STING/TUBA1B mediates neoliensinine-induced lysosomal disorders and lysosome-dependent cell death in T-cell malignancies (IMAGE)
Caption
(A) Gal3-GFP-expressing Jurkat cells were treated with 4 μmol/L of NeoL for 1 h, and immunolabeled for STING (red). Two cells in the same field under NeoL treatment were imaged by confocal microscopy. The white arrowhead indicates a cell with damaged lysosomes, and the yellow arrowhead shows a cell without obvious lysosome damage. The cell with lysosome damage was shown with an enlarged image to show co-localization of Gal3-GFP puncta with STING.
(B) Jurkat cells with the stable expression of Gal3-GFP were pretreated with 10 μmol/L of H151 (a STING inhibitor) or 10 μmol/L Dynasore (Dyn, a dynamin-dependent endocytosis blocker) for 1 h and then treated with 4 μmol/L of NeoL for 1 h, and imaged by confocal microscopy. The Gal3-GFP aggregates were labeled with 1–7 corresponding to increasing sizes of the puncta. Both the size and number of NeoL-induced Gal3-GFP puncta were reduced by H151 or Dyn treatment.
(C) Jurkat, Hut102, or Molt4 cells were pre-treated with 10 μmol/L of H151 for 1 h and then treated with 4 μmol/L of NeoL for 4 h. The cell death rates were determined by annexin V/PI staining (n ≥ 3). The STING inhibitor was shown to significantly attenuate cell death in three T-cell malignancy cell lines.
(D) The lysosomal membrane permeabilization (LMP) phenomenon was determined by immunofluorescence assay and co-localization of CTSL and LAMP1. STING KO Jurkat cells (#1 and #3) were treated with 0 or 4 μmol/L of NeoL for 2 h. STING KO was shown to partially reverse NeoL's effect on CTSL distribution.
(E) STING KO Jurkat cells (#1, #2, and #3) were treated with 0 or 4 μmol/L of NeoL for 4 h. The cell death rates were determined by annexin V/PI staining (n = 3). NeoL-induced cell death was decreased in three STING KO Jurkat clones.
(F) Co-localization of CTSL and LAMP1 was used to determine LMP phenomena in Jurkat cells pre-treated with 10 μmol/L of microtubule destabilizers, colchicine (Colc) or vinblastine sulfate (Vinb), for 1 h and then treated with 0 or 4 μmol/L of NeoL for 2 h. Microtubule destabilizers were shown to ameliorate NeoL-induced lysosome leakage.
(G) Jurkat cells were pre-treated for 1 h with 10 μmol/L of Colc or 10 μmol/L of Vinb, and then treated with 0 or 4 μmol/L NeoL for 4 h. The cell death rates were determined by annexin V/PI staining (n = 3). Both Colc and Vinb were shown to reduce NeoL-induced Jurkat cell death.
(H) Jurkat, Hut102, or Molt4 cells were pre-treated with 10 μmol/L of Dyn for 1 h and then treated with 4 μmol/L of NeoL for 4 h. The cell death rates were determined by annexin V/PI staining (n ≥ 3). Blocking dynamin-dependent lysosome endocytosis partially reduced NeoL-induced death rates in T-cell malignancy cells.
(I) The LMP phenomenon was determined by reduced co-localization of CTSL and LAMP1 immunofluorescence. Jurkat cells transfected with TUBA1B shRNA #1 showed improved co-localization of CTSL and LAMP1 under NeoL for 2 h.
(J) Jurkat cells transfected with TUBA1B shRNA #1, #2, or #3 were treated with 0 or 4 μmol/L of NeoL for 4 h. The cell death was determined by annexin V/PI staining (n = 3). TUBA1B knockdown reduced NeoL-induced death of Jurkat cells. P values were indicated in the graph, and P < 0.05 indicates statistical significance.
Credit
Po Hu, Yuxuan Huang, Zipeng Yu, Xiaolong Zhang, Guangming Yang, Yang Pan
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