In the first episode you have: Cliffjumper's death, the Autobot's reactions (especially Arcee's) and Optimus' speech in his memory. It's very clear that he's trying to keep Arcee from running off and doing something stupid in grief, which is sad in itself.
Optimus: We must not allow our anger over the loss of Cliffjumper to impair our judgement. As of today, only we five Autobots remain on this earth. We owe it to ourselves... to the memory of Cybertron... to any Autobots in any galaxy seeking safe harbor... to humankind... and we owe it to the memory of our fallen comrade... to survive.
And seeing poor dead Cliffjumper being dragged into the room in the second episode, ready to be made a zombie. The sight of his mangled corpse strapped down, Energon still leaking from his mouth is a harsh one indeed.
During the battle in the Energon mine, when Arcee sets out to recover Cliffjumper's remains not yet knowing that he's already dead.
Arcee: Let's get you home, partner!
Her description of him after the battle makes it that much worse.
Optimus: Arcee, what did you see?
Arcee: Not Cliff... At least, not anymore. He was mutated, butchered... like something from those 'Con experiments during the war...
Skyquake's death in "Masters and Students." We see a shot of one of his eyes closing for the last time before a visibly saddened Optimus really drives it home.
Optimus: Had Skyquake chosen to stray from his master's path... we might not be burying him today.
In "Scrapheap", Arcee and Optimus are stuck dying of robo-hypothermia in the Arctic. As they begin to succumb to the cold, Arcee reaches up and holds Optimus's hand.
Arcee's terrifying flashback in "Predatory" of Tailgate's murder at the hands of Airachnid, especially the part when she helplessly cries his name after he gets killed.
Miko and Bulkhead trapped underground in "Rock Bottom", with Miko having a major breakdown.
In particular, seeing Miko rendered speechless out of worry, fear and sheer exhaustion is particularly touching, even for some viewers who don't like her. On top of that, she cried!
Some viewers actually started liking Miko from this moment, since she nearly killed herself trying to dig Bulkhead out of the fix and almost refused to leave him to his fate, even when Jack rescued her. Sometimes, you really do have to wonder just how bad Miko's life with her parents in Japan might actually have been, because based on this and the Hurt episode below, her attachment to her new robotic overseer truly runs much deeper than suicidal attempts at thrill-seeking.
On a more meta level for the episode, the dedication at the end of the episode to the late Captain H.L. Larry Cullen, brother of Peter Cullen, Shell-Shocked Veteran of The Vietnam War, and the inspiration for the voice of Optimus Prime. Truly understated. His influence on the series is not often thought of, but is truly incalculable. Before Peter auditioned for Optimus, Larry told him, "Don't be a tough hero; you are strong enough to be gentle."
Episode 21: Miko, first when she realizes that Bulkhead's losing his memory of her, and later when it looks like he's been left brain dead.
Miko crying in the parking lot made it even more of a tearjerker. The others were just there, hearing her cry.
Episode 22- Ratchet confessing his sins while almost bleeding to death.
Ratchet: Optimus... I've been a fool...
Raf being held on life support after being shot by Megatron. He was teetering on the edge of life.
Bumblebee's face when he saw him was heartbreaking. Not to mention it's one of the few times in which he almost rushes out to try and kill Megatron for doing it.
At the end of the series, Bumblebee finally does just that.
Add to that how Ratchet berates himself for being too prideful to bother learning about human science and medicine as he cannot find a way to treat Raf, and then pretty much confessing how attached he's gotten to Raf, which could also be seen in the previous episode, when Ratchet invited Raf to watch as he tested synthetic energon.
Bumblebee and Raf looking sadly at one another as Raf is driven away in "One Shall Rise, Part 1" is quite heart-wrenching. Thankfully, it's not too long before they're reunited.
June proves to be a Mama Bear for all three of the kids, chewing out the Autobots for placing them in such danger on a regular basis. Despite the best intentions of the Autobots, and that they are visibly crumbling, everyone agrees that June is not lying at all, and Optimus himself apologizes to June, saying "it deeply grieves me that I have failed". It's a very adult moment in which Optimus takes responsibility for Raf nearly dying and for always endangering June's child, and even June is moved and softens a little.
When Ratchet describes Optimus and Megatron's backstory, there's a scene where Orion Pax and Megatronus are standing next to each other smiling on a vibrant Cybertron. Megatron once genuinely wanted a better world, but both that Megatron and the world is now dead.
The end of "One Shall Rise, Part 3". For using the Matrix to take Unicron down, Optimus is completely unable to remember his life, save for his time as Orion Pax. Megatron tells him the Autobots are his mortal enemies and then summons a GroundBridge to the Nemesis. Before Optimus goes through, he sees the faces of the other Autobots, and is completely unable to recognise them. Bumblebee's face is once again the saddest part.
Especially the way Bumblebee hopelessly beeps "Optimus!"
The final moments of the episode in general are guranteed to tug at the heartstrings.
Seeing the Band of Brothers broken into an arguing mess after losing Optimus is a tear jerker in a "man, they really did need him" sort of way.
Arcee's reaction to being back on her dead homeworld of Cybertron and seeing the ruins of its pre-war civilization was extremely powerful.
Arcee: This isn't how I wanted you to see my home.
The way Orion Pax is treated throughout "Orion Pax, Part 3", culminating in him being beaten up by two Vehicons who talk about how good it feels, while he's preaching to their better nature. This leads to a Moment of Awesome (and a possible Moment Of Funny) when Orion realizes that he has guns and kills the Vehicons.
To elaborate on his treatment a little further: he's eventually forced to work at gunpoint, tries to reason with the Vehicons for the sake of others' safety, gets beaten up for his efforts and doesn't fight back (believing himself to be unarmed), then briefly pleads with them to stop before he finds his guns. Simply seeing the normally strong, in-control and completely badass leader of the Autobots reduced to begging fearfully, however briefly, will certainly strike the heart of any Transformers fan.
In Operation Bumblebee, Ratchet telling Raf about how the field medic who saved Bumblebee's life after Megatron tore out his voicebox and left him for dead wasn't able to fix Bumblebee's voice. Of course, he's speaking about his OWN inability to completely repair Bumblebee, since he was the "field medic" who saved Bee's life.
Ratchet: Yes, well... the medic could have done better.
Bumblebee's optics, all the time he's miserable over losing his T-Cog, and how everything reminds him of it. Those puppy-dog optics, just so big and sad, constantly in this episode. Oh, Bumblebee, we still love you!
When Bumblebee first realizes that he can't transform, the look in his eyes shows that he's almost terrified of what could be wrong with him.
Ratchet's heartwrenching plea to donate his T-Cog to Bumblebee, saying he rarely needs it anyways. Both the viewers and Optimus instantly see this as Ratchet's way of trying to atone for not being able to fix Bumblebee before.
A subtle one in "Loose Cannons" when Bulkhead has a Hope Spot that another Wrecker is alive. Then Wheeljack says that Dreadwing killed him.
"Crossfire" gives us Breakdown's death, which manages to be horrific and heartbreaking at the same time. His arm is pinned with webbing and Airachnid viciously tears him apart offscreen. What makes it so sad is that Breakdown was a relatively decent villain who treated his underlings well and, while a boisterous brute, generally avoided kicking the dog. The worst part of all, though, is that MECH rebuilds and eventually uses his corpse to use as a mech suit for a heavily injured Silas later in the season. Even Breakdown deserved better than that.
Optimus being gutted by Silas controlling Nemesis Prime. Thankfully, it wasn't fatal.
For all "Triage" Wheeljack had been gently teasing Ratchet by referring to him by nicknames, "Doc", "Sunshine" and the like. But the moment he apologies, Ratchet knows something's terribly wrong.
Ratchet: Wheeljack? I told you not to call me that!
In "Toxicity", Bulkhead slowly dying while trying to destroy the Tox-En throughout the episode can be pretty hard to watch. Also Hardshell's seemingly fatal attack at the end, leaving his fate uncertain.
Bulkhead's flashbacks. At the time, they made fans believe that Toxicity was going to be his last episode.
The ending of "Hurt" with Miko sitting atop the alive, but most likely permanently damaged Bulkhead.
And just before that, Wheeljack takes off because he can't stand to see Bulkhead like that, despite still being badly wounded from his fight with Hardshell.
Then they twisted the knife; the camera zooms in on Bulkhead's face, as if to show that his eyes are going to open again and everything will just revert to normal. They don't open...
"Out of the Past" has Arcee and Miko bonding at Cliffjumper's grave.
Arcee: At first I didn't think I'd ever get used to Cliff's constant chatter. Now, there's nothing louder to me than the silence.
In "New Recruit", we have Bulkhead's slowly growing jealousy of Smokescreen, not even bothering to listen to Miko for comfort. His resentment and self-loathing at his inability to help due to his injuries is just painful to watch.
Especially at the end when he limps off, without speaking, into the depths of the base, not even looking at Miko as she tries to reassure him.
There's also Ratchet's scorn towards Wheeljack due to events from "Hurt", it's especially sad since they had gained respect for one another a few episodes ago.
Smokescreen himself has a moment of genuine sadness when he admits that he's unaware of Alpha Trion's ultimate fate. He tries to cover it up seconds later, and is pretty good about not bringing it up anymore. But you can tell his failure to protect his mentor dose weigh on him more than he'll admit.
In "The Human Factor", Smokescreen offers the Apex Armor to Bulkhead in an effort to help him recover. Bulkhead immediately rages and smashes one of Ratchet's monitors in anger. Of course, that was the point.
Knock Out's initial reaction to meeting Breakdown!Silas, he goes from happiness, to concern, to rage.
By the end of "Legacy" you really want to give Jack and Smokescreen a big hug, especially Smokescreen. That dejected look Smokescreen wears while everybody chews him out doesn't help the situation, but it sure makes you feel for him.
Arcee and Optimus each come dangerously close to the Despair Event Horizon in "Alpha Omega" (ESPECIALLY Arcee). Seeing either of those two on their knees is a real hit to the gut.
In "Hard Knocks", Arcee chewing Smokescreen out yet again. To the point that Smokescreen promptly does a Rage Quit.
Optimus's cry to the heavens in "Inside Job" after Starscream successfully makes off with the Omega Keys. We've never seen him suffer a complete breakdown and lose his cool until now.
Starscream's Jerkass Woobie status is on full display in "Patch". There's a brief moment when we pan by his unconscious body and see him twitching, as though trapped in a nightmare. Which of course, he is.
Also, the moment where he begs to Megatron to just beat him instead of outright killing him.
That's nothing! After that he begs Megatron to just finish him off quickly because he can't stand the torture anymore. Think about that for a moment... Starscream was actually begging for Megatron to kill him.
Starscream: (this being one of his memories, seeing Megatron as the cave starts collapsing) I did not abandon you, Master! I returned to save you! Look into my spark, Lord Megatron! My intentions were true!
Megatron: (to Starscream, with both of them outside his memories) Were they, Starscream?! Were they EVER?!
Dreadwing's calm resignation towards the inevitability of his death as he sets forth to avenge his brother Skyquake's disgrace at the hands of Starscream; handing in The Forge of Solus Prime to Optimus Prime (whom he knows will bring peace and justice to Cybertron) as a final point of no-return. Though a warrior defined by honor and loyalty, family was more important still. What a terrible waste that such a noble spirit had become a Decepticon.
Part Tear Jerker, part Nightmare Fuel: when the Decepticons capture the human children and threaten to kill them via Cybertron's toxic atmosphere, Jack and Raf look absolutely terrifed. Miko, for her part, just looks pissed off, which is kind of badass of her.
For that matter, the very visible worry on the Autobots' faces when they see what's happening.
"Darkest Hour" may as well be renamed "Wettest Hour", given the sheer tears that will be flowing:
Even if he wasn't there and is just lashing out, you have to feel bad for Ratchet as he laments the permanent loss of Cybertron.
"Optimus... we needed that."
Made all the worse by the fact that he isn't just lashing out...his grief is a palpable thing, and very, very raw. When the Autobots are departing through the ground bridge, Ratchet doesn't even look back.
Ratchet: I...never imagined it would end like this.
'Optimus: Neither did I, old friend.
The evacuation of the Autobot base. It especially hits home when you see the look Miko gives Jack. Whenever Miko has her spirits crushed, you can't help but feel your own following along...
Starscream shoots down the Jackhammer. While Wheeljack has survived being shot down before, the way Starscream snarls the victory is brutal and saddening. Wheeljack might have been a loner, but he bought the team precious time to evacuate, and even rescued Fowler in the fighting.
Starscream, of all 'bots, gets a moment just after the Autobots escape back to Earth. The look on his face when he takes in the fact that his home may now be permanently dead. Then he starts screaming about how Optimus will pay for dooming Cybertron. No boasting or bragging, no snide remarks...he was just plain heartbroken. Hard to believe he was capable of that emotion.
To anyone who read the Season 2 synopsis during its debut and finished Fall of Cybertron will find "Darkest Hour" to be especially nerve-crushingly sorrowful. From the synopsis, we were hopeful that Cybertron would finally be revived. Not to mention, those of us from Fall Of Cybertron will feel especially heartbroken knowing the world we left behind may never come back again.
June's horrified, "no" when the base is destroyed, as she believes that Jack was inside. Fowler also is clearly heartbroken by it, before forcing himself to look away and retreat.
The base being destroyed and the build-up to it. On top of everything that's already occurred, the most haunting Ominous Latin Chanting imaginable begins, as the Nemesis prepares to open fire on the base. The camera cuts between that, June and Fowler's reactions, and Megatron watching from the nose of the Nemesis. One can only imagine the sheer hatred within him towards our heroes as he prepares to do this. All the while, Optimus has this expression of fear, and very shaky resolve on his face as he readies the Star Saber to destroy the Groundbridge so his team won't be found. He has no idea he's directly in the line of fire and his staying behind is meaningless. Then the Nemesis fires on the base, destroying it. As the horror of what just happened sinks in, the viewer feels the same pain as June and Fowler. The Autobots' home, and the viewers' home, has just been destroyed in cold blood. Even worse, a very heart-wrenching remix of the show's main theme plays as June and Fowler react in pain. We just lost the place we've called home for two years.
The absence of Alpha Trion after the War on Cybertron is vaguely hinted at, and the pieces to his mysterious fate are left to the viewer to put together. As we later find out, near the end of the war, the Decepticons discovered the location of Alpha Trion and the artifacts and began to storm the building. He used the Covenant of Primus to see a hazy glimpse into the future where the Autobots arrived on Earth, and in a last ditch effort, sent every artifact to the distant planet in hopes that they would find them all and a encoded message in the Star Saber. It's heavily implied that not long after he sends them off, the Decepticons were finally able to break into the facility and killed Alpha Trion.
Ratchet's bout of depression when Raf and Bumblebee first find him is sad to watch. While he never was the most optimistic of bots to begin with, the destruction of the Autobot base and loss of Optimus (who he quite understandable believes is dead) really seemed to hit him hardest of all the heroes.
Watching Smokescreen panic as Optimus starts to die.
Smokescreen: This isn't how the story's supposed to end!
Luckliy, he uses the forge to save Optimus.
After Optimus' Big Damn Heroes made possible by using the last of the power in The Forge of Solus Prime, you realize there's no hope left to repair the Omega Lock, and that Cybertron is truly dead.
Unable to take working with Ultra Magnus anymore, Wheeljack once again leaves the team. Mercifully, he's persuaded to stay later by Arcee, but it's revealed that he never truly got over Bulkhead transferring out of the Wreckers. Thankfully, Arcee helps him snap out of it.
In Thirst, Silas, the vile, murderous terrorist who tormented Autobots and Decepticons alike, ends up as a heavily tortured guinea pig for Knock Out's synthen experiments. It's initially satisfying to watch, but by the time Airachnid kills him, he's outright grateful, the one time he showed any genuine humanity. Even if Cylas was an amoral scumbag,the thought of losing your humanity in a horrific accident, going mad with power,and then getting tortured to the point of suicide is truly horrible.
In "Evolution", we're given reason to feel sympathy for Predaking of all bots, who, just after gaining self awareness, loses all of his newfound brethren created by Project Predacon. The way the cloned Predacons cry out just before they're blown to smithereens is as heartwrenching as it is terrifying, and Peter Mensah's performance totally nails the anguish Predaking feels over their demise. And on top of all that? The Decepticons set the whole thing up, and Predaking has absolutely no idea about the truth.
Predaking: What have you done to my brethren!? WHAT HAVE YOU DONE!?
At the beginning of the episode, everything is business as usual at the Autobot base. Optimus is out so Ultra Magnus starts trying to scold the other bots for one reason or another. When Optimus returns everyone rushes to get away from the commander (greeting Optimus as an excuse). Optimus looks up to see Magnus awkwardly standing alone far in the back. The thing that really drives it home is the fact that Prime himself obviously feels sympathy for Magnus.
Ultra Magnus eventually does win over the other Autobots while working together with Wheeljack... but the battle with Predaking ended with him brutally losing his right hand, along with his weapon, the Forge of Solus Prime.
Wheeljack:(injured) And here I was just beginning to tolerate you...
In addition to the seemingly hopeless situation, Optimus failing to protect the tracking beacon from Starscream. Giving us the impression that there was nothing the Autobots could do to save Ratchet now.
Bumblebee once again ends up on the business end of Megatron's cannon. Only this time, he actually dies and would've remained that way had it not been for the cybermatter. It's only made worse by the slow-motion it goes into after the first shot connects, the music, and the reactions of the other 'Bots.
Especially Optimus the horror in his eyes as he tries to stop Megatron, and as Bumblebee falls is enough to bring tears to your eyes.
Optimus was no doubt experiencing serious flashback horror for seeing the scout suffer at Megatron's hands again.
Then we get Optimus's Big "NO!", which puts his anguished cry from "Inside Job" to shame. A moment later we see him suffer a complete breakdown and let lose on Megatron like never before.
Megatron's death. Awesome as it was, watching the Decepticon Leader slowly die like a bug on a stick was utterly wrenching at the same time. All the good he originally set out to do as Megatronus isn't worth a thing at the end. The wasteful loss of life, home, and friendship. It's sad watching as the light in his eyes fades as he falls off the lock and into the atmosphere.
Starscream's rage over Megatron's death. In its own way, it really shows how Starscream's character has developed over the course of the series. It also shows how honest he really was when he decided to pledge his loyalty to Megatron after returning to the Decepticons. The fact that he of all bots attempts a Last Villain Standin spite of the fact that Megatron was horrible to him, and that Shockwave of all bots has to stop him, Starscream cursing at him all the while, just makes the moment even sadder.
If you look carefully even Optimus seems sad as Megatron dies. You can see that it does sadden him that in the end he couldn't convince his old friend to repent and he does have to be put down
Raf, Miko and Jack's final farewell to their Transformer Companions Bumblebee, now with voice, Bulkhead and Arcee respectively. It really tugs at heartstrings, seeing how reluctant they all are to see their Autobot Protectors leave Earth (after the course of 3 seasons) to go back to Cybertron. The final words between each exchange is what really sells the tear-jerking moments.
Yes but none of the humans from Prime appear, meaning that the first statement stands, the humans do never see Optimus again, indeed it's implied that because of the new council prevent Team Prime from returning to earth, so the humans never see any of the Autobots again, and don't know why.
Predaking lamenting his status as the Last of His Kind. Mercifully, he finds out that there are two more, even if they aren't very bright.
Megatron's Heel Realization and subsequent HeelFace Turn. That's right, the Big Bad ends up being broken and comes to understand the pain that he's been inflicting on others without a second thought... until now, and he simply has no idea what to do with himself. That last look he and Optimus share really says a lot.
Megatron: I now know the true meaning of oppression, and have thus lost my taste for inflicting it.
Optimus telling the team that he has to sacrifice himself to restore the AllSpark. None of them took it very well.
Ratchet seemed to take it the hardest.
Ratchet: Optimus, I didn't return to save a life only to lose the one I care most about...
Grimlock's flashbacks to being captured and tortured by Shockwave.
The Covenant of Primus
The murder of Solus becomes (if possible) even more tragic in the true version of the events. As it turns out, her murder was actually orchestrated by Liege Maximo. Turns out Megatronus and Solus had a strong bond of trust and faith in each other. But Maximo set the two against each other by sowing distrust among the pair, culminating in Megatronus unintentionally killing Solus during an argument.