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* Minion's DisneyDeath.
** Especially Megamind's reaction to it. At first, he doesn't realize anything is wrong. Once he figures out that Minion's tank is empty of water and his friend is unconscious, there's just this sheer panic and desperation from him. And when he thinks that he's failed and that he was too late getting Minion back in the water, he just breaks down...
* Megamind apparently becoming convinced by Owen's "evidence" that Roxanne is cheating on him with Wayne and the two are laughing at him behind his back. In the end, it turns out to be a ruse to deceive Owen and win some time but still, it's pretty heartbreaking to watch him being tricked into thinking he's well and truly alone.