The last short, Conclusion, is the short that focuses on Uncle Ben's death and Peter's rage against his killer. Peter tells it best;
Peter: [Uncle Ben] would've been proud of this trophy... but he'll never get a chance to see it. Because you know what else is made up of three different kinds of metal?
(cut to a shot of a gun being fired and a bullet appearing on a black screen)
This is actually the first animated version of Ben's death where Peter cries, which makes it all the more sad.
There's also Peter's gut-wrenching "WHAT HAVE I DONE?!" when he catches the burglar who killed his Uncle Ben and recognizes him as the guy he let escape earlier that night.
Series
Seeing Harry appear to be going down a slippery slope as he is making weapons at Horizon and many things are to come with this...and we always see the connection with Peter. It comes with many concerns...
And his dad treating him like crap, and limiting his time with Peter... poor guy.
Seeing the Jackal being taken away, as he's Gwen's uncle. It's on equal grounds to Uncle Ben's demise in that both Gwen and Peter have to lose a beloved uncle that inspired them.
Sandman and his daughter, full stop. After thinking he'd murdered Sandman, Hammerhead took in his daughter and raised her as his own, convincing her that her father never really cared about her, all the while transforming her into a weapon for his own purposes. It reaches a head when she murders him onscreen while he sobs and says he always loved her.Ouch.
"The Rise Of Doc Ock" has this in spades. Poor Gwen is convinced that her uncle is dead, and then has to relive his betrayal. Harry and Peter break off their friendship for a while after they watch Norman Osborn die. Worst of all, Doc Ock wins and escapes with 4 mind-controlled commandos and the Jackal.
"Spider Island Part Four": Whatever had to be going through Peter's head when he saw Aunt May turn into a Spider Monster.
"Venom Returns" has Eddie Brock, mid- Villainous Breakdown, admit that he feels like a total nobody without the Symbiote, which after all of J. Jonah Jameson's constant verbal tormenting of him makes it clear that Eddie has suffered from very poor self-esteem. His entire villainous scheme, and his somewhat jokingly crappy apartment, becomes a lot harsher in hindsight when you realize it's just a sad man lashing out at his own inadequacy; this can be Truth in Television as many people with a poor self-image have a hard time taking control of their life. It becomes even worse when Peter remarks that the Symbiote stays with Brock because it finally found the perfect host—someone as bitter and angry as it was.
"Hobgoblin Part Two": Norman frames Harry for attacking Spider-Man, trying to mold him into the hero he wants that will uphold the Osborn name. In the end, he forces Harry to choose between him and Spider-Man, which clearly tears up Harry inside.
"My Own Worst Enemy": Even after Otto takes over Peter's body, several of Peter's memories still remain and haunt Otto in flashes. The most prevalent of these? The night Uncle Ben died. We get to see Spider-Man's origin story play out all over again, but literally through Peter's eyes this time. It's telling that Peter's most stubborn memory that refuses to leave even when someone else is inhabiting his body is his guilt over Uncle Ben's death. And Otto ends up experiencing the full emotional weight of it, breaking down into tears.
"A Troubled Mind" reveals Otto Octavius' backstory, and it isn't pretty. Besides having to deal with the usual bullies, his own father physically and emotionally abused him because he was jealous of his son's intelligence. Seeing his father abuse him first hand in particular is really hard to watch. After having his accomplishments constantly overlooked or even outright belittled, it's honestly not hard to see how Otto became the way he is.
Otto's death in the Season 2 finale. He finally made a Heel–Face Turn, had a relationship starting with Anna-Maria, was helping the Spiders out in major ways, and even got a new job... only for him to make a Heroic Sacrifice to save the city from Vulture's mech. Made worse by the Hope Spot beforehand, as he himself said there was a 2% chance he'd live, only for him to dissolve into nothing in Peter's arms. They didn't even have anything to bury at the funeral, which itself was only attended by Peter, Ann-Maria, Harry, Max, Miles, Gwen, and Anya.
In "Vengeance of Venom", when the entire city is taken over by the symbiotes and Peter appears watching at distance, and ends up breaking down in tears.
