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To block lysosomal acidification we used bafilomycin A1 (BafA1). A, LN-308 or LNT-229 cells were co-treated with 10 nM BafA1 and 10 µM B10 under hypoxic conditions (1% O 2 ) for 48 h. Cell density was assessed by CV (n = 4, SD, *p<0.05). B, cell death was quantified by LDH-release after treating LN-308 or LNT-229 cells with BafA1 and B10 under hypoxic conditions (0.1% O 2 ) for 19 h (LN-308) or 27 h (LNT-229) (n = 4, SD, * = p<0.05). To inhibit cathepsin B we used the cathepsin inhibitor <t>CA074-Me.</t> C, LN-308 or LNT-229 cells were co-treated with 10 µM CA074-Me and 10 µM B10 under hypoxic conditions (1% O 2 ) for 48 h. Cell density was assessed by CV (n = 4, SD, *p<0.05). D, cell death was quantified by LDH release after treating LN-308 or LNT-229 cells with CA074-Me and B10 under hypoxic conditions (0.1% O 2 ) for 19 h (LN-308) or 25 h (LNT-229) (n = 4, SD, *p<0.05).
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To block lysosomal acidification we used bafilomycin A1 (BafA1). A, LN-308 or LNT-229 cells were co-treated with 10 nM BafA1 and 10 µM B10 under hypoxic conditions (1% O 2 ) for 48 h. Cell density was assessed by CV (n = 4, SD, *p<0.05). B, cell death was quantified by LDH-release after treating LN-308 or LNT-229 cells with BafA1 and B10 under hypoxic conditions (0.1% O 2 ) for 19 h (LN-308) or 27 h (LNT-229) (n = 4, SD, * = p<0.05). To inhibit cathepsin B we used the cathepsin inhibitor <t>CA074-Me.</t> C, LN-308 or LNT-229 cells were co-treated with 10 µM CA074-Me and 10 µM B10 under hypoxic conditions (1% O 2 ) for 48 h. Cell density was assessed by CV (n = 4, SD, *p<0.05). D, cell death was quantified by LDH release after treating LN-308 or LNT-229 cells with CA074-Me and B10 under hypoxic conditions (0.1% O 2 ) for 19 h (LN-308) or 25 h (LNT-229) (n = 4, SD, *p<0.05).
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To block lysosomal acidification we used bafilomycin A1 (BafA1). A, LN-308 or LNT-229 cells were co-treated with 10 nM BafA1 and 10 µM B10 under hypoxic conditions (1% O 2 ) for 48 h. Cell density was assessed by CV (n = 4, SD, *p<0.05). B, cell death was quantified by LDH-release after treating LN-308 or LNT-229 cells with BafA1 and B10 under hypoxic conditions (0.1% O 2 ) for 19 h (LN-308) or 27 h (LNT-229) (n = 4, SD, * = p<0.05). To inhibit cathepsin B we used the cathepsin inhibitor CA074-Me. C, LN-308 or LNT-229 cells were co-treated with 10 µM CA074-Me and 10 µM B10 under hypoxic conditions (1% O 2 ) for 48 h. Cell density was assessed by CV (n = 4, SD, *p<0.05). D, cell death was quantified by LDH release after treating LN-308 or LNT-229 cells with CA074-Me and B10 under hypoxic conditions (0.1% O 2 ) for 19 h (LN-308) or 25 h (LNT-229) (n = 4, SD, *p<0.05).

Journal: PLoS ONE

Article Title: Hypoxia Enhances the Antiglioma Cytotoxicity of B10, a Glycosylated Derivative of Betulinic Acid

doi: 10.1371/journal.pone.0094921

Figure Lengend Snippet: To block lysosomal acidification we used bafilomycin A1 (BafA1). A, LN-308 or LNT-229 cells were co-treated with 10 nM BafA1 and 10 µM B10 under hypoxic conditions (1% O 2 ) for 48 h. Cell density was assessed by CV (n = 4, SD, *p<0.05). B, cell death was quantified by LDH-release after treating LN-308 or LNT-229 cells with BafA1 and B10 under hypoxic conditions (0.1% O 2 ) for 19 h (LN-308) or 27 h (LNT-229) (n = 4, SD, * = p<0.05). To inhibit cathepsin B we used the cathepsin inhibitor CA074-Me. C, LN-308 or LNT-229 cells were co-treated with 10 µM CA074-Me and 10 µM B10 under hypoxic conditions (1% O 2 ) for 48 h. Cell density was assessed by CV (n = 4, SD, *p<0.05). D, cell death was quantified by LDH release after treating LN-308 or LNT-229 cells with CA074-Me and B10 under hypoxic conditions (0.1% O 2 ) for 19 h (LN-308) or 25 h (LNT-229) (n = 4, SD, *p<0.05).

Article Snippet: CA074-Me was purchased from Enzo Life Sciences (Lörrach, Germany).

Techniques: Blocking Assay

Using a pLKO.1-based lentivirus LN-308 cathepsin B (CTSB) knockdown cells and control cells (non-targeting sequence, Scrsh) were generated. CTSB gene suppression was confirmed by qRT-PCR (A) and CTSB activity (B) (n = 3, SD, *p<0.05). The effect of the cathepsin inhibitor CA074-Me (10 µM) on cathepsin B activity in control cells (Scrsh) is shown in B. C, B10-mediated cytotoxicity under 0.1% O 2 in LN-308 control cells and CTSBsh cells was quantified by LDH-release assay (n = 4, SD, *p<0.05).

Journal: PLoS ONE

Article Title: Hypoxia Enhances the Antiglioma Cytotoxicity of B10, a Glycosylated Derivative of Betulinic Acid

doi: 10.1371/journal.pone.0094921

Figure Lengend Snippet: Using a pLKO.1-based lentivirus LN-308 cathepsin B (CTSB) knockdown cells and control cells (non-targeting sequence, Scrsh) were generated. CTSB gene suppression was confirmed by qRT-PCR (A) and CTSB activity (B) (n = 3, SD, *p<0.05). The effect of the cathepsin inhibitor CA074-Me (10 µM) on cathepsin B activity in control cells (Scrsh) is shown in B. C, B10-mediated cytotoxicity under 0.1% O 2 in LN-308 control cells and CTSBsh cells was quantified by LDH-release assay (n = 4, SD, *p<0.05).

Article Snippet: CA074-Me was purchased from Enzo Life Sciences (Lörrach, Germany).

Techniques: Sequencing, Generated, Quantitative RT-PCR, Activity Assay, Lactate Dehydrogenase Assay