Atf4 siRNA(m)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	siRNA conentration-50 nm Add X-tremeGENE HP Reagent : DNA complexes to cells and incubate for 48h. Post 48h, induce cells for differentiation and again transfect to achieve persistent inhibition.

Protocol tips
siRNA conentration-50 nm Add X-tremeGENE HP Reagent : DNA complexes to cells and incubate for 48h. Post 48h, induce cells for differentiation and again transfect to achieve persistent inhibition.

Publication protocol

Predesigned mouse Stealth Select RNAi™ siRNA (catalog number MSS202234; Invitrogen) was used to specifically knockdown ATF4 in 3T3‐L1 cells. The Stealth RNAi™ siRNA Negative Control Med GC (catalog number 12935300; Invitrogen) was carried out as a control. 3T3‐L1 cells were transfected with each siRNA (50 nm) using X‐tremeGENE HP transfection reagent (Roche, Basel, Switzerland) when reaching approximately 90% confluence. Two days after transfection, cells were induced to differentiate as described above. Two days after the induction of differentiation, another transfection was performed to achieve persistent inhibition of ATF4 protein synthesis as described by Cignarelli et al. [42].

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