(A) Two types of human neurons were generated and analyzed. Td-Tomato⁺ motor neurons (iMNs) were derived from human iPSCs by small molecule treatment and purified using fluorescence-activated cell sorting (FACS) under the control of the HB9 promoter. In parallel, i³Neurons (i³Ns) were produced by doxycycline-induced NGN2 expression from isogenic, integrated human H1 ESCs (Supplemental Table 1). Both iMNs and i³Ns were subsequently transduced with lentivirus carrying shRNA targeting SMN1/2 (SMN-KD), followed by RNA-seq. (B and C) Volcano plots showing differentially expressed genes (DEGs) in iMNs (B) and i³Ns (C) following SMN-KD. Each plot displays log₂ fold change (x axis) versus negative log10 adjusted P-value (y axis). Genes significantly upregulated (red dots) or downregulated (blue dots) are defined by adjusted P < 0.01 and |log₂ fold change| > 1. (D and E) Venn diagrams integrating RNA-seq datasets from iMNs (red), i³Ns (blue), and laser-microdissected motor neurons from SMA model mice (yellow) (37). Downregulated DEGs (D) and upregulated DEGs (E) were defined by adjusted P < 0.01 and |log₂ fold change| > 1. Gene lists are provided in Supplemental Table 2. KIF5A was identified as a commonly downregulated gene. (F–I) Western blot analysis of i³N lysates following SMN-KD (F) and of primary mouse cortical neurons treated with siRNAs targeting Smn (H), confirming reduced SMN and KIF5A protein levels. Quantification of SMN and KIF5A protein levels relative to scrambled controls (SCR or Scr) is shown in G and I. Data represent 3 independent experiments. Statistical analysis was performed using an unpaired 2-tailed t test with Welch’s correction.