Journal: Experimental and Therapeutic Medicine

Article Title: Predominant control of PDGF/PDGF receptor signaling in the migration and proliferation of human adipose‑derived stem cells under culture conditions with a combination of growth factors

doi: 10.3892/etm.2024.12444

Figure Lengend Snippet: Activation of growth factor receptors and signal enzymes in growth factor-treated hASCs. hASCs were cultured in DMEM containing 10% FBS, followed by starvation for 16 h. The cells were then incubated in DMEM containing PDGF-BB (20 ng/ml), VEGF (1 ng/ml), HGF (1 ng/ml), PDGF-BB (20 ng/ml)/VEGF (1 ng/ml) or PDGF-BB (20 ng/ml)/HGF (1 ng/ml) for 20 min. The cells then were washed, collected and lysed. Next, cellular proteins were analyzed by SDS-PAGE using 4-15% gels, followed by (A) immunoblotting with the indicated primary antibodies. (B) Ratio of phospho-PDGFRb versus total PDGFRb, (C) ratio of phospho-VEGFR2 versus total VEGFR2, (D) ratio of phospho-c-Met versus total c-Met, (E) ratio of phospho-ERK1/2 versus total ERK1/2 and (F) ratio of phospho-p38 versus total p38 were calculated. Data are presented as the mean ± SD (n=3). ** P<0.01 vs. control. hASCs, human adipose-derived stem cells; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor.

Article Snippet: After blocking with Blocking One-P reagent (cat. no. 05999-84; Nacalai Tesque, Inc.) for 60 min at room temperature, the membranes were incubated overnight at 4˚C with the following primary antibodies: Anti-phospho-Erk1/2 (1:1,000; cat. no. #4370; Cell Signaling Technology, Inc.), anti-Erk1/2 (1:1,000; cat. no. #4695; Cell Signaling Technology, Inc.), anti-phospho-PDGFRb (1:1,000; cat. no. GTX133525; GeneTex, Inc.), anti-PDGFRb (1:1,000; cat. no. 134491AP; Proteintech Group, Inc.), anti-phospho-c-Met (1:1,000; cat. no. 600401989S; Rockland Immunochemicals Inc.), anti-c-Met (1:1,000; cat. no. GTX631992; GeneTex, Inc.), anti-phospho-VEGFR2 (1:1,000; cat. no. CSBPA000703; Cusabio Technology, LLC), anti-VEGFR2 (1:1,000; cat. no. CSBPA008334; Cusabio Technology, LLC), anti-phospho-p38 MAPK (1:1,000; cat. no. #4511; Cell Signaling Technology, Inc.), anti-p38 MAPK (1:1,000; cat. no. #8690; Cell Signaling Technology, Inc.) and anti-β-actin (1:1,000; cat. no. #4970; Cell Signaling Technology, Inc.).

Techniques: Activation Assay, Cell Culture, Incubation, SDS Page, Western Blot, Derivative Assay

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: a Neural plate stage Xenopus laevis embryos were processed for co-immunoprecipitation (IP) assays. Example of Western blot assay from immunoprecipitates with FOLR1 or GFP (control) antibodies and probed for CD2AP or FOLR1. Similar results were observed in N = 3 independent experiments. b Neural plate stage Xenopus laevis embryos were fixed and processed for immunostaining. Images are transverse single z-sections of immunostained neural plate showing apical colocalization of phospho-CD2AP (p-CD2AP) and p-c-Cbl with C-cadherin. Scale bar, 10 μm. Similar results were observed in N = 3 independent experiments. c Two-cell stage Xenopus laevis embryos were bilaterally microinjected with 9.9 pmol of Control-morpholino (Control, Control-MO), 2.6 pmol CD2AP-MO1 (CD2AP KD1) or 7.4–9.9 pmol CD2AP-MO2 (CD2AP KD2/KD) per blastomere until neural tube closed in control embryos, when they were fixed and photomicrographed. Examples of whole embryos in each group. Arrowheads indicate open neural tube (neural tube defect, NTD). Numbers are embryos presenting closed (green) or open (purple, NTD) neural tube. Bar graph represents proportion of phenotypes in each group. d Two-cell stage Xenopus laevis embryos were unilaterally microinjected with 9.9 pmol Control-MO (Control) and 7.4–9.9 pmol CD2AP-MO2 (CD2AP KD) along with GFP and mCherry mRNA, respectively, and allowed to develop until they reached mid-neural plate stages, when they were fixed and processed for immunostaining. Image is a transverse section of the neural plate, immunostained for GFP (Control), mCherry (CD2AP KD) and C-cadherin. Double arrows indicate apical surface length of medial superficial Control (white) and CD2AP KD (magenta) neural plate cells. Scale bar, 20 μm. Graph shows individual and mean ± SD apical length of superficial neural plate cells per embryo, n of cells = 75 in each half of the neural plate, N of embryos = 4. **** p < 0.0001, two-tailed paired t -test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: Immunoprecipitation, Western Blot, Control, Immunostaining, Two Tailed Test

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: a Two-cell stage Xenopus laevis embryos were bilaterally microinjected with hEEA1-GFP and membrane mCherry mRNAs and unilaterally microinjected with 9.9 pmol CD2AP-MO (CD2AP KD) per blastomere along with fluorescent tracer and allowed to develop until they reached early neural plate stages (stage 13-14), when they were time-lapse imaged with an acquisition rate of 1 frame/6 min. Image is maximum intensity projection of single time frame. Dashed line indicates border between morpholino-injected and wild-type (WT) neural plate. Inset shows neural plate injected side showing tracer in blue. Graphs show distribution between both halves of the neural plate (in %) of the number of EEA1-GFP vesicles and area fraction of labeled endosomes per neural plate cell surface. Two-tailed paired t -test, n = 28 cells analyzed in each group from N = 5 embryos per group. Scale bar, 20 μm. b Two-cell stage Xenopus laevis embryos were bilaterally microinjected with 7.4 pmol Control-MO (Control) or CD2AP-MO (CD2AP KD) per blastomere and allowed to grow until they reached mid-neural plate stages (stage 15–17) when neural plate was dissected and processed for Western blot assays. Image is an example of Western blot assay. Graph shows individual and mean ± SD percent of optical density (OD) for C-cadherin immunoblot band normalized with GAPDH protein band OD and compared to controls. Two-tailed ratio t -test, n = 28 and 24 neural plates for Control and CD2AP KD groups, respectively, N = 5 independent experiments. In ( a , b ), * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: Membrane, Injection, Labeling, Two Tailed Test, Control, Western Blot

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: Two-cell stage Xenopus laevis embryos were microinjected with 14.8 pmol Control-morpholino (MO, Control, a , b ), 3.2 pmol FOLR1-MO (FOLR1 KD, a ) or 14.8 pmol CD2AP-MO2 (CD2AP KD, b ) per embryo and incubated with saline or proteasome and lysosome inhibitors at the end of gastrulation (stage 12) until neural plate stages (stage 17) when they were processed for Western blot assays. Images are examples of Western blot assays. Graphs show individual and mean ± SD percent of optical density (OD) for CD2AP ( a ) or FOLR1 ( b ) immunoblot band normalized with GAPDH protein band OD and compared to controls. In ( a ), n = 34 and 42 neural plates for Control and FOLR1 KD, respectively, N = 7 independent experiments; n = 20 and 26 neural plates for Control+inhibitors and FOLR1 KD+inhibitors groups, respectively, N = 3 independent experiments. In ( b ) n = 16 and 20 for Control and CD2AP KD groups, respectively and n = 16 and 18 neural plates for Control+inhibitors and CD2AP KD+inhibitors, respectively, N = 3 independent experiments. In ( a , b ) * p < 0.05, *** p < 0.001, ns: not significant, two-tailed ratio t -test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: Control, Incubation, Saline, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: a – c Neural plate from mid neural plate stage Xenopus laevis embryos was dissected and dissociated cells plated in vitro. After 2 h, cells were loaded with the Ca 2+ sensor Fluo4-AM and time-lapse imaged. a Example of 1-h recording of neural plate cell Ca 2+ activity. b , c Folinic acid ( b , c ), folic acid ( c ) or vehicle was added to neural plate cells in culture during time-lapse imaging and the Ca 2+ response was recorded in the first minute post addition. b Example of acute transient elicited by 100 μM folinic acid. c Graph shows mean ± SEM folinic- or folic acid-responsive neural plate cells compared to total number of cells with spontaneous Ca 2+ transients in 30 min recording, N = 3 independent experiments. Two-tailed one sample t and Wilcoxon test. d Two-cell stage embryos were bilaterally microinjected with GCaMP6s mRNA and grown until early and mid-neural plate stages when they were time-lapse imaged before and after addition of vehicle or 300 μM folinic acid. Image shows example of embryo with cells exhibiting Ca 2+ transients indicated with circles. Graph shows individual and mean ± SD percent change in Ca 2+ transient frequency before and after addition of vehicle or folinic acid, n = 4, 5, 6 and 7 embryos for Early-Vehicle, Early-Folinic acid, Mid-Vehicle and Mid-Folinic acid groups, respectively. One-sample two-tailed t -test, compared to hypothetical value of 100%. e – h Two-cell stage embryos were bilaterally microinjected with GCaMP6s mRNA ( e – h ) and unilaterally microinjected with 9.9 pmol FOLR1-MO1 (FOLR1 KD1/KD) or 1.6 pmol FOLR1-MO2 (FOLR1 KD2) per blastomere ( e ) and grown until mid-neural plate stages when they were time-lapse imaged. Image in ( e ) shows example of embryo with cells exhibiting Ca 2+ transients indicated with circles in WT and FOLR1 KD1 halves. Graphs show individual Ca 2+ transient frequency (transients/5 min) in WT and FOLR1 KD1 or KD2 halves ( e , n = 6 embryos) and in WT embryos before and after addition of vehicle ( f , n = 7 embryos), 50 μM folic acid ( g , n = 6 embryos), Na + and voltage-gated Ca 2+ channel blockers (VGC block : 0.02% tricaine+10 μM nitrendipine+25 μM TTA-2, h , n = 5 embryos), or a mix of folic acid and VGC block ( h , n = 5 embryos). Two-tailed paired t -test ( e – g ) and 1-way ANOVA-Tukey multiple comparisons test ( h ). In ( c – h ), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant. i Model of FOLR1 and CD2AP regulation of neural tube formation. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: In Vitro, Activity Assay, Imaging, Two Tailed Test, Blocking Assay

Journal: Frontiers in Aging Neuroscience

Article Title: Increased tau-induced inflammatory responses are associated with a greater degree of atherosclerosis in progressive supranuclear palsy

doi: 10.3389/fnagi.2025.1608631

Figure Lengend Snippet: Greater atherosclerotic burden and elevated plasma total tau, p-tau181 levels in patients with PSP. Using carotid artery Doppler ultrasound to assess the degree of carotid artery atherosclerosis by evaluating (A) max-CIMT, (B) max-CPT, (C) TPN, (D) CPS, and (E) carotid stenosis% in HCs ( n = 56) and patients with PSP ( n = 56). Using MRA to evaluate the degree of intracranial arterial atherosclerosis by recording (F) ICS vascular territory and (G) MRA GSS in HCs ( n = 56) and patients with PSP ( n = 56). (H) Heatmap of multivariable linear regression heatmap showing the association between disease duration/clinical scores H&Y staging scale, UPDRS-III, PSPRS, MMSE and atherosclerosis measures (max-CIMT, max-CPT, TPN, CPS, carotid stenosis% and MRA GSS) in patients with PSP ( n = 56). Peripheral (I–L) blood lipid, (M) glycosylated hemoglobin, (N) homocysteine parameters, and plasma levels of (O) total tau, (P) p-tau181, (Q) p-tau396 in HCs ( n = 56) and patients with PSP ( n = 56). (R) Heatmap of multivariable linear regression heatmap showing the association between tau levels (total tau, p-tau181, p-tau396) and atherosclerosis measures (max-CIMT, max-CPT, TPN, CPS, carotid stenosis% and MRA GSS) in patients with PSP ( n = 56). Results are expressed as mean and SD. Statistical approaches: (A–E, G, I–Q) covariance (ANCOVA), with age, gender, smoking status and stroke history as covariates, (F) chi-square test, (H, R) multivariable linear regression. In heatmaps, each cell represents the standardized regression coefficient (β) derived from multivariable linear regression models, where each Y -axis was regressed on X -axis while adjusting for age, gender, smoking history, hypertension, diabetes, and dyslipidemia (For MMSE, one more correction for period of education). The color intensity of heatmap indicates the strength and direction of association (β), with positive associations shown in red and negative in blue. P < 0.05 indicates statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001), and ns indicates p ≥ 0.05.

Article Snippet: The following ELISA kits were used: Human interleukin-6 (IL-6) ELISA kit (E-HSEL-H0003, Elabscience Biotechnology, Wuhan, China), Human interleukin-1β (IL-1β) ELISA kit (E-HSEL-H0001, Elabscience Biotechnology, Wuhan, China), Human interleukin-10 (IL-10) ELISA kit (E-EL-H0005, Elabscience Biotechnology, Wuhan, China), Human interferon γ (IFN-γ) ELISA kit (E-HSEL-H0007, Elabscience Biotechnology, Wuhan, China), Human tumor necrosis factor-α (TNF-α) ELISA kit (E-HSEL-H0109c, Elabscience Biotechnology, Wuhan, China), Human total tau ELISA kit (ml057755, mlbio, Shanghai, China),and Human P-tau181 ELISA kit (ml057691, mlbio, Shanghai, China), Human P-tau396 ELISA kit (E-EL-H5314c, Elabscience Biotechnology, Wuhan, China), Mouse interleukin-6 (IL-6) ELISA kit (ml098430, mlbio, Shanghai, China), Mouse interleukin-1β (IL-1β) ELISA kit (ml098416, mlbio, Shanghai, China) and Mouse tumor necrosis factor-α (TNF-α) ELISA kit (mIC50536-1, mlbio, Shanghai, China).

Techniques: Clinical Proteomics, Derivative Assay

Journal: Frontiers in Aging Neuroscience

Article Title: Increased tau-induced inflammatory responses are associated with a greater degree of atherosclerosis in progressive supranuclear palsy

doi: 10.3389/fnagi.2025.1608631

Figure Lengend Snippet: Elevated peripheral blood macrophage ratio and plasma inflammatory cytokine levels in patients with PSP. Representative flow cytometry analysis of (A) macrophages (CD14 + CD68+), and (B) classically activated macrophages (CD86 + CD206-M1) subpopulations from peripheral blood (left) and quantifications (right) in HCs ( n = 10) and patients with PSP ( n = 10). Plasma levels of inflammatory cytokine (C) CRP, (D) IL-6, (E) IL-1β, (F) IL-10, (G) TNF-α, and (H) IFN-γ in HCs ( n = 56) and PSP with patients ( n = 56). (I) Heatmap of multivariable linear regression heatmap showing the association between inflammatory cytokine levels (CRP, IL-6, IL-1β, IL-10, TNF-α, and IFN-r) and atherosclerosis measures (max-CIMT, max-CPT, TPN, CPS, carotid stenosis% and MRA GSS) in patients with PSP ( n = 56). (J) Heatmap of multivariable linear regression heatmap showing the association between tau levels (total tau, p-tau181, p-tau396) and inflammatory cytokine levels (CRP, IL-6, IL-1β, IL-10, TNF-α, and IFN-r) in patients with PSP ( n = 56). Results are expressed as mean and SD. Statistical approaches: (A, B) unpaired non-parametric Mann-Whitney U tests, (C–H) covariance (ANCOVA), with age, gender, smoking status and stroke history as covariates, (I, J) multivariable linear regression. In heatmaps, each cell represents the standardized regression coefficient (β) derived from multivariable linear regression models, where each Y -axis was regressed on X -axis while adjusting for age, gender, smoking history, hypertension, diabetes, and dyslipidemia. The color intensity of heatmap indicates the strength and direction of association (β), with positive associations shown in red and negative in blue. P < 0.05 indicates statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001), and ns indicates p ≥ 0.05.

Article Snippet: The following ELISA kits were used: Human interleukin-6 (IL-6) ELISA kit (E-HSEL-H0003, Elabscience Biotechnology, Wuhan, China), Human interleukin-1β (IL-1β) ELISA kit (E-HSEL-H0001, Elabscience Biotechnology, Wuhan, China), Human interleukin-10 (IL-10) ELISA kit (E-EL-H0005, Elabscience Biotechnology, Wuhan, China), Human interferon γ (IFN-γ) ELISA kit (E-HSEL-H0007, Elabscience Biotechnology, Wuhan, China), Human tumor necrosis factor-α (TNF-α) ELISA kit (E-HSEL-H0109c, Elabscience Biotechnology, Wuhan, China), Human total tau ELISA kit (ml057755, mlbio, Shanghai, China),and Human P-tau181 ELISA kit (ml057691, mlbio, Shanghai, China), Human P-tau396 ELISA kit (E-EL-H5314c, Elabscience Biotechnology, Wuhan, China), Mouse interleukin-6 (IL-6) ELISA kit (ml098430, mlbio, Shanghai, China), Mouse interleukin-1β (IL-1β) ELISA kit (ml098416, mlbio, Shanghai, China) and Mouse tumor necrosis factor-α (TNF-α) ELISA kit (mIC50536-1, mlbio, Shanghai, China).

Techniques: Clinical Proteomics, Flow Cytometry, MANN-WHITNEY, Derivative Assay