Hi there, I have two-time points(4 and 24), two groups (D and N) with three samples in each group, and five experimental conditions (untreated, treatment1, treatment 2, treatment 3, treatment4), so a total of 223*5=60 samples.

The questions are:

Does the treatment have an effect compared to untreated controlling by time point and group? I wrote design = ~group+time+condition. Should I include here nested samples since the treated-untreated are paired? How do you do that?

Are there differences between treatment and control at 4 (treatment/untreated at 4) or 24 (treatment/untreated at 24), or from 4 to 24(treatment/untreated at 4)/(treatment/untreated at 24) ? For this, I wrote design = ~group+stim.time after creating a variable combining stim and time. Is this correct? Should I include here nested samples since the treated-untreated are paired? How do you do that?

Are there differences in response to treatment (treatment/untreated) between groups at a specific time (either 4 or 24)? No Idea how to write this one. Should I include here nested samples since the treated-untreated are paired? How do you do that?

Are there differences between groups' response to treatment at 24 and 4 hours (treatment/untreated at 4)/(treatment/untreated at 24)? No Idea how to write this one. Should I include here nested samples since the treated-untreated are paired? How do you do that?

Thank you so much in advance

I unfortunately only have time to field software-related questions on the support site. For statistical consultation, I recommend contacting a local statistician at your institute, or someone familiar with setting up linear models in R.