Supplementary Figure 6: Efficient knockdown of OSMR in mouse astrocytes and human BTSCs

(a) EGFRvIII-expressing BTSC73 was targeted by 3 different shRNA spanning the OSMR gene using lentiviral-mediated system as described in the Methods section. Representative panels of untransduced BTSC73 and BTSC73 transduced with either GFP-expressing scramble control or GFP-expressing-OSMR-knockdowns (KD) are shown. The brightest GFP positive cells in BTSC73 were FACS-sorted (right panel) following 1 week in culture. (b) RT-q-PCR analyses of OSMR-KD1, -KD2, and -KD3-BTSC73 and scrambled control-BTSC73 are shown. Gene expression was normalized to GAPDH. (c) Protein lysates of BTSC112 (expressing EGFRvIII and high level of phosphorylated STAT3), BTSC139 (not expressing EGFRvIII or high level of phosphorylated STAT3) and BTSC145 (not expressing EGFRvIII but expressing high level of phosphorylated STAT3) were subjected to immunoblotting analyses using antibodies indicated on the blot. Tubulin was used as loading control. (d) Correlation of phosphorylated STAT3 (Y705) with EGFR (Y1068) was obtained by performing Pearson analyses on clinical glioblastoma samples at TCGA for which proteomic data was available for these phosphorylation sites. (e) EGFRvIII/Stat3^loxP/loxP astrocytes were transduced with lentiviruses carrying either short hairpin RNAs (shRNA) against OSMR or a scrambled shRNA control. Transduced cells were selected with blasticidin. mRNA for EGFRvIII-expressing astrocytes that were transduced with either of shOSMR1 and 2 or scrambled shRNA was analyzed by q-RT-PCR using 5 different primer pairs (denoted 1, 2, 3, 4, 5 on the X axis) that were designed against different regions of Osmr gene. Expression was normalized to GAPDH (f) Protein lysates of EGFRvIII-expressing astrocytes were subjected to immunoblotting analyses using the OSMR antibody in the absence and presence of an OSMR blocking peptide (top panel) to confirm the specificity of the OSMR antibody. Tubulin was used as loading control. Lysates of OSMR-KD1, -KD2 and scrambled control EGFRvIII-expressing astrocytes were subjected to immunoblotting analyses using an OSMR antibody. (bottom panel). Tubulin was used as loading control. (g) OSMR-KD1, -KD2 and scrambled control were subjected to immunostaining with an antibody against OSMR and Hoechst. (h) OSMR-KD1, -KD2 and scrambled control were subjected to immunostaining with an antibody against ki67 and Hoechst.