Fig 2: Comparative assessments of the CD133, PDGFRa and ALDH1 isoforms in isogenic GSCs by Western blot. “R1”, “R2” and “R3” designate different tumor regions from which isogenic GSCs were isolated. Isogenic GSCs show preferential expression of ALDH1A3 but not ALDH1A1. Considerable intratumoral variations in the levels of CD133 or PDFGRα do not mirror variations in the levels of ALDH1A3.

Fig 3: Assessments of ALDH1A1 and ALDH1A3 expression in GSCs by immunofluorescence staining. (a) GSCs cultured under self-renewal-promoting conditions and co-stained with anti-ALDH1A1 or anti-ALDH1A3 (red) and anti-nestin (green) antibodies. Counterstaining with DAPI. ALDH1A3 but not ALDH1A1 expression is readily detectable in self-renewing GSCs. Magnification 20×. Scale bars corresponds to 50 µM. (b) ALDH1A3 expression persists in GSCs under differentiation-inducing condition. GSCs were propagated under differentiation-inducing condition (withdrawal of self-renewal-promoting factors bFGF and EGF) and co-stained with anti-ALDH1A3 (red) and anti-nestin (green) antibodies. Counterstaining by DAPI. Magnification 20×. Scale bars correspond to 50 µM. Insets show enlarged images of individual cells demarcated by broken lines. The ALDH1A3 signal is readily seen in both the nuclear and cytoplasmic compartments.

Fig 4: Western blot assessments of ALDH1A3 by internally binding anti-ALDH1A3 antibodies: (a) pattern revealed by antibody PA5-29188 (aa126-423); (b) pattern revealed by antibody ab129815 (aa100-200). The abundance of truncated ALDH1A3 proteins relative to the full-length ALDH1A3 (ALDH1A3-FL) is much greater in GSCs than in non-stem glioma cells U87 and LN229.

Fig 5: Effects of proteasome inhibition by MG132 on the steady-state levels of ALDH1A3-FL and its truncated variants. (a) Western blot assessments of whole lysates of GSC#726/R1 cells treated with MG132 or DMSO (mock control). Increasing protein amounts (40, 60 and 80 µg per well) were loaded in lanes 1&4, 2&5 and 3&6, respectively. After probing with ALDH1A3 antibodies, the membrane was stripped and probed for actin. (b) Graphical presentation of the normalized data. The intensity of the bands corresponding to ALDH1A3-FL, ALDH1A3∆17 or ALDH1A3∆12 was quantified by densitometry and normalized to actin. The mock-treated control was set to 100 (black bars). Grey bars show the mean change in the abundance of ALDH1A3-FL, ALDH1A3∆17 or ALDH1A3∆12 after MG132 treatment compared to mock-treated control samples. Proteasome inhibition leads to a decrease in the abundance of truncated ALDH1A3 proteins and increase of the full-length ALDH1A3.