Toll-Like Receptor Signalling Pathways Regulate Hypoxic Stress Induced Fibroblast Growth Factor but Not Vascular Endothelial Growth Factor-A in Human Microvascular Endothelial Cells
and Winfried M. Amoaku *Round 1
Reviewer 1 Report
It was a pleasure to read this manuscript. This experimental study was obviously carefully planned and executed. The presented results are relevant for further clinically orientated research projects.
Author Response
thank you very much for your comments
Reviewer 2 Report
In the manuscript 'Toll-like Receptor Signaling Pathways Regulate Hypoxic Stress Induced Fibroblast Growth Factor but not Vascular Endothelial Growth Factor-A in Human Microvascular Endothelial Cells', Akhtar et al investigated the effects of hypoxia on TLR activation in HMEC-1 cells. The authors found time-dependent increases in TLR activation and cell death which opens up avenues for future research and the targeting of TLR signaling as therapeutic in ophthalmic conditions.
I only have a few queries before I can recommend publication:
- Please add RRIDs to the antibodies on Page 5.
- Space too many on line 36, 237, 252, 286, 349
- Add gene/target names to individual panels of Figure 2, Figure 3, Figure 5 to enhance readability.
- Do the different colours in Figure 4 represent individual experiments?
Author Response
In the manuscript 'Toll-like Receptor Signaling Pathways Regulate Hypoxic Stress Induced Fibroblast Growth Factor but not Vascular Endothelial Growth Factor-A in Human Microvascular Endothelial Cells', Akhtar et al investigated the effects of hypoxia on TLR activation in HMEC-1 cells. The authors found time-dependent increases in TLR activation and cell death which opens up avenues for future research and the targeting of TLR signaling as therapeutic in ophthalmic conditions.
I only have a few queries before I can recommend publication:
Query.1 Please add RRIDs to the antibodies on Page 5.
Response.1 Many thanks for indicating this. We have added the RRIDs for each antibody on page 4 and page 5.
Query.2 Space too many on line 36, 237, 252, 286, 349
Response.2 Formatting will be applied by the journal at typesetting stage. We have removed the unwanted spaces where appropriate.
Query.3 Add gene/target names to individual panels of Figure 2, Figure 3, Figure 5 to enhance readability.
Response.3 Thank you. We have added the gene/target names in Figure 2. Please see the revised Figure 2 in the main text body.
Figure 3: Y-axis of each plot show the protein name. We did not amend this figure 3 as requested to avoid the redundancy of labelling.
Figure 5 has been clearly labelled. As detailed in the legend of figure, ‘A’ represents the western blot image and ‘B’ semi-quantitative signal intensity of bands in figure 5A.
Query.4 Do the different colours in Figure 4 represent individual experiments?
Response.4 Thank you for indicating this. We have explained the different colours in legend of the Figure 4. Please see the revised legend below:
Figure 4. Increased expression of TLR3 and TLR4 in HMEC-1 cells under hypoxia. Intracellular levels of TLR3 (A and B) and TLR4 (C and D) in HMEC-1 under hypoxia and normoxia for 24 and 48 hours, respectively was analyzed by flow cytometry. Green: Isotype control Ab staining; Blue: TLR Ab staining of normoxic cells; and Red: TLR Ab staining of hypoxic cells. Note: Magenta colour was a result of overlapping histogram of Normoxia (red) and Hypoxia (blue). All data is representative of three independent experiments.
