mean fluorescence intensity  (Celigo Inc)

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: Mean fluorescence intensity (MFI) was analyzed using Imaris software (v9.9).

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

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: Ned-19 blocked gp120-induced increases in endolysosome ROS, lipid peroxidation, and nitric oxide. (A, C, E) Representative images of SH-SY5Y cells treated with vehicle (control), Ned-19 (1 μM, 1 h), gp120 (500 pM, 4 h) and Ned-19 plus gp120, and stained for ROS (CellROX, red), lipid peroxidation (Bodipy C11, green), nitric oxide (SiRNO, red), endolysosomes (LysoTracker, green or red), and nuclei (Hoechst 33342, blue). Scale bars were 15 µm (A) and 10 µm (C, E) . (A, C, E) Representative Imaris-generated surface rendering of Lysotracker-positive endolysosomes containing ROS, lipid peroxidation, and nitric oxide fluorescence. Scale bar = 1 µm. (B, D, F) Images were analyzed with Imaris software, and data were presented as fold-changes of mean fluorescence intensity (MFI) for CellROX, Bodipy C11 or SiRNO fluorescence within LysoTracker-positive endolysosomes. Each data point represents the fluorescence MFI within Lysotracker-positive endolysosomes from one field of view with a minimum of 100 cells analyzed per condition across at least two biological replicates. Data were shown as means and SEM with individual data points (n = 6 to 7). 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: Mean fluorescence intensity (MFI) was analyzed using Imaris software (v9.9).

Techniques: Control, Staining, Generated, Fluorescence, Software, Comparison

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: Ned-19 blocked gp120-induced decreases in endolysosome GSH and H 2 S, but not increases in H 2 S n . (A,C,E) Representative images of SH-SY5Y cells treated (1 h) with vehicle (control), Ned-19 (1 μM), gp120 (500 pM), and Ned-19 plus gp120. Cells were stained for GSH (RT-AM-GSH, blue/green), H 2 S (P3, green), sulfane sulfur (SSP4, green), endolysosomes (LysoTracker, red), and nuclei (Hoechst 33342, blue). Scale bars = 10 µm. (A,C,E) Representative Imaris-generated surface rendering of LysoTracker-positive endolysosomes containing GSH, H 2 S, or H 2 Sn. Scale bar = 1 µm. (B,D,F) Images were analyzed with Imaris software and data were presented as fold-changes of mean fluorescence intensity (MFI) of RT-AM GSH, P3 and SSP4 fluorescence within LysoTracker-positive endolysosomes. Each data point represents MFI within Lysotracker-positive endolysosomes from one field of view with a minimum of 100 cells analyzed per condition across at least two biological replicates. Data were shown as means and SEM with individual data points (n = 6 to 7). 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: Mean fluorescence intensity (MFI) was analyzed using Imaris software (v9.9).

Techniques: Control, Staining, Generated, Software, Fluorescence, Comparison

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: Ned-19 blocked gp120-induced increases in endolysosome protein cysteine oxidation. (A) Representative images of SH-SY5Y cells pre-treated for 1 h with Ned-19 (1 μM) or vehicle before treatment for 1 h with gp120 (500 pM). Cells were stained for protein cysteine oxidation (PCO; DCP-Rho-1, red), endolysosomes (LysoTracker, green) and nuclei (Hoechst 33342, blue). Scale bars = 10 µm. The bottom panels showed representative Imaris-generated surface rendering of Lysotracker-positive endolysosomes containing PCO fluorescence. Scale bar = 1 µm. (B) Quantification of the fold changes of mean fluorescence intensity (MFI) of DCP-Rho-1 staining within LysoTracker-positive endolysosomes showed that Ned-19 significantly decreased PCO, gp120 significantly increased PCO, and Ned-19 blocked gp120-induced increases in endolysosome PCO. Each data point represents the MFI of DCP-Rho1 staining within Lysotracker-positive endolysosomes from one field of view with a minimum of 133 cells analyzed per condition across at least two biological replicates. Data were means and SEM with individual data points (n = 7). Two-way ANOVA with Tukey’s multiple comparison tests were used for statistical analyses. **** p <0.0001

Article Snippet: Mean fluorescence intensity (MFI) was analyzed using Imaris software (v9.9).

Techniques: Staining, Generated, Fluorescence, Comparison

Journal: Journal of Advanced Research

Article Title: Airway basal stem cell-derived extracellular vesicles drive ECM remodeling and suppress fibroblasts activation via the miR-30a-5p/FAP axis in benign tracheal stenosis

doi: 10.1016/j.jare.2025.08.014

Figure Lengend Snippet: Quantification, characterization, and internalization assays of BSC-EVs. (a) Representative fluorescent immunocytochemistry images showing KRT5 (green), MUC5B (green), AC-TUB (green), P63 (red), and KI67 (red) expression in BSCs, with nuclei counterstained using DAPI (blue). Scale bars, 100 μm. (b) HE staining demonstrates the formation of a pseudostratified epithelium by BSCs, comprising ciliated and mucus-producing cells under air–liquid interface differentiation. Scale bar, 30 μm. (c) Nano-flow cytometry analysis reveals the particle size distribution of BSC-EVs. (d) Representative transmission electron microscopy images of BSC-EVs. Scale bar, 300 μm. (e) Western blot analysis confirms the presence of EV markers, including TSG101, HSP70, CD81, CD9, and CD63. #1, #2, #3, #4, and #5 represent BSC-EVs from three different human samples, respectively. (f) Tracking of intratracheally injected PKH-26-labeled BSC-EVs (red) in the rabbit trachea over 14 days, with representative gross, fluorescent stereomicroscope, and fluorescence microscopy images presented sequentially.

Article Snippet: Mean fluorescence intensity was quantified using Celigo software (top right).

Techniques: Immunocytochemistry, Expressing, Staining, Flow Cytometry, Transmission Assay, Electron Microscopy, Western Blot, Injection, Labeling, Fluorescence, Microscopy

Journal: Journal of Advanced Research

Article Title: Airway basal stem cell-derived extracellular vesicles drive ECM remodeling and suppress fibroblasts activation via the miR-30a-5p/FAP axis in benign tracheal stenosis

doi: 10.1016/j.jare.2025.08.014

Figure Lengend Snippet: Assessment of fibroblasts activation and ECM remodeling of miR-30a-5p-containing BSC-EVs in an indirect co-culture system. (a) Schematic representation of the co-culture model for AFs and BSCs transfected with NC or miR-30a-5p, with either DMSO or GW4869 as treatments. (b) Representative fluorescent immunocytochemistry images showing FAP expression in AFs treated with NC inhibitors + DMSO, NC inhibitors + GW4869, miR-30a-5p inhibitors + DMSO, and miR-30a-5p inhibitors + GW4869 (n = 3 per group). Scale bar = 100 μm (top left). Mean fluorescence intensity was quantified using Celigo software (top right). Collagen contractility assays showing representative collagen gel images post-release (bottom left) were quantified for contraction area using ImageJ software (bottom right). (c) Western blot analysis of protein levels for Collagen I, FAP, α-SMA, MMP2, MMP7, MMP9, TIMP1, TIMP2, and TIMP3 in the four treatment groups. (d) Representative images of collagen gels post-release in AFs treated with NC mimics + DMSO, NC mimics + GW4869, miR-30a-5p mimics + DMSO, and miR-30a-5p mimics + GW4869. (e) Corresponding western blot analysis of Collagen I, FAP, α-SMA, MMP2, MMP7, MMP9, TIMP1, TIMP2, and TIMP3 protein levels in these four groups.

Article Snippet: Mean fluorescence intensity was quantified using Celigo software (top right).

Techniques: Activation Assay, Co-Culture Assay, Transfection, Immunocytochemistry, Expressing, Fluorescence, Software, Western Blot