Fig 2: Localization of SIDT2 to the plasma membrane. (A) mRNA levels in HeLa cells were analyzed by RT-PCR. Expression of SIDT2 mRNA was detected. In contrast, SIDT1 mRNA was not detected in Hela cells. (B) HeLa cells were incubated with (+) or without (-) biotin, and a biotin-streptavidin pull-down assay was performed to purify cell surface proteins as described in the Materials and Methods section. Proteins were analyzed by western blotting using anti-SIDT2 (Abnova), anti-ß-actin (ACTB), anti-N-cadherin and anti-cathepsin D antibodies. (C) HeLa cells expressing EGFP-tagged SIDT2 were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Arrows indicate plasma membrane localization.

Fig 3: Effect of overexpression of SIDT2 on endocytosis. (A) Experimental paradigm for the confocal microscope analysis. (B and C) HeLa cells were transfected with empty vector, pCI-neo/mouse SIDT2 or pCI-neo/human SIDT2 plasmids, and cultured for 48 hours. Cells were washed with PBS, and incubated with or without 500 nM of Rhodamine B (B) or Rhodamine B isothiocyanate-dextran (C). Cells were washed with PBS again, and analyzed by confocal microscopy. Quantification of fluorescence intensity was performed. Error bars indicate s.d. (n = 3). *, n.s., not significant.

Fig 4: Knockdown of SIDT2 reduces the uptake of naked ssOligos. (A) Experimental paradigm for the confocal microscopic analysis. (B and C) HeLa cells were transfected with 10 nM of control siRNA (siControl) or siRNA against SIDT2 (siSIDT2#1 or #2). At 72 hours after transfection, SIDT2 mRNA levels (B) and SIDT2 protein levels (C) were analyzed. In the qRT-PCR analysis, SIDT2 mRNA levels were normalized against ß-actin mRNA levels (B). In the western blot analysis, anti-SIDT2 (custom antibody) and anti-ß-actin antibodies were used (C). Error bars indicate s.d. (n = 3). *, P < 0.05. (D–F) HeLa cells were transfected with siControl or siSIDT2#1 (D and E) and siControl or siSIDT2#2 (F). Seventy-two hours after transfection, cells were washed with PBS, and incubated with or without 1000 nM (D) or 500 nM (E and F) of the naked Alexa568-ssOligos for 6 hours. Cells were washed with PBS again, and analyzed by confocal microscopy. Quantification of fluorescence intensity was performed with ImageJ. Error bars indicate s.d. (n = 3). *, P < 0.05.

Fig 5: Knockdown of SIDT2 inhibits the antisense effect induced by gymnosis.(A) Experimental paradigm for the qRT-PCR analysis. (B and C) HeLa cells were transfected with siControl or siSIDT2#1 (B) and siControl or siSIDT2#2 (C). Seventy-two hours after transfection, cells were washed with PBS, incubated with (oligo+) or without (oligo–) 20 nM of the naked antisense oligonucleotides targeting miR–16 for 6 hours, and RNA levels were analyzed by qPCR. The miR-16 levels were normalized against ß-actin mRNA levels. The miR-16 levels were expressed as percentages compared to the levels of oligo– samples (expressed as 100%). Error bars indicate s.d. (n = 6). *, P < 0.05.