1 2 3 M M M Stab1 315-HARE 190-HARE 170 kDa 130 kDa 100 kDa 70 kDa Vinculin Stabilins A. B. C. * ** Fig. S1 * Relative expression Fig. S1. Endocytosis of 125 I-ASO in cell lines treated with scrambled and specific anti-Stabilin siRNAs. (A) All cell lines were cultured in 24-well plates at least 3 days prior to the endocytosis experiment. Cells at 50% confluency were transfected with 5 pmol scrambled or Stab-specific siRNAs with RNAiMAX transfection reagent followed by a 2 day recovery period. The control and treated cells were then incubated with 0.1 mM 125 I-ASO alone or with 200- fold excess competitor for 1, 3, and 6 hrs. Cell associated radioactivity was measured and normalized to protein levels which were measured by the Bradford assay. The data is expressed as the mean±SD, n=3, *p≤0.05. (B) Cell lysates were separated by 6% SDS-PAGE electrophoresis, blotted to nitrocellulose, and probed with antibodies against the V5 epitope tag of the Stabilin receptors (red) and a load control protein, Vinculin (green). Lanes are labeled as 1) untreated, 2) scrambled control, 3) Stabilin siRNA, M) protein marker. (C) Densitometry of the Western blots was normalized to the untreated control set at 100% expression level.

Fig. S2 Fig. S2. Direct binding assay to test lot to lot s190 variation and negative controls. 200 ng of two batches of s190 (secreted form of the receptor) preparations, BSA, and DJ-1 were placed in polysorp wells with increasing concentrations of 125 I-ASO, washed with TBS-tween and the amount bound was measured by a gamma counter.

Fig. S3 A. Legend and Part B on next page

B. Fig. S3 Fig. S3. Confocal imagery of Stabilin-1 cells using ERTracker and LysoTracker. Stab1 cells were plated on glass coverslips and incubated media containing 0.05 µM Cy-3 ASO (excitation-emission: nm) for the indicated time points followed by a 30 minute incubation of 1.0 µM of LysoTracker ( nm) or ER-Tracker ( nm). Cells were fixed in 2% paraformaldehyde for 25 min and imaged by Olympus FV500 Inverted (Olympus IX-81) Confocal Microscope. Quantitative analysis of Cy3- ASO (red) vs. green (ER-Tracker and blue (LysoTracker) was based on channel splitting and overlay using Fiji Coloc-2 software. Data is expressed as the mean of at least 6 images at each time point for each fluor and statistically analyzed using Pearson’s correlation coefficient. The data is expressed as the mean±SD, n≥6, *p=0.021, **p≤0.001, NS=not significant.

Positive Mode ESI-MS Fig. S4 A.A. B.B. Fig. S4. Verification of ASO degradation using gel electrophoresis and electrospray ionization mass spectrometry (ESI-MS). (A) 33 mg (lanes 1 and 2) and 66 mg (lanes 3 and 4) of untreated/intact ASO (lanes 1 and 3) and acid treated ASO (lanes 2 and 4) were separated by 2% agarose gel electrophoresis (95 V, 1 hr) and the gel was stained with 0.005% Stainz-all in 50% ethanol. The bands indicated by the arrow are intact ASO as visualized by the stain. Degraded ASO remains unstained. (B) Both intact and acid treated ASOs diluted to 20 mM in 50% acetonitrile with 1% dipropylammonium acetate (5 mM) ion pairing reagent were directly infused at 5 mL/min. 20 averaged spectra were obtained in positive mode. ESI conditions: capillary voltage 4500V, drying gas 4L/min. Spectra obtained over m/z with an estimated resolving power of 49,000 at m/z of 400. Multiple peaks of the intact ASO (arrow) are indicative of the number of sodium atoms paired with the ASO molecule.

Fig. S5 A. B. C. D. Fig. S5. Acid treated (degraded) ASO vs intact ASO for evaluation of degraded ASO secreted from cells. As a control to evaluate degraded vs intact ASO, we incubated 125 I-labeled or unlabeled ASO in 2 M HCl for 1 hr at 90°C to degrade the ASO. In (A), both intact (red) and degraded (blue) preparations were processed by DEAE liquid chromatography and washed with 0.4 M NaCl (first 10 fractions collected of highly degraded ASO) and then washed with 2.0 M NaCl (fractions of moderately degraded ASO). Each fraction was measured by a gamma counter. The 14 th fraction represents the material (intact) that remained on the column which is 87% of untreated ASO and 21% of acid treated ASO. (B) Cells were pulsed for 2 hrs with 0.1 mM 125 I-ASO, washed 5x in HBSS, followed by a 6 hr chase in fresh Endocytosis media. At 6 hrs, the medium was centrifuged and the clarified media was passed over a DEAE column with the indicated fractions collected. (C) The experiment was repeated with EV and Stab1 cells revealing that the wash and elution profiles are nearly identical, (D) yet when the liquid chromatography trace was quantitatively analyzed, there was nearly twice as much degraded than intact ASO in the Stabilin-1 cells when compared with EV cells.

Fig. S6 Fig. S6. siRNA inhibition of Stab1 and Stab2 affect malat-1 expression. Both Stabilin-1 and 315-HARE (Stab2) cells were cultured with DMEM + 8% FBS + 50  g/mL hygromycin B in 24- well plates for at least 2 days prior to the experiment. Cells at 50% confluency were transfected with 5 pmol scrambled or Stab-specific siRNAs with RNAiMAX transfection reagent followed by a 2 day recovery period. On the day of the internalization experiment, cells were incubated with 10 nM malat-1 ASO for either 3 or 6 hours, washed once with HBSS, and incubated for a total of 24 hrs post ASO exposure in DMEM + 8% FBS, washed once with PBS, and harvested in RLT buffer containing beta-mercaptoethanol. Cells were assessed by qPCR for both malat-1 RNA and GAPDH RNA as a load/housekeeping control and the data is expressed as a percentage of malat-1 to GAPDH expression levels, mean±SD, n=3, *p=0.05.

Fig. S7 Fig. S7. Endocytosis assay of 125 I-ASO with glycosaminoglycan competitors. 315-HARE cells were cultured in 24-well plates 2 days prior to the experiment in which the cells were incubated with assay medium containing 0.1  M 125 I-ASO with 200-fold excess competitor for 1.5 hr. Cell associated radioactivity was measured and normalized to protein levels which were measured by the Bradford assay. The data is expressed as the mean±SD, n=3, *p=0.002, **p≤0.001.