Hematogenous metastasis is essential for the progression of advanced hepatocellular carcinoma (HCC) and can occur even after patients receive multidisciplinary therapies, including immunotherapy and hepatectomy; circulating tumor cells (CTCs) are one of the dominant components of the metastatic cascade. However, the CTC capture efficiency for HCC is low due to the low sensitivity of the detection method. In this study, epithelial cell adhesion molecule (EpCAM)/vimentin/Glypican-3 (GPC3) antibody-modified lipid magnetic spheres (LMS) were used to capture tumor cells with epithelial phenotype, mesenchymal phenotype and GPC3 phenotype, respectively, in order to capture more CTCs with a more comprehensive phenotype for monitoring tumor metastasis.P = 0.037), guiding the application of clinical individualized therapy.The novel CTC detection system of Ep-LMS/Vi-LMS/GPC3-LMS was characterized by low toxicity, strong specificity (96.94%), high sensitivity (98.12%) and high capture efficiency (98.64%) in vitro. A sudden increase in CTC counts accompanied by the occurrence of lung metastasis was found in vivo, which was further validated by a clinical study. During follow-up, the rapid increase in CTCs predicted tumor progression in HCC patients. Additionally, genetic testing results showed common genetic alterations in primary tumors, CTCs and metastatic tissues. The proportion of patients predicted to benefit from immunotherapy with the CTC detection method was higher than that for the tissue detection method (76.47% vs. 41.18%, The Ep-LMS/Vi-LMS/GPC3-LMS sequential CTC capture system is convenient and feasible for the clinical prediction of HCC progression. CTCs captured by this system could be used as a suitable alternative to HCC tissue detection in guiding immunotherapy, supporting the clinical application of CTC liquid biopsy.The online version contains supplementary material available at 10.1186/s12951-023-01783-9. Hepatocellular carcinoma (HCC), one of the most common malignant tumors, has the sixth-highest incidence of all cancers worldwide. It is also the third leading cause of cancer-related death . HCC is Among many cell and molecular markers, circulating tumor cells (CTCs) are more reliable due to noninvasive sampling, real-time information representation, and dynamic reflection of the tumor gene spectrum. CTCs are tumor cells that fall off from tumor lesions into the peripheral blood circulation and play an important role during tumor metastasis –11. CompIn previous studies on CTC separation, EpCAM and vimentin were often used as capture targets . However−1 has the characteristic absorption peak of the C = O stretching vibration in the ester group, and 1112.7 cm−1 is the stretching vibration absorption peak of C–O–C in the ester bond. 2849.55 cm−1 was the stretching vibration absorption peak of -CH-, and 1652.8 cm−1 was the stretching vibration absorption peak of –CO–NH– in the amide bond, but the EpCAM, vimentin and GPC3 antibodies did not have these absorption peaks. The UV test results of Ep-LMS, Vi-LMS and GPC3-LMS are shown in Fig. 2/kg, 25 Am2/kg and 35 Am2/kg, respectively, which were lower than that for Fe3O4 (66.5 Am2/kg).The basic principle of the immune lipid magnetic sphere method is that the antibody with the magnetic sphere binds to the CTC surface antigen, and then CTCs are separated from blood under the action of a magnetic field. Since the existing CTC sorting methods based on the epithelium or EMT are far from sufficient, a novel CTC sorting method in combination with high GPC3 expression for capturing multiple subsets of CTCs is needed. In this study, EpCAM/vimentin/GPC3 antibody-modified LMS were used to capture tumor cells with epithelial phenotype, mesenchymal phenotype and GPC3 phenotype, respectively, a clinically feasible CTC sorting scheme of LMS coupled with different antibodies  was designed and constructed to monitor HCC progression for the first time , with a sensitivity of 98.12% , but there was no significant difference between the sham operation group (SO group) and PR group at the same time points after surgery. This may be due to residual lesions after tumor debarking, which kept the number of CTCs high Fig. F. When t+/DAPI+/CD45− and obvious cellular morphology were identified as CTCs.The separated CTCs Fig. A and CTCP < 0.001, Fig. P < 0.001, Fig. P < 0.001, Fig. P < 0.001, Fig. P > 0.05; Fig. During multidisciplinary comprehensive treatments, 3 patients withdrew from the HCC group, 13 cases had hepatectomy following conversion therapy 13/17, 76.5%), and 4 cases with TACE could not receive liver resection due to failed comprehensive therapies . Therefore, 17 patients with advanced HCC included in this study were followed up, and CTCs were detected at different time points, as shown in Fig. .5%, and P = 0.002) and tumor metastasis . As seen from the above results, both primary tissues and CTCs could detect mutations in targetable genes with clinical significance. The number of gene mutations in CTCs was lower than that in primary tissues, which may be due to the greater number of tumor cells in tissue samples. However, more high-frequency gene mutations were found in CTCs.In this study, multiple subsets of CTCs were captured from HCC patients by the combination scheme of Ep-LMS/Vi-LMS/GPC3-LMS. To assess the suitability of CTCs in analyzing the tumor mutational landscape, we conducted NGS on matched CTC samples and tumor tissues from 17 HCC patients. Figure TP53, CTNNB1 and ATRX were the most commonly mutated genes . The results showed that nes Fig. B. Furthenes Fig. C. The abHCC is a common and highly malignant tumor. Without timely diagnosis and treatment, the survival time of primary liver cancer patients may be only 3–6 months. Surgical resection is one of the most effective methods for HCC. However, the liver has a rich blood supply and is prone to metastasis after hepatectomy due to hematogenous metastasis. Moreover, when the portal vein is invaded by tumor cells, tumor thrombi will form, resulting in focal metastasis. Additionally, the entry of tumor cells into systemic circulation through the blood will lead to extrahepatic metastasis. To limit such metastases, effective prevention, treatment, and monitoring of tumor cell metastasis are vital.In recent years, CTCs have attracted extensive attention as potential “seeds” of tumor metastases. CTCs play an important role during tumor metastasis . The botHepatectomy is still the most effective treatment for HCC, and CTC counts can be used as an effective method in evaluating postoperative efficacy and monitoring tumor progression , 29. PreAn expanding body of evidence indicates the efficacy of immunotherapy in both second-line and first-line settings for advanced HCC. Therefore, it is necessary to detect corresponding mutated genes and then select the most effective therapeutic schedule. Compared with traditional detection methods, NGS has the advantage of offering a single-time high-throughput detection of the status and changes of multiple genes in vivo, enriching the rigid requirements for clinical diagnosis, treatment, and continuous monitoring of HCC prognosis , 36. BioIt has been shown that tumor mutation burden (TMB) is related to the effectiveness of immunotherapy; the higher the TMB is, the greater the tumor inhibition effect and clinical benefits achieved from immunotherapy; conversely, the lower the TMB is, the worse the clinical benefits acquired from immunotherapy . In thisIn this study, EpCAM/vimentin/GPC3 antibody-modified LMS were used to capture tumor cells with epithelial phenotype, mesenchymal phenotype and GPC3 phenotype. For the first time, a therapeutically practical CTC sorting method based on sequential enrichment of Ep-LMS/Vi-LMS/GPC3-LMS (the combination scheme) was established, and this method captures several subpopulations of CTCs effectively and sensitively for dynamically monitoring HCC progression. In addition, this study confirms that the gene mutations in matched primary tissues, CTCs and metastatic tissues are complementary, CTCs and metastatic tissues have a higher rate of shared mutations, and gene detection in multiple subsets of CTCs can provide a more comprehensive mutation spectrum for HCC, which provides a solid foundation for clinical immunotherapy and, thus, facilitate liquid biopsy-based applications.n = 39), including primary lesions of HCC (n = 17), preoperative CTC samples (n = 17) and metastatic tissues (n = 5), were used for 610 gene mutation detection by NGS. HCC patients were followed up every month until September 30, 2022, by monitoring serum AFP levels, abdominal ultrasonography, chest X-ray or computed tomography depending on the patient’s condition. General data, metastatic characteristics, pathologic characteristics and survival were compared among the groups. This study was approved by the ethics committee of our hospital , and informed consent was obtained from participants in accordance with respective committee regulations.A total of 44 participants were considered for inclusion in this study from May 2021 to September 2022, including 24 healthy volunteers without any hepatitis (blank control) and 20 advanced HCC patients evaluated by clinical history, AFP and imaging findings. The inclusion criteria for HCC patients in this study were (a) availability of CTCs, frozen biopsy and/or resected primary and metastatic HCC tissues; (b) pathologically proven HCC based on WHO criteria; (c) gene mutation detection for CTCs and HCC tissues; and (d) availability of follow-up data. Advanced HCC patients received conversion therapies, including immunotherapy, targeted therapy (sorafenib/lenvatinib), transcatheter arterial chemoembolization (TACE), traditional Chinese medicine (Huaier granule) , and liv3O4 solution, carboxymethyl chitosan hexadecyl quaternary ammonium salt (HQCMC), CK8/18/19 (CK)-FITC (Fluorescein Isothiocyanate), CD45-PE, DAPI, Ep-LMS, and Vi-LMS were purchased from Huzhou Lieyuan Medical Laboratory Co., Ltd. A Prussian blue staining kit was purchased from Solarbio. DMEM, fetal bovine serum and trypsin were purchased from Gibco, and GPC3 antibody was purchased from Abcam. 1,2-Dioleoyl phosphatidylcholine (DOPC), dimethyl octadecyl epoxy propyl ammonium chloride (GHDC), cholesterol, dichloromethane, N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and other commonly used reagents were all purchased from Sinopharm Group. The TIANamp Genomic DNA Kit was purchased from Tiangen Biotech (Beijing).Fe2/95% air at 37 °C.At the authors’ institution, a stepwise metastatic human HCC model system was established, which included a metastatic HCC model in nude mice (LCI-D20) , an HCC 3O4 solution and 20 mL 0.1 mol/L phosphate buffer saline (PBS) (pH 7.4) were added, followed by ultrasonic oscillation under a power of 100 W at 25 °C for 6 min and rotary evaporation for 30 min to remove dichloromethane and obtain LMS solution (1.8 mg/mL). Subsequently, 0.1 mg surfactant GHDC was dissolved in 1 mL isopropyl alcohol, and 60 µg GPC3 was dissolved in 1 mL GHDC solution (GPC3-GHDC), which was added to the coupling agents NHS (0.1 mg) and EDC (0.1 mg). After incubation overnight at 4 °C, 1 mL GPC3-GHDC was added into 1 mL LMS solution, which was subjected to vortex oscillation for 5 min and stored at 4 °C. Finally, the sample was taken out of the vortex every 1 h for 5 min for 24 h to obtain GPC3-LMS (2.16 mg/mL).Cholesterol (6 mg), DOPC  and emulsifier HQCMC  were codissolved in dichloromethane as the solvent. Then, 1 mL 20 mg/mL FeThe sample (10 μL) was diluted in 1 mL of distilled water, and the particle size and potential of the magnetic spheres were measured by using a BI-90Plus laser particle sizer/Zeta potentiometer (Brookhaven), 50 μL of which was coated on mica plates. The morphology of LMS was observed under an AFM. Then, 50 μL of diluent was dropped on a copper mesh, and the morphology of LMS was observed under TEM after drying. After freeze-drying of a 1 mL sample, the infrared spectrum obtained by the KBr tablet method was measured on a Bio-Rad FTS 6000 spectrometer using FT-IR spectroscopy. Afterward, 10 μL samples were taken for magnetic separation and diluted in 1 mL distilled water, and the LMSwere scanned by ultraviolet absorption spectroscopy with an ultraviolet spectrophotometer. After a 1 mL sample was freeze-dried into powder, the magnetization curve was determined using a vibrating sample magnetometer (VSM) at room temperature.2 incubator for 3 h. Finally, 150 μL dimethyl sulfoxide (DMSO) was added to dissolve the prepared blue and purple crystalline formazans. The experimental results were read in a microplate reader and counted , and 3 parallel tests were carried out in each group.The cells were prepared into a single-cell suspension with trypsin and cultured in 96-well plates at 8000 cells per well. When the cells grew to 80% confluence, the medium was replaced with 100 μL of complete DMEM containing different amounts of Ep-LMS, Vi-LMS and GPC3-LMS. In the blank control group, 100 μL of complete DMEM was added for further culture. After adding 20 μL MTT reagent (5 mg/mL), incubation was performed in a COSingle-cell suspensions of Hep3B, Huh7, MHCC97L and MHCC97H cells were prepared. After counting, 100 cells were added to 7.5 mL PBS solution to simulate CTC suspension by separation with LMS, Ep-LMS, Vi-LMS and GPC3-LMS. The isolated cells were then stained with Prussian blue dye and observed under a fluorescence microscope after fixation on porous slides. Additionally, the captured cells were smeared on the sample mirror, sprayed with gold after drying, and observed under a SEM.Using the Ep-LMS/Vi-LMS/GPC3-LMS capture scheme, HUVECs, Hep3B, Huh7, MHCC97L and MHCC97H cells were adjusted to different cell gradients of 10, 50, 100, 500, 1000, 5000 and 10,000 cells, respectively. The sensitivity and specificity of this scheme were determined in the PBS system and the simulated blood system, respectively. Finally, magnetic spheres with antibodies at 0, 20, 40, 60, 80 and 100 μg/mL were used to capture Huh7 cells. The cell capture efficiency of magnetic spheres was investigated in the above systems.4) were incubated in a culture dish, which was supplemented with 1 mL cell culture medium and then cultured in a constant-temperature 5% CO2 incubator at 37 °C for 24 h. After replacing the culture medium, 20 μL Ep-LMS-FITC or Vi-LMS-FITC, GPC3-LMS-FITC, 100 μL DAPI and 100 μL Dil were added. Finally, the culture dish was fixed on a fluorescence microscope and photographed at 0, 5, 10, 15 and 20 min.Huh7 cells , 3 nude mice were randomly selected at the end of weeks 0, 2, 3, 4 and 5 for weighing and then sacrificed for sample collection and CTC capture. Tumor volume was calculated by the formula V = a × b2/2, where a is the long tumor axis and b is the short tumor axis. Then, 72 mice were randomized into 4 groups . Three nude mice in each group were randomly sacrificed for sample collection and CTC isolation at the end of the 0, 1st, 2nd, 3rd, 4th and 5th weeks. Resection started on Day 14 after HCC tissue implantation. In the blank control group , the mice underwent liver exposure without resection; in the RR group (n = 18), the mice underwent radical HCC resection with a negative surgical margin; in the PR group (n = 18), mice underwent partial HCC resection with preservation of 2 mm of tumor pedicles [n = 18), mice underwent exposure of the liver but no resection.In the subcutaneous implantation tumor model group  were taken at different time points, and each sample was divided into five 2 mL aliquots. Then, 15 μL Ep-LMS, Vi-LMS, and GPC3-LMS was added to the Ep-LMS group, Vi-LMS group, and GPC3-LMS group, respectively, and incubated for 15 min. The Ep-LMS/Vi-LMS group was treated with 7.5 μL of Ep-LMS and Vi-LMS successively and incubated for 15 min. The Ep-LMS/Vi-LMS/GPC3-LMS group was treated with 5 μL of Ep-LMS, Vi-LMS and GPC3-LMS successively (the combination scheme). After incubation, the centrifuge tube was inserted into the magnetic separation rack for adsorption for 10 min. Subsequently, blood was removed, and 20 μL of CK-FITC, DAPI staining solution and CD45-PE were added for staining in the dark for 15 min. After staining, washing was performed twice using 1 mL of distilled Hn = 610) were detected via target region capture and NGS technology [Total DNA from tumor tissues and CTCs was extracted by the TIANamp Genomic DNA Kit . The samples  ± standard deviation (SD). One-way ANOVA was used for comparisons between different time points, and the SNK test was used for pairwise comparisons. The results were considered statistically significant at Additional file 1: Figure S1. Flow chart for preparation of magnetic spheres.Additional file 2: Figure S2. Effects of LMS (A), Ep-LMS (B), Vi-LMS (C) and GPC3-LMS (D) on the viability of different cell lines.Additional file 3: Figure S3. Optimal rendering of DAPI, FITC and Dil staining after incubation with cells for 15 min.Additional file 4: Figure S4. HE staining of various organs suspected of tumor involvement in nude mice.Additional file 5: Figure S5. Immunofluorescence-based identification of animal blood CTCs. CTCs were captured by Ep-LMS, Vi-LMS and GPC3-LMS and identified by immunofluorescence staining with DAPI, CK-FITC and CD45-PE.Additional file 6: Figure S6. Relationship between tumor volume and the number of tumor cells.Additional file 7: Figure S7. Gene mutation analysis. Heatmaps showing the frequency and distribution of gene mutations in primary tissues (A), metastatic tissues (B) and CTCs (C) and the rate of shared mutations between primary tissues and CTCs (D).Additional file 8: Figure S8. TMB detection for tissues and CTCs. The reference TMB value was 3.6 Muts/Mb. If the detection value was higher than the reference value, was recommended.Additional file 9: Table S1. Preoperative and postoperative clinical parameters.Additional file 10: Table S2. Comparisons among different CTC enrichment techniques


TP53 tumor suppressor gene is one of the most studied gene in virtue of its ability to prevent cancer development by regulating apoptosis, cell cycle arrest, DNA repair, autophagy and senescence. Furthermore, the modulation of metabolism by P53 is fundamental for tumor suppressor activity. Studies in mouse models showed that mice carrying TP53 mutations affecting the acetylation in the DNA binding domain still retain the ability to transactivate genes involved in metabolism. Noteworthy, mice expressing the triple 3KR or the single K117R mutant do not show early on-set tumor development in contrast to TP53−/− mice. Interestingly, the mouse K117R mutation corresponds to the human tumor-derived K120R modification, which abrogates P53-dependent activation of apoptosis without affecting growth arrest. In this study, we investigated the property of the human P53 K120R mutant in the regulation of metabolism by analyzing the transcriptional specificity in yeast- and mammalian-based reporter assays, the metabolic phenotype associated to its expression in colon cancer HCT116TP53−/− cells and the induction of P53 targets and proteins involved in the antioxidant response. These properties were analyzed in comparison to wild type P53 protein, the human triple mutant corresponding to mouse 3KR and the cancer hot-spot R273H mutant. We confirm the selective functionality of P53 K120R mutant, which shows a transcriptional activity on cell cycle arrest but not on apoptotic targets. Interestingly, this mutant shows a partial transactivation activity on p53 response element belonging to the metabolic target TIGAR. Moreover, we observe a significant uncoupling between oxygen consumption and ATP production associated with higher lipid peroxidation level in all P53 mutants carrying cells with respect to wild type P53 expressing cells. Noteworthy, in the absence of a pro-oxidative challenge, cells expressing K120R mutant retain a partial capacity to modulate glucose metabolism, limiting lipid peroxidation with respect to the other P53 mutants carrying cells. Lastly, especially in presence of human 3KR mutant, a high expression of proteins involved in the antioxidant response is found. However, this response does not avoid the increased lipid peroxidation, confirming that only wild type P53 is able to completely counteract the oxidative stress and relative damages.The  TP53 tumor suppressor gene, encoding the P53 tetrameric transcription factor, is the most commonly altered gene in human cancer, highlighting its key role in maintaining cellular homeostasis. The majority of TP53 alterations found in tumors are missense mutations affecting six codons located in the central DNA binding domain (DBD)  that typically result in the complete loss of tumor suppressor functions. However, the spectrum of missense mutations is extremely broad along the TP53 coding sequence, with more than 2,000 different amino-acid changes reported; indeed, TP53 mutations can be classified not only as loss of function (LoF) but also as partial function, with altered specificity or gain of function (GoF), highlighting the functional heterogeneity of the encoded P53 mutant proteins  to Arginine (R) substitution at codon 120 (here indicated as p53K120R), is an example of a DNA contact alteration found in cancers able to maintain some DNA binding capacity in virtue of the positive charge preservation of the amino acid side chain, which allows a heterogenous transcriptional activity  is able to bind and activate the transcription of the pro-survival TNFAIP8 gene, that is critical for escape from apoptosis in tumor cells  and comprising the K117 site that corresponds to K120 codon of human P53, have impaired P53 function in apoptosis, cell cycle arrest, and senescence. However, these mice are not prone to early tumor development as the TP53 null mice  was identified from microarray analysis as a P53 target repressed by mouse p533KR mutant and p53WT , according to the inability of this mutant protein to induce the apoptotic targets BBC3 (PUMA), TNFRSF10B (KILLER) and PMAIP1 (NOXA) and the ability to induce the cell-cycle inhibitor CDKN1A (P21). As for p53WT mice, p53K117R animals did not develop early-onset tumors  its transcriptional specificity in yeast- and mammalian-based reporter assays, ii) the metabolic phenotypes of human colon cancer HCT116TP53−/− cells expressing the mutant protein, and iii) the induction of P53 targets and proteins involved in the antioxidant response. The p53K120R features were analyzed in comparison not only to p53WT but also to the human P53 mutant resembling the triple mutant previously analyzed in mouse models. Furthermore, since the mutation giving rise to R273H DNA contact mutant protein (here indicated as p53R273H) is one of the main hot spot mutation found in human tumors, generating a mutant P53 protein unable to transactivate all type of P53 response elements (REs) or promoters   , the metS. cerevisiae LUC1 reporter strains were used: yLFM-P21-5’, yLFM-MDM2P2C, yLFM-BAX A + B, yLFM-KILLER, yLFM-AIP1, yLFM-NOXA, and yLFM-TIGAR (LUC1); the REs belong to apoptotic , cell cycle arrest [CDKN1A (P21)], P53 regulation (MDM2), and metabolic (TIGAR) targets. Yeast plasmids expressing the different TP53 mutations were constructed exploiting in vivo yeast homologous recombination and all manipulations involving the reporter assay and yeast transformations were performed as previously described  under a galactose inducible expression ; pRS314 was used as empty vector. Regarding the construction of the human P53 mutant that resembles the mouse p533KR mutant, since the mouse position K162 is replaced in human by Q165, an acetyl-mimetic amino acid, we constructed the human triple P53 K120R + K164R + Q165R mutant and named it p533KR for the analogy with the mouse mutant . Cells were grown in RPMI containing 10% foetal bovine serum, l-glutamine, penicillin-streptomycin antibiotic mixture , and maintained at 37 °C, in 5% CO2, at 100% humidity.Human colon carcinoma HCT116WT, p53K120R, p533KR, p53R273H) in mammalian cells. Plasmids expressing P53 mutants were constructed as previously described . After 24 h, cells were collected and washed with cold phosphate buffer saline (PBS). Lysis was performed in 1X PLB buffer . Luciferase assays were conducted, as previously described (WT (100%). For western blot and metabolic analysis, HCT116TP53−/− cells were transfected as reported above with 1 µg of pCIneo-based P53 expression vector. After 16 h of transfection, cells were treated with 100 µM hydrogen peroxide (H2O2) for further 6 h. The percentage of living cells following this treatment did not differ among cells carrying different P53 proteins (A pCIneo-based vector (Promega) was used to express P53 proteins  was evaluated in 100,000 cells for each experiment by a thermostatically controlled oxygraph apparatus equipped with an amperometric electrode . Cells were resuspended in PBS and permeabilized with 0.03% digitonin. To evaluate the OCR performed by Complexes I, III and IV pathway, 10 mM pyruvate +5 mM malate were added, while 20 mM succinate were used to activate the pathways composed by Complexes II, III and IV; after the addition of respiratory substrates, 0.1 mM ADP was added in each experiment. The respiratory rates were expressed as nmol O/min/106 cells .o-F1 ATP synthase activity was measured in 100,000 cells incubated for 10 min at 37°C in a medium containing 10 mM Tris-HCl pH 7.4, 100 mM KCl, 5 mM KH2PO4, 1 mM EGTA, 2.5 mM EDTA, and 5 mM MgCl2, 0.6 mM ouabain, 0.3 mM P1, P5-Di (adenosine-5′) pentaphosphate, and 25 mg/ml ampicillin. The ATP synthesis was induced by the addition of 10 mM pyruvate +5 mM malate or 20 mM succinate, to stimulate the pathways composed by Complexes I, III and IV or Complexes II, III and IV, respectively. The reaction was monitored for 2 min, every 30 s, in a luminometer , by the luciferin/luciferase chemiluminescent method , with ATP standard solutions between 10−8 and 10−5 M. Data were expressed as nmol ATP produced/min/106 cells  efficiency was calculated as the ratio between the produced ATP concentration and the consumed oxygen amount (P/O ratio).By measuring the OCR and the ATP synthesis trough F6 cells  and glucose six phosphate dehydrogenase (G6PD) coupling system. The assay medium contained 100 mM Tris-HCl pH 7.4, 2 mM ATP, 10 mM NADP, 2 mM MgCl2, 2 International Units (IU) of hexokinase, and 2 IU of glucose 6-phosphate dehydrogenase. Data were normalized on the cell number and expressed as mM consumed glucose/106 cells .6 cells . Data were normalized on the cell number and expressed as mM released lactate/106 cells .Once the glucose consumption and lactate release amounts have been obtained, the lactate fermentation yield was calculated by comparing the values of lactate produced with those of theoretical lactate calculated as twice the glucose consumed.Lactate dehydrogenase activity was evaluated as a marker of lactate fermentation, following the NADH oxidation, at 340 nm. The reaction mixtures contained: 100 mM Tris-HCl pH 7.4, 0.2 mM NADH, and 5 mM pyruvate . For theMalondialdehyde (MDA) concentration was assayed as a lipid peroxidation marker, by the thiobarbituric acid reactive substances (TBARS) assay . 600 μl T student test, ordinary One-way anova and Two-way anova followed by comparisons test were performed by using GraphPad Prism version 8.0. For Windows, GraphPad Software, San Diego, California, United States .K120R, p533KR and p53R273H mutants was examined using a yeast-based functional assay. Our analyses confirm that p53K120R maintains high functionality on P21 and MDM2 REs but shows partial (BAX and KILLER) or poor  activity on the REs of apoptotic genes  on the same REs. The p53R273H mutant, used as a control of LoF mutant, shows no transactivation activity in any yeast strain tested. The p53K120R and p533KR mutants were also evaluated on the RE of the metabolic target TIGAR; they show a partial activity (p53K120R > p533KR) and not a complete loss of transactivation ability as displayed by p53R273H mutant , but not in cells expressing the other P53 mutant proteins.In the same mammalian cells, the level of P21, MDM2, BAX, and TIGAR endogenous proteins was also assessed . In keepproteins , whereasK120R mutant protein with respect to p533KR and p53R273H mutants.All together these data confirm the selective functionality of p53K120R, p533KR, and p53R273H), the OxPhos activity and the lactate fermentation rate were investigated in HCT116TP53−/− cells transiently transfected as previously described.To evaluate the metabolic phenotype induced by the expression of P53 mutant proteins , converting pyruvate to lactic acid by LDH activity . Thus, tWT glucose consumption, lactate release, and LDH activity are significantly lower than in cells transfected with the empty vector, while cells expressing p53K120R, p533KR, and p53R273H proteins show a significant increment (WT (that is comparable to empty cells) with respect to P53 mutant proteins  are characterized by a lower activation of these two metabolic pathways with respect to p53WT, despite maintaining a higher activity than the empty control. In detail, p533KR displays lower enzymatic activities compared to p53K120R carrying cells, which, in turn, appear slightly but significantly lower than those observed in p53WT cells. These results are in line with the decreasing efficiency of aerobic metabolism and the need to increase glucose consumption and anaerobic glycolysis in cells expressing these P53 mutant proteins since both the conversion of glutamine to alpha-ketoglutarate and beta-oxidation are two metabolisms that provide substrates for the Krebs cycle.We also determined the level of glutaminase (GLS), glutamate dehydrogenase activity (GDH), as well as 3-hydroxyacyl-CoA dehydrogenase as markers of amino acid metabolism and beta-oxidation, respectively. Data reported in o OxPhos . Convers2O2) as pro-oxidative stress. The addition of H2O2 determines an increase of OCR values independently from the substrate in empty vector, p53K120R, and p53R273H expressing cells compared to untreated cells but a decrease in cells with p533KR mutant . No other significant P53-dependent modulations are evident with exception of a downregulation of GCLM level in p53K120R carrying cells with respect to empty sample . Moreover, while p53WT cells are characterized also by an upregulation of GCLC, p533KR cells show a significant induction of all analyzed proteins . In addition, the human P53 triple mutant (p533KR), that resembles the mouse mutant carrying Lysine to Arginine missense mutations at the three acetylation sites of the DBD  or promoters  but poorly or partially BAX, KILLER, AIP1, NOXA targets, confirming its transcriptional specificity  in HCT116TP53−/− cells transfected with p53WT, p53K120R, p533KR and p53R273H proteins  , suggestironment . Thus, tK120R mutant also in comparison to the human triple p533KR mutant. Li and others, by using a mouse model found that the mouse p533KR protein failed to activate P53 target genes of cell cycle arrest or apoptosis, but it retained the ability to regulate metabolism as p53WT by repressing xCT, coding for the main transporter for cystine  protein, involved in lipid peroxidation, favors its nuclear localization; inside the nucleus it was supposed that DDP4 acts as P53 transcription cofactor, stimulating xCT expression , a surrogate measure of lactate export, appeared significantly enhanced, suggesting a metabolic switch to the anaerobic energy metabolism. Specifically, the inducible expression of p53R237H caused the increment of both glycolytic capacity and OCR, in agreement with our data.The effect of expression of different types of P53 mutant proteins on glycolysis and mitochondrial metabolism has been previously addressed . By analK120R are characterized by a metabolic phenotype that is intermediate between p53WT and p53R273H expressing cells, indicating a partial activity by the peculiar TP53 K120R cancer mutation in modulation of cellular metabolism. On the other hand, p53R273H cells showed the most evident metabolic change, which could compromise the cellular environment in agreement with its LoF and GoF activities. Such diversity can rely not only on P53 transcriptional specificity, as emerging from our results, but also from the interconnection with the cellular environment in which the P53 mutant protein can operate.In conclusion, our results underline that not all P53 mutant proteins can be considered metabolically equivalent. We demonstrated that cells carrying p53


Enterolobium contortisiliquum trypsin inhibitor (EcTI), a protease inhibitor, on cell viability, cell migration, invasion, cell adhesion, and cell death  in vitro using human melanoma cells (SK-MEL-28 and CHL-1). Although EcTI did not affect non-tumor cells, it significantly inhibited the proliferation, migration, invasion, and adhesion of melanoma cells. Investigation of the underlying mechanisms revealed that EcTI triggered apoptosis and nuclear shrinkage, increased PI uptake, activated effector caspases-3/7, and produced reactive oxygen species (ROS). Furthermore, EcTI disrupted the mitochondrial membrane potential, altered calcium homeostasis, and modified proteins associated with survival and apoptosis/autophagy regulation. Acridine orange staining indicated acidic vesicular organelle formation upon EcTI treatment, demonstrating a cell death display. Electronic microscopy corroborated the apoptotic pattern by allowing the visualization of apoptotic bodies, mitochondrial cristae disorganization, and autophagic vesicles. Taken together, these results provide new insights into the anti-cancer properties of the natural EcTI protein, establishing it as a promising new therapeutic drug for use in melanoma treatment.Cell adhesion and migration are crucial for cancer progression and malignancy. Drugs available for the treatment of metastatic melanoma are expensive and unfit for certain patients. Therefore, there is still a need to identify new drugs that block tumor cell development. We investigated the effects of  S. miltiorrhiza roots and the polyphenol of Olea europaea leaves , followed by the generation of ROS, which modulates cell death due to the initiation of apoptosis mediated by BAX/BCL-2 pro-apoptosis/apoptosis balancing protein. Since malignant melanoma is characterized by a poor prognosis and resistance to agents inducing apoptosis, the ability of EcTI to reduce BCL-2 and increase BAX confers on these protein-relevant characteristics to sensitize cancer cells to death.Thus, EcTI altered the metabolic state of cells by the loss of mitochondrial membrane potential [CaAutophagy is another pathway that culminates in cell death and alters essential proteins of autophagosome formation. This includes the inhibition of BECLIN, the increase of ULK protein expression, and an increased LC3-II/LC3-I ratio. Upregulation of ULK may trigger increased autophagic activity by activating LC3. Confirming our previous findings, inhibition of BECLIN may result from the cleavage of caspases, such as caspase-3 and caspase-8, inducing the mitochondrial membrane permeability of cells, leading to apoptosis . InteresInitially, apoptosis and necrosis were considered individual and exclusive; however, there is an interplay between apoptosis, necrosis, and autophagy as a complementary style that allows cancer cell damage . The forIt is clear that the mechanisms involved in the harm to melanoma between the SK-MEL-28 and the CHL-1 cell lines are different, which is not surprising since they are genetically heterogeneous cells. However, in all the melanoma lines treated with EcTI, patterns of activated cell death, often by different routes, showed a non-standard effect. The cell morphology obtained by transmission electron microscopy confirmed our findings as apoptotic bodies, chromatin condensation, disorganized mitochondrial crest, active pinocytosis, formation of multivesicular bodies, and autophagic vesicles. Melanoma cells treated with EcTI showed the appearance of cell death events in different stages, with cells in the early or late apoptosis phase and autophagic vesicles, still containing sparse healthy cells resistant to the effect of the compound.E. contortisiliquum seeds was briefly precipitated using acetone (80% (v/v), 4 °C), loaded onto a DEAE–Sepharose column in Tris/HCl (0.1 M), pH 8, and eluted with 0.15 M of NaCl added to the equilibrium buffer  was deposited. The inhibitor was purified following the procedure described by de Paula et al. . The prom buffer . Furtherm buffer  and sizem buffer . Inhibitm buffer  and SDS–m buffer  were per2 and 95% air, supplemented with 10% fetal bovine serum , 100 mg/mL streptomycin, and 100 U/mL penicillin . The medium was refreshed every three days, and cells were used for experiments or cryopreservation before reaching 90% confluence. All cells previously tested negative for mycoplasma contamination  (SK-MEL-28 (BRAFV600E positive) and CHL-1 human cell lines were purchased from ATCC  and cultured in high-glucose DMEM. B16F10-nex cells were generously provided by Dr. Luiz Rodolpho R. G. Travassos . All cells were cultured after 20–30 passages at 37 °C in a sterile humidified atmosphere with 5% COAldrich) .2. Then, 100 µL of DMSO was added, and the absorbance was monitored at 540 nm .Cell viability was evaluated by the MTT assay . An aliqTo evaluate the effects of some modulators of specific types of cell death in EcTI-treated cells, Z-VAD (20 µM), necrostatin-1 , and necrosulfonamide  were preincubated with cells for 24 h. Then, EcTI (5–100 µM) was added and the reaction proceeded for an additional 24 h . The eff2 for adherence. After initial incubation, the medium was replaced with EcTI (5–100 µM) in the experimental groups, but not in the control group. After incubation for 24 h, 10 mM BrdU was used , according to the manufacturer’s instructions. A spectrophotometer  at 405 nm was used to measure the light emission of the samples [Cells (5000 cells/well) were incubated for 24 h in a 96-well plate at 37 °C and 5% CO samples .2. Then, heat-denatured BSA (1% w/v) was added, incubated for 1 h at 37 °C, and washed with PBS buffer . Adherent cells were fixed using cold methanol for 40 min, washed with PBS, and stained with 1% toluidine blue (w/v) for 30 min. The cells were washed three more times with PBS and solubilized in 100 µL of 1% SDS (w/v) for 30 min at 37 °C. To measure the flattening of adherent cells, a microplate reader spectrophotometer (Spectra Max PLUS) was used to record the absorbance at 540 nm. The data are expressed as the percentage of adherent cells normalized to the percentage of the control cells [The adhesion assay was performed using collagen I (8 µg/well), collagen IV (4 µg/well), fibronectin (4 µg/well), vitronectin (0.2 µg/well), and laminin (4 µg/well) in a 96-well plate at 4 °C overnight. Approximately 5000 cells plus EcTI  were seeded on the attached substrates per well and incubated for 4 h at 37 °C and 5% COol cells .2 for adherence and scratched with a 200 µL pipette tip. Images were acquired immediately after gap creation (0 h). After the cells were washed with PBS, fresh medium containing EcTI  was added and incubated for 24 h. Images were acquired by Evosfl Inverted Digital Microscope in phase contrast, using a 4× objective lens (Sony® ICX285AL CCD Camera) to assess the closure rate over time. The scratched area was analyzed using the ImageJ MRI Wound Healing Tool, and the percentages of cell migration were calculated by the wound ratio at the beginning and at the end of the wound [To analyze the coordinated movement of melanoma cells, 50,000 cells were seeded in a 24-well plate at 37 °C and 5% CO2 for 24 h. The inserts were washed with PBS, and the non-invaded cells were carefully removed with a cotton swab from the upper surface of the chamber. The cells were fixed with cold methanol for 30 min and stained with 1% (w/v) toluidine blue. They were washed again in PBS, and the invaded cells were counted in at least 10 visual fields under an inverted microscope using a 10× objective lens  [Boyden chamber invasion was evaluated in 8.0 µm pore size. Melanoma cells  were seeded onto the inserts in a 24-well plate, after polymerization with Matrigel coating (1:6 serum-free medium) for 30 min at 37 °C. Cells were pre-incubated with EcTI (5–100 µM) exclusively in the experimental groups for 15 min in a fresh, FBS-free medium. Then, 400 µL of 10% FBS medium was added to the lower chamber, followed by incubation at 37 °C and 5% COGermany) . The per2. After this period, EcTI  was added to each well and incubated for another 24 h, washed thoroughly with PBS, and incubated for 10 min with Hoechst staining solution . Nuclear staining was observed with an inverted fluorescence microscope (Leica) using a 10× objective lens and a set of blue filter cubes (excitation: 394–410 and emission: 435–485). The nuclear area (µm2) was measured using ImageJ software, using the Make Binary Tool and Measure. The percentages of the nuclear area were calculated using the control group compared to the EcTI treatment groups [Hoechst 33,342 nuclear staining  was used to detect the nuclear changes. In total, 5000 cells/well were seeded in a 96-well black plate for 24 h at 37 °C and 5% CO2. EcTI  was added, and the cells were incubated for another 24 h and washed with PBS. To monitor pore formation, PI  was added. Triton X-100 (0.01%), incubated for 24 h, was used as the positive control. Images were acquired using an inverted fluorescence microscope (Leica) with a 10× objective lens and a set of red filter cubes (excitation: 542/12 nm BP and emission: 575–640 nm BP) [To analyze the internalization of propidium iodide (PI), 5000 cells were seeded in a 96-well black plate for 24 h at 37 °C and 5% CO0 nm BP) .2. Then, EcTI was added (50 and 100 µM/well) and incubated for an additional 24 h. After the addition of 100 nM TMRE to the CHL-1 cells and 500 nM to the SK-MEL-28 cells, the cells were incubated for another 45 min at 37 °C and washed with PBS to replace TMRE. Images were acquired using an inverted fluorescence microscope , using a 10× objective lens and a red filter cube set (excitation: 515–560 nm and emission: 590 nm). Next, 50 µM FCCP (carbonyl cyanide 4-((trifluoromethoxy) phenylhydrazone) was incubated for 2 h at 37 °C before TMRE staining was used as a positive control. The fluorescence of the cells was calculated using ImageJ, considering CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of FCCP readings), in which CTCF is corrected for the total fluorescence of the cells.To quantify changes in mitochondrial membrane potential (MMP), the cell-permeant fluorescent lipophilic dye tetramethylrhodamine ethyl ester (TMRE)  was used in live cells . In tota2. Thereafter, EcTI  was added and the mixture was incubated for another 24 h. Then, FLUO-4-AM (5 µM) was loaded and incubated for 1 h at 37 °C. Images were acquired using an inverted fluorescence microscope , using a 10× objective lens, and absorbance was read with the Spectrophotometer FlexStation Multi-Mode Microplate Reader (Molecular Devices) at 485 nm for excitation and 530 nm for emission. Data were analyzed as a relative fluorescence unit (RFU) [The intracellular calcium level in melanoma cells was measured using the FLUO-4-AM Direct Calcium Assay Kit according to the manufacturer’s instructions . In total, 5000 cells/well were incubated in a 96-well black plate for 24 h at 37 °C and 5% COit (RFU) ,39.2DCFDA, a general indicator of reactive oxygen species (ROS), was used to analyze ROS production. A total of 50,000 cells (SK-MEL-28 and CHL-1)/well were incubated in a 24-well plate for 24 h at 37 °C and 5% CO2. Subsequently, EcTI (50 and 100 µM/) or H2O2 (500 µM) was added to the wells for an additional 24 h incubation period. After this, cells were stained with 5 µM of the fluoroprobe CM-H2DCFDA for 40 min, except for the negative control (unstained cells) group. The medium was discarded. Cells were washed with PBS and collected to determine ROS production using the FL1-H channel in the BD Accuri C6 flow cytometer  and Flow Jo 10 software.CM-H® Caspase-3/7 Green reagent was used according to the manufacturer’s instructions . A total of 5000 cells/well were seeded into a 96-well black plate and cultivated for 24 h at 37 °C and 5% CO2. EcTI was added at concentrations of 25, 50, and 100 µM/for 24 h, and fresh medium was added to the control group. After this period, the fluorogenic substrate (1 µM) was loaded per well, followed by a 30 min incubation period at 37 °C. Images were acquired using an inverted fluorescence microscope  using a 10× objective lens, and 494 nm for excitation and 516 nm for emission.For caspase-3 and -7 activity, the CellEvent2 for complete adherence and treated with EcTI (5–100 µM) in the experimental groups or fresh medium in the control group. For cytometric analysis, cells were detached using Versene solution  and washed with PBS. The suspension was labeled with cell death staining solution ) according to the manufacturer’s instructions . The cells were incubated for 15 min at room temperature, protected from light, and at least 10,000 events were collected for acquisition. Dot plot analyses were performed in a BD Accuri C6 Cytometer using C6 Accuri Software . Staurosporine (1 µM) was used as the apoptosis control, and Triton X-100 (0.1%) was used as the necrosis control [To evaluate cell death, 50,000 cells were seeded in a 24-well plate for 24 h at 37 °C and 5% CO control .2, 60 mM Pipes, pH 6.9). Afterwards, cells were fixed with PFA (5% (w/v) diluted in PHEM plus 5% sucrose (w/v) and 0.25% Triton X-100 (v/v)) for 15 min and washed thoroughly with PHEM containing 0.75% glycine (w/v). Cells were then stained with 5 µM Hoechst for 15 min at room temperature and washed 3 times with PHEM. Subsequently, ImageXpress Micro Confocal High-Content Screening equipment was used to analyze labeled cells with fluorescent compounds. The MetaXpress high-content image acquisition and analysis software was used to analyze EcTI internalization of at least 60 cells/field and 9 sites/well.The conjugation of EcTI to AlexaFluor488  was carried out as recommended by the manufacturer for 3 h with constant agitation. Thereafter, the solution was subjected to molecular exclusion chromatography (Superdex75 10/300GL) to remove excess unbound fluorophores. For the internalization assay, 8000 human melanoma cells were seeded into a black 96-well plate for 24 h. In sequence, cells were incubated with 12.5 µM EcTI-AlexaFluor488 for 10 and 30 min, 1, 2, and 4 h, and then washed with PHEM , an additional 24 h incubation period occurred. Cells were then mechanically detached using RIPA buffer  plus fresh phosphatase and protease inhibitors. Total lysate protein was quantified using a MicroBCA Assay Kit . Next, 30 µg was loaded onto SDS-PAGE gel (10%–5%), transferred to PVDF membranes (0.2 µm) using a Trans-Blot Turbo Transfer System , and blocked with BSA (2 h at room temperature) to receive primary antibodies, after washing with TBST solution . Then, PVDF membranes were incubated overnight, at 4 ºC, with primary antibodies, diluted 1:1000 : anti-FAK (#3285), anti-phospho FAK , anti-SRC (#2109), anti-phospho SRC , anti-PI3K (#BD 610045), anti-phospho PI3K (#4228 Y458/Y199), anti-MTOR (#2983), anti-phospho MTOR (#2971 S2448), anti-ERK (#4695), anti-phospho ERK (#4370 T202/Y204), anti-BCL-2 (#2876), anti-BAX (#2772), anti-BECLIN (#3495), anti-ULK (#8054), anti-AMBRA1 (#24907), anti-LC3A/B (#12741), and anti-GAPDH (#5174). Thereafter, membranes were incubated for 1 h at room temperature with secondary antibodies, anti-rabbit (#7074) or anti-mouse (#7076), diluted 1:1000, detected by chemiluminescence  using the Gel System Uvitec—Alliance 4.7. Densitometric analysis was performed using ImageG software, with GAPDH as a control for each sample, and the phosphorylated protein to total protein ratio was calculated after GAPDH normalization.SK-MEL-28 and CHL-1  were incubated in a 6-well plate for 24 h at 37 °C and 5% COTo further study the autophagy process, we evaluated the formation of acidic vesicular organelles, a morphological feature of autophagy . An acidv/v) formaldehyde and 2.5% (v/v) glutaraldehyde for 30 min at room temperature and diluted in the buffer cacodylate . Cells were then embedded in 4% (v/v) agar, and fixed with 2% osmium tetroxide in sodium cacodylate  for 2 h. Then, the cells were dehydrated in increasing ethanol concentrations , and absolute). A mixture of propylene oxide and Epon 612 resin (1:1) was added to the samples overnight. Then, pure Epon was added to the mixture for an additional 2 h under vacuum and polymerized in an oven at 60 °C for 48 h. In the fenestrated copper metallic grid, ultra-thin cuts (70 nm) were placed, and uranyl acetate/plumb citrate was used as a contrast. The images were examined using transmission electron microscopy  operated at 80 kV. Micrographs were acquired with a GATAN 791 camera (USA), while brightness, contrast levels, and image resolution were adjusted using ADOBE Photoshop 7.Cells  were seeded in a 24-well plate before treatment with EcTI (100 µM) for 24 h, washed with PBS, and incubated with Versene solution for 5–7 min. Cells were fixed with 2% , and statistical analyses were performed using GraphPad Prism version 8 . Comparisons between experimental groups were conducted using one-way ANOVA followed by Tukey’s test. Statistical significance is indicated by * With EcTI treatment, a sustained response against human melanoma cells has been observed by inhibiting FAK, SRC, and PI3K proteins and reducing functions essential for tumor regulation, such as cell proliferation, migration, invasion, and cell adhesion to multiple matrix molecules commonly upregulated in melanoma. EcTI still modified cell morphology, as observed by pyknosis and nuclear condensation, in addition to enhancing PI uptake, suggesting a pattern of cell death. In addition, EcTI increased caspase-3/7 activity, which altered the mitochondrial transmembrane potential and intracellular calcium concentration, stimulated cell death, which was also observed by marked BAX/BCL-2 expression, and generated ROS. This apoptotic/autophagic evidence was confirmed by electron microscopy encompassing non-canonical signaling pathways involving autophagic proteins such as ULK and LC3-II/LC3-I with amplified expression in SK-MEL-28 cells. Conversely, in CHL-1, EcTI stimulated BECLIN expression, pointing to the differences in the mechanism of action between these human cell lineages .Taken together, our studies have shown that in addition to the inhibition of proteolytic enzymes, plant inhibitors can be used to block cancer-related events, suggesting the potential of this protein in the investigation of melanoma skin cancer. Even with some questions posed in carrying out this study, and with the awareness that there is much to be investigated further, this work aims to contribute to the understanding of EcTI activity, evaluate the relationship of its functional properties and mechanisms of action, and encourage further studies on the development of promising compounds for melanoma.


Citrus maxima Merr. cv. Jinshayou) during the room temperature storage at 20 ± 2°C for 90 d.The loss of postharvest storability of pummelo fruit reduces its commodity value for long run. To maintain its storability, the effects of postharvest dipping treatment by salicylic acid (SA) with different concentrations  were investigated on pummelo fruit (2O2), and malondialdehyde (MDA) content of the 0.3% SA-treated pummelo fruit were reduced compared to the control (dipped in distilled water). Pummelo fruit treated with 0.3% SA exhibited the most outstanding ability to excess reactive oxygen species (ROS) accumulation, as evidenced by promoted the increases in glutathione (GSH), total phenolics and flavonoids contents, delayed the AsA decline, and enhanced the activities of antioxidant enzymes and their encoding genes expression.Among all treatments, pre-storage SA treatment at 0.3% demonstrated the most significant ability to reduce fruit decay incidence, decrease weight loss, delay peel color-turned process, and inhibit the declines in total soluble solids (TSS) as well as titratable acid (TA) content. The increases in electrolyte leakage, hydrogen peroxide (HPre-storage treatment dipped with SA, particularly at 0.3%, can be used as a useful and safe preservation method to maintain higher postharvest storability and better overall quality of ‘Jinshayou’ pummelo fruit, and thus delaying postharvest senescence and extend the storage life up to 90 d at room temperature. Due to nces)    (2 . As the nces) , an important plant phenolic compound, acts as an important signaling molecule with a role in enhancing postharvest resistance to senescence stress and pathogen invasion in horticultural products during long-term storage and shipment . RecentlAlthough pre-storage SA treatment has been shown to be useful for the postharvest preservation of various fruits and vegetables, as far as we know, there is rarely information on the alleviatory effects of SA on pummelo postharvest senescence and the overall quality of ‘Jinshayou’ pummelo fruit during long-term storage. The aim of this study was to evaluate the preservative effect of SA on postharvest fruit quality, oxidative stress, antioxidant capacity, and ROS-scavenging system in ‘Jinshayou’ pummelo fruit during the room temperature storage, and provide theoretical evidence to develop SA as a green and efficacious preservative for postharvest pummelo fruit.22.13), polyvinylpyrrolidone (PVP), and iron sulfate were purchased from Sinopharm Chemical Reagent Co. Ltd. . Folin–Ciocalteu reagent, gallic acid (GA), rutin, L-ascorbic acid (L-AsA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and dithiothreitol (DTT) were obtained from Sigma-Aldrich, Chemical Co. .SA, sodium hydroxide (NaOH), thiobarbituric acid (TBA), hydrochloric acid (HCl), trichloroacetic acid (TCA), methanol, ethanol, aluminum chloride (AlCl2.2C. maxima Merr. cv. ‘Jinshayou’) fruit, at mature-green stage (about 195 d after anthesis), were harvested from a commercial orchard in Ji’shui city . Fruit with uniformity of weight, shape, color, maturity , and without visible defects were selected, washed with sterile water, and randomly divided into four groups . Each group was dipped in 0 (control), 0.1%, 0.2%, 0.3% SA solution for 5 min. After being naturally air-dried, each fruit was individually film packaged, and stored at room temperature condition  for 90 d. Fruit color change, decay rate, weight loss, and other biochemical quality parameters were measured at 15 d intervals. For each sample, the tissues of juice sacs  randomly taken from the control and three SA-treated groups were frozen in liquid nitrogen, ground into powder and then stored at -80°C.Pummelo (2.3The CIE parameters of L* (dark to light), a* (green to red) and b* (blue to yellow) on two opposite equatorial sites of ‘Jinshayou’ pummelo peel were measured directly using a CR-400 colorimeter  following the protocol described by h° = arctan (b*/a*)0, a0, and b0 were the readings at harvest (0 d), and L*, a*, and b* were the readings of each sampling time during storage period.Where L2.4The detailed method for evaluation of fruit decay rate has been previously described by 2.5At 15-day intervals during storage at 20 ± 2°C, the weight loss of ‘Jinshayou’ pummelo fruit was recorded according to the protocol described by 2.6-1 NaOH solution, and the results were calculated based on the NaOH consumption and expressed as %.Total soluble solid (TSS) content in pummelo juice extracted from 10 fruit was determined with a digital refractometer , calibrating with deionized water before each reading, and the results were expressed as a percentage (%). Titratable acidity (TA) content was analyzed in terms of citric acid by adding 4.0 grams extracted juice with two drops of 1% phenolphthalein in 40 mL of distilled water, and then it was titrated with 0.1 mol LTo estimate cell membrane permeability, electrolyte leakage (EL) was determined using a DDS-307A conductivity meter  as described by 2O2) content assay, a total of 2.0 g frozen sample was extracted with 5 mL of pre-cooled acetone and the homogenate was centrifuged (10 000 × g at 4°C for 20 min) to discard the residue. H2O2 content was determined using a specific detection kit  by monitoring the absorbance at 412 nm (-1) on a frozen weight (FW).For hydrogen peroxide  TCA solution and centrifuged (10 000 × g at 4°C for 20 min). Afterwards, 2.0 mL of the supernatant was mixed by adding the same volume of 0.67% TBA (dissolved in 50 mM NaOH) solution, followed by boiling water bath for 20 min, and then quickly cooled in an ice bath. Finally, the absorbance of the supernatant was recorded at three specific wavelengths  using a UV-Vis spectrophotometer , with the results were reported as millimole per gram (mmol g-1) FW.Malondialdehyde (MDA) content was assayed using the TBA method as described by L-AsA as the standard, where the AsA content was expressed as mg of AsA equivalent per 100 g of juice sac FW.Quantitative determination of AsA content in pummelo fruit was carried out on juice sac samples according to the 2,6-dichlorophenol–indophenol (DPIP) dye titration method described by -1) FW.The glutathione (GSH) content of pummelo juice sac was determined using the 5,5’- dithiobis-(2-nitrobenzoic acid) reaction method, a protocol described by 3 colorimetric method outlined by -1) of juice sac FW.Two grams of pummelo juice sac were homogenized with 8 mL of 1% HCl-methanol solution, followed by an extraction step at 4°C in the dark for 20 min, and vacuum filtered to remove the pumice. Following the Folin–Ciocalteu method and the AlCl·OH) scavenging capacity assays. Determination of DPPH scavenging capacity was performed as described by 517 - sample OD517)/control OD517 ×100.Two different assays were applied to assess the total antioxidant capacity in the juice sac of pummelo fruit: DPPH and hydroxyl radical (·OH scavenging capacity was determined based on the SA–Fenton method as described by v/v) ethanol. The reaction system consisted of 0.5 mL of 9 mM iron sulfate, 0.5 mL of 9 mM SA (dissolved in ethanol), 0.5 mL of juice supernatant, 2.5 mL of deionized water and 0.5 mL of 8.8 mM H2O2. After the water bath at 37°C for 20 min, the absorbance of the mixture reaction was determine at 410 nm. The ·OH scavenging capacity was expressed as percentage (%) and calculated with the formula as follows: (control OD410 - sample OD410)/control OD410 ×100.The 2.7g for 30 min, and the supernatant was collected for assaying ROS-scavenging enzymes  activities.For the enzyme extraction and activity assay, all steps were carried out at 4°C. The extraction of crude enzyme was obtained by homogenizing 2.0 g of frozen juice sac powder with 8 mL of pre-cooled 100 mM phosphate buffer  and removing the sediment by the centrifugation at 12 000 × via a specific SOD test kit  by detecting the absorbance of the reaction system at 560 nm. SOD activity was reported as U g-1, where one unit (U) of SOD activity was equal to the photochemical reduction of nitroblue tetrazolium inhibited by 50% per minute. The activities of CAT (EC 1.11.1.6), APX (EC 1.11.1.11), and GR (EC 1.8.1.7) were measured as the description of -1 FW, where one unit (U) of CAT, APX, and GR activities was defined as an increase in the absorbance by 0.01 per minute at 240 nm, 290 nm, and 340 nm, respectively. POD (EC 1.11.1.7) activity was monitored in accordance with the guaiacol oxidation method as reported by 2O2 (diluted with 50 mM phosphate buffer) was adding to trigger the reaction mixture for POD activity containing 3 mL of 25 mM guaiacol solution and 0.3 mL of crude enzyme. POD activity was expressed as U g-1, where one unit (U) of POD activity was equal to the absorbance at 470 nm increased by 1 per minute.SOD (EC 1.15.1.1) activity was determined 2.8Actin (Cg8g022300) gene was used as the internal control gene. Specific primers of CmSOD (Cg7g011780), CmCAT (Cg3g025260), CmAPX (Cg6g002810), CmGR (Cg5g018970), CmPOD (Cg2g001370), and Actin were designed with Primer 5.0 and listed in -ΔΔCt method  method described by t method .2.9P< 0.05 or **P< 0.01) in All physico-biochemical parameters and gene expression data were acquired from three biological replicates (n = 3), each comprising a separate mixture of 10 pummelo fruit sampled per replicate. Duncan’s multiple range test was applied to analyze the data using SPSS version 20.0. Significant differences among the control and SA treatments are highlighted with lowercase letters in 33.1Color is a pivotal factor which influences the acceptability of the fruit by consumers. Surface color of ‘Jinshayou’ pummelo fruit turns from green to yellow during room temperature storage , pummelo fruit showed the highest TA content (0.46%) which dropped continuously with the stretch of storage period. A rapid degradation of TA was recorded in the control fruit as compared to its slow degradation in 0.2% and 0.3% SA-treated fruit from 30 to 90 d , ROS accumulation could lead to a decline in storage quality due to oxidative stress, as could the enzymatic antioxidant system, including SOD, CAT, APX, GR, and POD. As can be seen in CmCAT during the late 30 d of storage, which resulted in a higher overall CmCAT expression level by 42.7% compared to the control pummelo juice sac from 60 d to 90 d , respectively , both of them perform a pivotal role in the AsA-GSH system together with other enzymatic antioxidant systems to maintain redox homeostasis in postharvest fruits, and their amount can directly indicate the fruits’ ability to scavenge ROS . FigureAs the main secondary metabolites in plants, both phenolics and flavonoids are not only closely associated with the color conversion, flavor formation, and stress resistance of harvested fruits, but also protect them from over-produced ROS-caused oxidative damage . TPC is 3.52O2 content, MDA content, TPC, POD activity, and CmPOD expression (P< 0.05 or 0.01). The loss of fruit postharvest storability is due to the imbalance of ROS homeostasis, and result in an increase of defective fruit as well as the degradation of fruit quality. Pre-storage SA treatment was effective to enhance the antioxidant capability and reduce postharvest loss of pummelo fruit during room temperature storage. A similar finding was reported by 2O2 content, and MDA content, with the coefficients being 0.983, 0.966, and 0.974, respectively (P< 0.01), because it is highly related to oxidative stress and ROS accumulation. Moreover, ROS-scavenging enzymes activities  and expression levels of their encoding genes  exhibited high negative correlations with fruit decay rate and oxidative stress-related parameters (P< 0.05 or 0.01). Other studies have also proved that ROS accumulation leads to membrane lipid deterioration and is highly negatively correlated with the activities of antioxidant enzymes during longan fruit storage  and correlation analysis were applied to identify the ROS metabolism-related parameters that functionally referred to these measured parameters in pummelo fruit after 0.3% SA treatment. All 25 parameters linked to fruit postharvest ROS metabolism were mainly clustered into two main components (PC1: 66.719%; PC2: 20.72%,  storage . These f4Pummelo fruit, being a typical non-climacteric variety, is prone to a rapid decline in nutritional quality when stored at room temperature, thus limiting its storability. Numerous studies have revealed that pre-storage SA treatment prior to storage can delay postharvest senescence and quality deterioration of a variety of horticultural fruits, including apricot, blueberry, cornelian cherry, goji berry, papaya, pear, winter jujube, and tomato, as well as improve their resistance to abiotic stress . In thisThe deterioration of horticultural fruits’ nutritional quality, mainly due to their own respiration rate, is a major cause for fruit flavor loss and is regarded as a key metric to evaluate their storability and postharvest freshness during storage . As harv2O2 production (2O2, can cause the oxidation of unsaturated fatty acids, disrupting the cell membrane’s integrity, eventually leading to membrane lipid peroxidation (2O2 content and MDA content in both treatments increased dramatically from 30 d onwards (2O2, and MDA during room temperature storage was also reported by Citrus fruits are stored at room temperature for too long, resulting in a loss of cell membrane integrity, which can be determined through EL, MDA accumulation and Hoduction . Prolongxidation . In pumm onwards Figure 22 and H2O2; subsequently, the disproportionated H2O2 is directly or indirectly decomposed into O2 and H2O2 with the concerted effort from CAT, APX, GR, and POD (CmCAT, CmAPX, CmGR, and CmPOD were up-regulated by 0.3% SA treatment in pummelo fruit (2O2 content and the lower MDA accumulation (2O2 and the less level of oxidative stress in pummelo juice sacs are dependent on the improvement of SOD, CAT, POD, APX, and GR activities, and the up-regulation of their encoding gene expressions. Other reports also indicated that elevated ROS-scavenging enzyme activities and up-regulated their encoding gene expressions contributed to the balance of ROS homeostasis and the maintenance of postharvest storability in ‘Eureka’ lemon (It is a well-established notion that the maintenance of ROS homeostasis could decrease excess ROS accumulation and protects living cells from oxidative stress damage, which is vital for enhancing postharvest storability and maintaining overall quality in harvested fruits . SOD, CA and POD . Numerou and POD . To redulo fruit Figure 3a’ lemon , ‘Dahuana’ lemon , ‘Ningqia’ lemon , ‘Cornela’ lemon , and ‘Doa’ lemon , and ‘Maa’ lemon . Above fIn addition to enzymatic antioxidant system, the non-enzymatic antioxidant system also has its irreplaceable role in reducing the oxidative senescence in harvested fruits . In high5In summary, pre-storage treatment of 0.3% SA dipping was found be to effective in reducing postharvest fruit loss and maintaining the overall quality of ‘Jinshayou’ pummelo fruit. This was mainly due to 0.3% SA enhancing the activities of ROS-scavenging enzymes, along with up-regulating their corresponding gene expressions, maintaining higher levels of TSS, TA, AsA, GSH, TFC, and free radical scavenging capacity, and decreasing fruit decay incidence. All of these contributed to reducing oxidative stress and stabilizing the ROS homeostasis in pummelo juice sacs, thereby maintaining postharvest storability and overall quality in ‘Jinshayou’ pummelo fruit (The original contributions presented in the study are included in the article/QH: methodology, formal analysis, data curation, and writing-original draft. LH: methodology, investigation, and validation. JC: conceptualization, supervision, and funding acquisition. YZ: methodology, software, and investigation. WK: investigation and formal analysis. CC: conceptualization, supervision, project administration, and writing-review and editing. All authors contributed to the article and approved the submitted version.


Modulation of lipid metabolism is a well-established cancer hallmark, and SCD1 has been recognized as a key enzyme in promoting cancer cell growth, including in glioblastoma (GBM), the deadliest brain tumor and a paradigm of cancer resistance. The central goal of this work was to identify, by MS, the phospholipidome alterations resulting from the silencing of SCD1 in human GBM cells, in order to implement an innovative therapy to fight GBM cell resistance. With this purpose, RNAi technology was employed, and low serum-containing medium was used to mimic nutrient deficiency conditions, at which SCD1 is overexpressed. Besides the expected increase in the saturated to unsaturated fatty acid ratio in SCD1 silenced-GBM cells, a striking increase in polyunsaturated chains, particularly in phosphatidylethanolamine and cardiolipin species, was noticed and tentatively correlated with an increase in autophagy (evidenced by the increase in LC3BII/I ratio). The contribution of autophagy to mitigate the impact of SCD1 silencing on GBM cell viability and growth, whose modest inhibition could be correlated with the maintenance of energetically associated mitochondria, was evidenced by using autophagy inhibitors. In conclusion, SCD1 silencing could constitute an important tool to halt GBM resistance to the available treatments, especially when coupled with a mitochondria disrupter chemotherapeutic. LC-MS/MS was performed for each ion to obtain information on fatty acyl composition. The molecular species of each phospholipid class, identified from the interpretation of the MS/MS spectra of each ion, are listed in The different PL species were then identified in order to evaluate how the changes in the FA profile , observeThe PC profile of U87 cells C showed The molecular analysis of the SM class F showed Regarding CL molecular composition G, the moTaken together, the overall effects of SCD1 silencing in U87 cells led to a decrease in the abundance of PL species containing MUFAs. Consistently, this lipid composition change was translated into an increase in hydrocarbon chain conformational order, as assessed by the increase in fluorescence polarization of the DPH probe incorporated in liposomes prepared from the lipids extracted from the cells .Since SCD1 silencing affected CL molecular composition G and CL A primary requisite of cancer cells to ensure their rapid proliferation regards an enhanced supply of nucleic acids, proteins and lipids. With regard to lipid requirements, cancer cells have the capacity to switch from the uptake of fatty acids from the external medium (exogenous fatty acids) to the synthesis de novo of saturated fatty acids  that areThe rationale for the choice of these proteins to be analyzed in the context of the present work was as follows: ACC catalyzes a rate-limiting reaction in the biosynthesis of long-chain fatty acids, generating malonyl-CoA from acetyl-CoA, acting hence upstream of SCD1 and being indirectly regulated by this enzyme . Thus, wOther proteins also analyzed in U87 cells and NHAs were Akt, PI3K and HK-II, whose rationale is as follows. Akt is a serine/threonine kinase that regulates the activity and expression of multiple enzymes involved in glycolysis and fatty acid synthesis in cancer cells, thus contributing to their survival, with high levels of phosphorylated Akt being found in GBM patients with severe prognosis . In ordeFollowing these initial studies on the characterization of U87 cells grown in the standard and low-serum-content medium, all of the subsequent experiments were performed with cells grown in the latter medium, so that nutrient-starvation conditions were mimicked in vitro, and cellular outcomes of SCD1 silencing could be investigated and exploited from a therapeutic perspective.U87 cells grown in starvation conditions (2% serum) and transfected with siSCD1 presented significantly lower viability as compared to siNT-transfected cells 72 h after transfection B. When tA central aim of the present work was to identify the alterations in the phospholipid composition of the human GBM U87 cells promoted by SCD1 silencing, in order to highlight the contribution of lipid remodeling to the resistance of these cells to adverse conditions. Although SCD1 has been considered as a promising target for the treatment of a broad spectrum of human malignancies , as far As a first approach, an analysis of fatty acid relative contents in lipid extracts from cells grown in different conditions was performed . It is iCuriously, although the UFA (MUFA+PUFA)/SFA decreased as a consequence of SCD1 downregulation, the levels of PUFAs, per se, showed a significant increase, probably in an attempt to counteract the increased membrane order resulting from the predominance of SFAs over MUFAs, for example at the ER level, promoting lipotoxicity. Such putative PUFA-induced compensation of membrane biophysical properties could explain the relatively modest inhibition of cell proliferation induced by SCD1 silencing. On the other hand, the limited supply of lipids by the low-serum-culture medium suggests that PUFAs might have been provided by LDs in a lipophagy process. It is noteworthy that mammalian cells lack the ability to produce fatty acids of the n3 and n6 series; this is the reason why they need to obtain, from the diet, linoleic acid (18:2) and linolenic acid (18:3), which are precursors of n6 PUFAs  and n3 PUFAs , respectively. Since autophagy was promoted by SCD1 silencing, as evidenced by the observed increase in LC3BII/I ratio A, it couHowever, in terms of the heterogeneous membrane structure, the changes in the fatty acid profile of each PL class emerge as being much more meaningful than the overall alterations in membrane lipid fatty acid composition, the same assumption being applicable to the stability of the lipid monolayer of LDs. In fact, depending on its fatty acid composition, each PL class reveals a particular behavior in hydrophobic-driven lipid/protein associations , reflected by different lipid/lipid and lipid/protein interactions.It is remarkable that, although SCD1 downregulation had not modified the relative proportions of PL classes as well as the cholesterol/phospholipid molar ratio in U87 cells, fatty acid composition within each PL class was significantly altered, with a predictable impact on cell functioning, namely regarding mitochondrial bioenergetics and autophagy.PC species with a total of one or two unsaturations  were significantly decreased, which was accompanied by an increase in disaturated species  and those having a monounsaturated fatty acid but longer chains, i.e., 18:0/18:1. Such fatty acid composition changes in PC molecules might translate into more ordered lipid domains, thus favoring, for example, lipid raft stability .In contrast, the PE phospholipid, which is prone to adopt a cone shape when incorporated into polyunsaturated fatty acids, showed, upon SCD1 silencing, an increase in the species containing a saturated chain (18:0) and a polyunsaturated chain, namely 20:4, 22:5, 22:6 and, in the case of plasmenyl-ethanolamine, 20:5, thus anticipating a high propensity to generate lipid packing defects . RemarkaRegarding the SM species, a significant relative abundance of very asymmetric SM species containing a very long fatty acid chain with 24 carbon atoms, whose molecular geometry fits well with that of cholesterol in lipid rafts , was fouIt is not surprising that the predominant PI species in GBM contains a stearoyl and an arachidonoyl chain (18:0/20:4), since this combination of acyl chains is the most represented (≥70%) in the PI class in mammalian cells, the reason for such preference being a controversial issue . Other aAdditionally, at the level of the molecular species of CL, the pattern of decrease in MUFAs, accompanied by an increase in PUFAs, after SCD1 silencing, was quite evident. Interestingly, the level of CL with linoleic acid and polyunsaturated chains, and of symmetrical and, therefore, mature CLs  increaseCL plays a crucial role in the quality control of mitochondria, which increasingly appears to be a pressing need for cancer cells, despite the fact that their energy metabolism is of the glycolytic type . Two pro3, 10% (v/v) heat-inactivated fetal bovine serum , 100 U/mL penicillin and 100 µg/mL streptomycin. Normal human astrocytes (NHAs) were generously donated by Dr Anne Régnier-Vigouroux . NHAs were maintained in DMEM-HG supplemented with 10 mM NaHCO3, 2% (v/v) heat-inactivated FBS, 1% N-2 (Thermo Fisher Scientific), 1% non-essential amino acids, 100 U/mL penicillin and 100 µg/mL streptomycin. Cells were plated 24 h before any experiment at densities of 1.85 × 104 cells/cm2 for U87 and 1.55 × 104 cells/cm2 for NHA. All cells were cultured at 37 °C under a humidified atmosphere containing 5% CO2.U87 and U373 human glioblastoma (GBM) cells, kindly donated by Dr. Peter Canoll  and Dr. Ernst Wagner , respectively, were maintained in Dulbecco Modified Eagle Medium-high glucose  supplemented with 10 mM Hepes, 12 mM NaHCOThe siRNA duplexes targeting stearoyl-CoA desaturase-1  and a non-targeting siRNA duplex  were synthesized by GeneCust . 3-Methyladenine (3-MA) and wortmannin (WM) were obtained from Selleckchem , and stock solutions were prepared in DMSO  and stored at −20 °C. Stock solution of chloroquine  was freshly prepared in sterile water. Oligomycin A, rotenone and antimycin A were obtained from Santa Cruz Biotechnology , and stock solutions were prepared in DMSO and stored at −20 °C. Antibodies for SCD1 , ACC , pACC , AMPK , PI3K , AKT , pAKT , HK-II , LC3B  and α-tubulin (#3873) were purchased from Cell Signaling Technology . Antibody for β-actin (A5441) was purchased from Sigma. The secondary antibodies anti-rabbit IgG-AP (NIF1317) and anti-mouse IgG + IgM-AP (NIF1316) were obtained from GE Healthcare . All other reagents were obtained from Sigma unless stated otherwise.Complexes of siRNA with Lipofectamine RNAiMAX  were prepared as described previously . Twenty-CQ (25 µM), WM (5 µM) or 3-MA (5 mM) were added to U87 cells 20 h after siRNA transfection in medium containing 2% FBS followed by 48 h of incubation at 37 °C.w/v, 4 °C, overnight) at several timepoints for at least 5 days. Each well was washed with water, dried, stained using 200 µL SRB solution (0.057% w/v in 1% v/v acetic acid) for 1 h at 37 °C, washed with 1% (v/v) acetic acid, and dried. Cell-bound SRB was solubilized with 200 µL of Tris buffer , and the absorbance was measured at 510 nm in a microplate reader . The doubling time was calculated using the GraphPad Prism exponential growth curve equation.Cell proliferation was measured by the sulforhodamine B (SRB) colorimetric assay, as described previously ,55. U87 A570 and A600 are the absorbances of the samples and Cell viability was assessed by a modified Alamar Blue assay, using resazurin dye in growth medium, as described previously . The absg, 4 °C, 5 min) in ice-cold phosphate-buffered saline . Cells were resuspended in 100 µL of PBS, and 1 µL propidium iodide (PI) solution (50 µg/mL in PBS) was added to cells. After 15 min incubation in the dark at room temperature, 200 µL of PBS were added to each sample, and cells were immediately analyzed in a Becton Dickinson FACSCalibur flow cytometer  using a 488 nm argon excitation laser, and the emitted PI fluorescence was measured on FL3 with a 670 nm longpass filter. A minimum of 10,000 events were collected for each sample. PI positive staining cells were considered as dead cells.For total cell death assessment, U87 cells were collected and washed by centrifugation (300× E = 10S) and quantified using a NanoDrop 2000 (Thermo Fisher Scientific). For each sample, 500 ng of total RNA were converted into cDNA in the thermal cycler VWR Unocycler  using the NZY First-Strand cDNA Synthesis Kit . qRT-PCR was performed in a StepOnePlus Real-Time PCR System  using diluted cDNA (1:20 in RNase-free water) and the SsoAdvanced Universal SYBR Green Supermix . The reaction conditions were previously described . The pri2EDTA, 1 mM EGTA, 0.1% SDS, 1% NP40, 1% sodium deoxycholate, supplemented with a protease inhibitor cocktail , 0.5 mM DTT, 1 mM PMSF, 1 mM NaF and 1 mM Na3VO4 at 4 °C, followed by three freeze/thaw cycles in liquid nitrogen. Lysates were centrifuged at 16,000× g at 4 °C for 15 min, and the total protein content of the supernatants quantified using the Bio-Rad DC protein assay (Bio-Rad). Forty micrograms of total protein were diluted in Laemmli buffer, denatured upon 5 min incubation at 95 °C, and loaded onto a 7.5 or 10% polyacrylamide gel. After electrophoresis, proteins were blotted onto a PVDF membrane at 750 mA for 2 h  and blocked with 5% (w/v) non-fat milk in Tris-buffered saline with Tween-20  at room temperature for 1 h. Membranes were incubated overnight at 4 °C with rabbit anti-SCD1 (1:1000), anti-ACC (1:1000), anti-pACC (1:1000), anti-AMPK (1:1000), anti-PI3K (1:1000), anti-AKT (1:1000), anti-pAKT (1:1000), anti-HK-II (1:1000) or anti-LC3B (1:1000) primary antibodies. The membrane was then washed thrice with TBST (10 min each) and incubated with anti-rabbit alkaline phosphatase-labeled secondary antibody  at 4 °C for 2 h. After incubation with ECF substrate  at room temperature for a maximum of 5 min, protein bands were detected using a ChemiDoc Touch Imaging System (Bio-Rad). As an internal control for protein loading, the membrane was re-probed with mouse anti-α-tubulin (1:1000) or mouse anti-β-actin (1:5000) antibody and, thereafter, with anti-mouse alkaline phosphatase-labeled secondary antibody . Band intensities were obtained using the Quantity One software (Bio-Rad) and normalized to the corresponding α-tubulin or β-actin levels for each condition.Cells were lysed using lysis buffer containing 20 mM Tris-HCl (pH 7.5) 0.15 M NaCl, 1 mM NaCell pellets were resuspended in 1 mL of ice-cold water and sonicated. Aliquots (4 μL) were taken for protein quantification using the Bio-Rad DC protein assay (Bio-Rad). Then, total lipids were extracted from cells using the Bligh and Dyer procedure, as described previously .v/v) perchloric acid was added to the dried lipids, followed by incubation at 180 °C for 2 h in a heat block, with a glass marble covering each tube. Then, 0.825 mL water, 0.125 mL of 2.5% (w/v) ammonium molybdate, and 0.125 mL of 10% (w/v) ascorbic acid (freshly prepared) were added to each tube, followed by incubation for 10 min in a boiling water bath, and the absorbance of each sample was then measured in a microplate reader (SPECTRAmax PLUS 384) at 800 nm. The amount of phosphorus was determined from a standard curve obtained by using aliquots of a Phosphorus Standard Solution (Sigma), containing 3.25 to 156 nmol of phosphorus, which were submitted to the same treatment as the samples.Aliquots of lipid extract (50 μL) were placed into glass tubes and allowed to air dry. The total amount of phospholipid was quantified using a phosphorus assay, according to Rouser et al.  with somTotal cholesterol was quantified using the Amplex Red Cholesterol Assay Kit (Thermo Fisher Scientific). Briefly, aliquots of lipid extract (5 μL) were allowed to air dry and then suspended in the reaction buffer (160 μL) from the kit. Reactions took place in a black 96-well plate by the addition of 50 μL of Amplex Red working solution with 50 μL of assay sample, according to the manufacturer’s instructions. Fluorescence was measured in a fluorescence microplate reader  at excitation/emission of 560/590 nm.m/z 50–550 in a 1 s cycle in a full scan mode acquisition. The initial oven temperature was 80 °C, the first ramp rate was 25 °C·min−1 to 160 °C, then from 160 to 210 °C at 2 °C·min−1, and finally until 250 °C at 30 °C·min−1. The injector and detector temperatures were 220 and 250 °C, respectively. Helium was used as carrier gas at a flow rate of 1.3 mL·min−1. FAMEs were identified by comparison of spectra with those available in the Wiley275 database and the FAME archive from AOCS Lipid Library. The relative amounts of FAs were calculated by the percent area method, considering the sum of areas of the identified FAs.Fatty acid profiling was evaluated by the analysis of fatty acid methyl esters (FAMEs) obtained by transesterification . Brieflyv/v) and treated with boric acid in ethanol (1.8% w/v) and activated at 100 °C for 10 min. Then, the plates were spotted with 40 μg of phospholipid extract, and TLC plates were developed in a mixture of solvents consisting of chloroform/ethanol/water/triethylamine . To reveal the phospholipid spots, TLC plates were sprayed with a primuline solution  and visualized under UV light (λ = 340 nm). PLs were identified taking into consideration the distances of the spots from the point of origin and the front of the solvent , as compared to those of the spots of standard PLs run in the same plate. Two spots per sample were scraped off from the plates directly into tubes and quantified using the phosphorus assay, as described above. Prior to spectrophotometric determination, samples were centrifuged for 5 min at 500× g to promote silica sedimentation. The percentage of PL class was calculated relative to the total amount of phosphorus in the sample.Separation of PL classes by TLC from the lipid extract was performed using Whatman LK5 thin-layer plates and according to Vaden et al. . Brieflyv/v) with 10 mM ammonium acetate) and mobile phase B (acetonitrile:methanol 60:40 (v/v) with 10 mM ammonium acetate). Initially, 0% of mobile phase A was held isocratically for 10 min, followed by a linear increase to 100% of A within 10 min and a maintenance period of 25 min, returning to the initial conditions in 10 min. Aliquots of 25 μg of total phospholipid were diluted in 90 μL of mobile phase B, filtered, and 10 μL was introduced into the Ascentis Si column  with a flow rate of 40 µL min−1 and at 30 °C. The LXQ was operated in both positive (electrospray voltage +5 kV) and negative (electrospray voltage −4.7 kV) modes at 275 °C capillary temperature with a sheath gas flow of 8 U. Normalized collision energy (CE) was 27 (arbitrary units) for MS/MS. Data acquisition was carried out on an Xcalibur™ data system . Relative quantification of individual phospholipid species was achieved by determination of the ratio between the area of reconstructed ion chromatogram of a given m/z value against the sum of the reconstructed areas considered for each class.To perform LC-MS analysis of lipid extracts, a high-performance LC (HPLC) system (Thermo scientific Accela) coupled online to a linear ion trap  mass spectrometer was employed. The solvent system consisted of two mobile phases, as follows: mobile phase A  according to a protocol described previously , to whic2 incubator for 1 h. During the incubation time, 50 μL of 10 μM oligomycin, 55 μL of 10.1 μM FCCP, and 60 μL of 10.3 μM rotenone/10.3 μM antimycin A, prepared in assay medium, were sequentially loaded into the injection ports in the XFe24 sensor cartridge. Four OCR measurement cycles were performed before the addition of inhibitors (baseline OCR) and after the sequential addition of oligomycin (to inhibit ATP synthase), FCCP , and a mix of rotenone and antimycin A . Each OCR measurement cycle consisted of 2 min of mixing, 30 s of waiting, and 4 min of OCR recording. Mitochondrial respiration parameters, namely non-mitochondrial respiration (OCR after rotenone/antimycin A injection), mitochondrial basal respiration (baseline OCR minus rotenone/antimycin A response), ATP-linked respiration (baseline OCR minus oligomycin response), proton leak (oligomycin response minus rotenone/antimycin A response), maximal respiration (FCCP response minus rotenone/antimycin A response), spare respiratory capacity (maximum OCR minus baseline OCR) and coupling efficiency , were determined after OCR measurement normalization in terms of the protein content in each well, evaluated by the SRB assay.Real-time oxygen consumption rates (OCRs) were determined using the Seahorse XFe24 extracellular flux analyzer . Briefly, 96 h after plating the cells in XFe24 microplates, cells were washed in pre-warmed assay medium . Cells were then maintained in 450 μL/well of assay medium at 37 °C in a non-COp < 0.05.Results from all experiments were represented as mean ± SD. The value “n” refers to the number of independent experiments. Data were analyzed using GraphPad Prism 6 Software . Statistical significance was set at