The 'Trigger Once' is stalking you remember that runs only one time an not anymore during the runtime of the layout until restarted. And for the Event on line 5 isn't working because you made it a subevent of line 4 Event so it won't trigger.
1. "Trigger Once" do just that trigger one time and will NEVER EVER EVER EVER EVER run again until the program is restarted or layout restarted. This is usually used for setting things up or something that absolutely does not have to run again.
2. Get better and knowing when something is a subevent of something else. So Line 4 Event 'If Sprite Animation Frame=2' has a sub-event of 'If Sprite Animation Frame not = 2' will never be true while it is a sub-event of 'if Sprite Animation Frame =2'. what you can just do is right click Sprite Animation = 2 and select the 'Else' Statement and destroy the sprite in there.