The four questions
To decide whether SS-31 blocks BAX, the team broke the question into four parts. For each cell, they asked:
- Could BAX still gather on the mitochondria at all?
- Did BAX start gathering later than normal?
- Did BAX gather more slowly?
- Did less BAX gather in total?
If SS-31 truly blocks BAX, at least one of those should show a clear effect.
Question one: could BAX still gather?
Yes. Every cell treated with SS-31 and staurosporine was still able to recruit BAX to its mitochondria and go on to the leak stage.
And every single cell that showed BAX gathering also released cytochrome c, rapidly, right at the moment BAX started to gather. The same happened in untreated cells. The paper notes this is a typical feature of cells treated with staurosporine.
So SS-31 did not prevent the trigger being pulled.
Question two: did BAX start later?
The team wondered whether the earlier brain study's finding, less BAX on mitochondria, might be explained by a delay. If SS-31 made BAX start later, a snapshot at a fixed time would catch fewer cells that had reached that stage, and it would look like less BAX overall.
To test this, they recorded when BAX started gathering in every cell they imaged, across 13 imaging sessions. They then compared the share of imaged cells that went into BAX recruitment during the main window, 14 to 21 hours after staurosporine.
No significant difference. Not for any individual SS-31 dose against vehicle, and not for all SS-31 doses combined against vehicle.
So SS-31 did not delay the start.
Question three: did BAX gather more slowly?
This is where SS-31 did show an effect, and it needs to be read carefully.
When the team looked at the maximum speed of BAX gathering for each dose group separately, none of them was significantly slower than vehicle, although there was a trend toward lower speeds with SS-31.
They then combined all the SS-31 groups and compared the first 20 minutes of BAX gathering against vehicle cells over the same period. In that combined, paired analysis, BAX gathered significantly more slowly with SS-31. The statistical result was very strong.
They also looked at how long the whole gathering phase lasted:
- At the lowest dose, 0.01 micromolar, it lasted on average 9.6 minutes longer than in vehicle cells, a significant difference.
- With all doses combined, it lasted on average 4 minutes longer, also significant.
So SS-31 did slow BAX down. Modestly, but measurably.
Question four: did less BAX gather in total?
No. Because BAX gathered a bit more slowly but kept going a bit longer, the total amount that ended up on the mitochondria was the same in SS-31 and vehicle cells.
Picture two people filling identical buckets. One pours slightly slower but keeps pouring a little longer. Both buckets end up equally full.
Putting it together
| Question | Result with SS-31 |
|---|---|
| Could BAX still gather? | Yes, in every cell |
| Did cytochrome c still escape? | Yes, in every cell with BAX gathering |
| Did BAX start later? | No |
| Did BAX gather more slowly? | Slightly, when doses were combined |
| Did the gathering phase last longer? | Slightly, about 4 minutes on average |
| Did less BAX gather overall? | No |
The authors' conclusion follows directly: SS-31's protective effect is not at the level of BAX activity once staurosporine-induced apoptosis has started.
Why the slowdown is still interesting
It would be easy to dismiss the slowdown as too small to matter. Four extra minutes in a process that ends in cell death anyway does not save the cell.
But it is a real, measurable change. Something about SS-31 makes the mitochondrial surface slightly less welcoming to BAX. The authors suggest one possibility: SS-31 might partly reduce the movement of cardiolipin from the inner to the outer membrane, making the landing pad from Part 3 a little less effective. They are clear that this idea is untested.
That is the kind of clue that points future research in a useful direction.
How to square this with the brain study
These results do not prove the brain bleed study wrong. That study used a different model, rat brain tissue after an actual injury, and measured BAX at one moment 24 hours later.
There are reasonable ways both can be true. In a living brain, SS-31 might protect mitochondria earlier, before the death decision is made, so fewer cells ever reach the BAX stage. That would show up as less BAX in a snapshot, without SS-31 blocking BAX directly once the process starts.
What this paper rules out is the simple version: SS-31 standing in BAX's way once the trigger has been pulled.
Next, what the results say about how SS-31 does not work, and what that implies about how it might.
This series discusses a peer-reviewed laboratory study in cultured cells. The results have not been shown in people. Nothing here is medical advice, and SS-31 is discussed for research purposes only.
