the oligomannose content of constructs produced in cho ‐ k1 cells was generally found to be higher than in cho ‐ s cells, except for constructs containing the asn site ( d221n / c309l / n297a / c575a and d221n / c575a ). however, the overall pattern in oligomannose content across the constructs and cell lines is unclear. in fact, high ‐ mannose n ‐ glycans are mostly present in low abundances ( 0. 8 % – 6. 1 % ), yet the control fc from cho ‐ k1 cells ( 17. 3 % ) and the d221n / c309l / n297a / c575a construct from cho ‐ s cells ( 15. 4 % ) contain unusually high levels of oligomannose structures. we cannot say why such differences in oligomannose processing emerged between the constructs; however, redox ‐ sensitive cysteine residues have been suggested as a possible influence on glyco sylation ( blundell et al. 2020; capellari et al. 1999 ). hypersialylated igg1 ‐ fc constructs produced in cho ‐ k1 cells could eventually serve as a basis for novel biopharmaceuticals that target autoimmune diseases via sialic acid receptor binding and treat viral influenza infections via hemagglutinin engagement. in light of the lower overall sialylation levels for constructs produced in cho ‐ s cells relative to cho ‐ k1 cells, cho ‐ s cells might not be a suitable production platform for such hypersialylated therapies. the general conclusions of this study are that cho ‐ k1 cells have a greater ability to process n ‐ glycans into larger and more complex structures than cho ‐ s cells. this is reflected particularly in the higher abundance of sialylated n ‐ glycans and the increased level of branching in cho ‐ k1 cells. the endo ‐ β ‐ galactosidase digests provided further confirmation of these patterns and also revealed that antibod y production was associated with increased branching in both cho ‐ k1 and cho ‐ s cells and reduced sialylation in cho ‐ k1 cells. additionally, the differences in the cellular n ‐ glyco mes were mirrored in a comparison of n ‐ glycans from a panel of igg1 ‐ fc constructs produced in cho ‐ k1 and cho ‐ s cells. our results suggest that cho ‐ k1 cells may be better suited for the manufacture of biopharmaceuticals where more complex, branched, or sialylated n ‐ glycans are beneficial. ultimately, we have shown that decades of genetic divergence in industrial cho cell lines are likely to have impacted glyco sylation capabilities. looking forward, further studies could elucidate whether natural variation, cell line ‐ specific glyco sylation capabilities or specific productivity are responsible for some of the trends observed in the data. in future, it would also be of interest to determine whether cell line ‐ specific differences are present in the o ‐ glyco sylation profiles of industrially relevant cho cells, which could help to inform the choice of cell line for o ‐ glyco sylated biologics. finally, understanding the glyco sylation machinery of cho cells is essential for the development of predictive in silico models for cellular and biopharmaceutical glyco sylation, and will ultimately help improve the effectiveness of glyco engineering strategies and the development of biobetters. discussion cell surface receptors and their coupled intracellular signaling pathways have been intensively studied in many physiological and pathological processes over five decades. due to the critical feature of these receptor - mediated signaling in promoting malignant proliferation, invasion, and metastasis, designing / seeking small molecules and monoclonal antibod ies that target receptors and downstream pathways has become a pivotal strategy in anti - cancer drug discovery. therefore, a better understanding of the key players in the signaling pathways may offer opportunities for therapeutic intervention. this study reveals a positive feedback dna - pk / myt1l - cxcr1 proliferative signaling loop that contributes to the progression of glioblastoma via the erk1 / 2 pathway and provides a novel insight into the role of dna - pk in the myt1l - mediated transactivation of cxcr1. myt1l is predominantly expressed in human brains, in particular fetal brains [ 32 ]. several key studies have demonstrated the pivotal role of myt1l in the direct conversion of human non - neuronal cells to neurons [ 33, 34, 35 ]. however, the contributing role of myt1l in carcinogenesis remains poorly understood. our previous studies have uncovered that mir - 141 was down-regulate d in glioblastoma and was able to suppress cell proliferation by directly targeting myt1l, implicating an oncogenic role of myt1l in glioblastoma. in this study, we found that in normal dna - pk glioblastoma m059k cell lines, knockdown of myt1l attenuated cell proliferation and induced apoptosis and s-phase cell cycle arrest ( figure 1a – d ). in contrast, the ectopic expression of myt1l promoted cell proliferation, inhibited apoptosis, and shortened the s phase ( figure 1e, f, h, j ). similar phenotypic alterations were also noted in hek293 cells in response to ectopic myt1l ( figure 1e – g, i ). these results indicated that in cooperation with dna - pk, myt1l may function as an oncogene in the progression of glioblastoma. however, in the loss - of - function dna - pk m059j cell line, the knockdown of myt1l promoted cell proliferation, attenuated apoptosis, and shortened the s phase ( figure 2a – d ). conversely, the enforced expression of myt1l inhibited cell proliferation and induced a g1 cell cycle arrest ( figure 2e – g ), suggesting a tumor suppressor role of myt1l in dna - pk - deficient glioblastoma cells, although it suppressed apoptosis ( figure 2h ). mechanically, the sustained activation of the akt pathway in both hek293 and m059k cells and the activation of the erk1 / 2 pathway in m059k cells ( figure 1k ) may contribute to cell proliferation induced by ectopic expression of myt1l, which supports the key role of these two well - studied oncogenic signaling pathways in the development of human malignancies [ 36 ]. however, the proliferative inhibition mediated by ectopic expression of myt1l in m059j cells may not be attributed to the activation of these two pathways ( figure 2i ). a total of 13 % apoptotic cells were also found in the negative control sirna group ( figure 1c ), which may reflect the cytotoxic effect of either transfection reagent ( lipofect amine 3000 ) or negative control sirna. the cell cycle is tightly regulated by cyclins, cdks, and cdk inhibitors. although cyclin e1 was down-regulate d in hek293, while it was up - regulated in m059k cells in response to myt1l expression ( figure 1k ), that may play a role in shortening the s phase ( figure 1g, h ) because of the critical requirement of cyclin e in the s phase progression [ 37, 38 ]. furthermore, p21, a well - known cdk inhibitor, was down-regulate d in hek293 and m059k cells in response to myt1l ( figure 1k ), which may also contribute to shortening the s phase. moreover, p21 has been shown to promote cell death in cancer cells via the activation of autophagy [ 39, 40, 41 ]; the down-regulate d p21 may be a causal player in attenuating apoptosis in hek293 cells that express myt1l ( figure 1i, j ). interestingly, cdk6 was down-regulate d in m059j cells in response to myt1l ( figure 2i ), which may play a role in a g1 cell cycle arrest ( figure 2g ). ectopic myt1l induced down - regulation of caspase 3 ( figure 1k, supplementary figure s1 ) in both hek293 and m509k cells, which may play a contributing role in apoptotic inhibition ( figure 1i, j ). however, this is not the case in m059j due to the absence of caspase 3 in both gfp - and myt1l - m059j cells ( figure 2i ). as a transcription factor, the transcriptional targets of myt1l, unfortunately, remain largely unknown. here, we discovered that myt1l could directly regulate the transcription of cxcr1 through a novel cis - acting element ( −2114 / −2103 ) at the cxcr1 promoter.