Proteomics characterisation of the L929 cell supernatant and its role in BMDM differentiation

L929 cell supernatant is commonly used to differentiate murine macrophages from bone marrow. The supernatant and its effect on macrophage phenotype was characterised by proteomics and the authors identified novel immunoregulatory proteins.

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Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance.
Sincerely, Shachi Bhatt, Ph.D. Executive Editor Life Science Alliance https://www.lsajournal.org/ Tweet @SciBhatt @LSAjournal Interested in an editorial career? EMBO Solutions is hiring a Scientific Editor to join the international Life Science Alliance team. Find out more herehttps://www.embo.org/documents/jobs/Vacancy_Notice_Scientific_editor_LSA.pdf The manuscript by Heap et al describes in-depth proteomic characterization of the L929 cell supernatant (secretome) used to differentiate macrophages from bone marrow followed by comparative proteomic analysis of bone marrow-derived macrophages using L929 secretome or M-CSF as the differentiating agent.
The authors identified more than 2000 proteins from L929 secretome and did additional bioinformatics analysis for these proteins. The data shows that there are many immune-regulatory proteins secreted and also point towards possible extracellular vesicle-mediated protein secretion. Following proteomic analysis of BMDMs upon three different differentiation process showed that L929 secretome induces slightly stronger anti-inflammatory M1 phenotype that those differentiated with M-CSF or M-CSF+MIF.
In general I think the manuscript is mostly well written and data is clearly presented, and the experiments are technically sound.
I have some comments that the authors should address in the revision: Table1: what was the selection criteria for proteins to be included in this? Suppl Table 1: the columns AA-AA show REF!, please correct Fig 1B: 'extracellular exosome' is the main GO class for the proteins identified; is there any evidence on extracellular vesicles present in the secretome? This possibility should be at least discussed The numbers of identified and quantified proteins in the secretome and BMDM do not match in mat+met and results (e.g lines 231-232, 247 and 341-342 as well as 239 and 268-269), these should be corrected. Fig 2B: are the up-and down-regulated proteins in the comparisons the same? That should be clearly shown; all the data in the rest of the figure is only from one comparison The paragraph starting at line 368 and following paragraph (results in Fig 3A): the text is slightly confusing/difficult to follow and should be clarified Reviewer #2 (Comments to the Authors (Required)): Dear editior Thanks for inviting me to evaluate this article entitled "Proteomics characterisation of the L929 cell supernatant and its role in BMDM differentiation".
In this paper, the researchers used quantitative mass spectrometry to characterise the kinetics of protein secretion from L929 cells over a two-week period. The results showed that there were a large number of M-CSF in LCCM and some of immune-regulatory proteins. In addition, macrophages differentiated with LCCM induced a stronger anti-inflammatory M1 phenotype. These results have certain reference value.
The structure of the article is well arranged and the logic is clear. But here are a few mistakes in this manuscript that need to be fixed: 1.Line424: please change "may by phagocytosed" to "may be phagocytosed". 2.Line 450: "L929 differentiated BMDMs" This expression is inconsistent with "l929-differentiated BMDMs" in line 447. 3.Line 454: This is also supported by the considerable higher number of BMDMs obtained "This"is ambiguous. 4.Line 460: This indicates that M-CSF differentiated macrophages are possibly more dendritic celllike. "This" is ambiguous.

Response to reviewers:
We thank both reviewers for their positive reviews.

Reviewer #1 (Comments to the Authors (Required)):
Table1: what was the selection criteria for proteins to be included in this? -Added that "selected for known immunoregulatory functions" Suppl Table 1 The paragraph starting at line 368 and following paragraph (results in Fig 3A): the text is slightly confusing/difficult to follow and should be clarified -we slightly changed the text to make it more clear.