Dear Dr. Blackburn,

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Shuo Qie

Academic Editor

PLOS One

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“National Institutes of Health, Grant/Award Numbers R37CA227656 (to J.S.B.) and F99CA294265 (to V.D.O.) and the Kentucky Pediatric Cancer Research Trust Fund PON27282400002665. This research is also supported by the Flow Cytometry and Immune Monitoring Shared Resource of the Markey Cancer Center (P30CA177558).”

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Reviewer #1: Yes

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2. Has the protocol been described in sufficient detail??>

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: The Genetically Encoded Death Indicator (GEDI) is a ratiometric, dual-fluorescence biosensor that enables real-time detection of cell death through calcium influx. It was originally developed to study neurodegeneration (PMC8421388). In this manuscript, the authors modified GEDI to assess whether it can be used to assess cell death in cancer cells in vitro in cell culture. In order to show GEDI efficacy, the authors expressed the GEDI dual reporter in an H3K27M-pDMG (diffuse midline glioma) cell line and tested whether the GEDI reporter can accurately distinguish dying cells treated with radiation and JQ1, a bromodomain inhibitor that has been shown previously to potentiate killing in combination with radiation (PMC11213469). Radiation therapy is used to treat this disease. Overall, this protocol represents an innovation over existing approaches to assess cell death in live cells via microscopy.

The strengths of this manuscript are:

1. This protocol provides a way to assess the effect of treatment on apoptosis in live cancer cells using live imaging.

2. The data generated from this protocol can be analyzed and quantified using ImageJ software**,** which is free and easily accessible.

3. Based on the data provided, it appears that the GEDI reporter system may be more sensitive than the traditional caspase and CellTiter-Glo protocols.

4. Since the morphology of the cells is captured, if there are changes in the morphology of the resistant and sensitive cells, this protocol can assess these differences. Second, since the cells express a fluorescent protein, these cells could, in principle, be tracked over time in vitro and in vivo.

The limitations of this protocol stem from the following, which will need to be addressed by the authors.

Major considerations:

1. Use of a single cancer cell line in the analysis. Since this is a new cell death reporter that has not been used before in cancer cells, comparing among different cancer cell lines would greatly increase the utility and clarify the limitations for using this approach. Can the authors select another cell line from the same tumor type or another solid tumor to show that the data generated are reproducible?

2. The comparisons of the GEDI reporter to the CellTiter-Glo and caspase 3/7-based staining in Figures 3Band C that are routinely used to assess cell death are not "apples to apples" comparisons. There are live cell dyes to detect caspase 3/7 and annexin V staining in live cells using microscopy-imaging-based protocols. This would be a true comparison needed to assess whether the GEDI system is more sensitive than caspase fluorescence detection in cells. This analysis would be needed to show that the GEDI system is equivalent to or better than equivalent assays used in laboratories. Even if the GEDI system is equivalent, it could be advantageous for screening as the caspase and annexin V staining dyes, which are used in conjunction with nuclear-red or other color stains, tend to be expensive, limiting their use in screening.

3. The use of 25 and 8 Gy in the experiments is a problem as patients generally are not given single 25 or 8 Gy radiation doses but rather 25 or 8 Gy are given as cumulative 2-4 Gy daily doses of radiation. Hence, the non-physiological doses of radiation used in the initial characterization may not be representative of the conditions that typical screens may occur under. 4 Gy or cumulative dosing of 4 or 2 Gy over time might be more relevant.

4. Can the authors provide a more in-depth description in the materials and methods section of how the imaging of the cells was conducted? I.e., how often were the cells imaged, how many cells were plated, is the time point gap between serial images sufficient to track cells?

Minor comments:

1. Can the authors provide a figure in Figure 5 that can correlate GEDI ratios to absolute numbers of cells dying over the number of live cells?

2. In Figure 6, the claim that this protocol can be used to perform lineage tracing is not justified by the experiments shown. For lineage tracing, some kind of barcoding system would be needed as all the cells are labelled with the same color and there is no way to distinguish between cells.

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Reviewer #1: No

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A live cell biosensor protocol for high-resolution screening of therapy-resistant cancer cells

PONE-D-25-50553R1

Dear Dr. Jessica S. Blackburn,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Shuo Qie

Academic Editor

PLOS One

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