Journal: bioRxiv

Article Title: HIV-1 gp120-induced lysosomal stress responses are controlled by TRPML1 redox sensors

doi: 10.64898/2026.03.02.709165

Figure Lengend Snippet: gp120-induced decreases in endolysosome Fe 2+ and increases in cytosolic Fe 2+ were mediated by endolysosome TRPML1 channels (A, B) Endolysosome Fe 2+ levels were measured with LysoRhoNox-1 by flow cytometry and data were presented as fold changes of mean fluorescence intensity (MFI). gp120-induced decreases in endolysosome Fe 2+ levels were significantly decreased by pre-treatment (1 h) with the TRPML1 inhibitors Ned-19 ( A , 1 μM) and ML-SI1 ( B , 10 μM). (C) Cytosolic Fe 2+ levels were measured with Phen Green FL DA by flow cytometry and data were presented as reciprocals of MFI (1/MFI). gp120-induced increases in cytosolic Fe 2+ levels were significantly decreased by the TRPML1 inhibitors ML-SI1 (10 µM), YM201 (5 µM), and Ned-19 (1 µM). YM201, but not ML-SI1 or Ned-19 significantly decreased basal cytosolic Fe 2+ levels. (D, E) Representative Western blot image ( D ) and quantification of ferritin H (FTH1) protein expression levels showed that gp120-induced increases in protein expression levels of FTH1 were blocked by 1 h treatments with Ned-19 (1 µM, 1 h). (F) Representative immunofluorescence images of TRPML1 (green) and nuclei stained with Hoechst 33342 (blue) in U87MG cells transfected with control shRNA or TRPML1 knockdown (TRPML1 KD); TRPML1 KD significantly decreased protein expression levels of TRPML1 by ∼41%. (G) TRPML1 KD significantly decreased basal cytosolic Fe 2+ levels and significantly decreased gp120-induced increases in cytosolic Fe 2+ levels. (H) TRPML1 KD significantly reduced ferric ammonium citrate (FAC)-induced increases in cytosolic Fe 2+ levels. (I) Pre-treatment (1 h) with the TRPML1 agonist NAADP-AM (1 µM) potentiated gp120-induced increases in cytosolic Fe 2+ levels. Data were shown as means and SEM with individual data points (n = 4-11) included on each bar. Two-way ANOVA with Tukey’s multiple comparison tests were used for statistical analyses. * p <0.05, ** p <0.01, *** p <0.001, **** p < 0.0001

Article Snippet: U87MG cells were transfected with either TRPML1 shRNA plasmid (Santa Cruz, Sc-44519) or control shRNA plasmid (Sc-108060) using Jet prime reagent as per manufacturer’s instructions.

Techniques: Flow Cytometry, Fluorescence, Western Blot, Expressing, Immunofluorescence, Staining, Transfection, Control, shRNA, Knockdown, Comparison

Journal: Journal of Translational Medicine

Article Title: Artemisinin conferred cytoprotection to human retinal pigment epithelial cells exposed to amiodarone-induced oxidative insult by activating the CaMKK2/AMPK/Nrf2 pathway

doi: 10.1186/s12967-024-05593-x

Figure Lengend Snippet: The Nrf2 pathway was involved in the cytoprotective effect of artemisinin on amiodarone-induced cell death in D407 cell cultures. ( a-b ) The cells were treated with artemisinin at various concentrations for 1 h. The Nrf2 and GAPDH protein levels were detected by western blotting, and protein bands were quantified by Image J ( n = 3). ( c ) The cell cultures were incubated with or without the Nrf2 inhibitor ML385 at concentrations ranging from 2.5 to 15 µM for 24 h, and cell viability was measured using MTT ( n = 4). ( d-e ) Cells attached to 12-well plates were treated with 5 or 10 µM ML385 for 24 h. The Nrf2 and GAPDH protein levels were detected by western blotting, and protein bands were quantified by Image J ( n = 4). ( f-g ) The cell cultures were pretreated with 5 µM ML385 for 6, 12, or 24 h, followed by 20 µM artemisinin for 1 h and 5 µM amiodarone for another 24 h. Cell viability was determined by the MTT assay ( n = 4), and cell death was detected through the LDH cytotoxicity assay ( n = 4). ( h-j ) Cells attached to 24-well plates were transfected with control shRNA (shCTL) or Nrf2 shRNA (shNrf2) plasmids (0.5 µg/well) for 72 h. The Nrf2, HO-1, and GAPDH protein levels were detected by western blotting, and protein bands were quantified by Image J ( n = 3). ( k ) Cells transfected with shCTL or shNrf2 plasmids were attached to 96-well plates, and pretreated with artemisinin for 1 h, then treated with amiodarone for another 24 h and processed for LDH assay ( n = 4)

Article Snippet: The human AMPKɑ2 shRNA plasmids (target sequence: TGTGAAAGAAGTGTGTGAA), control shRNA plasmids (CON207, target sequence: TTCTCCGAACGTGTCACGT), the AMPKɑ2-overexpressing plasmids, and control overexpressing plasmids (CON238) were purchased from GenePharma Co., Ltd. (Shanghai, China).

Techniques: Western Blot, Incubation, MTT Assay, LDH Cytotoxicity Assay, Transfection, Control, shRNA, Lactate Dehydrogenase Assay

Journal: Journal of Translational Medicine

Article Title: Artemisinin conferred cytoprotection to human retinal pigment epithelial cells exposed to amiodarone-induced oxidative insult by activating the CaMKK2/AMPK/Nrf2 pathway

doi: 10.1186/s12967-024-05593-x

Figure Lengend Snippet: AMPK-mediated activation of the Nrf2/HO-1 pathway was linked to the cytoprotective effect of artemisinin on amiodarone-induced apoptosis in D407 cell cultures. ( a-d ) The cell cultures were pretreated with 1 µM STO-609 for 30 min or 1 µM Compound C for 30 min, followed by 20 µM artemisinin for 1 h and 5 µM amiodarone for another 24 h. The HO-1, cleaved caspase-3, and GAPDH protein levels were detected by western blotting, and the protein bands were quantified by Image J ( n = 3). ( e-h ) Cells attached to 12-well plates were transfected with shCTL or AMPKɑ2 shRNA (shAMPKɑ2) plasmids (2 µg/well) for 48 h. The AMPKɑ2, HO-1, cleaved caspase-3, and GAPDH protein levels were detected by western blotting, and protein bands were quantified by Image J ( n = 3). ( i-j ) Cells attached to 24-well plates were transfected with the AMPKɑ2-overexpressing (OE-AMPKɑ2) or control-overexpressing (OE-CTL) plasmids (0.5 µg/well) for 24 h. The AMPKɑ2 and GAPDH protein levels were detected by western blotting, and protein bands were quantified by Image J ( n = 3). ( k ) The cell cultures transfected with OE-CTL or OE-AMPKɑ2 plasmids for 24 h were attached to 96-well plates and treated with 5 µM amiodarone for 24 h, and processed for the LDH assay ( n = 4)

Article Snippet: The human AMPKɑ2 shRNA plasmids (target sequence: TGTGAAAGAAGTGTGTGAA), control shRNA plasmids (CON207, target sequence: TTCTCCGAACGTGTCACGT), the AMPKɑ2-overexpressing plasmids, and control overexpressing plasmids (CON238) were purchased from GenePharma Co., Ltd. (Shanghai, China).

Techniques: Activation Assay, Western Blot, Transfection, shRNA, Control, Lactate Dehydrogenase Assay