Journal: bioRxiv

Article Title: Decoding subtype development and function in human pluripotent stem cell-derived midbrain dopaminergic neurons

doi: 10.64898/2026.09.23.753784

Figure Lengend Snippet: a , Experimental schematic of TGFβ pathway modulation during mDA progenitor patterning. b, PITX3::GFP reporter design (top), flow cytometry of PITX3 CRISPRa activation (middle), and immunofluorescence (IF) for PITX3 and TH at D30 (bottom). c, Flow cytometry of PITX3, CD171 and CD99 at D25. d, Flow cytometry of ALDH activity and CD99 at D25. e, Flow cytometry of PITX3 expression and ALDH activity in the indicated gated subpopulations, with quantification of the percentage of PITX3+ALDH+ cells in each subpopulation. One-way ANOVA; n = 4. f, RT–qPCR of the indicated genes in D25 control and Activin A/BMP7-patterned populations. Student’s t -test; n = 3. g, Flow cytometry of PITX3 and ALDH activity in the indicated CD99-CD56+ populations. h, IF for PITX3, ALDH1A1 and CALB1 at D26. Scale bars, 100 μm. i, Schematic illustrating mDA subtype patterning through modulation of TGF-β signaling. j, Schematic of the day 25 sorting strategy and RT–qPCR analysis of the indicated genes in sorted day 25 populations. One-way ANOVA; n = 3–4. k, IF for ALDH1A1, PITX3 and CALB1 at D26 in populations derived from D25 sorted cells (left), with quantification of relative ALDH1A1 expression (right). Scale bars, 100 μm. One-way ANOVA; n = 4–8. l, Flow cytometry of TH and ALDH activity in the indicated CD99− and CD99+ populations, with quantification of the percentage of CD99-TH+ALDH+ cells. One-way ANOVA; n = 3. m . Schematic of hPSC-A9/A10 transplantation into the mouse striatum (top); IF staining for GIRK2, TH and CALB1 in A9 and A10 grafts at 6 months post-transplantation, with quantification of GIRK2+TH+ and CALB1+TH+ cells in the grafts. Student’s t -test; n = 3. All error bars in represent s.e.m.

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against GIRK2 (1:100; Alomone Labs, APC_006-GP) and calbindin (1:200; Cell Signaling Technology, 13176).

Techniques: Flow Cytometry, Activation Assay, Immunofluorescence, Activity Assay, Expressing, Quantitative RT-PCR, Control, Analysis, Derivative Assay, Transplantation Assay, Staining

Journal: Science Advances

Article Title: Synaptic vesicles that store monoamines and glutamate differ in protein composition

doi: 10.1126/sciadv.adz6836

Figure Lengend Snippet: ( A ) Schematic of HA-tagged VMAT2 and striatal images from HA-VMAT2 KI mice showing HA-VMAT2 in TH + axons. Scale bar, 5 μm. ( B ) SV isolation workflow. Synaptosomes (P2) were osmotically lysed to release vesicles (LS1) which were separated from plasma membranes (LP1) and sedimented in LP2. Light organelles from S2 were collected to isolate axonal vesicles not enriched in varicosities (P4). ( C ) Immunoblot on whole-brain samples showing HA-VMAT2, VGLUT2, and synaptophysin in LP2 and synaptophysin in P4 fractions in 5 to 25% glycerol gradients. ( D ) Normalized HA-VMAT2 and VGLUT2 signals across gradient fractions from whole-brain LP2 and P4 samples and comparison of HA-VMAT2 versus VGLUT2 in LP2 fractions ( n = 4 biological replicates for VGLUT2 LP2 and P4, 3 for HA-VMAT2 LP2, and 2 for HA-VMAT2 P4; mean ± SEM; * P < 0.05, unpaired t test). ( E ) Immunoblots showing HA-VMAT2 or VGLUT2 with synaptophysin in whole-brain LP2, gradient purified SVs, and immunoisolation ± antibody. ab, antibody. ( F ) Immunoblots and quantification of HA-VMAT2 and VGLUT2 in vesicles from striatal LP2 immunoisolated with anti-VGLUT2 or anti-HA antibodies ( n = 4 biological replicates; mean ± SEM; **** P < 0.0001, unpaired t test). Example proteins quantified in VMAT2 and VGLUT2 SVs from whole brain ( G ) or striatum ( H ) shown as heatmaps of median log 2 VGLUT2/VMAT2 ratios normalized to total protein. Columns represent biological replicates. Dashed lines signify ±0.5 cutoff of log 2 VGLUT2/VMAT2 ratio. Gray proteins are not detected in striatum; red proteins are significantly enriched in one SV population ( P < 0.05). Volcano plots showing proteins in VMAT2 versus VGLUT2 SVs from striatal LP2 ( I ) and P4 samples ( J ) in three independent experiments. Red data points have P values of <0.05 and log 2 VGLUT2/VMAT2 ratio of ±0.5. Dashed lines mark the ±0.5 log 2 VGLUT2/VMAT2 ratio and P = 0.05. Blue points are associated with labels.

Article Snippet: The following antibodies were used in the study: VGLUT2 Gp (1:1000; Synaptic Systems, 135404); VGLUT2 Ch (1:5000; Synaptic Systems, 135416); HA Rt (1:500; Roche, 11867423001); TH Ms (1:1000; EMD Millipore, MAB5280); Syt1 Ms (1:5000; Synaptic Systems, 105011); Syt2 Gp (1:1000; Synaptic Systems, 105225); Syp Ms (1:500; Synaptic Systems, 101011); Syp Gp (1:500; Synaptic Systems, 101308); Syg Rb (1:100; Synaptic Systems, 103002); VAMP2 Rb (1:500; Synaptic Systems, 104202); CSP Rb (1:500; Synaptic Systems, 154003); CSP Gp (1:500; Synaptic Systems, 154004); Slc10A4 Rb (1:200; Sigma-Aldrich, HPA028835); and SCAMP5 Rb (1:50; Invitrogen, PA5-61269).

Techniques: Isolation, Clinical Proteomics, Western Blot, Comparison, Purification

Journal: Science Advances

Article Title: Synaptic vesicles that store monoamines and glutamate differ in protein composition

doi: 10.1126/sciadv.adz6836

Figure Lengend Snippet: ( A to D ) Confocal images of midbrain cultures from HA-VMAT2 KI mice showing the distribution of Slc10A4 (A), syt1 (B), CSPα (C), and SCAMP5 (D) in VMAT2 and VGLUT2 varicosities and the quantification of mean fluorescence intensity of the proteins of interest in the two types of release sites. White arrowheads point to VMAT2 and orange arrowheads to VGLUT2 varicosities. Scale bars, 10 μm. Insets show magnification of areas indicated by white boxes and line ROIs with corresponding fluorescence profiles in three channels shown below. Inset scale bar, 2 μm. Each data point represents the average fluorescence intensity of ROIs per image [ n = 24/26 (A), 30/27 (B), 21/22 (C), or 18/22 (D)], and quantifications were performed on images of three [(A) and (D)] or four [(B) and (C)] independent cultures; mean ± SEM; ** P = 0.0028, *** P < 0.001, and **** P < 0.0001, by unpaired t test [(B) and (D)] or Mann-Whitney test (A and C). Quantification of Pearson’s and Manders’ colocalization coefficients between Slc10A4 ( E ), syt1 ( F ), CSPα ( G ), or SCAMP5 ( H ) and HA-VMAT2 versus VGLUT2 in images that contain both dopamine and glutamate neurons. Each data point represents an image [ n = 23 (E), 18 (F), 16 (G), or 19 (H) from three independent cultures; mean ± SEM; * P < 0.05, *** P < 0.001, and **** P < 0.0001, by paired t test]. ( I to L ) Presynaptic varicosities stained for HA-VMAT2, syt1, and CSPα (I) or VGLUT2, syt1, and CSPα (J). Scale bars, 1 μm. (K) Scatterplot of CSPα and syt1 signal intensities quantified in individual HA-VMAT2 or VGLUT2 varicosities where each data point represents an individual presynaptic varicosity. (L) Quantification of syt1/CSPα ratio in presynaptic varicosities averaged per image ( n = 28/25 from two independent cultures; mean ± SEM; **** P < 0.0001, by Mann-Whitney test). a.u., arbitrary units.

Article Snippet: The following antibodies were used in the study: VGLUT2 Gp (1:1000; Synaptic Systems, 135404); VGLUT2 Ch (1:5000; Synaptic Systems, 135416); HA Rt (1:500; Roche, 11867423001); TH Ms (1:1000; EMD Millipore, MAB5280); Syt1 Ms (1:5000; Synaptic Systems, 105011); Syt2 Gp (1:1000; Synaptic Systems, 105225); Syp Ms (1:500; Synaptic Systems, 101011); Syp Gp (1:500; Synaptic Systems, 101308); Syg Rb (1:100; Synaptic Systems, 103002); VAMP2 Rb (1:500; Synaptic Systems, 104202); CSP Rb (1:500; Synaptic Systems, 154003); CSP Gp (1:500; Synaptic Systems, 154004); Slc10A4 Rb (1:200; Sigma-Aldrich, HPA028835); and SCAMP5 Rb (1:50; Invitrogen, PA5-61269).

Techniques: Fluorescence, MANN-WHITNEY, Staining

Journal: Science Advances

Article Title: Synaptic vesicles that store monoamines and glutamate differ in protein composition

doi: 10.1126/sciadv.adz6836

Figure Lengend Snippet: ( A ) Example of 3D surfaces generated in a striatal z-stack of a staining for HA-VMAT2 or VGLUT2. The same surfaces are transferred to the channel showing the staining for a protein of interest for quantification of its mean intensity in HA-VMAT2 and VGLUT2 surfaces. Scale bar, 4 μm in all directions. Confocal image of a single plane in a striatal slice from a HA-VMAT2 KI mouse stained for HA-VMAT2, VGLUT2, and Slc10A4 ( B ) or CSPα ( C ) together with quantification of the mean intensity of the protein of interest in 3D surfaces generated on the basis of HA-VMAT2 or VGLUT2 signal. Scale bar, 10 μm. White arrowheads point to HA-VMAT2 and orange arrowheads to VGLUT2 varicosities. Quantification graphs show mean values of varicosities per image in gray and mean values per mouse in black [ n = 5 mice; mean ± SEM; **** P < 0.0001 for (B) and ** P = 0.006 for (C) by paired t test on mouse averages]. Insets show the magnified view of the area highlighted by a white box in the larger image and the plot profile of fluorescence intensities in three channels along the line ROI. Inset scale bar, 2 μm.

Article Snippet: The following antibodies were used in the study: VGLUT2 Gp (1:1000; Synaptic Systems, 135404); VGLUT2 Ch (1:5000; Synaptic Systems, 135416); HA Rt (1:500; Roche, 11867423001); TH Ms (1:1000; EMD Millipore, MAB5280); Syt1 Ms (1:5000; Synaptic Systems, 105011); Syt2 Gp (1:1000; Synaptic Systems, 105225); Syp Ms (1:500; Synaptic Systems, 101011); Syp Gp (1:500; Synaptic Systems, 101308); Syg Rb (1:100; Synaptic Systems, 103002); VAMP2 Rb (1:500; Synaptic Systems, 104202); CSP Rb (1:500; Synaptic Systems, 154003); CSP Gp (1:500; Synaptic Systems, 154004); Slc10A4 Rb (1:200; Sigma-Aldrich, HPA028835); and SCAMP5 Rb (1:50; Invitrogen, PA5-61269).

Techniques: Generated, Staining, Fluorescence

Journal: Science Advances

Article Title: Synaptic vesicles that store monoamines and glutamate differ in protein composition

doi: 10.1126/sciadv.adz6836

Figure Lengend Snippet: ( A ) A single plane of a confocal image of a fixed brain slice from a HA-VMAT2 KI mouse acquired in the DLG. The slice is stained for HA-VMAT2, syt2, and VGLUT2. White arrowheads point to VMAT2 and orange arrowheads to VGLUT2 varicosities. Scale bar, 10 μm. ( B ) Magnified view of the area highlighted by white box in (A) and plot profile of fluorescence intensities in three channels along the line ROI. Inset scale bar, 3 μm. ( C ) Quantification of mean syt2 intensity in HA-VMAT2 and VGLUT2 surfaces across brain regions. Str., striatum; Hipp., CA2 region of the hippocampus; VPN, ventral posteromedial nucleus. Each data point represents a mouse ( n = 4). Data indicate mean ± SEM; * P < 0.05, by unpaired t test. ( D ) Scatterplots showing the distribution of syt2 abundance across VGLUT2 + and VMAT2 + varicosities in the hippocampus, PO, and VPN.

Article Snippet: The following antibodies were used in the study: VGLUT2 Gp (1:1000; Synaptic Systems, 135404); VGLUT2 Ch (1:5000; Synaptic Systems, 135416); HA Rt (1:500; Roche, 11867423001); TH Ms (1:1000; EMD Millipore, MAB5280); Syt1 Ms (1:5000; Synaptic Systems, 105011); Syt2 Gp (1:1000; Synaptic Systems, 105225); Syp Ms (1:500; Synaptic Systems, 101011); Syp Gp (1:500; Synaptic Systems, 101308); Syg Rb (1:100; Synaptic Systems, 103002); VAMP2 Rb (1:500; Synaptic Systems, 104202); CSP Rb (1:500; Synaptic Systems, 154003); CSP Gp (1:500; Synaptic Systems, 154004); Slc10A4 Rb (1:200; Sigma-Aldrich, HPA028835); and SCAMP5 Rb (1:50; Invitrogen, PA5-61269).

Techniques: Slice Preparation, Staining, Fluorescence

Journal: Science Advances

Article Title: Synaptic vesicles that store monoamines and glutamate differ in protein composition

doi: 10.1126/sciadv.adz6836

Figure Lengend Snippet: ( A ) Confocal image of a VGLUT2 + dopamine neuron in primary midbrain culture from HA-VMAT2 KI mice. Scale bar, 10 μm. ( B ) Magnified regions from white boxes in (A) and fluorescence intensity profiles of HA-VMAT2, VGLUT2, and TH along a line ROI, showing overlapping VGLUT2 and HA-VMAT2 peaks. Scale bars, 3 μm. ( C ) Scatterplot of HA-VMAT2 and VGLUT2 signal in individual presynaptic varicosities from VGLUT2 + dopamine neurons (colors indicate individual neurons). ( D ) Quantification of HA-VMAT2 in the presynaptic terminals of dopamine-only versus VMAT2 + /VGLUT2 + neurons and VGLUT2 in glutamate-only versus VMAT2 + /VGLUT2 + neurons. DA, dopamine; Glu, glutamate. Each data point represents mean of ROIs per image ( n = 19/21 for HA-VMAT2 and 11/20 for VGLUT2 from two independent cultures). Data indicate mean ± SEM; *** P = 0.0006 by Mann-Whitney test. Confocal images of a dopamine/glutamate neuron ( E ) or dopamine-only neuron ( F ) stained for TH (not shown), synaptophysin, HA-VMAT2, and VGLUT2. Gray arrowheads in (E) point to VGLUT2 + /VMAT2 + varicosities and white arrowheads in (F) to VMAT2-only terminals. In both images, orange arrowheads point to VGLUT2-only terminals likely from glutamate neurons. Scale bars, 10 μm. Quantification of VAMP2 ( G ), Slc10A4 ( H ), syt1 ( I ), syt2 ( J ), SCAMP5 ( K ), synaptophysin ( L ), and CSPα ( M ) in presynaptic terminals of dopamine-only or VMAT2 + /VGLUT2 + neurons. Each data point represents mean of individual ROIs per image [ n = 17/11 (G), 15/8 (H), 8/10 (I), 11/6 (J), 11/8 (K), 24/17 (L), or 22/14 (M)]. Quantifications were performed on images from two [(G), (H), (J), and (K)] or three [(I), (L), and (M)] independent cultures. Data indicate mean ± SEM. ** P < 0.01 by unpaired t test (K and M) or Mann-Whitney test (L). Scatterplots showing correlations between Slc10A4 ( N ) and synaptophysin ( O ) with HA-VMAT2 (magenta) or VGLUT2 (orange) in presynaptic terminals containing both transporters.

Article Snippet: The following antibodies were used in the study: VGLUT2 Gp (1:1000; Synaptic Systems, 135404); VGLUT2 Ch (1:5000; Synaptic Systems, 135416); HA Rt (1:500; Roche, 11867423001); TH Ms (1:1000; EMD Millipore, MAB5280); Syt1 Ms (1:5000; Synaptic Systems, 105011); Syt2 Gp (1:1000; Synaptic Systems, 105225); Syp Ms (1:500; Synaptic Systems, 101011); Syp Gp (1:500; Synaptic Systems, 101308); Syg Rb (1:100; Synaptic Systems, 103002); VAMP2 Rb (1:500; Synaptic Systems, 104202); CSP Rb (1:500; Synaptic Systems, 154003); CSP Gp (1:500; Synaptic Systems, 154004); Slc10A4 Rb (1:200; Sigma-Aldrich, HPA028835); and SCAMP5 Rb (1:50; Invitrogen, PA5-61269).

Techniques: Fluorescence, MANN-WHITNEY, Staining

Journal: Science Advances

Article Title: Synaptic vesicles that store monoamines and glutamate differ in protein composition

doi: 10.1126/sciadv.adz6836

Figure Lengend Snippet: ( A ) Dopamine neuron from an HA-VMAT2 culture double stained for HA-VMAT2 using secondary antibodies coupled with Alexa 488 and Alexa 647 fluorophores. Scale bar, 2 μm. Quantification shows Pearson’s correlation coefficient (PCC) in individual varicosities for HA imaged in two channels versus for HA and VGLUT2 measured in datasets shown in (B) and (C). Each data point represents the mean of individual varicosities per image (mean ± SEM; * P = 0.0265 by unpaired t test; n = 10 HA/HA and 15 HA/VGLUT2 images from two independent cultures). Insets show magnification of areas indicated with white boxes together with line ROIs and fluorescence profiles in HA-488 and HA-647 channels. VGLUT2 + DA neuron from an HA-VMAT2 midbrain culture stained for TH, HA, VGLUT2, and synaptophysin ( B ) or for TH, HA, VGLUT2, and syt1 ( C ). Scale bars, 2 μm. Quantifications show Pearson’s correlation of synaptophysin (B) or syt1 (C) with HA versus VGLUT2 quantified in varicosities that contain both transporters. Each data point represents the mean of individual varicosities per image (mean ± SEM; **** P < 0.0001 by paired t test; n = 14 images of synaptophysin and 13 images of syt1 from two independent cultures). Insets show magnification of areas indicated with white boxes with line ROIs and fluorescence profiles in HA, VGLUT2, and synaptophysin (B) or HA, VGLUT2, and syt1 (C) channels. Sides of all insets measure 860 nm. ns, not significant.

Article Snippet: The following antibodies were used in the study: VGLUT2 Gp (1:1000; Synaptic Systems, 135404); VGLUT2 Ch (1:5000; Synaptic Systems, 135416); HA Rt (1:500; Roche, 11867423001); TH Ms (1:1000; EMD Millipore, MAB5280); Syt1 Ms (1:5000; Synaptic Systems, 105011); Syt2 Gp (1:1000; Synaptic Systems, 105225); Syp Ms (1:500; Synaptic Systems, 101011); Syp Gp (1:500; Synaptic Systems, 101308); Syg Rb (1:100; Synaptic Systems, 103002); VAMP2 Rb (1:500; Synaptic Systems, 104202); CSP Rb (1:500; Synaptic Systems, 154003); CSP Gp (1:500; Synaptic Systems, 154004); Slc10A4 Rb (1:200; Sigma-Aldrich, HPA028835); and SCAMP5 Rb (1:50; Invitrogen, PA5-61269).

Techniques: Staining, Fluorescence

Journal: Science Advances

Article Title: Synaptic vesicles that store monoamines and glutamate differ in protein composition

doi: 10.1126/sciadv.adz6836

Figure Lengend Snippet: ( A ) Confocal images and quantification of the fluorescence intensity of syt1 and SCAMP5 in uninfected hippocampal neurons or cultures infected with lentiviruses containing a scrambled control sequence, shRNA targeting SCAMP5, or shRNA together with a SCAMP5 rescue construct. Scale bar, 10 μm. Each data point represents mean presynaptic SCAMP5 or syt1 intensity per image from two independent cultures ( n = 13 uninfected, 11 scramble, 11 shRNA, and 8 rescue images). Data indicate mean ± SEM, **** P < 0.0001 by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Average response curves and quantification of peak fluorescence intensity of VGLUT2-pHluorin ( B ) or VMAT2-pHluorin ( C ) to a 60-s stimulation at 10 Hz. Data indicate mean ± SEM, * P = 0.0225 and ** P = 0.017 by one-way ANOVA with Tukey’s multiple comparisons. N = 13 scramble, 17 shRNA, and 17 rescue coverslips for VGLUT2-pHluorin and 11 scramble, 10 shRNA, and 9 rescue coverslips for VMAT2-pHluorin from three independent cultures.

Article Snippet: The following antibodies were used in the study: VGLUT2 Gp (1:1000; Synaptic Systems, 135404); VGLUT2 Ch (1:5000; Synaptic Systems, 135416); HA Rt (1:500; Roche, 11867423001); TH Ms (1:1000; EMD Millipore, MAB5280); Syt1 Ms (1:5000; Synaptic Systems, 105011); Syt2 Gp (1:1000; Synaptic Systems, 105225); Syp Ms (1:500; Synaptic Systems, 101011); Syp Gp (1:500; Synaptic Systems, 101308); Syg Rb (1:100; Synaptic Systems, 103002); VAMP2 Rb (1:500; Synaptic Systems, 104202); CSP Rb (1:500; Synaptic Systems, 154003); CSP Gp (1:500; Synaptic Systems, 154004); Slc10A4 Rb (1:200; Sigma-Aldrich, HPA028835); and SCAMP5 Rb (1:50; Invitrogen, PA5-61269).

Techniques: Fluorescence, Infection, Control, Sequencing, shRNA, Construct

Journal: Science Advances

Article Title: Synaptic vesicles that store monoamines and glutamate differ in protein composition

doi: 10.1126/sciadv.adz6836

Figure Lengend Snippet: ( A ) A single plane of a confocal image of a fixed brain slice from a HA-VMAT2 KI mouse acquired in the DLG. The slice is stained for HA-VMAT2, syt2, and VGLUT2. White arrowheads point to VMAT2 and orange arrowheads to VGLUT2 varicosities. Scale bar, 10 μm. ( B ) Magnified view of the area highlighted by white box in (A) and plot profile of fluorescence intensities in three channels along the line ROI. Inset scale bar, 3 μm. ( C ) Quantification of mean syt2 intensity in HA-VMAT2 and VGLUT2 surfaces across brain regions. Str., striatum; Hipp., CA2 region of the hippocampus; VPN, ventral posteromedial nucleus. Each data point represents a mouse ( n = 4). Data indicate mean ± SEM; * P < 0.05, by unpaired t test. ( D ) Scatterplots showing the distribution of syt2 abundance across VGLUT2 + and VMAT2 + varicosities in the hippocampus, PO, and VPN.

Article Snippet: The following antibodies were used in the study: VGLUT2 Gp (1:1000; Synaptic Systems, 135404); VGLUT2 Ch (1:5000; Synaptic Systems, 135416); HA Rt (1:500; Roche, 11867423001); TH Ms (1:1000; EMD Millipore, MAB5280); Syt1 Ms (1:5000; Synaptic Systems, 105011); Syt2 Gp (1:1000; Synaptic Systems, 105225); Syp Ms (1:500; Synaptic Systems, 101011); Syp Gp (1:500; Synaptic Systems, 101308); Syg Rb (1:100; Synaptic Systems, 103002); VAMP2 Rb (1:500; Synaptic Systems, 104202); CSP Rb (1:500; Synaptic Systems, 154003); CSP Gp (1:500; Synaptic Systems, 154004); Slc10A4 Rb (1:200; Sigma-Aldrich, HPA028835); and SCAMP5 Rb (1:50; Invitrogen, PA5-61269).

Techniques: Slice Preparation, Staining, Fluorescence

Journal: Science Advances

Article Title: Synaptic vesicles that store monoamines and glutamate differ in protein composition

doi: 10.1126/sciadv.adz6836

Figure Lengend Snippet: ( A ) Confocal image of a VGLUT2 + dopamine neuron in primary midbrain culture from HA-VMAT2 KI mice. Scale bar, 10 μm. ( B ) Magnified regions from white boxes in (A) and fluorescence intensity profiles of HA-VMAT2, VGLUT2, and TH along a line ROI, showing overlapping VGLUT2 and HA-VMAT2 peaks. Scale bars, 3 μm. ( C ) Scatterplot of HA-VMAT2 and VGLUT2 signal in individual presynaptic varicosities from VGLUT2 + dopamine neurons (colors indicate individual neurons). ( D ) Quantification of HA-VMAT2 in the presynaptic terminals of dopamine-only versus VMAT2 + /VGLUT2 + neurons and VGLUT2 in glutamate-only versus VMAT2 + /VGLUT2 + neurons. DA, dopamine; Glu, glutamate. Each data point represents mean of ROIs per image ( n = 19/21 for HA-VMAT2 and 11/20 for VGLUT2 from two independent cultures). Data indicate mean ± SEM; *** P = 0.0006 by Mann-Whitney test. Confocal images of a dopamine/glutamate neuron ( E ) or dopamine-only neuron ( F ) stained for TH (not shown), synaptophysin, HA-VMAT2, and VGLUT2. Gray arrowheads in (E) point to VGLUT2 + /VMAT2 + varicosities and white arrowheads in (F) to VMAT2-only terminals. In both images, orange arrowheads point to VGLUT2-only terminals likely from glutamate neurons. Scale bars, 10 μm. Quantification of VAMP2 ( G ), Slc10A4 ( H ), syt1 ( I ), syt2 ( J ), SCAMP5 ( K ), synaptophysin ( L ), and CSPα ( M ) in presynaptic terminals of dopamine-only or VMAT2 + /VGLUT2 + neurons. Each data point represents mean of individual ROIs per image [ n = 17/11 (G), 15/8 (H), 8/10 (I), 11/6 (J), 11/8 (K), 24/17 (L), or 22/14 (M)]. Quantifications were performed on images from two [(G), (H), (J), and (K)] or three [(I), (L), and (M)] independent cultures. Data indicate mean ± SEM. ** P < 0.01 by unpaired t test (K and M) or Mann-Whitney test (L). Scatterplots showing correlations between Slc10A4 ( N ) and synaptophysin ( O ) with HA-VMAT2 (magenta) or VGLUT2 (orange) in presynaptic terminals containing both transporters.

Article Snippet: The following antibodies were used in the study: VGLUT2 Gp (1:1000; Synaptic Systems, 135404); VGLUT2 Ch (1:5000; Synaptic Systems, 135416); HA Rt (1:500; Roche, 11867423001); TH Ms (1:1000; EMD Millipore, MAB5280); Syt1 Ms (1:5000; Synaptic Systems, 105011); Syt2 Gp (1:1000; Synaptic Systems, 105225); Syp Ms (1:500; Synaptic Systems, 101011); Syp Gp (1:500; Synaptic Systems, 101308); Syg Rb (1:100; Synaptic Systems, 103002); VAMP2 Rb (1:500; Synaptic Systems, 104202); CSP Rb (1:500; Synaptic Systems, 154003); CSP Gp (1:500; Synaptic Systems, 154004); Slc10A4 Rb (1:200; Sigma-Aldrich, HPA028835); and SCAMP5 Rb (1:50; Invitrogen, PA5-61269).

Techniques: Fluorescence, MANN-WHITNEY, Staining

Journal: Frontiers in Immunology

Article Title: HSP70-mediated regulation of P-glycoprotein SUMOylation suppresses ferroptosis to drive thermoresistance in non-small cell lung cancer

doi: 10.3389/fimmu.2026.1844793

Figure Lengend Snippet: HSP70 enhances P-gp protein stability by regulating SUMOylation of P-gp. (A, B) P-gp expression evaluated by RT-qPCR and western blot; (C) SUMOylation sites of P-gp predicted using the online GPS-SUMO database; (D) Interaction between P-gp and HSP70 and SUMOylation levels of P-gp assessed by Co-IP assay; (E) The SUMOylation levels of wild-type and mutant P-gp measured with Co-IP assay; (F) P-gp protein stability evaluated by CHX chase assay. All cell experiments were independently repeated three times. Data were presented as mean ± SD. Comparisons among multiple groups were performed using one-way ANOVA, followed by Tukey’s multiple comparisons test. ** P < 0.01, *** P < 0.001.

Article Snippet: Beginning in the second week after hyperthermia initiation, mice received tail vein injections of the P-gp inhibitor verapamil (25 mg/kg; HY-14275, MCE) thrice per week for three weeks , wherein verapamil was dissolved sequentially in 10% DMSO, 40% polyethylene glycol 300, 5% Tween-80, and 45% saline.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Co-Immunoprecipitation Assay, Mutagenesis

Journal: Frontiers in Immunology

Article Title: HSP70-mediated regulation of P-glycoprotein SUMOylation suppresses ferroptosis to drive thermoresistance in non-small cell lung cancer

doi: 10.3389/fimmu.2026.1844793

Figure Lengend Snippet: Knockdown of P-gp facilitates ferroptosis, which partially reverses the promoting effect of HSP70 on the resistance of NSCLC cells to hyperthermia. (A, B) RT-qPCR and western blot to evaluate P-gp expression in cells; (C) LDH to assess cell death; (D) BODIPY staining to examine lipid peroxidation; (E–G) Test kit to detect Fe 2+ , MDA, and GSH levels in cells; (H) Western blot to assess ferroptosis-related proteins (GPX4 and SLC7A11) in cells; (I) CCK-8 to examine cell viability; (J) Transwell to assess cell invasion and migration. Cell experiments were repeated thrice. Data were exhibited as mean ± SD. One-way ANOVA was utilized for data comparisons among multiple groups, and Tukey’s multiple comparison test was employed for the post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Beginning in the second week after hyperthermia initiation, mice received tail vein injections of the P-gp inhibitor verapamil (25 mg/kg; HY-14275, MCE) thrice per week for three weeks , wherein verapamil was dissolved sequentially in 10% DMSO, 40% polyethylene glycol 300, 5% Tween-80, and 45% saline.

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Expressing, Staining, CCK-8 Assay, Migration, Comparison

Journal: Frontiers in Immunology

Article Title: HSP70-mediated regulation of P-glycoprotein SUMOylation suppresses ferroptosis to drive thermoresistance in non-small cell lung cancer

doi: 10.3389/fimmu.2026.1844793

Figure Lengend Snippet: HSP70 suppresses oxidative stress and mitochondrial dysfunction by upregulating P-gp. (A, B) Measurement of intracellular ROS and SOD levels using commercial kits; (C) Assessment of MMP assessed by JC-1 fluorescent probe; (D) Measurement of mtDNA levels; (E–H) Real-time monitoring curves of OCR in cells, along with quantitative analyses of basal respiration, ATP synthesis, and maximal respiration levels. All cell experiments were independently repeated three times. Data were presented as mean ± SD. Comparisons among groups were performed using one-way ANOVA, followed by Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Beginning in the second week after hyperthermia initiation, mice received tail vein injections of the P-gp inhibitor verapamil (25 mg/kg; HY-14275, MCE) thrice per week for three weeks , wherein verapamil was dissolved sequentially in 10% DMSO, 40% polyethylene glycol 300, 5% Tween-80, and 45% saline.

Techniques:

Journal: Frontiers in Immunology

Article Title: HSP70-mediated regulation of P-glycoprotein SUMOylation suppresses ferroptosis to drive thermoresistance in non-small cell lung cancer

doi: 10.3389/fimmu.2026.1844793

Figure Lengend Snippet: HSP70-mediated SUMOylation regulates P-gp to inhibit ferroptosis and thus boost resistance to hyperthermia and tumor growth in vivo . (A) Tumor images and weights; (B) Lipid peroxidation assessed by BODIPY staining; (C–F) Intratumoral ROS, Fe 2+ , MDA, and GSH levels; (G, H) Expression of ferroptosis-related proteins (GPX4 and SLC7A11), SUMO1, and SUMO2/3 detected by western blot; (I) HSP70 level in tumor tissues measured by RT-qPCR. n = 6. Data were presented as mean ± SD. Comparisons among groups were performed using one-way ANOVA, followed by Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Beginning in the second week after hyperthermia initiation, mice received tail vein injections of the P-gp inhibitor verapamil (25 mg/kg; HY-14275, MCE) thrice per week for three weeks , wherein verapamil was dissolved sequentially in 10% DMSO, 40% polyethylene glycol 300, 5% Tween-80, and 45% saline.

Techniques: In Vivo, Staining, Expressing, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: HSP70-mediated regulation of P-glycoprotein SUMOylation suppresses ferroptosis to drive thermoresistance in non-small cell lung cancer

doi: 10.3389/fimmu.2026.1844793

Figure Lengend Snippet: HSP70-mediated SUMOylation modulates P-gp in vivo . (A, B) IHC analysis of HSP70-positive cells and P-gp-positive cells in nude mouse tumor tissues. n = 6. Data were presented as mean ± SD. Comparisons among groups were performed using one-way ANOVA, followed by Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Beginning in the second week after hyperthermia initiation, mice received tail vein injections of the P-gp inhibitor verapamil (25 mg/kg; HY-14275, MCE) thrice per week for three weeks , wherein verapamil was dissolved sequentially in 10% DMSO, 40% polyethylene glycol 300, 5% Tween-80, and 45% saline.

Techniques: In Vivo