Journal: Scientific Reports

Article Title: Unraveling the structure–activity relationships of organometallic ferrocene-pyrazole and ferrocene-pyrimidine curcumin analogues in amyloid-β aggregation and glioblastoma treatment

doi: 10.1038/s41598-025-23467-0

Figure Lengend Snippet: Super-resolution fluorescence images of U87MG cells immuno-stained against α-tubulin excitation 488 nm, green fluorescence anti-α-tubulin conjugated with AlexaFluor 488, ab185031, 1:100), and ubiquinol-cytochrome c reductase (excitation 555 nm, orange fluorescence of anti- ubiquinol-cytochrome c reductase (ab110252, 1:100), secondary anti-mouse AlexaFluor 546 (ThermoFisher Scientific, 2:1000). Nuclei were counterstained with Hoechst 33342 (excitation 405 nm; blue fluorescence). Typical structures are shown in the lower right. Cells were exposed to 12.5 µM of ferrocene-containing analogues of curcumin for 48 h. White arrows indicate the perinuclear localization of mitochondria.

Article Snippet: Fig. 9 Super-resolution fluorescence images of U87MG cells immuno-stained against α-tubulin excitation 488 nm, green fluorescence anti-α-tubulin conjugated with AlexaFluor 488, ab185031, 1:100), and ubiquinol-cytochrome c reductase (excitation 555 nm, orange fluorescence of anti- ubiquinol-cytochrome c reductase (ab110252, 1:100), secondary anti-mouse AlexaFluor 546 (ThermoFisher Scientific, 2:1000).

Techniques: Fluorescence, Staining, Analogues

Journal: Scientific Reports

Article Title: Unraveling the structure–activity relationships of organometallic ferrocene-pyrazole and ferrocene-pyrimidine curcumin analogues in amyloid-β aggregation and glioblastoma treatment

doi: 10.1038/s41598-025-23467-0

Figure Lengend Snippet: Super-resolution fluorescence images of U87MG cells immunostained against Giantin (excitation 488 nm, green fluorescence of secondary antibody AlexaFluor 488, primary antibody anti-Giantin (ab80864, 1:100)) and ubiquinol-cytochrome c reductase (excitation 555 nm, orange fluorescence of anti- ubiquinol-cytochrome c reductase (ab110252, 1:100), secondary anti-mouse AlexaFluor 546 (ThermoFisher Scientific, 2:1000). Nuclei were counterstained with Hoechst 33342 (excitation 405 nm, blue fluorescence). Typical structures are shown in the lower right. Cells were exposed to 12.5 µM of ferrocene-containing analogues of curcumin for 48 h. White arrows indicate the localization of the Golgi apparatus.

Article Snippet: Fig. 9 Super-resolution fluorescence images of U87MG cells immuno-stained against α-tubulin excitation 488 nm, green fluorescence anti-α-tubulin conjugated with AlexaFluor 488, ab185031, 1:100), and ubiquinol-cytochrome c reductase (excitation 555 nm, orange fluorescence of anti- ubiquinol-cytochrome c reductase (ab110252, 1:100), secondary anti-mouse AlexaFluor 546 (ThermoFisher Scientific, 2:1000).

Techniques: Fluorescence, Analogues

Journal: iScience

Article Title: The node of Ranvier influences the in vivo axonal transport of mitochondria and signaling endosomes

doi: 10.1016/j.isci.2024.111158

Figure Lengend Snippet:

Article Snippet: Briefly, H C T (residues 875–1315) fused to an improved cysteine-rich region was expressed in bacteria as a glutathione-S-transferase fusion protein, cleaved and subsequently labeled with Alexa Fluor 555 C2 maleimide (Thermo Fisher Scientific, A-20346).

Techniques: Recombinant, Mutagenesis, Software, Fluorescence

Journal: Scientific Reports

Article Title: Conformational change of Syntaxin-3b in regulating SNARE complex assembly in the ribbon synapses

doi: 10.1038/s41598-022-09654-3

Figure Lengend Snippet: Native state conformations of syntaxin-1a and syntaxin-3b. ( A ) The disorder tendencies of syntaxin-1a and -3b are analyzed by PONDR VLXT . The protein domain diagram illustrates the common domains of syntaxin-1a and syntaxin-3b (top panel) and the syntaxin-3b construct used in this experiment (bottom panel). ( B ) Schematic of a surface-tethered syntaxin molecule labeled with FRET dye pairs on a functionalized surface of a microscope slide in a closed and open conformation leading to high and low FRET efficiency, respectively. ( C ) Representative single-molecule fluorescence intensity time traces of sytnaxin-1a (SX-1a) and syntaxin-3b (SX-3b) in the closed and open conformations (top panels). The donor and acceptor intensities were converted to FRET efficiency time traces (bottom panels). ( D ) smFRET efficiency histograms of isolated SX-1a and SX-3b. ( E ) Percent closed populations of SX-1a and SX-3b were extracted from (D) by fitting two Gaussian functions to the FRET efficiency histograms. Shown are means ± SD ( n = 3). ( F ) Bulk fluorescence anisotropy measurements of interactions between Alexa 488 labeled syntaxins and unlabeled SNAP-25 at 0 μM, 0.009 μM, 0.045 μM, 0.090 μM, 0.450 μM, 0.900 μM concentrations. The anisotropy curves are fit with Hill equations to estimate the disassociation constant K d . Shown are means ± SD ( n = 3).

Article Snippet: Syntaxin molecules containing intra-molecular FRET labeling sites, i.e., double cysteine mutations, were stochastically labeled with Alexa 555 C 2 and Alexa 647 C 2 maleimide (Thermo Fisher Scientific, Waltham, MA) fluorescent dyes at 10 × molar excess of the protein concentration in TBS overnight on a rotating platform at 4°C.

Techniques: Construct, Labeling, Microscopy, Fluorescence, Isolation

Journal: Scientific Reports

Article Title: Conformational change of Syntaxin-3b in regulating SNARE complex assembly in the ribbon synapses

doi: 10.1038/s41598-022-09654-3

Figure Lengend Snippet: The phosphomimetic T14E and the LE mutations alter the conformation of syntaxin-3b. ( A ) The disorder tendencies of WT, T14E, and LE (L165A, E166A) mutation of syntaxin-3b are analyzed by PONDR VLXT . The protein domain diagram illustrates the common domains in syntaxin-1a and syntaxin-3b (top panel) and the syntaxin-3b construct used in this experiment (bottom panel). ( B ) Schematic of a surface-tethered syntaxin-3b molecule labeled with FRET dye pairs on a functionalized surface of the microscope slide in a closed and open conformation leading to high and low FRET efficiency, respectively. ( C ) Representative single-molecule fluorescence intensity time traces of sytnaxin-3b wild-type (WT) (left panels), T14E (middle panels), and LE (right panels) mutants. The donor and acceptor intensities were converted to FRET efficiency time traces (bottom panels). ( D ) smFRET efficiency histograms of WT, T14E and LE syntaxin-3b mutants. ( E ) Percent closed populations of syntaxin-3b WT, T14E, and LE mutant were extracted from ( D ) by fitting two Gaussian functions to the FRET efficiency histograms. Shown are means ± SD ( n = 3). ( F ) Bulk fluorescence anisotropy measurements of interactions between Alexa 488 labeled syntaxins and unlabeled SNAP-25 at 0 μM, 0.009 μM, 0.045 μM, 0.090 μM, 0.450 μM, 0.900 μM concentrations. The anisotropy curves are fit with Hill equations to estimate the disassociation constant K d . Shown are means ± SD ( n = 3).

Article Snippet: Syntaxin molecules containing intra-molecular FRET labeling sites, i.e., double cysteine mutations, were stochastically labeled with Alexa 555 C 2 and Alexa 647 C 2 maleimide (Thermo Fisher Scientific, Waltham, MA) fluorescent dyes at 10 × molar excess of the protein concentration in TBS overnight on a rotating platform at 4°C.

Techniques: Mutagenesis, Construct, Labeling, Microscopy, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Investigation of Stoichiometry of T4 Bacteriophage Helicase Loader Protein (gp59) *

doi: 10.1074/jbc.M109.029926

Figure Lengend Snippet: Structures of the forked DNA substrates used in this study. 1, forked DNA (10/40/40-mer) with biotin at the 5′-end of the leading strand for the smPB study using unlabeled substrate (unlabeled forked DNA); 2, forked DNA (34/62/50-mer) with Cy5 dye at the 5′-end of the primer and biotin at the 3′-end of the lagging strand (Cy5-labeled forked DNA) for the smPB study with labeled substrate and gp59-Alexa 555.

Article Snippet: In brief, the protein was dialyzed in labeling buffer (20 m m Tris (pH 7.5), 150 m m NaCl, and 10% glycerol) for 8 h and incubated with a 5-fold excess of Alexa 555 (Alexa Fluor 555 C 2 maleimide, Invitrogen) for ∼4 h. The unreacted dye was removed by chromatography using a cation-exchange resin and a salt gradient.

Techniques: Labeling

Journal: The Journal of Biological Chemistry

Article Title: Investigation of Stoichiometry of T4 Bacteriophage Helicase Loader Protein (gp59) *

doi: 10.1074/jbc.M109.029926

Figure Lengend Snippet: Histogram plot of smPB events. The histogram represents the smPB experiment done with Cy5-labeled forked DNA (FkD-Cy5) with gp59-Alexa 555 and other proteins. Histograms show the normalized events versus number of photobleaching steps. Similar histograms were built for smPB experiments with unlabeled forked DNA with the same proteins (data not shown). The experiments were done in triplicate, and the errors assigned. The population of the hexamer seen is indicated by the circle.

Article Snippet: In brief, the protein was dialyzed in labeling buffer (20 m m Tris (pH 7.5), 150 m m NaCl, and 10% glycerol) for 8 h and incubated with a 5-fold excess of Alexa 555 (Alexa Fluor 555 C 2 maleimide, Invitrogen) for ∼4 h. The unreacted dye was removed by chromatography using a cation-exchange resin and a salt gradient.

Techniques: Labeling