BAX activation in mouse retinal ganglion cells occurs in two temporally and mechanistically distinct steps

Table 1 Comparison of BAX translocation kinetics between tissue culture cells and retinal ganglion cells (RGCs) in ex vivo retinal imaging experiments

Cell Type	Number of Cells	Number of BAX Loci	Apoptotic Stimulus	Initiation of Translocation (Minutes)	Coefficient of Variation (Initiation)	Translocation Rate (Log₂RFU/min)	Coefficient of Variation (Rate)	Y-Maximum (RFU)
D407^a	42	682	STS	174 ± 32	2.5%	-1.8 ± 0.8	55%	3.8
HeLa^a	16	145	STS	158 ± 24	2.9%	-1.0 ± 0.6	154%	3.4
HCT116^a	36	339	STS	551 ± 52	1.0%	-1.8 ± 0.6	55%	4.1
661W	20	342	HDAC3 Overexpression	795 ± 63	0.6%	-2.2 ± 0.9	46%	3.2
RGC ONC	22	160	ONC	99 ± 24	12%	-5.1 ± 0.9	22%	1.5
RGC STS	10	52	STS	113 ± 34	14%	-6.0 ± 0.9	13%	1.4
RGC Retro – ON	5	22	ONC	77 ± 24	9%	-4.7 ± 0.8	30%	1.3
RGC Retro – OFF	5	16	ONC	n/a^b	n/c^c	+ 9.0 ± 4.4	n/c^c	-1.3

Apoptotic stimuli included staurosporine (STS), overexpression of histone deacetylase 3 (HDAC3), or optic nerve crush (ONC). “Zero times” for the initiation of translocation are based on either the addition of STS (D407, HeLa, HCT116 cells, and the RGC STS cohort), time from transfection of cells (661W cells), or time of animal euthanasia (RGC ONC and Retro cells). Retinal explants were harvested from eyes at 7 days after ONC (naïve retinas were used for STS experiments). The initiation times and translocation rates were calculated from the translocation data after fitting with a sigmoid function as previously described [11]. Means and standard deviations are derived from cell populations, where multiple loci within a cell were first averaged, and cells with fewer than 3 loci were omitted. Values for Coefficient of Variation within the cell population were calculated as described in the Methods. Y-Maximum refers to the number of relative fluorescence units (RFU) of fluorescently tagged-BAX that had accumulated at individual loci during an experiment. Values of RFU intensity were set to 1.0 at baseline. All data represents experiments using GFP-BAX
^aData for D407, HeLa, and HCT116 cells were described previously in reference [11]
^bNot applicable (n/a). Retrotranslocation was found to occur immediately after completion of maximum translocation in these cells
^cNot calculated (n/c) due to the low number of data points available

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