Figure 5
Figure 5. A-I06 suppresses the expression of functional XBP-1, and A-I06–treated B cells phenocopy B cells deleted with XBP-1 gene. (A) Structures and chemical synthesis of A-I06, A-I07, STF-083010, and B-A05, including the x-ray structure of STF-083010 with hydrogens omitted for clarity. (B) Assessment of STF-083010 aqueous stability using RP-HPLC. Injection of the freshly prepared DMSO stock solution of crystalline STF-083010 showed only minimal decomposition. STF-083010 subjected to multiple freeze-thaw cycles showed significant breakdown. Dissolution of STF-083010 in a 1:1 DMSO:water mixture resulted in complete conversion to A-I06 and A-I07 after 1 hour. Results shown here are representative of 3 independent experiments. (C) Wild-type B cells were stimulated with LPS (20 μg/mL) for 48 hours to allow the expression of XBP-1, and subsequently treated with DMSO (control), STF-083010 (50μM), A-I06 (50μM), or A-I07 (50μM) for 24 hours. Cells were lysed and analyzed for the expression of XBP-1, IRE-1, calreticulin, p97, and actin by immunoblots using specific Abs. Data shown in immunoblots are representative of 3 independent experiments. For each experiment, wild-type B cells were purified and pooled from 2 mouse spleens. (D) CLL cells isolated from 8-month-old Eμ-TCL1 mice were cultured in the presence of LPS. Simultaneously, these cells were treated with DMSO (control), STF-083010 (50μM), A-I06 (50μM), or A-I07 (50μM) for 48 hours. Cells were lysed and analyzed for the expression of XBP-1, p97, and actin by immunoblots using specific Abs. Data shown in immunoblots are representative of 3 independent experiments. For each experiment, CLL cells were purified and pooled from 2 Eμ-TCL1 mouse spleens. (E) CLL cells isolated from 8-month-old Eμ-TCL1 mice were cultured in the presence of LPS. Simultaneously, these cells were treated with DMSO (control), STF-083010 (50μM), A-I06 (50μM), or A-I07 (50μM) for 48 hours. Cells were lysed in TRIzol reagent to extract RNA. Unspliced and spliced forms of mouse XBP-1 mRNA, and mouse actin mRNA were detected by reverse transcription followed by PCR using specific primers. Results are representative of 3 independent experiments. For each experiment, CLL cells were purified and pooled from 2 Eμ-TCL1 mouse spleens. (F) WaC3 cells were treated with DMSO (control), STF-083010 (50 μM), A-I06 (50μM), or A-I07 (50μM) for 72 hours, and subsequently lysed for RNA extraction. Unspliced and spliced forms of human XBP-1 mRNA, and human actin mRNA were detected by reverse transcription followed by PCR using specific primers. Results are representative of 3 independent experiments. (G) Wild-type B cells were cultured in the presence of LPS and A-I06 (50μM) for indicated times and lysed for analysis by immunoblots using Abs against μ heavy chain, p97 and actin. Data shown in immunoblots are representative of 3 independent experiments. For each experiment, wild-type B cells were purified and pooled from 2 mouse spleens.

A-I06 suppresses the expression of functional XBP-1, and A-I06–treated B cells phenocopy B cells deleted with XBP-1 gene. (A) Structures and chemical synthesis of A-I06, A-I07, STF-083010, and B-A05, including the x-ray structure of STF-083010 with hydrogens omitted for clarity. (B) Assessment of STF-083010 aqueous stability using RP-HPLC. Injection of the freshly prepared DMSO stock solution of crystalline STF-083010 showed only minimal decomposition. STF-083010 subjected to multiple freeze-thaw cycles showed significant breakdown. Dissolution of STF-083010 in a 1:1 DMSO:water mixture resulted in complete conversion to A-I06 and A-I07 after 1 hour. Results shown here are representative of 3 independent experiments. (C) Wild-type B cells were stimulated with LPS (20 μg/mL) for 48 hours to allow the expression of XBP-1, and subsequently treated with DMSO (control), STF-083010 (50μM), A-I06 (50μM), or A-I07 (50μM) for 24 hours. Cells were lysed and analyzed for the expression of XBP-1, IRE-1, calreticulin, p97, and actin by immunoblots using specific Abs. Data shown in immunoblots are representative of 3 independent experiments. For each experiment, wild-type B cells were purified and pooled from 2 mouse spleens. (D) CLL cells isolated from 8-month-old Eμ-TCL1 mice were cultured in the presence of LPS. Simultaneously, these cells were treated with DMSO (control), STF-083010 (50μM), A-I06 (50μM), or A-I07 (50μM) for 48 hours. Cells were lysed and analyzed for the expression of XBP-1, p97, and actin by immunoblots using specific Abs. Data shown in immunoblots are representative of 3 independent experiments. For each experiment, CLL cells were purified and pooled from 2 Eμ-TCL1 mouse spleens. (E) CLL cells isolated from 8-month-old Eμ-TCL1 mice were cultured in the presence of LPS. Simultaneously, these cells were treated with DMSO (control), STF-083010 (50μM), A-I06 (50μM), or A-I07 (50μM) for 48 hours. Cells were lysed in TRIzol reagent to extract RNA. Unspliced and spliced forms of mouse XBP-1 mRNA, and mouse actin mRNA were detected by reverse transcription followed by PCR using specific primers. Results are representative of 3 independent experiments. For each experiment, CLL cells were purified and pooled from 2 Eμ-TCL1 mouse spleens. (F) WaC3 cells were treated with DMSO (control), STF-083010 (50 μM), A-I06 (50μM), or A-I07 (50μM) for 72 hours, and subsequently lysed for RNA extraction. Unspliced and spliced forms of human XBP-1 mRNA, and human actin mRNA were detected by reverse transcription followed by PCR using specific primers. Results are representative of 3 independent experiments. (G) Wild-type B cells were cultured in the presence of LPS and A-I06 (50μM) for indicated times and lysed for analysis by immunoblots using Abs against μ heavy chain, p97 and actin. Data shown in immunoblots are representative of 3 independent experiments. For each experiment, wild-type B cells were purified and pooled from 2 mouse spleens.

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