Supplementary Figure 15: Investigation of the effects of JAGN1 knockdown on ER stress and the glycosylation, localization and functionality of G-CSF-R (CSF3R).

(a) The induction of ER stress was studied in HeLa cells by transfecting the cells first with JAGN1-specific oligonucleotides (numbered 1 and 2) and then treating them with thapsigargin. In protein blotting, BIP levels were detected as an indicator of ER stress, and knockdown of JAGN1 is shown. Actin was used as a loading control. No differences in induced BIP levels were observed between control siRNA and JAGN1-silenced samples. (b) Glycosylation of G-CSF-R was studied in more detail in JAGN1-silenced HeLa cells. Cells were first transfected with either control siRNA or JAGN1-specific siRNAs and were then transfected with construct encoding HA-tagged G-CSF-R. The samples were then either left untreated or treated with EndoH or PNGase enzymes. Protein blotting analysis shows HA-tagged G-CSF-R and actin as a loading control. No differences in glycosylation between control siRNA and JAGN1-silenced samples were detected. (c) The localization of G-CSF-R was further studied by immunofluorescence staining in JAGN1-silenced HeLa cells. The cells were first transfected with siRNAs as explained above and were then transiently transfected with a construct encoding GFP-tagged G-CSF-R (in the pMMP vector). G-CSF-R fused to GFP is detected on a green channel, and anti-G-CSF-R is detected on a red channel. DAPI was used to stain nuclei (blue). No differences in the localization of G-CSF-R between control siRNA and JAGN1-silenced samples were detected. Scale bar, 10 μm. (d) To study the effect of JAGN1 knockdown on G-CSF-R–dependent signaling, G-CSF-R–GFP was expressed in JAGN1-silenced HeLa cells. In contrast to cells treated with control siRNA, JAGN1 knockdown HeLa cells showed reduced phosphorylation of STAT3 upon exposure to rhG-CSF.