If Zatsune and Arakawa didn't cross this with their uncalled for attack on Miku and Rin, or when they activated an electric barrier around the stage, they definitely crossed this line when they trapped the audience, of which nearly a third of whom are children, in a wall of fire.
Zatsune crosses the line even further when she escapes from prison and decides to eliminate Miku, once and for all.
Miku and Rin, the way they are falselyblamed and endure a brutalbeatdown. It's especially bad for Rin, for she ended up passing out on the way to the hospital, then she learned she would have to undergo surgery and was reduced to a nervous crying wreck when she found out (She had never had surgery before). It's hard not to feel sorry for her during that scene. Miku gets another moment when she realizes that a homicidal Zatsune has broken out of prison and is rapidly making her way to the hospital to kill her.
The poor people in the audience during the concert, especially the 864 people that were hurt. They had merely come to see the Vocaloids perform live in the park, but soon they ended up basically forced to fight for their lives.