Mega Man: Defender of the Human Race has so many sad moments the author calls them "Defender Moments."
The first major one is in Episode 3, with the death of Doc the Metool. Roll's reaction to it makes it worse.
At the end of Episode 3, Mega Man has to talk Roll down from going after Wily herself after Doc's death.
Doc's funeral in the interlude to Episode 4.
Roll's horrified reaction when Gamma blasts Mega Man and blows his arm off in Episode 4.
ProtoMan going through with the plan to betray Mega Man in Episode 5. His internal conflict over it makes it worse, as does the scene where he lashes out at Mega, nearly killing him.... before they save each other's lives.
Roll and Dr. Light's reactions to Mega Man being framed for Kalinka's kidnapping and taken away for questioning in Episode 6.
Dr. Cossack's near-heroic sacrifice in Episode 6. He tells his deceased wife to keep the gates of heaven open for him, before being saved by Mega Man.
Episode 7 has Mega Man's life flashing before his eyes as Bass nearly kills him. He's saved by ProtoMan giving him an energy can and helping him take out Bass.
Episode 8 has Mega's reaction to Roll being trapped in the tower. He screams her name and pounds frantically on the walls, to no avail.
ProtoMan being demoted in the Episode 9 epilogue, as well as explicitly ordered to never leave the fortress without Wily's say-so.
Episode 10 has Mega lamenting that after the war his friendships might fade. Then later he discovers Kalinka's cutting scar, showing how badly she was hurting after her mom died.
Everything post home invasion in Episode 10, especially Roll and Peekaboo, and Kalinka's second kidnapping.
The climax of Episode 10, with Ballade's death and an innocent girl's paralyzation.
Everything in the epilogue of Episode 10. Tiesel finds out the hard way that he can't back out of his deal with the Conduit, Mr. Black is tormented over memories of his wife, Janet is paralyzed and hurt to such a degree that she can't be operated on, Mega Man is horribly traumatized over this and cries with Kalinka and his family over it, and ProtoMan is cold-bloodedly tortured by Wily even after begging him to stop.
The first chapter of Episode 11 continues the sad train, especially the last scene, featuring ProtoMan.
The second chapter of Episode 11 has Wily rigging a conversation between Mega Man and ProtoMan to hit them where it hurts, and tear apart their shaky familial bond. What makes it worse is when Proto deviates from Wily's script and says how he really does feel about Mega, and his resentment over getting no respect and love from anyone. It ends with Mega crying, and Centaur Man mockingly imitating him as he reports it to Wily.
Some choice quotes:
ProtoMan: ...Every time I see you, it's just a reminder of everything I'll never be... I hate you for it. Things would have been a lot better if... if you were dead.
Mega Man: When you felt bad for blasting that ion bomb in Times Square, I could tell you wanted a family to love you! You're just going to throw that away now?
ProtoMan: Maybe I do want a family, but no family wants me!
Mega Man: You might hate me and my family for who are and what we stand for... but, no matter what we went through, I never forgot that you were my brother! I always loved you!
The third chapter of Episode 11 has Mega Man coming home from the above scene and telling his family about it.
It also has both Proto and Mega reflecting on the park conversation from Episode 5, Dr. Light and Roll trying to help Mega Man through his depression, and Mr. Black remembering good times with his wife before she died.
Mega Man: I just want everything to be normal again... I don't want to fight anymore.
The fifth chapter of Episode 11 has Roll's life flashing before her eyes as she's buried in a collapsed building. While she survives, how she does can also bring tears, as Mega Man thanks Bass for saving her... in front of ProtoMan, who'd been forced to stop her from being saved.
The sixth Episode 11 chapter has Mega seeing the full video of ProtoMan's activation, Roll trying and failing to get Bass to change his ways, and Wily invoking and weaponizing a heartwarming moment between himself and Bass to hurt ProtoMan.
Episode 11, chapter 7, has ProtoMan's continued downward spiral and the slaughter of the entire army sent to fight with Mega Man.
Episode 11 chapter 8 has Rock deciding to quit being Mega Man, shattered over the trauma he and others have suffered.
It also has ProtoMan finally regaining his courage and confidence back after they were literally taken from him. And Wily burning the photograph of Proto and Mega together.
Episode 11, chapter 9, has Dr. Light giving in to despair. Roll desperately tries to convince Mega Man to rethink his decision to retire, but despite pressure he's unsure... so she slaps him and says if he lets Wily take over she'll never forgive him. Mega Man then reviews the footage and memories of all of Wily's prior attacks so he has a clearer mindset of what he's fighting against... and the humans he's fighting for.
Mr. Black's full memory of Tonya's death.
Episode 11, chapter 11 has the big moment. ProtoMan is forced to kill Mega Man via the Mad Grinder, and seems helpless to do so due to Wily's override command. But memories of his brother, Nomad, and Mega Man never doubting that he loves ProtoMan convince him to turn his back on Wily, saving his brother's life and the world.
The entirety of Episode 11, chapter 12. The whole chapter deals with the fallout from the previous one and Dr. Light, Roll, Rush, and ProtoMan doing their best to save a critically injured Mega Man. Some choice scenes:
Mega Man's injury is the big national news story.
Dr. Light being forced to sop up Mega's oil/blood with his lab coat.
Roll tearfully asking ProtoMan how he could hate her brother.
ProtoMan allowing his own internal regulator to be removed and swapped to help Mega...and that doesn't work until two hours later.
Proto taking off his visor to look on Mega Man with his own eyes. Dr. Light notices his face hasn't changed a bit since he left.
ProtoMan: ... I don't want you to die... I don't hate you, I never did... I love you, little brother. Dr. Light: There has to be one more miracle left in this old body of mine, Rock. And I intend to find it in time to save you.
Everyone from the Bonnes, the President, Governor Deacon, the New York police, and Kalinka Cossack reacting to the news of Mega's condition.
The symbolism of ProtoMan literally giving Mega Man his heart.
The chapter ends with happy tears, as Mega Man wakes up, remembers his family, and gives ProtoMan a real, brotherly hug.
After everything he's done, Dr. Wily gets one of these moments in the Episode 11 epilogue. Genuinely devastated over ProtoMan leaving, Wily considers why it happened... and realizes that it's too late to fix anything, it's his own fault, and by hurting ProtoMan he was no better than his own abusive father. He even contemplates giving up the war at one point. Even though his rage takes over soon after, it was a humanizing, surprising moment for a character many readers had come to see as a monster.
In the interlude to Episode 12, part 1, Wily is still down over the events of the previous chapters, which makes Bass declare him weak.
Also, 68 percent of New York distrusts ProtoMan. Both Mega and Proto see this news on TV.
The interlude to Episode 12, part 2, has a lot of sad or sobering moments involving ProtoMan, particularly how everyone cheering for the New Year stops when he steps forward.
Episode 12, chapter 2 has Miss T destroying Tonya's wedding dress.
The climax of Episode 12 has Mega Man calling the ambulance to help a possibly fatally-wounded Mr. Black even after the latter betrayed him.
Mega Man: I promised to defend the human race...even him.
The Episode 12 epilogue has the final scene, involving Mega Man and Mr. Black. Barton lives, and Mega Man convinces him to let go of his hatred and desire for revenge and help people. He agrees to, starting with Janet, and admits that thanks to Mega Man, he's changed his views on robots. To fully let go of his quest for revenge, he finishes the chess game he and Tonya were playing before she died—and has her piece win the match.
Splash Woman brainwashes Mega Man to love her and not Kalinka. This is played to heartbreaking effect.
A deleted scene would have made it even worse: Originally, Kalinka was going to see Splash Woman controlling Mega Man to kiss her, and Splash would have kissed him to hurt her. Kalinka would have to later be convinced that Mega wasn't acting that way of his own free will. It was scrapped because it would cast doubt on their relationship from that point on.
Episode 13 reveals that Nomad will try to kill Mega Man on sight due to Wily's programming. Both she and ProtoMan are distraught by this.
Episode 13 has a fake-out where it appears Plum is dead when her body is recovered from the ocean. She revives a few paragraphs later thanks to intravenous energy transmission, but it's a very tense, sad scene even after she wakes up.
In the Episode 13 epilogue, Roll meets Nomad and bonds with her. While heartwarming, it's also sad because of how Roll must keep it a secret from her family, and what Wily did to Nomad in the first place.
In the interlude to Episode 14, Tiesel Bonne gives footage of Mega and Kalinka's kiss to the Conduit, opening Mega Man and Kalinka up to tabloid scrutiny like never before should the footage be leaked.
In the interlude to Episode 14, Brain Bot is overjoyed when he sees the Stardroids, not knowing that the invaders do not come in peace.
At the start of Episode 14, Dr. Light realizes he hasn't spent much time with Mega Man and Roll even as their birthdays approach, and resolves to make it up to them. This is promptly derailed when the Stardroids invade.
After Mega Man, his siblings, and Bass are defeated by the Stardroids' initial attack, Mega Man asks Dr. Light if he's okay. Despite him assuring him that he's fine, the narration says Light is very worried that the war will cost him one or all of his children.