Figure 2. NILV‐S/MAR‐mediated gene knockdown.

• A
  Schematic drawing of the lentiviral vectors used. Abbreviations as in Fig 1; RFP: red fluorescent protein; Luc2: codon‐optimized (for Homo sapiens) firefly luciferase; H1p: H1 RNA polymerase III promoter; shRFP: small hairpin RNA targeting the RFP transcript; shNT: small hairpin RNA with nontargeted control sequence.
• B
  The experimental setting for proof‐of‐concept experiment showing that a NILV‐S/MAR‐based vector can yield long‐term gene silencing.
• C
  Flow cytometry histograms of RFP expression (x‐axis) of A549/RFP ⁺Luc⁺ cells at days 20 and 27 after NILV‐S/MAR(GFP, shNT) or NILV‐S/MAR(GFP, shRFP) transduction.
• D
  Luciferase activity of the A549/RFP ⁺Luc⁺ cells at days 20 and 44 after NILV‐S/MAR(GFP, shNT) or NILV‐S/MAR(GFP, shRFP) transduction.
• E–H
  Representative fluorescence microscopy images show the expression of RFP from either NILV‐S/MAR‐transduced or untransduced A549/RFP ⁺Luc⁺ cells with cell nuclei stained with Hoechst 33342 (blue). RFP signal (orange) was collected with a fixed exposure time (300 ms) for equal comparison.

Data information: Data are shown as mean ± SEM. Statistical analysis was performed using one‐way ANOVA with Tukey's multiple comparisons test. Exact P‐values are reported in Appendix Table S1.