that the 3.3.4.G° (kJ mol−1), enthalpy ΔH° (kJ mol−1), and entropy ΔS° (J mol−1 K−1) were obtained from 65,66R (8.314 J mol−1 K−1), T (K), and Kd are the universal gas constant, absolute temperature, and distribution coefficient, respectively.The effect of temperature on sorption experiments was also investigated. Gibbs free energy ΔG° suggest that the adsorption process of SiPy for Cu(ii), Zn(ii), Cd(ii), and Pb(ii) is thermodynamically favorable and spontaneous. Meanwhile, the positive value of ΔH° indicates that the adsorption process has an endothermic nature. On the other hand, the obtained negative values of ΔS° points out an increase in the randomness and disorder at the surface of the adsorbent All thermodynamic parameters are listed in 3.3.5.ii), Zn(ii), Cd(ii), and Pb(ii) with optimum concentration of each metal ion , Zn(ii), Cd(ii), and Pb(ii), respectively). According to ii) ions. Furthermore, given this selectivity, our adsorbent could be tested for its applicability in the removal of Cu(ii) from river waters, which is discussed in the next section.The adsorption selectivity of SiPy was examined using aqueous solutions containing Cu(3.3.6.ii) adsorption using HCl (2 mol L−1) as the eluent. After five regeneration cycles, the adsorption efficiency of the adsorbent for Cu(ii) can still reach 93.81%  Touissit–Boubekker river (in the Jerada–Oujda region) where: pH = 7.1, TDS = 2031 mg L−1 and σ = 2301 μS cm−1. The adsorption efficiency of SiPy (10 mg) was investigated under optimal conditions by the batch method using 10 mL of the river water. The percentage removal efficiency of Cu(ii) ion was found to be high, up to 92% and 94% from Ghis and Touissit–Boubekker rivers, respectively, for SiPy  from wastewater.In order to test its feasibility in a real application, an adsorption experiment was carried out on real water samples originating from (i) Ghis river (located next to Al Hoceima) where: pH = 7.7, total dissolved solids (TDS) = 1297 mg Lfor SiPy . Thus, S3.3.8.ii) with other literature adsorbents. It is clear that SiPy has a higher adsorption efficiency for Cu(ii) ion (90.25 mg g−1).3.4.3.4.1.ii) indicate that the absorption peak at 1462 cm−1 belonging to the NC group disappeared after the adsorption of Cu(ii) (77 The results demonstrate that the NC group exhibits marked binding of Cu(ii).The organic fraction of the surface of the material plays an important role in the removal of metal ions. The FTIR spectra of SiPy before and after the adsorption of Cu(f Cu(ii) .77 The r3.4.2.78Theoretical investigation was undertaken to get insights into the mechanism of adsorption of metal ions with SiPy. The following theoretical methods were used: DFT, QTAIM, LOL, NCI, and dual descriptor.11⋯H29), (O11⋯H25), (O22⋯H13) as well as a repulsive interaction within the pyridine ring  method afforded all possible structures of (Cu(ii)–SiPy, Zn(ii)–SiPy, Cd(ii)–SiPy, and Pb(ii)–SiPy (ii)–SiPy) structures form stable complexes but these complexes do not have the same stability, given the bond lengths and the type of coordination . For instance, the bond lengths of Cu(ii)–SiPy (Cu33–O12 = 1.95 Å) are smaller compared to those for Zn(ii)–SiPy (Zn33–O12 = 2.08 Å) and even smaller for Cd(ii)–SiPy Cd–O12 = 2.31 Å) and Pb(ii)–SiPy (Pb33–O12 = 2.47 Å).Dual descriptors are a useful function used to reveal the reactive sites. ii)–SiPy ). The boii), Zn(ii), Cd(ii))–SiPy, whereas another coordination site is occupied by a water molecule. A bidentate coordination mode is however noticed for Pb(ii)–SiPy, which supports the stability order Cu(ii)–SiPy > Zn(ii)–SiPy > Cd(ii)–SiPy > Pb(ii)–SiPy.A tridentate coordination mode is noticed for –N10, M(ii)–O12, M(ii)–O17) with the use of E(2) (second-order stabilization energy) and EC (electronic configurations).In order to better understand metal coordination, we have used the NBO E(2) are useful to indicate the binding strength, which is found to be higher for the Cu(ii) complex with a charge transfer LP(O12) → LP*(Cu(ii)) of 16.63 kcal mol−1 compared to LP(O12) → LP*(Zn(ii)) = 16.11 kcal mol−1 and LP(O12) → LP*(Zn(ii)) = 6.03 kcal mol−1 for Zn(ii) (ii)–SiPy complex, a lower E(2) value was found, indicating a low interaction strength of Cu(ii)–O10. Thus, the computational investigation completely replicates the experimental selectivity  . For theectivity .802ρcp values, and a low value of the electron density, ρcp ≈ 0 (ii) complex is ρcp = 0.08 (Cu33–O10), which is higher compared to the ones found for the Zn(ii) complex with ρcp=0.05 (Zn33–O10), as well as for the Cd(ii)complex with ρcp=0.02 (Cd–O10) and the Pb(ii) complex. Thus, the stability of the four complexes follows the order Cu(ii) > Zn(ii) > Cd(ii) > Pb(ii).All the interatomic areas have positive ∇ ρcp ≈ 0 , which sii)33–N10, M(ii)33–O12, M(ii)33–O17, and M(ii)33–O19), which confirms the stability of the complexes. In the contour map, the blue color represents a low electron density region, whereas the red color shows a high electron density. The purple color means the absence of electron density . Such a color is not found for the Cu(ii) complex, whereas a small area is detected for the Zn(ii) complex. However, a larger area is found for the Cd(ii) complex, and the Pd(ii) complex has a much larger purple area. Thus, the stability of our four complexes is confirmed as follows: Cu(ii) > Zn(ii) > Cd(ii) > Pb(ii).The structures of the complexes shown in 5.−1 for Cu(ii), Zn(ii), Cd(ii), and Pb(ii) respectively, with an optimal sorption pH of 7. The adsorption kinetics process is dominated by the pseudo-second-order model, which indicates a homogeneous character. The adsorption properties indicated monolayer adsorption, chemisorption binding mechanism, and an endothermic and spontaneous process. The SiPy adsorbent was able to maintain 93.81% of Cu adsorption capacity even after five cycles of adsorption and desorption experiments. A computational study suggested that the removal of the metal ions is the synergistic effect of CN, R–O–R, and R–OH groups functionalized onto the SiPy material. The CN group plays a major function during the complexation of metal ions. Furthermore, the computational investigation confirmed the affinity of our hybrid material for Cu(ii). The results suggested that the SiPy adsorbent displays great advantages of high adsorption capacity toward Cu(ii), rapid response, high selectivity, and good reusability, which makes it as a good candidate for wastewater treatment applications.In conclusion, a new simple and low-cost method for the highly efficient cleanup of heavy metal ions in water has been presented, using silica organically modified with pyridine, which has been synthesized and fully characterized. The maximum sorption capacity after reaching the equilibrium (30 min) was 90.25, 75.38, 55.23, and 35.12 mg gThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.RA-012-D1RA06640D-s001


The p53 family of proteins comprises p53, p63, and p73, which share high structural and functional similarity. The two distinct promoters of each locus, the alternative splicing, and the alternative translation initiation sites enable the generation of numerous isoforms with different protein-interacting domains and distinct activities. The co-expressed p53/p73 isoforms have significant but distinct roles in carcinogenesis. Their activity is frequently impaired in human tumors including colorectal carcinoma due to dysregulated expression and a dominant-negative effect accomplished by some isoforms and p53 mutants. The interactions between isoforms are particularly important to understand the onset of tumor formation, progression, and therapeutic response. The understanding of the p53/p73 network can contribute to the development of new targeted therapies.The p53 tumor suppressor protein is crucial for cell growth control and the maintenance of genomic stability. Later discovered, p63 and p73 share structural and functional similarity with p53. To understand the p53 pathways more profoundly, all family members should be considered. Each family member possesses two promoters and alternative translation initiation sites, and they undergo alternative splicing, generating multiple isoforms. The resulting isoforms have important roles in carcinogenesis, while their expression is dysregulated in several human tumors including colorectal carcinoma, which makes them potential targets in cancer treatment. Their activities arise, at least in part, from the ability to form tetramers that bind to specific DNA sequences and activate the transcription of target genes. In this review, we summarize the current understanding of the biological activities and regulation of the p53/p73 isoforms, highlighting their role in colorectal tumorigenesis. The analysis of the expression patterns of the p53/p73 isoforms in human cancers provides an important step in the improvement of cancer therapy. Furthermore, the interactions among the p53 family members which could modulate normal functions of the canonical p53 in tumor tissue are described. Lastly, we emphasize the importance of clinical studies to assess the significance of combining the deregulation of different members of the p53 family to define the outcome of the disease. TP53 have been found in nearly all tumor types and are estimated to contribute to more than 50% of all cancers.p53 has a central role in tumorigenesis; it regulates cellular response to stress signals or oncogenic cellular stimuli by inducing transient or permanent cell-cycle arrest, DNA repair, apoptosis, or senescence . The inaTP73 . T. TTP53 gt manner ,13. Tran(ATG160) ,14, resuTP53 mRNA transcripts translate at codon 1 and/or codon 40 to encode the long isoforms (p53/Δ40p53), while transcribed from the internal P2, translation can start at codons 133 and/or 160, giving rise to the short isoforms (Δ133p53/Δ160p53). The transcription from the canonical P1 generates intron 2-spliced  or intron 2-retained  TP53 mRNA transcripts. Translation at the codon 1 of intron 2-spliced TP53 mRNA transcript encodes the full-length p53 isoforms [TP53 mRNA, IRES-mediated translation can produce Δ40p53 isoforms from the codon 40 [TP53 mRNA transcripts with retained intron 2 that are translated only at codon 40 [TP53 mRNA transcript that can be translated either at codons 133 or 160, encoding the Δ133p53 or Δ160p53 isoforms, respectively [TP53 mRNA transcripts that encode β or γ isoforms, respectively. Since both exons 9β and 9γ contain premature termination codons (PTCs), exons 10 and 11 remain untranslated in β and γ isoforms [TP53 mRNA transcripts encoded by the TP53 gene can give rise to 12 protein isoforms, known as p53α/β/γ, Δ40p53α/β/γ, Δ133p53α/β/γ, and Δ160p53α/β/γ [Consequently, the p53 isoforms can be classified as long or short depending on the initiation of transcription and translation. Transcribed from the canonical P1, the isoforms ,14. Howecodon 40 ,16,17. Icodon 40 ,11,14. Iectively ,14,18. Tp53α/β/γ ,20,21,22p53α/β/γ ,24. Furtp53α/β/γ ,26. Intep53α/β/γ ,27.TP73 gene situated in the region 1p36.33 that is often deleted in neuroblastoma and various other human tumors. Frequent occurrence of loss of heterozygosity (LOH) and structural homology with the canonical tumor suppressor p53 placed p73, together with almost concomitantly discovered p63 protein, into the common p53 protein family [TP73 gene consists of 15 exons  and it can be transcribed into different mRNAs, subsequently producing several different protein isoforms , ΔEx2/3p73 (lacking exons 2 and 3), and ΔN′p73  ,30,31. ITP73 mRNA representing another possible mechanism of p73 expression regulation via CAP-independent translation [Similar to p53, an IRES element was discovered in exon 2 of the nslation . Alternanslation ,34,35. Cnslation ,37,38.PTP4A1, PLK2) depends on the transactivation activity of the TAD2 [The canonical, full-length p53 (p53α) isoform displays a modular domain structure and contains 393 residues that are organized in seven functional domains B. The N-the TAD2 .Following TA domains at the N-terminus lies a proline-rich domain  that contains 15 proline residues, some as part of five PXXP motifs (where P represents proline and X any residue) important for growth suppression. These motifs have the ability to bind SH3 (Src homology 3) domains of proteins such as c-Src and PI3K (phosphatidylinositol 3-kinases) and mediate signal-transduction pathways ,45. The The p53 protein binds to DNA through its core domain, the conserved sequence-specific DBD (residues 94–290). The DBD contains four conserved regions  and inclBAX promoter, but do not affect the transactivation of p21 promoter [The p53 protein possesses a nuclear-localization signal  that facilitates the shuttling of p53 between the nucleus and cytoplasm. It has been shown that the nuclear import of p53 is negatively regulated by ubiquitination of lysine residues 319–321 within the NLS, causing the retention of p53 in the cytoplasm. Upon stress, the level of ubiquitination reduces, and the positive charges of NLS residues are rendered unmasked and recognized by importin α3, an adaptor molecule that facilitates nuclear import of non-ubiquitylated p53 ,58. The promoter , which wpromoter .BAX and p21), thereby acting as dominant-negative mutants [To fulfill its function as a tetrameric transcription factor, p53 needs to exhibit its capacity to form tetramers, an active form needed for binding to RE and gene transactivation, dependent on the OD. It has been revealed that the monomer, which consists of a β-strand and an α-helix, connects with the second monomer to form a dimer in an antiparallel manner by means of their β-strands and α-helices. Two dimers connect through their α-helices to form a tetramer. On the other hand, β-strands are not included in the interaction between the dimers since they are located outside of the tetramer . In addi mutants .The C-terminal domain (CTD) (residues 357–393) is an arginine- and lysine-rich basic domain that recognizes and binds nonspecifically to DNA and RNA. The CTD is intrinsically disordered and contains NLSs, as well as the sites with the most frequent posttranslational modifications (PTM) that include phosphorylation, acetylation, ubiquitination, methylation, neddylation, and sumoylation ,67,68. HTo summarize, different p53 isoforms, except the canonical full-length p53, lack part of the N- and/or C-termini and, consequently, are deficient in some of the functional domains B. The isThe full-length p73 and p53 proteins show substantial degree of homology in the composition of the main functional protein domains  B. The lep21, 14-3-3σ, and GADD45 causing cell-cycle arrest, growth suppression, and apoptosis in different cell lines. This transactivation activity was dependent on the presence of 13 unique residues at the N-terminus of the ΔNp73 isoforms and the adjacent region with PXXP motifs (PRD), which together form a novel transactivation domain specific for the ΔNp73 isoforms [S isoform, possibly contributing to pro-survival mechanisms in tumors. The induction of caspase-2S expression was dependent on direct TAp73β/ΔNp73β binding to a specific 18 bp site in the CASP-2S promoter region. Neither ΔNp73α nor TAp73α was able to induce caspase-2S expression under the same experimental conditions [EGR1 (early growth response 1) and CDC6 (cell division cycle 6), independently of p53 in microarray analysis [PAI-1 and Col1a1, possibly through its interaction with Smad transcription factors and subsequent binding to Smad-binding elements (SBEs) [Only the TAp73 isoforms transcribed from P1 that are not subjected to additional splicing at the 5′-end contain a complete TAD at the N-terminus, which makes them strong transcriptional activators B. At firnditions . In contanalysis . In addis (SBEs) . In mores (SBEs) .The PRD of the p73 protein comprises two PXXP motifs situated between residues 84–87 and 103–106 . BetweenThe structure of the p73 DBD is highly similar to that of the p53 protein but differs in the L2 loop important for protein–protein interactions . The DBDα-helix located C-terminally of the core OD (homologous to p53) whose deletion causes a conformational instability and dissociation of the p73 homotetramer into dimers. The absence of the second α-helix is reflected in the reduced p73 transcriptional activity [The OD of p73 comprises residues 352 to 390 and enables tetramerization of p73 and its activity as a transcription factor. In spite of the high degree of similarity in amino-acid sequence with the OD of the p53 protein, the heterotetramers composed of the full-length p73 and p53 have not been identified. This was explained by the discovery of an additional activity . As intaNext to the OD, there is a long stretch of 109 amino acids connecting it to an SAM domain which is specific for the C-terminus of p73 . The SAMThe differential structure of multiple p53/p73 isoforms and their potential interactions offer a broad area for investigation of the molecular basis for their diverse biological functions.The expression and function of the p53 isoforms can be regulated on the transcriptional, posttranscriptional, translational, and posttranslational levels .TP53 promoter usage (P1 or P2) and the alternative splicing of intron 2 or 9, which can be modulated by different factors. The expression of p53 isoforms is tissue-specific and can be precisely regulated. In addition to epigenetic events that influence the activity of TP53 promoters [Helicobacter pylori-infected gastric epithelial cells [As previously mentioned, the expression of p53 isoforms on the transcriptional and posttranscriptional levels is regulated by romoters , severalromoters . In addiromoters . Furtherromoters ,90,91,92al cells .TP53 mRNA β and γ variants and decrease the level of TP53 mRNA α variants, which would suggest that SRSF1 regulates alternative splicing of TP53 intron 9 and favors its complete exclusion  [TP53 mRNA β variants and induced replicative senescence in early passages of normal human fibroblasts, thus revealing that SRSF3 regulates alternative splicing of TP53 intron 9 and modulates the p53-mediated cellular senescence [TP53 pre-mRNA. This allows the recruitment of the splicing factor SRSF7 that prompts the alternative splicing of TP53 pre-mRNA to generate the p53β isoform, shown to be involved in the regulation of IR-induced cellular senescence [TP53 pre-mRNA [+ ion. The G4 structures can arise in both DNA and RNA sequences and, thus, can affect gene transcription, mRNA splicing, and translation. Indeed, it has been shown that G4 structures promote the splicing of intron 2, resulting in fully spliced TP53 mRNA. In contrast, mutations that abolished G4 formation prefer retention of the intron 2 and, consequently, increase the expression of intron 2-retained TP53 mRNA. Therefore, the G4 structures influence the splicing of intron 2 and regulate the ratio between intron 2-spliced and intron 2-retained mRNAs [TP53 pre-mRNA [On the posttranscriptional level, several factors have been shown to regulate the alternative splicing of intron 2 or 9. The splicing factors, such as SRSF1 and SRSF3, which are members of serine/arginine-rich (SR) proteins, have been shown to negatively regulate the expression of p53β and p53γ isoforms ,94. SR p and 9γ) . Furthernescence ,98. In anescence . The Δ40pre-mRNA . The G4 ed mRNAs . In addied mRNAs ,102,103.pre-mRNA . It has pre-mRNA ,105.TP53 can cause reduced binding of PTB to the IRES element and weaker IRES activity [TP53 translation is controlled by several other proteins such as RPL26 or nucleolin, which interact with each other and utilize 5′ or 3′UTRs of TP53 mRNA to enhance or suppress the p53 translation after stress, respectively [On the translational level, p53 isoforms, such as full-length p53 and Δ40p53, can be regulated by IRES-mediated translation ,17,106, activity . Furtherectively ,116. In ectively ,118 thatectively ,119,120.ectively ,118.TP53 mRNA; however, the underlying mechanism still needs to be elucidated [Many different PTMs, such as phosphorylation, acetylation, ubiquitination, methylation, neddylation, and sumoylation, can occur at more than 50 sites located within the TAD, DBD, OD, and CTD of the p53 protein where the TAD and CTD are known to be the most affected ,68. The ucidated .To conclude, we present some regulators and mechanisms that, on different levels, can modulate the expression and stability of p53 isoforms, thereby influencing their biological activities and functions.The p73 expression and activity are regulated at multiple levels, including the previously described usage of multiple promoters, alternative splicing, and translation initiation sites, involving epigenetic and PTMs, as well as interactions with other proteins .TP73 gene is E2F-1, which affects only P1-driven transcription. This regulation is crucial for the induction of apoptosis in the absence of functional p53 and is not dependent on posttranscriptional regulation by p14ARF and MDM2 [+-dependent histone deacetylase hSirT1 through its interaction with PCAF. The interaction of hSirT1 and PCAF represses TAp73 expression as they are co-recruited with E2F-1 on the P1 promoter [TP73 gene just upstream of the initiating codon ATG in exon 2, which contains six consensus ZEB-binding sites [TP73 expression in the tumors [TP73 promoter activity [The p73 expression is intensively regulated transcriptionally in response to different stimuli such as DNA damage, viral oncogenes, proliferative signals, and epigenetic modifications ,130,131.and MDM2 ,133. Hisand MDM2 . The sampromoter . The E2Fpromoter . More pre tumors . Moreovee tumors . In addie tumors . More ree tumors . GemC1  by its ligand amphiregulin in human hepatocellular carcinoma cells and normal hepatocytes. Generation of the ΔEx2p73 isoform is dependent on the activation of JNK1  and the downregulation of the mRNA splicing factor SLU7 . RegardiThe p73 protein is subjected to numerous PTMs (mainly phosphorylations and acetylations) throughout its functional domains, which modulate its activity and stability ,145,146.Numerous regulators and mechanisms of the p73 isoforms’ expression and stability have been discovered to have an impact on their activity and biological functions. Of particular interest are the mechanisms involved in differential isoform regulation, especially those affecting the TAp73/ΔNp73 ratio, which could be used as a potential biomarker and basis for targeted therapy.p53 is a transcription factor, which, via multiple mechanisms, regulates the transcription of a plethora of target genes whose products exert various biological functions ,163. TheTo investigate biological activities of p53 isoforms, several small interfering RNAs have been designed that specifically target a subgroup of p53 splice variants. Furthermore, different antibodies that recognize the epitopes within different functional domains of p53 have been developed which can detect specific subgroups of p53 protein isoforms .Drosophila, mouse) have been developed to investigate the biological activities of p53 isoforms and their involvement in different cellular processes that are associated with tumorigenesis [Due to the highly conserved structure of p53 through evolution, different in vivo models , in addition to Zp53α, encodes Zp53β, ZΔNp53α, and ZΔ113p53α, homologous to human p53β, Δ40p53α, and Δ133p53α, respectively. In response to stress signals, ZΔ113p53α is transactivated by the full-length Zp53 and interferes with Zp53 functions by modulating the expression of p53 target genes, thereby antagonizing apoptotic activity via induction of antiapoptotic bcl2L expression and by interacting with p53 [p21, MDM2, and Bcl-2 [p21, miR-34a, BAX, and PUMA [rad51/RAD51, lig4/LIG4, and rad52/RAD52) [PIDD and cell-cycle arrest gene p21, thereby promoting apoptosis over cell-cycle arrest upon γ-irradiation [p21 or MDM2 promoters rather than the BAX promoter, endogenous p53β preferentially binds to BAX or p21 promoters rather than the MDM2 promoter. In addition, when co-expressed with p53, p53β enhances p53 transcription activity on BAX, but not p21. Contrary to Δ133p53α and Δ40p53, p53β enhances p53-mediated apoptosis [It has been shown that p53 isoforms can regulate cell fate outcome in response to stress signals by differentially modulating target gene expression in a p53-dependent and p53-independent manner. For example, the zebrafish with p53 ,174. Comnd Bcl-2 . In addiand PUMA . Interes2/RAD52) ,180. Com2/RAD52) ,195,196.2/RAD52) . Howeveradiation . The p53p21, miR-34a, INK4ap16), increased senescence-associated β-galactosidase activity, and a senescence-associated secretory phenotype (SASP) that includes secretion of proinflammatory SASP cytokines such as IL-6 and IL-1β. Several studies have reported the specific senescent-associated signature of p53 isoform expression that is characterized by reduced expression of Δ133p53 and elevated expression of p53β [Increasing evidence implicates Δ133p53α and p53β as regulators of cellular senescence ,177,178. of p53β ,177,178. of p53β ,177,178. of p53β ,166,177. of p53β ,177,178. of p53β ,177.RAD51 expression and inhibit RAD51 foci formation in response to DNA damage [rad51/RAD51 (needed for HR), lig4/LIG4 (needed for NHEJ) and rad52/RAD52 (needed for SSA) [Upon DNA damage, full-length p53 regulates the expression of its target genes involved in cell-cycle arrest, DNA damage repair, apoptosis, or senescence to maintain genome stability. However, p53α promotes only some DDR pathways, such as excision repair pathways , while inhibiting double-strand break (DSB) repair mechanisms, such as homologous recombination (HR), nonhomologous end joining (NHEJ), and single-strand annealing (SSA) ,202,203.A damage . In addiA damage , a positA damage . Interesfor SSA) . Althougfor SSA) . It has for SSA) ,180 and,for SSA)  or by enfor SSA) .p21, miR-34a, PAI-1, IGFBP7) [The p53 isoforms are also involved in induced pluripotent stem cell (iPSC) and embryonic stem cell (ESC) regulation. It has been previously reported that overexpression of p44 (homologous to human Δ40p53) impairs the regenerative capacity of adult stem cells while enhancing the regenerative capacity of ESC in mouse models ,183. Spe IGFBP7) . In line IGFBP7) ,181. Sin IGFBP7) ,181 and MYC, HIF, and KRAS, as well as PI3K/AKT/mTOR axis, are known to play a role in deregulated cellular energetics, an established hallmark of cancer [BRAF or KRAS mutant CRC cells. Once inside the cell, vitamin C elevates reactive oxygen species (ROS) levels that inactivate glycolytic enzyme GAPDH , ultimately leading to an “energy crisis” and cancer cell death [Recent research has shown that the p53 family of proteins is involved in the transactivation of metabolic enzymes included in glucose, nucleotide, amino-acid, and lipid metabolism, as well as mitochondrial metabolism and autophagy ,208,209.f cancer ,211,212.f cancer . It has ll death ,214. Accll death . The Δ40ll death .+ macrophages). Interestingly, Δ133p53β was shown to directly regulate the mRNA expression of CD274 that encodes the PD-L1 immune checkpoint marker. Furthermore, prostate cancers with a high level of Δ133p53β mRNA were associated with poor patient outcome [SOX2, OCT3/4, and NANOG, key cell pluripotency/reprogramming genes, resulting in increased mammosphere-forming ability and metastatic potential of cancer cells. Furthermore, etoposide treatment of breast cancer cells increased CSC formation and elevated the level of Δ133p53β, as well as SOX2, OCT3/4, and NANOG, which can increase the risk of resistance and cancer recurrence [Many biological activities of the p53 isoforms have been described in the context of cancer biology. Here, we present only some of the studies that investigated such cellular processes. One is angiogenesis that is inhibited by full-length p53. Bernard and coworkers investigated the role of Δ133p53 in tumor angiogenesis and tumor progression using human glioblastoma cell line U87. The results showed that Δ133p53α and Δ133p53γ but not Δ133p53β promote angiogenesis, while the ratio between p53 and Δ133p53α is important for its regulation. Furthermore, depletion of Δ133p53 isoforms inhibited angiogenesis and glioblastoma tumor growth in in vivo models  . The rol outcome . Elevate outcome . Both chcurrence . In addicurrence . Both Δ1currence .Here, we presented some of the p53 isoforms and their biological activities associated with carcinogenesis and age-related diseases; however, their biological functions in cancer, especially CRC are described later in more detail.Trp73−/−) mice that exhibit severe neurological, pheromonal, reproductive, and inflammatory abnormalities [Trp73 gene encoding the DBD and were, thus, deficient for the expression of all p73 isoforms. As a consequence, it was impossible to attribute specific biological functions observed to the activity of a particular isoform. Moreover, the initial experiments on Trp73−/−-mice failed to show increased susceptibility to spontaneous tumorigenesis, most probably due to their short lifespan [TP73 gene mutations in cancer and initial experiments on Trp73−/−-mice failed to support its potential tumor suppressor role [Trp73+/− mutant mice showed increased incidence of diverse spontaneous tumors often manifesting LOH. Moreover, mice heterozygous for both p53 and p73 mutation exhibited an even more aggressive tumor phenotype than the Trp53+/− mice bearing only mutation of p53 [TAp73−/−) mice lacking exons 2 and 3 that encode the TAD showed increased incidence of spontaneous tumors  and genomic instability (aneuploidy) [The p73 protein exerts its functions mainly by acting as a transcription factor affecting the expression of numerous target genes related to tumor suppression, as well as to various non-oncogenic cellular processes . Multiplmalities . These mlifespan . Since isor role . Later dn of p53 . These rn of p53 . The roluploidy) . At the uploidy) .Maintenance of genomic stability is crucial in cancer prevention, especially upon DNA damage. In parallel with work on knockout models, numerous expression studies on cell models supported the role of p73 isoforms in molecular processes related to tumor suppression such as cell-cycle regulation, DNA repair, apoptosis, and senescence ,222,223.CDC25B, CDC25C, CDC2, CCNB1, CCNB2, and TOP2A) and as inducers of p21, p57, and GADD45 [Rad51, BRCA2, Mre11, and Rad50 [SMUG1 and MUTYH [The TAp73 isoforms as transcriptional factors are involved in the regulation of the G1/S and G2/M cell-cycle checkpoints, acting as repressors of a plethora of genes  or permanent cell-cycle exit (senescence). Very early after its discovery, p73 was recognized as an inducer of DNA damage-induced apoptosis, mainly via the transcriptional activation of the components of the extrinsic  and intrinsic  apoptotic pathways ,231. In poptosis .TAp73−/−-mice, ΔNp73-knockout mice expectedly were not prone to tumor formation in agreement with its potentially oncogenic function [MDR1 gene, also known as ABCB1, and its product p-glycoprotein (p-gp) by inhibiting p53 function [The functions of ΔNp73 isoforms are mostly exerted through their dominant-negative effect on TAp73 and wt p53, impeding their activity on numerous target genes. In contrast to function . ΔNp73 ifunction . It was function . There afunction ,238. In function .Vegf-A [During the last decade, an important role of p73 in the regulation of angiogenesis emerged . While oVegf-A .Cox4il (complex IV subunit cytochrome C oxidase subunit 4 isoform 1) involved in mitochondrial metabolism, preventing ROS accumulation and premature aging in mice [p73 is extensively implicated in normal and tumor cell metabolism, contributing to adaptation to stress conditions, mainly by regulating the expression of different target genes. TAp73 directly transactivates mitochondrial  in mice . Interes in mice . High-th in mice ,249,250. in mice . However in mice ,252. Con in mice , and itsΔNp73−/− mice neurons showed slightly reduced glycolysis and reduced amounts of membrane phospholipids, long-chain fatty acids, sphingolipids [N-acetylaspartylglutamate, and neurotransmitter glycine, suggesting a delay in neuronal differentiation [The ΔNp73 isoforms have also been implicated in glucose metabolism. Deletion of the ΔNp73 isoforms was shown to inhibit the expression of the glycolysis enzyme hexokinase 2, through upregulation of peptide hormone amylin. This resulted in the inhibition of glycolysis, as well as the induction of ROS and apoptosis, consequently leading to regression of p53-deficient tumors in mice . Accordigolipids , N-acetyntiation . Interesntiation .In addition to the numerous functions of p73 more or less closely related to tumorigenesis, during the last decade, many non-oncogenic functions of p73 emerged mainly due to extensive research on the isoform-specific knockout models ,256. p73Trp73−/− mice, as multiciliated cells are crucial for fluid and germ cell transport across tissue surfaces [The TAp73 isoforms are recognized as main transcriptional regulators of multiciliogenesis in airways, oviduct, and brain ependyma. This role explains the airway infections and infertility observed initially in surfaces ,265,266.Different p73 isoforms participate in various cellular processes, many of which are related directly or indirectly to tumorigenesis. Some of these functions are shared between several distinct p73 and p53 isoforms, revealing their potential complementary activities, and some of them are attributed only to a specific isoform. Thus, p53/p73 isoforms form a complex network of different functions and activities which are very often based on protein interactions.Protein interactions are important events in a variety of cellular responses, sometimes causing the loss of protein activity. This is particularly relevant in the context of cancer cells expressing the wt p53 protein (or TAp73), whose functionality is needed for an efficient response to anticancer therapies. Vilgelm et al. showed that p73 and p53 proteins are co-expressed in colon carcinoma tissues from the same patient .The transactivation function of isoforms of the p53 protein family is executed through the correct formation of a tetramer through the OD of the monomers A. More pThe formation of mixed heterocomplexes between oncogenic and antioncogenic family members correlates with the lack of capacity to transactivate their target genes B. It is There is an autoregulatory feedback loop among p53, TAp73, and ΔΝp73. Both p53 and TAp73 induce the expression of the ΔΝp73 isoforms via direct binding to the p73-specific RE within P2 . InducedWe have demonstrated the complex formation between various p53 isoforms  and TAp73β, while only Δ133p53α/β/γ form a complex with TAp73α. All p53 isoforms counteract the TAp73β transactivation function but with a different efficiency and in a promoter-dependent manner. Interestingly, the TAp73β apoptotic activity was augmented by p53β . It has BAX and GADD45 in p53-null cells but an oligomeric complex is still not confirmed  exhibit a dominant-negative potential toward p53α, as well as transcriptionally inactive ΔNp73α and ΔNp73β. The transcriptionally inactive mutants and isoforms have an intact OD and are able to form heterotetramers [In addition, the direct interaction between family members has been shown in the model organisms. Ou and coauthors have shown that protein–protein interaction between the zebrafish ∆113p53 (a homolog of Δ133p53) and the full-length p53 is essential for the antiapoptotic function of Δ113p53 . Billantetramers .PUMA and p21 promoters. Additionally, the transcription activity of the entire network of p53 family proteins contributes to the cellular response to chemotherapeutic drug treatment in colon and esophageal cancer cells [−/− double-knockout MEFs), there is no binding of p53 to the promoters of the proapoptotic BAX, NOXA, and PERP genes. However, p73, on the other hand, can induce apoptosis in the cells without functional p53 [Noteworthily, the p53 family members interact with each other to regulate common target genes. In HCT116 colon cancer cells, it was found that p53 and TAp73 isoforms bind simultaneously to er cells . Δ133p53er cells . Flores onal p53 ,281.However, some works support a prion-like p53 aggregation related to the dominant-negative effect of some hotspot mt p53 . The mutTo conclude, there is a significant crosstalk between family members in the tumors. They can bind specifically to the p53RE and modulate the expression of the p53 target genes. Through binding to the p53RE, some family members can exert dominant-negative effects on the activity of p53, p63, and p73.The p53/p73 isoforms have been identified in many different normal and cancer cells and tissues, including, but not restricted to, ovarian, melanoma, bladder, sarcoma, breast, kidney, and colon cancer. Their expression has often been found deregulated in tumors when compared to healthy tissues, which points to their involvement in tumorigenesis ,145,285.Research on ovarian cancer, for example, has shown that the expression of different p53 isoforms is altered in around 50% of ovarian cancer cell lines and primary ovarian cancers. More so, expression of p53δ, a splice variant caused by a splice site mutation in intron 9, was associated with impaired response to platinum-based chemotherapy and has been identified as a prognostic marker for the recurrence-free and overall survival of ovarian carcinoma patients. p53β was also correlated with adverse clinical features and worse recurrence-free survival in patients exhibiting functionally active p53. Among p73 isoforms, ΔN′p73 was the preferentially expressed ΔN isoform in ovarian cancer cells with functionally active p53 . ΔN′p73 The p53 family is expressed as an intricate network of isoforms in both normal and tumor colorectal tissue. Recently conducted genome-wide alternative splicing analysis on the CRC cohort from TCGA database emphasized the importance of alternative splicing events in the p53 signaling pathway in CRC metastasis and recurrence . SeveralA high expression of the canonical p53, Δ133p53α and β, a moderate expression of Δ133p53γ, and a lower expression of p53β and γ have been reported in normal colon tissue . Colon aIn normal colon, a low gene and protein expression of both TAp73 and ΔNp73 was found compared to the colorectal adenocarcinomas. Interestingly, the prevalence of p73α and β gene expression was shown by nested PCR, compared to p73γ, δ, ϕ, and ε in both normal and tumor tissue . AnotherΔNp73β mRNA has also been identified as an exosomal cargo from ΔNp73β ectopically expressing HCT116 colon cancer cells, and it is selectively packed into exosomes from CRC patients when compared to exosomes from healthy controls. The transmission of exosome cargo to epithelial, fibroblast, and endothelial cells and its significant effect on proliferation and drug resistance in the acceptor cells were observed. This finding supports the potential prognostic value of the exosomal ΔNp73 in colon cancer patients .These data paint a complex image of the p53/p73 isoform network in colon healthy tissues, as well as colorectal tumors.Since the p53 family proteins are involved in a plethora of cell signaling pathways that regulate processes such as survival, proliferation, differentiation, apoptosis, and motility of both healthy and cancer cells ,297,298,−/− cells [RAD51, LIG4, and RAD52 by binding to their promotors. Furthermore, the knockdown of TAp73 causes accumulation of DNA DSBs, which leads to G2 cell-cycle arrest and promotes cell senescence upon γ-irradiation. Interestingly, mutations in the Δ133p53 promoter region reduce its activation in response to DNA damage, which may be related to tumorigenesis [Among the few papers tackling the role of p53 isoforms in colorectal cancer, Katoch and colleagues showed that ∆40p53 downregulates the expression of the transcriptional repressor protein YY1 via upregulating miR-186 which consequently inhibits cell proliferation of HCT116/− cells . There i/− cells . Δ133p53igenesis . Diaz anigenesis . On the igenesis . The ectigenesis .Further experiments confirming the oncogenic role of the ΔNp73 isoforms in CRC showed that ΔNp73 overexpression increased the colonosphere formation of CRC cells DLD1 and significantly reduced cell death in HCT116 and DLD1 caused by a small-molecule prodigiosin, which restores the p53 pathway . InteresExperiments have revealed that the p53 isoforms, particularly Δ133p53, are involved in the invasion of CRC. Campbell and colleagues showed that all three Δ133p53 isoforms  promote invasion of HCT116 through matrigel via RhoA and ROCKA (Rho-associated protein kinase A). Furthermore, cells transfected with Δ133p53α display a more rounded phenotype . SimilarThe p73 isoforms may have a role in modifying the expression levels of the angiogenesis-related factors in CRC, which is yet to be understood. Díaz and coauthors found correlations between the expression levels of the proangiogenic VEGF-A  and ΔNp73 and TAp73 in a cohort of CRC patient samples. Interestingly, the expression of ∆Ex2p73, ∆Ex2/3p73, ∆Np73, or TAp73 correlated with the expression of a putative antiangiogenic isoform VEGF165b. However, the expression of ΔEx2p73 inversely correlated with the antiangiogenic protein PEDF (pigment epithelium-derived factor) .−/− HCT116 [p53 isoforms have been implicated in the cellular metabolism of CRC. For example, glucose starvation promotes IRES in lung and colon cancer cells that is regulated via the SMAR1 (scaffold/matrix attachment region-binding protein 1) protein and elevates the expression of TIGAR (TP53-induced glycolysis regulatory phosphatase) whose product is involved in glycolysis . In addi− HCT116 ,308.Furthermore, the ectopic expression of Δ133p53 in HCT116 cells increases the expression of the NF-κB targets interleukin 6, 8 and antiapoptotic protein Bcl-2, probably through inhibition of p53 .p53β and Δ133p53 show dynamic correlation in the development of colon carcinoma. Increased levels of p53β and reduced levels of Δ133p53 in colon adenoma tissues have been described as a senescence-associated p53 isoform signature that may represent a senescence barrier observed in premalignant tumors. Interestingly, in CRC tissues, this signature is lost and even reversed with cancer stage, which may indicate an escape from the senescence barrier during the progression from adenoma to carcinoma .The mutation status of an established tumor suppressor protein such as p53 has surprisingly limited relevance in cancer progression diagnostics. One reason might be the overly simplified image of its action, neglecting the existence of the p53 family isoforms which act in concert. Therefore, the question is whether the p53 family isoforms could have a better diagnostic ability for cancer patients than the canonical protein itself. In that context, Campbell and colleagues showed that the CRCs with elevated Δ133p53 mRNA are more aggressive and are associated with a shorter disease-free survival (DFS) . MoreoveFindings have shown that p53 autoantibodies are produced early during tumor development in CRC patients, making the p53 autoantibodies promising markers for early detection ,313. ResThese data point to the importance of investigating isoforms hand in hand with the canonical proteins themselves in cancer research.NOXA, PUMA, and p21 leads to an increase in apoptosis [Jun-dependent manner. This research highlighted the involvement of the p73 pathway in the maintenance of stem potential of CRCSC, as well as the potential of p53 pathway-activating therapeutics to target colorectal cancer [Since there is ongoing evidence that p53 family isoforms act as epigenetic p53 inhibitors in cancer, the potential of targeting them in colon cancer has been investigated, with the goal of reactivating the p53 pathway and getting a better response to therapy. Zhang and colleagues published an interesting article where they, after a functional cell-based assay screening of almost 2000 small molecules, identified NSC59984 as that which specifically restores the p53 pathway in mt p53 CRA cells SW480 and DLD-1 via activation of TAp73. Importantly, a favorable therapeutic index between normal and cancer cells was presented. The authors showed that NSC59984 not only induces TAp73-dependent cell death in cancer cells in vitro but also synergizes with the DNA-damaging agent CPT11 in inducing cell death of mt p53-expressing cells . Furtherpoptosis . Interesl cancer .Since p53 isoforms are differentially expressed in CRA and CRC and correlated to patient prognosis, their targeting to restore the p53 signaling pathway alone or in combination with already established anticancer agents presents an attractive strategy for CRC therapy.The p53 family members play a significant role in carcinogenesis and treatment response. In addition to the fact that p53 is often mutated in tumors, it has become evident that different expression patterns of its isoforms are relevant in carcinogenesis. Furthermore, in tumors with mutated or otherwise inactivated p53, substituting its tumor suppressor function by TAp73 is a possible therapeutic approach. However, the existence of p53-like and p53-inhibitory isoforms, as well as a close functional crosstalk among all family members, gives the p53 family isoforms both tumor suppressor and oncogenic roles. To completely understand and decipher the role of the p53 and p73 isoforms in cancer, it is important to explore the mechanism supporting the dysregulation of isoform balance, as well as perform clinicopathologic studies which correlate the co-expression of p53 family isoforms with p53 mutation status and clinical outcome. Once the complex regulation of the p53 family isoforms is fully understood, they can be used as biomarkers or as therapeutic options.


Although visceral adipocytes located within the body’s central core are maintained at approximately 37°C, adipocytes within bone marrow, subcutaneous, and dermal depots are found primarily within the peripheral shell and generally exist at cooler temperatures. Responses of brown and beige/brite adipocytes to cold stress are well studied; however, comparatively little is known about mechanisms by which white adipocytes adapt to temperatures below 37°C. Here, we report that adaptation of cultured adipocytes to 31°C, the temperature at which distal marrow adipose tissues and subcutaneous adipose tissues often reside, increases anabolic and catabolic lipid metabolism, and elevates oxygen consumption. Cool adipocytes rely less on glucose and more on pyruvate, glutamine, and, especially, fatty acids as energy sources. Exposure of cultured adipocytes and gluteal white adipose tissue (WAT) to cool temperatures activates a shared program of gene expression. Cool temperatures induce stearoyl-CoA desaturase-1 (SCD1) expression and monounsaturated lipid levels in cultured adipocytes and distal bone marrow adipose tissues (BMATs), and SCD1 activity is required for acquisition of maximal oxygen consumption at 31°C. Adipocytes in bone marrow, subcutaneous and dermal sites generally exist at temperatures below 37°C. This study identifies the molecular and metabolic program that adapts white adipocytes to these cooler environments. Survival of euthermic animals is dependent on tight regulation of body temperature and cellular function in environmental conditions below thermoneutrality. Although mammals have developed a complex system to defend core body temperature within a narrow range, the exterior and extremities remain much cooler. Whereas the body core is maintained at a relatively constant approximately 37°C across a broad range of environmental conditions, the body shell, which includes peripheral extremities and subcutaneous regions of the trunk, is characterized by a wider spectrum of temperatures. For almost a century, scientists have known that temperature gradients exist across the body, from central to distal, and superficial to deeper regions . For exaTemperature is detected by sensory neurons within skin, core, and brain, which transmit signals to the central thermoregulatory network. After signal integration, specific hypothalamic neurons regulate body temperature through a combination of behavioral and physiological mechanisms ,4. PhysiWhite adipocytes are distributed throughout the body in discrete depots and the intrinsic cellular and metabolic properties of different populations are shaped by the specific niches in which they reside ,13. WherTo explore the adaptation of white adipocytes to changing environmental temperature, we exposed cultured adipocytes to a “cool” temperature of 31°C, the temperature at which distal marrow and subcutaneous adipose tissues exist when animals and humans are at a room temperature of 22°C . We founScd1 expression and higher levels of monounsaturated lipids compared to those found in the proximal tibia or within white adipose depots -acetate  and 5 μM sodium acetate for 12 hours at either 37°C or 31°C. At the end of the indicated incubation time, adipocytes were harvested, and lipids were extracted for analyses by thin layer chromatography.Cultured adipocytes were incubated overnight in fresh serum-free DMEM:F12 medium prior to measurement of Cultured adipocytes were washed 1× with PBS and then incubated with DMEM  containing BSA-conjugated approximately 3 μCi/ml of either -palmitic acid, -oleic Acid (PerkinElmer) or n- octanoic acid  and 22 μM of unlabeled NEFAs, respectively (Sigma-Aldrich). For long-chain fatty acid oxidation, assay media was contained with 200 μM L-carnitine (Sigma-Aldrich), and a subset of wells were treated with 40 μM etomoxir  2 hours before and duration of the assay. After 3 hours, conditioned media from cells was passed through columns containing AG1-X8 Anion Exchange Resin  and collected in scintillation vials, then mixed with scintillation cocktail Bio-Safe II . Radioactivity in the supernatant was measured using a scintillation counter.2 incubator for 1.5 hour prior to the assay. The sensor cartridge was loaded with oligomycin (Port A), FCCP (Port B), and rotenone/antimycin A (Port C)  to achieve final concentrations of 1 μM, 1 μM, and 0.5 μM, respectively. Other procedures for the assay were performed by following the manufacturer’s instructions. OCR was measured and normalized to DNA. Isolated mitochondria (5 μg protein) from MSC-derived adipocytes were seeded to XF96 microplates, and OCR was assayed as described above. Final concentrations of reagents are the following: 1 mM adenosine 5′-diphosphate (ADP), 10 mM succinate, 2 μM rotenone, 10 mM pyruvate, 1 mM malate, 40 μM palmitoylcarnitine, For the parametric equations: Basal respiration = —; ATP-linked respiration = (last OCR before oligomycin)—(minimum OCR after oligomycin); Proton leak = (minimum OCR after oligomycin)—; Maximal respiration = (maximum rate after FCCP injection)—; SRC = —.Cellular and mitochondrial OCR were determined using the Seahorse XF96 Extracellular Flux Analyzer . To measure OCR on cool-adapted adipocytes, we used 2 different approaches and which gave similar results. In the first approach, a 96-well cell culture microplate for Seahorse was cut into half, and then approximately 480 MSCs were seeded into each well. Four days after seeding, adipogenesis was induced at 37°C as described above. Four days after induction of differentiation, adipocytes in half of the plate were cultured at 31°C, whereas the other half were incubated at 37°C for 12 days. At this point, the plate was rejoined in order to evaluate oxygen consumption at 31°C or at 37°C. In the second approach, differentiated adipocytes cultured at either 37°C or 31°C for 8 days (4 days before the assay) were trypsinized and isolated by centrifugation. Adipocytes were seeded on Cell-Tak–coated Seahorse XF96 microplates, then cultured overnight upside down at either 37°C or 31°C. One of the advantages of the first approach is that adipocytes are cultured on the same plates for the entire experiment. However, it is difficult in long-term culture to control cell numbers and thus basal OCR. Although we favor use of the second approach, and in our hands the replated adipocytes appear to maintain their differentiate morphology, one concern is the potential for adipocyte dedifferentiation. On the day of assay, the cells were washed and incubated with unbuffered XF assay medium  contained 5 mM glucose, 0.2 mM pyruvate, and 1 mM glutamine and placed the cell culture microplate into either 31°C or 37°C non-COg for 10 minutes at 4°C. The supernatant was then centrifuged for 15 minutes at 8,000g at 4°C, and the pellet was washed with ice-cold buffer. After centrifugation at 7,000g for 10 minutes at 4°C, the pellet containing mitochondria was resuspended for analyses.Isolation of mitochondria was as described previously . Briefly13C6 glucose (Sigma-Aldrich), 2,3-13C2 pyruvate , or 13C5 glutamate (Sigma-Aldrich) for 1 hour. Cells were then rapidly (<5 seconds) rinsed with 150 mM ammonium acetate and snap frozen by addition of liquid nitrogen directly to the cell plate. Metabolites from frozen cells were extracted by adding ice-cold 8:1:1 HPLC grade methanol:chloroform:water to the frozen cell plate, scraping to detach and lyse cells, transferring the suspension to microcentrifuge tubes, and centrifuging for 5 minutes at 15k RCF to remove pellet debris. Polar metabolites were analyzed in the supernatant by hydrophilic interaction chromatography-electrospray time of flight mass spectrometry (HILIC-ESI-TOF) as described previously [Adipocytes from MSC were preincubated with fresh DMEM  with unlabeled glucose (5 mM), pyruvate (0.2 mM), and glutamine (1 mM) for 2 hours. For incubation in tracer-labeled media, a media change was performed. Substrate concentrations were kept constant except for the substitution of unlabeled metabolites with either eviously .Cultured adipocytes were washed twice with PBS and then suspended in 500 μl of a 1:2.5 methanol/water mixture. The cell suspension was transferred to a borosilicate glass tube. Wells were rinsed with 500 μl of the 1:2.5 methanol/water mixture, and the volume was transferred to a glass tube, then vortexed after adding 375 μl chloroform. Then, another 375 μl chloroform and 375 μl 0.9% NaCl were added to each tube, which was then vortexed vigorously and centrifuged at 2,500 rpm for 20 minutes at 4°C. The lower organic chloroform layer containing total lipids was transferred to a new tube and stored at −20°C until analyzed.For the BMAT and WAT, tissue was transferred to a borosilicate glass tube after crushing on dry ice. Add 1,000 μl of the 1:2.5 methanol/water mixture 375 μl chloroform, then homogenize with immersion tissue grinder. Then, another 375 μl chloroform and 375 μl 0.9% NaCl were added to each tube, which was then vortexed vigorously and centrifuged at 2,500 rpm for 20 minutes at 4°C. The next step is the same as the process as described above for the cells.TAG and phospholipids were separated from the total lipid extract of cultured adipocytes and adipose tissue, by thin-layer chromatography applying the lipids as a band on a 20 × 20 cm silica gel thin-layer chromatography plate . The plate was developed with hexane-diethyl ether-acetic acid . The TAG and phospholipids were identified with respect to the retention flow (rf) of the authentic TAG and phospholipid standard applied on the same plate. TAG and phospholipid bands were scraped from the thin-layer chromatography plate, extracted with chloroform, and the lipids were subjected to derivatization as follows.trans-esterification with boron trifluoride-methanol. The derivatized methyl esters were redissolved in a small volume of hexane and purified by thin-layer chromatography using n-hexane-diethyl ether-acetic acid  as the developing solvents. After development, plates were dried and sprayed with Premulin. Fatty acid methyl ester bands were identified under ultraviolet light by comparing the rf of methyl heptadecanoate (C17:0) standard  applied side by side on the same plate. Methyl esters were extracted from thin-layer chromatography powder with diethyl ether concentrated under nitrogen and redissolved in 100 μl hexane. The lipids’ fatty acid compositions were analyzed by gas chromatography (GC) as follows. FAMEs were analyzed with 1 μl sample injection on an Agilent GC machine, model 6890N equipped with a flame ionization detector, an autosampler model 7693 and a ChemStation software for data analysis. An Agilent HP 88 with a 30-m GC column with a 0.25-mm inner diameter and 0.20-mm film thickness was used. Hydrogen was used as a carrier gas, and nitrogen was used as a makeup gas. The analyses were conducted with a temperature programming of 125 to 220°C. The fatty acid components within unknown samples were identified with respect to the retention times of authentic standard methyl ester mixtures run side by side. The fatty acid components were quantified with respect to the known amount of internal standard added, and the calibration ratio was derived from each fatty acid of a standard methyl ester mixture and methyl heptadecanoate internal standard. The coefficient of variation for GC analyses was within 2.3% to 3.7%.Fatty acids in the extracted lipids were derivatized into their methyl esters by g and 4°C. Then, 100 μL of the upper lipophilic layer was transferred to an amber glass autosampler vial with fused glass insert. This extract was dried by vacuum centrifugation for approximately 1 hour. Samples were resuspended in 50 μL of 9:1 methanol:toluene for analysis by LC–MS.Cell plates were rapidly frozen with liquid nitrogen and maintained at −80°C until extraction. Cell plates were extracted on dry ice. To each well, 200 μL of ice cold methanol was added, and cells were scraped using a plastic cell scraper, then sample was transferred to a 1.5-mL microcentrifuge tube. The cell culture well was then rinsed with 658 μL of methyl tert-butyl ether (MTBE), and this solvent was added to the same 1.5-mL microcentrifuge tube. The samples were then sonicated for 5 minutes using a program of 20 seconds on, 10 seconds off, and amplitude of 30 , chilled bath sonicator), temperature was maintained at 14°C. To induce phase separation, 164 μL water was added, and the solution was vortexed again for 10 seconds, then the sample was centrifuged for 2 minutes at 14,000Sample analysis was performed using a Vanquish Binary Pump coupled to a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific) as published previously ,42. For 6 automatic gain control (AGC) target, 100 ms ion accumulation time (max IT). MS2 scans were collected using 1 m/z isolation window, stepped normalized collision energy [5 AGC target and 50 ms max IT. A 30.0-second dynamic exclusion was employed.Eluent was introduced onto the mass analyzer using a HESI II ESI source operating at 275°C, with 30 units of sheath gas, 6 units of aux gas (300°C), |4.0 kV| spray voltage (positive mode and negative mode), and 60 units of S-lens RF. Full MS and top2 MS2 spectra were collected in alternating polarity; full MS 200 to 1,600 m/z were collected at 30,000 resolution with 1 × 10n energy , and acqRaw files were processed using Compound Discoverer 3.0 (Thermo Fisher Scientific) and LipiDex .DNA was prepared from cells using Gentra Puregene Kits including RNase A treatment . mtDNA copy number per nuclear genome in MSC-derived adipocytes was quantified as described previously . Primer RNA isolation, reverse transcription, and quantitative PCR were performed as previously described . Primer in vivo samples, although alignment was conducted using STAR (version 2.7.5a), and differential expression analysis was conducted using featureCounts/DESeq2. Plots were generated using variations or alternative representations of native DESeq2 plotting functions, ggplot2, plotly, and other packages within the R environment. The accession number for the raw sequencing data for cultured adipocytes is NCBI Gene Expression Omnibus: GSE159451 and for gluteal WAT is GSE169142.Total RNA was isolated and purified as described above. After DNase treatment, samples were submitted to the University of Michigan Advanced Genomics Core for quality control, library preparation, and sequencing using the Illumina Hi-Seq platform. Read files were downloaded and concentrated into a single fastq file for each sample. Quality of raw read data was checked using FastQC (version v0.11.30) to identify features of the data that may indicate quality problems . The Tuxedo Suite software package was used for alignment, differential expression analysis, and post-analysis diagnostics. Briefly, reads were aligned to the UCSC reference genome using TopHat (version 2.0.13) and Bowtie2 (version 2.2.1). FastQC was used for the second round of quality control post-alignment to ensure that only high-quality data were input to expression quantitation and differential expression analysis. Differential expression analysis was done using 2 distinct methods to check for analysis consistency: Cufflinks/CuffDiff and HTSeq/DESeq2, using UCSC build mm10 as the reference genome sequence. A similar approach was taken to analyze the RNA-seq data for the Pathway analysis was conducted on ranked lists of log2 fold change using GSEA v4.0.3 by the Broad Institute. Prior to analyses, mouse gene symbols were remapped to human ortholog symbols using chip annotation files. The mouse genes that did not have equivalent human orthologs were excluded from the analysis. The output from DESeq2 analysis was used to generate a list of genes ranked by the metric −log10 false discovery rate (FDR) * log2 fold change. The resulting list was run through preranked GSEA using the Molecular Signatures Database v7.1 . Enriched pathways were defined by an FDR < 0.05. The normalized enrichment score (NES) is the primary metric from GSEA for evaluating the magnitude of differentially expressed pathways. Pathway impact analysis was conducted on AdvaitaBio’s iPathwayGuide to identify enriched KEGG pathways. The ggplot2 package in R was used for further visualization of the enriched pathways. Further pathway analyses were performed with Enrichr for genes regulated by cool temperatures in cultured cells and adipose tissue.After lysis in 1% NP-40, 120 mM NaCl, 50 mM Tris-HCl (pH 7.4), 50 mM NaF, 2 mM EDTA, 1× protease inhibitor cocktail (Sigma-Aldrich), protein concentrations of lysates after centrifugation were measured by BCA protein assay (Thermo Fisher Scientific). Lysates were diluted to equal protein concentrations in lysis buffer and then boiled in SDS sample buffer (20 mM Tris (pH 6.8), 2% SDS, 0.01% bromophenol blue, 10% glycerol, 5% 2-mercaptoethanol) and subjected to SDS-PAGE and immunoblotting according to standard techniques. Separation of phosphorylated proteins using the Phos-Tag gel  was according to the manufacturer’s instructions. Quantification of protein expression was done using ImageJ software. Antibodies used were as follows: ACC , p-ACC(Ser79) , Adiponectin (Sigma Aldrich A6354), ACAA2 (MCKAT) (Thermo Fisher Scientific A21984), ATGL , β-actin , CPT1A (Abcam ab128568), CHREBP , ERK2 , FASN (Abcam ab22759), FABP4 (R&D #1443), HSL , p-HSL (Ser563) , p-HSL (Ser660) . HADHB (MTPβ) , HSP70 , HSP90 (BD Transduction Lab 610418), Laminin , OXPHOS Cocktail (Abcam ab110413), Perilipin (Abcam Ab3526), PMP70 (Thermo Fisher Scientific PA1650), PEX5 (Thermo Fisher Scientific PA558716), PPARg (Millipore MAB3872), SCD1 , SREBP1 (ThermoFisher Invitrogen 2A4), Tubulin (Invitrogen MA1-80017), UCP1 , and VDAC1 (Abcam ab15895).t test. When comparing multiple experimental groups, an analysis of variance (ANOVA) was followed by post hoc analyses with Dunnett or Sidak test, as appropriate. Differences were considered significant at p < 0.05 and are indicated with asterisks. For metabolomics data analyses, the proportion of labeling at each carbon position was calculated by dividing each species by total sum of peak areas of all labeled positions. The proportion of data is well known to follow a beta distribution. Beta regression model is an extension of the generalized linear model with an assumption that the response variable follows a beta distribution with values in standard unit interval  [All data are presented as mean ± SD. When comparing 2 groups, significance was determined using 2-tailed Student al  . Our flual  . We propal  . To applal  . All datS1 FigA) Rats were housed from birth to 11 weeks of age at room temperature (22°C) or thermoneutrality (29°C). Lipid composition of phospholipids for pTib and dTib, and CV8 was determined by GC (n = 6). Desaturation index at the top of graph is (16:1 + 18:1)/(16:0 + 18:0).  Mice were housed from birth to 13 weeks either at 22°C or at 29°C without posterior hair after weaning. Whereas Scd1 mRNA expression is elevated in subcutaneous WAT depots of mice at 22°C (B), Adipoq expression was not altered (C). Gene expression was normalized to geometric mean value of Hprt, Tbp, Gapdh, and Ppia and was expressed relative to 37°C control (n = 8–9). For panels (A–C), values are mean ± SD. *p < 0.05. Data shown are representative of at least 3 independent experiments. CV8, caudal vertebra-8; dTib, distal tibia; GC, gas chromatography; pTib, proximal tibia; SCD1, stearoyl-CoA desaturase-1; SFAs, saturated fatty acids; UFAs, unsaturated fatty acids; WAT, white adipose tissue.((PDF)Click here for additional data file.S2 FigA) Cultured MSC adipocytes adapt to 31°C without morphological changes. Four days after differentiation, adipocytes were moved to the indicated temperatures for 12 days. (B) Adipocyte markers are expressed similarly between control cells cultured at 37°C and those incubated at 37°C from days 4 to 16. For adipocyte samples, 11 μg of protein lysate was analyzed per lane, whereas for brown adipose tissue, 0.1 or 0.5 μg lysate was included as a positive control for UCP1. (C) Beige adipocyte markers were not induced by exposure of cultured adipocytes to 31°C for the indicated days (n = 6). Gene expression was normalized to geometric mean value of Hprt, Tbp, and Ppia and was expressed relative to 37°C control (31°C for 0 day) (n = 4). * indicates significance at p < 0.05. Fabp4, fatty acid-binding protein 4; Fgf21, fibroblast growth factor 21; MSC, mesenchymal stem cell; PPARγ, peroxisome proliferator–activated receptor gamma; Ppargc1a, peroxisome proliferator–activated receptor gamma coactivator 1 alpha; UCP1, uncoupling protein 1.((PDF)Click here for additional data file.S3 FigA) MA plot showing the log2-mean expression versus log2-fold change of mRNA transcript expression in 12-day cool exposed MSC adipocytes compared to day 0. Each dot represents a gene. Twelve days of cool temperature exposure induced 1,872 genes (red) and suppressed 2,511 genes (blue). Significance was defined by an FDR <0.05 and absolute fold change >1.5. (B) Heat map of top 50 enriched and top 50 depleted genes in 12-day cool exposed MSC adipocytes. Color key based on rlog-transformed read count values and significance was defined by an FDR <0.01. FDR, false discovery rate; MSC, mesenchymal stem cell; NS, not significant; RNA-seq, RNA sequencing.((PDF)Click here for additional data file.S4 FigA) Release of tritiated water from adipocytes treated with labeled octanoic acid. Adipocytes cultured at the indicated temperature for 12 days were incubated with tritiated octanoic acid for 3 hours in the presence and absence of etomoxir (n = 6).  Cool adaptation increases enzymes involved in synthesis and degradation of NEFAs. Lysates were collected after the indicated days of cool adaptation. SVCs from human (B) or eWAT from C57BL/6J mice (C) were differentiated into adipocytes. Human white preadipocytes . (D) OXPHOS genes are up-regulated at the mRNA level. Heat map of genes involved in complexes I, II, III, VI, and V were constructed from KEGG map of OXPHOS genes (mmu00190). CPT1ɑ, carnitine palmitoyltransferase 1 alpha; eWAT, epididymal white adipose tissue; FASN, fatty acid synthase; NEFA, nonesterified fatty acid; OXPHOS, oxidative phosphorylation; SCD1, stearoyl-CoA desaturase-1; SVC, stromal vascular cell.((PDF)Click here for additional data file.S5 FigA) Expression of UCP1 in adipocytes adapted to 31°C for the indicated days. A total of 11 μg of adipocyte or 0.5 or 1 μg BAT lysate was evaluated by immunoblot for UCP1. (B) Primary adipocytes isolated from eWAT or sWAT by collagenase digestion were cultured floating at either 37°C or 31°C for 2 days. BAT lysate  was used as a positive control for UCP1. (C) Cool adaptation increases enzymes involved in synthesis and degradation of NEFAs in adipocytes derived from UCP1 knockout mice. (D) Elevated basal OCR of adipocytes at 31°C is UCP1 independent. MSC adipocytes derived from WT or UCP1 KO mice were cultured at 31°C or 37°C for 12 days and basal OCR evaluated (n = 8). BAT, brown adipose tissue; CPT1ɑ, carnitine palmitoyltransferase 1 alpha; eWAT, epididymal white adipose tissue; FASN, fatty acid synthase; KO, knockout; MSC, mesenchymal stem cell; NEFA, nonesterified fatty acid; OCR, oxygen consumption rate; PPARγ, peroxisome proliferator–activated receptor gamma; SCD1, stearoyl-CoA desaturase-1; sWAT, subcutaneous white adipose tissue; UCP1, uncoupling protein 1; WT, wild-type.((PDF)Click here for additional data file.S6 FigA, B) Cool adaptation increases the expression of HSL and ATGL but decreases phosphorylation of perilipin and HSL in response to CL-316,243. Mature adipocytes were adapted to 31°C for 12 days or remained at 37°C. Adipocytes were treated with (A) vehicle, 1 μM or 10 μM forskolin, or (B) vehicle, 0.05 μM, 0.2 μM, or 10 μM CL-316,243 for 30 minutes. Lysates were collected for immunoblot analyses. (C) Induction of enzymes involved in de novo lipogenesis with adaptation to 31°C is independent of ChREBP. Adipocytes derived from WT or ChREBP KO mice were cultured at 31°C or 37°C for 4 days before collecting samples. Although ChREBP deficiency impairs adipocytes differentiation, addition of rosiglitazone to differentiation media resulted in robust adipogenesis of both sets of cells, as has been reported. (D) On day 6 of MSC differentiation, adipocytes were infected in serum-free medium with adeno-shLacZ control or adeno-shSreb1 to induce gene knockdown. Adipocytes were then allowed to recover following infection and were cultured at the indicated temperature from day 8 to day 12. ACC, acetyl-CoA carboxylase; ATGL, adipose triglyceride lipase; ChREBP, carbohydrate response element-binding protein; Fabp4, fatty acid-binding protein 4; FASN, fatty acid synthase; HSL, hormone-sensitive lipase; KO, knockout; MSC, mesenchymal stem cell; SCD1, stearoyl-CoA desaturase-1; WT, wild-type.((PDF)Click here for additional data file.S7 FigA) No effect of SCD inhibitors on SRC of adipocytes cultured at 37°C (n =  8–16 per group). (B) Increased SRC of adipocytes adapted to 31°C was inhibited with SCD inhibitors: A-939572, MF-438, or CAY10566 (n = 8–16 per group). FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; OCR, oxygen consumption rate; Oligo, oligomycin; Rot/Anti, rotenone and antimycin; SCD, stearoyl-CoA desaturase.Adipocytes were seeded on Cell-Tak–coated Seahorse XF96 Cell Culture Microplate, then cultured overnight upside down in order to attach to the plate at either 37°C or 31°C. Adipocytes were cultured with SCD inhibitors: A-939572, MF-438, or CAY10566 at the indicated temperature for 2 days before the assay. ((PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 DataAll numerical raw data associated with (XLSX)Click here for additional data file.S2 DataAll numerical raw data associated with (XLSX)Click here for additional data file.S3 DataAll numerical raw data associated with (XLSX)Click here for additional data file.S4 DataAll numerical raw data associated with (XLSX)Click here for additional data file.S5 DataAll numerical raw data associated with (XLSX)Click here for additional data file.S6 DataAll numerical raw data associated with (XLSX)Click here for additional data file.S7 DataAll numerical raw data associated with (XLSX)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.