PMCID string | Title string | Sentences string |
|---|---|---|
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Moreover, our results align with prior studies comparing Tau phosphorylation in the LAN-5 cell line (endogenous levels of Tau) and a CHO cell line with stable Tau overexpression. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Those studies similarly demonstrated high phosphorylation levels at S202 and S404 under conditions of stable overexpression of Tau . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Furthermore, these observations provide a plausible explanation for variations in mitosis-specific phosphorylation of the PHF1 epitope across different cell lines, as these variations likely depend on the baseline phosphorylation levels during interphase . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Second, by avoiding the use of microtubule polymerizing or depolymerizing drugs to arrest cells in M phase, we minimized potential indirect effects of these agents. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | This approach may explain some discrepancies between our findings and previously published data. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | For instance, we did not observe p-S214 as a mitosis-specific phospho-epitope, as reported by Illenberger et al. , who used nocodazole-treated cells. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Similarly, we did not identify AT100 as a mitosis-specific phospho-epitope, contrary to the findings of Delobel et al. , who studied progesterone-induced maturation of Xenopus oocytes and Tau-stably transfected neuroblastoma cells. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Given the strong Tau-specific staining we achieved with both p-S214 and AT100 antibodies under the same immunocytochemistry conditions on specific Drosophila tissues , we can exclude antibody inefficiency as a contributing factor. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Finally, among the tested phospho-epitopes, we identified novel mitosis-specific phosphorylation sites, which are p-T205, p-T217 and p-S416. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Additionally, we confirmed previously described mitosis-specific phosphorylation sites, such as AT8 and p-S422 . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | One important aspect of our study is the strategy used to validate in vitro findings in an in vivo system easily amenable to study cell division within an epithelial layer after three days of transient human Tau overexpression. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | We chose Drosophila melanogaster because this model proved useful to study human Tau phosphorylation in the nervous tissue and because the molecular mechanisms governing cell division are evolutionarily conserved across animal species . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | This model also enabled to validate the observed phospho-signature in non-cancerous cells. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | In this system, the phospho-epitopes AT8, p-T217, and p-S422 displayed clear and strong staining that was almost exclusively localized to mitotic cells, confirming their mitotic specificity. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | However, unlike the in vitro results, we observed distinct staining for p-T205 and p-S416 in non-mitotic cells. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | These findings are consistent with the increase in staining, upon Tau overexpression, observed during interphase under in vitro conditions: the fold increases in staining intensity for AT8, p-T217, and p-S422 were modest (1.42-fold, 1.48-fold, and 1.65-fold, respectively) compared to the more substantial increases observed for p-T205 and p-S416 (5.34-fold and 3.39-fold, respectively). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | This suggests that the slight increases in phosphorylation levels of p-T205 and p-S416 upon Tau overexpression during interphase in cell culture did not interfere with detecting additional phosphorylation at these sites during mitosis. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | However, in the Drosophila wing disc in vivo, this distinction was less apparent. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Whether this observation applies broadly to other tissues warrants further investigation. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Our study focused on investigating the physiological phosphorylation of Tau during the cell cycle. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | This work contributes to a broader effort to understand Tau phosphorylation under physiological conditions, such as during development or in response to hibernation and fluctuations in body temperature . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Here, we demonstrate that p-T217, a recently identified biomarker of early AD, is highly phosphorylated during mitosis, alongside the previously characterized AT8 and p-S422 phospho-epitopes. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | These findings lend support to the longstanding hypothesis that cell-cycle reentry occurs in degenerating neurons during AD . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Critically, these results may also provide mechanistic insight into the emerging significance of p-T217 as an AD biomarker. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Alongside the AT8 epitope, p-T217 is detected in post-mortem brains of both asymptomatic individuals (Braak stages 0–III) and symptomatic AD patients , suggesting that these phosphorylations occur early in disease progression, potentially during preclinical or mild cognitive impairment stages. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Furthermore, recent studies highlight that plasma p-T217 exhibits superior diagnostic accuracy for AD compared to other established tau biomarkers . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Hence, specific kinases implicated in phosphorylating T217 as well as AT8 epitope and S422, their differential activity during mitosis—and their potential link to AD pathogenesis—warrants further investigation. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Recent reviews and the Hanger lab’s updated database (bit.ly/2JyZTbS) indicate that kinases known to phosphorylate both S202 and T205 for AT8 epitope, as well as T217 and S422 are GSK3, p38, ERK1/2, JNK1-3, and CK1/2. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Some of these kinases (e.g., ERK) have emerging roles in G2/M transition or mitotic organelle distribution or in mitotic checkpoint (e.g., CK1, GSK3) . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Previous work on recombinant Tau and Lan5 cell extracts showed that the mitotic cdk1/cdc2 kinase can phosphorylate Tau within a T212/T217 peptide . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | To get further insight in the putative role of mitotic kinases like CDK1, Aurora, PLK and Nek , we used the GPS 6.0 software (https://gps.biocuckoo.cn/, accessed on 18 September 2025) to screen the 2N4R Tau sequence . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | We found that S202, T205, T217, and S422 match CDK1 and Nek2 consensus motifs (Supplementary Table S3). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | None of these aligned with Aurora or PLK consensus sequences. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Our work focused on the 4R isoform, which is more prone to aggregation , and overrepresented in insoluble fibrils in AD post-mortem tissues compared to the 3R isoform . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | While all phospho-sites tested are present in the 3R isoform, future studies should examine this isoform to determine whether the mitotic phospho-signature is isoform-dependent. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | A key unresolved question concerns the functional consequences of this mitotic phosphorylation pattern on Tau’s cellular roles. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | The most immediate hypothesis is that it triggers Tau detachment from microtubules, a known mitotic event . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Given Tau’s established roles in promoting microtubule growth and inhibiting microtubule shrinkage , its release from microtubules could facilitate the dynamic microtubule remodeling essential for spindle assembly and mitotic progression. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | However, whether this specific phospho-signature directly impairs Tau–microtubule binding remains to be experimentally validated. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Supporting this possibility, studies using a pseudo-phosphorylated TauE14 mutant—which mimics phosphorylation at all mitotic signature sites plus ten additional epitopes—demonstrate a dramatically reduced microtubule affinity . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Emerging evidence further suggests that modulating Tau phosphorylation in cancer cells can alter cell cycle progression . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Moving forward, it will be critical to dissect Tau’s functional contributions to mitotic microtubule dynamics and clarify how its phosphorylation state regulates these processes. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Such work may bridge critical gaps in our understanding of Tau’s dual roles in normal cell division and neurodegenerative pathology. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | MYCN and c-MYC are critical driver oncogenes in several childhood cancers, including neuroblastoma. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Currently, the clinical development of MYC inhibitors has been hindered by the intrinsically disordered structure of MYC proteins, which lack well-defined ligand-binding pockets. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Proliferation-associated protein 2G4 (PA2G4) directly binds to and stabilizes MYCN protein, leading to markedly increased MYCN levels in neuroblastoma cells. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Here, we demonstrate that PA2G4 is essential for MYCN-driven tumor growth in neuroblastoma in vivo. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Moreover, PA2G4 elevates c-MYC protein levels in neuroblastoma cells by inhibiting its ubiquitin-mediated degradation. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | In turn, c-MYC upregulates the transcription and protein expression of PA2G4, creating an oncogenic feed-forward expression loop. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | A small molecule PA2G4 inhibitor, WS6, directly disrupts the PA2G4-c-MYC protein–protein interaction, resulting in decreased levels of both PA2G4 and c-MYC. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | WS6 exhibited selective cytotoxicity in c-MYC-overexpressing cell lines. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Together, these findings identify PA2G4 as a shared cofactor for both the c-MYC and MYCN oncoproteins and highlight its interaction with MYC family oncoproteins as a promising therapeutic vulnerability in MYC-driven cancers. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | The MYC oncogene is deregulated in more than 50% of human cancers, including both adult and pediatric malignancies . |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | The MYC family of oncogenes (c-MYC, MYCN, and L-MYC) encode transcription factors, expressed ubiquitously across cells . |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | These transient “super-transcription factors” regulate over 15% of the human genome, controlling crucial cellular processes such as metabolism , proliferation , differentiation , and apoptosis . |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Despite their critical role in oncogenesis, direct therapeutic targeting of MYC proteins has proven difficult, as it is an intrinsically disordered protein without a specific active site and with no hydrophobic pockets . |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | As a transcription factor, MYC is located in the nucleus, making it inaccessible to monoclonal antibody therapies . |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Consequently, MYC has been labeled “undruggable,” and research has primarily focused on indirect approaches for targeting its function . |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | In recent years, extensive efforts have been made to inhibit MYC at various stages of its biogenesis and activity . |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | These include targeting MYC gene transcription, MYC mRNA translation, MYC/Max interaction , MYC protein stability , and MYC downstream pathways . |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | We previously identified a novel MYCN binding protein known as proliferation-associated protein 2G4 (PA2G4), which stabilizes MYCN and prevents its degradation . |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | One mechanism by which PA2G4 can cause MYC stabilization is by sequestering Fbxw7, a component of the ubiquitin–proteasome pathway , in the cytoplasm. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | In neuroblastoma cells, PA2G4 and MYCN act together in a forward feedback loop, driving continued tumorigenesis . |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | High PA2G4 expression independently predicts poor patient survival. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Targeting PA2G4/MYCN binding by WS6, a small molecule inhibitor, leads to reduced levels of both proteins and suppresses neuroblastoma tumorigenicity in vitro and in vivo . |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | While the PA2G4-MYCN interaction in neuroblastoma has been well characterized , little is known about its potential role in regulating c-MYC. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Notably, in high-risk neuroblastoma cases lacking MYCN amplification, c-MYC overexpression can serve as a functional driver, conferring similarly poor clinical outcomes . |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | MYCN and c-MYC share significant structural and functional homology , with both genes composed of three exons interrupted by two introns and showing 84% amino acid identity in common regions. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | In mouse models, MYCN can compensate for the normal functions of c-MYC, supporting survival, development, and reproduction in the absence of c-MYC . |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | This functional redundancy suggests a conserved oncogenic role for PA2G4 across MYC family members and raises the possibility that PA2G4 may also contribute to tumorigenesis through stabilization of c-MYC in c-MYC-driven malignancies. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | In this study, we reveal PA2G4 as a critical cofactor for both MYCN and c-MYC in neuroblastoma. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | We demonstrate that PA2G4 promotes MYCN-driven tumor growth in vivo and stabilizes c-MYC protein by preventing its proteasomal degradation. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Furthermore, we identify a feed-forward loop wherein c-MYC transcriptionally upregulates PA2G4 expression. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Targeting this oncogenic axis with the small-molecule inhibitor WS6 disrupts the PA2G4–c-MYC interaction, reduces oncoprotein levels, and selectively impairs the viability of c-MYC-overexpressing cells. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | These findings establish PA2G4 as a shared vulnerability in MYC-driven neuroblastoma and a promising therapeutic target. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Neuroblastoma cell lines (SH-SY5Y, SK-N-AS) and two normal lung fibroblast cell lines (MRC-5 and WI-38) were obtained from the American Type Culture Collection (ATCC). |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | SH-SY5Y and SK-N-AS cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen Life Technologies, Carlsbad, CA, USA) with 10% fetal Bovine serum (FBS). |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | MRC-5 and WI-38 were cultured in Alpha-MEM media (Invitrogen, Life Technologies) with 10% FBS. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Human B-Acute Lymphoblastic Leukemia (B-ALL) cell lines; KOPN-8, SEM-K2, and human Burkitt’s Lymphoma (BL) cell lines; and Raji and P493-6 were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies) supplemented with 10% FBS. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Murine B-cell ALL cell lines WEHI-47 and WEHI-116, derived from the Eµ-Myc mouse model (Walter and Eliza Hall Institute of Medical Research, Parkville, Australia), were maintained in DMEM supplemented with 10% FBS, 100 μM L-aspafbragine, and 55 μM β-metacaptoethanol. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Medulloblastoma cell line UW-288 was obtained from Dr Nick Gottardo (Telethon Kids Institute, Nedlands, Australia) and maintained in DMEM supplemented with 10% FBS. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | All cell lines used were authenticated by Cell Bank Australia (Westmead, Australia), free from mycoplasma, and cultured at 37 °C and 5% CO2 in a humidifier incubator. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | The P493-6 cell line was established from the stable transfection of EBNA2-conditional EREB2-5 cells (B-cells), with a c-MYC expression construct (pmyc-tet), and was provided by Stefania Purgato, Dipartimento di Farmacia e Biotecnologie (FABIT, Bologna, Italy). |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | To suppress the expression of c-MYC in P493-6 cells, 1 µg/mL doxycycline (Life Technologies) was added to cells growing in RPMI medium. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | P493-6 cells treated with doxycycline to repress c-MYC are referred to as P493-6 +Dox, and untreated cells are referred to as P493-6 -Dox. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Cell viability was assessed using resazurin. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | The reagent was prepared in phosphate-buffered saline (PBS) and contained 75 mg resazurin, 12.5 mg methylene blue, 164.5 mg potassium hexacyanoferrate (III), and 211 mg potassium hexacyanoferrate (II) trihydrate in 500 mL of PBS. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | After 6 h incubation with resazurin, the change in fluorescence was measured by Victor 3 multilabel Plate Reader (Perkin Elmer, Shelton, CT, USA) at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Protein was extracted from cell pellets using RIPA buffer (Sigma-Aldrich, Burlington, MA, USA) with 10% protease inhibitor (Sigma-Aldrich). |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Quantification of proteins was conducted using the Pierce BCA protein assay kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | A total of 30–50 μg protein samples were loaded onto Criterion TGX 10% precast gel (Bio-Rad, Gladesville, Australia) and transferred to a nitrocellulose membrane (Bio-Rad). |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | The membrane was blocked in 5% skim milk in Tris-buffered saline with Tween-20 (20 mM Tris-HCl (pH 7.6), 137 mM NaCl, and 0.1% Tween-20 for an hour before incubation at 4 °C overnight with the following primary antibodies: rabbit anti-cMYC (1:1000, Cell Signaling Technologies, Beverly, MA, USA), rabbit anti-PA2G4 (1:2000, Atlas Antibodies, Stockholm, Sweden, Cat# HPA016484), mouse anti-Vinculin (1:2000, Sigma-Aldrich, Cat# V9131), mouse anti-GAPDH (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc-365062), and rabbit anti-β-actin (1:5000, Sigma, Cat# SAB2100037). |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Anti-mouse or anti-rabbit horseradish peroxidase secondary antibodies (1:5000, Life Technologies, Cat# 31430, 31460) were added and incubated for 2 h at room temperature. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Immunoblots were visualized by Clarity ECL reagent (Bio-Rad) and ChemiDoc MP Imaging System (Bio-Rad). |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Quantification of protein expression was measured by Image Lab software v6.1 (Bio-Rad) and normalized to loading control. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | For siRNA-mediated knockdown, 40 nM of Qiagen (Millennium Science Australia, Victoria, Australia) custom PA2G4 duplex oligos (PA2G4 siRNA#1: 5′-GAGCAACAGGAGCAAACTATT-3′, PA2G4 siRNA#2: 5′-ACTGAGCCTGTGTGAGAAATT-3′, or cMYC siRNA#5: 5′-ATGCTATTGCTGTTCTAATTA-3′, cMYC siRNA #7 5′-GATGAGGAAGAAATCGATG-3′) were transfected with lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Dharmacon on-target plus control siRNA (Cat# D-001810-10-20) was used as siControl. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | Cells were transfected between 24, 48, 72, and 96 h, depending on the experimental requirements. |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | All experimental procedures involving mice were approved by the University of New South Wales Animal Care and Ethics Committee according to the Animal Research Act, 1985 (Australia), and the Australian Code of Practice for Care and Use of Animals for Scientific Purposes 2015 (Approval number: 20/68B, Approval date: April 2020). |
PMC12468391 | PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies | The TH-MYCN transgenic mouse model of neuroblastoma used has been previously described . |
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