Journal: Neurotherapeutics

Article Title: Continuous intravenous infusion of human mesenchymal stromal cell-derived small extracellular vesicles in spinal cord injured rat modulates extracellular matrix and has greater therapeutic efficacy than multiple single injections

doi: 10.1016/j.neurot.2026.e00892

Figure Lengend Snippet: Characterization of hMSC-sEVs and experimental timelines. (a) Transmission electron micrograph of hMSC-sEVs showing typical morphology. Scale bar = 100 nm. (b) Size distribution of hMSC-sEVs, with most vesicles between 80 and 200 nm. (c) Western blot analysis of three independent hMSC-sEV preparations and their corresponding parental hMSCs. Exosomal markers Alix, CD63, and CD9 were enriched in hMSC-sEVs relative to hMSCs. The endoplasmic reticulum marker Calnexin was strongly detected in hMSC lysates but showed only minimal expression in hMSC-sEV preparations, confirming negligible contamination with intracellular organelle-derived material. (d, e) Treatment timelines for 3-day (d) and 6-day (e) protocols starting on Day 7 post-SCI. Rats received (1) Daily PBS injections, (2) PBS administered via osmotic pump, (3) daily injections of human MSC-derived sEVs(hMSC-sEVs), and (4) hMSC-sEVs administered via osmotic pump. Treatments began on Day 7 post-SCI and continued for 3 (d) or 6 (e) days. Functional recovery (BBB score) and body weight were monitored for 10 weeks; tissues were collected on Day 70 for IHC and western blot. Abbreviations: hMSC-sEVs, human mesenchymal stem cell-derived small extracellular vesicles; BBB, Basso, Beattie, and Bresnahan locomotor score; IHC, immunohistochemistry; WB, western blot; PBS, phosphate-buffered saline; SCI, spinal cord injury.

Article Snippet: To prepare cells for extracellular vesicle (EV) isolation, hMSCs were washed three times with phosphate-buffered saline (PBS, Thermo Fisher) to remove residual FCS-derived vesicles and cultured in serum-free DMEM for 48 h. The conditioned medium was then harvested and subjected to differential ultracentrifugation for small EV (sEV) isolation, following MISEV2023 recommendations [ ] and the protocol by Théry et al., 2006 [ ].

Techniques: Transmission Assay, Western Blot, Marker, Expressing, Derivative Assay, Functional Assay, Immunohistochemistry, Saline

Journal: Neurotherapeutics

Article Title: Continuous intravenous infusion of human mesenchymal stromal cell-derived small extracellular vesicles in spinal cord injured rat modulates extracellular matrix and has greater therapeutic efficacy than multiple single injections

doi: 10.1016/j.neurot.2026.e00892

Figure Lengend Snippet: Effects of daily injections or continuous infusion of hMSC-sEVs on locomotor recovery after SCI. (a–b) Time course of BBB locomotor scores for SCI rats treated with (hMSC-sEVs) or PBS over 3 days (a) or 6 days (b). Animals received daily injections (blue symbols) or continuous pump infusion (red symbols) of hMSC-sEVs beginning at 7 days post-SCI. Grey diamonds represent PBS-treated groups (pooled data from daily injection and pump infusion conditions). Dashed lines at 8 and 10 were added to facilitate comparisons between final BBB scores for daily injection and pump infusion conditions in 3-day and 6-day treatment protocols. n = 15 for all hMSC-sEVs treatment groups, n = 7 or 8 for individual PBS treatment groups, and n = 15 for combined PBS groups (daily injection and pump infusion) with either 3-day or 6-day treatment. (c–e) Bar graphs Comparing BBB scores at: 14 days (c), 21 days (d), and 70 days (e) post-SCI. At 14 and 21 days post-SCI. Statistical significance: #p < 0.05, ##p < 0.01, ###p < 0.001 for comparisons between daily and continuous treatment groups (Daily vs. Pump); ◇◇p < 0.01, ◇◇◇p < 0.001 for comparisons between daily MSC-sEVs and PBS-treated groups; ◆◆◆p < 0.001 for comparisons between continuous MSC-sEVs and PBS-treated groups; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 for comparisons between other treatment groups. (f) Images showing typical body postures of locomoting rats at 70 post SCI after treatment with daily PBS injections, continuous PBS infusion, daily hMSC-sEVs injections, or continuous hMSC-sEVs infusion for 6 days. Abbreviations: SCI: Spinal Cord Injury, PBS: Phosphate-Buffered Saline, MSC: Mesenchymal Stem Cell, sEVs: Small Extracellular Vesicles, hMSC-sEVs: Human Mesenchymal Stem Cell-Derived Small Extracellular Vesicles, BBB score: Basso, Beattie, and Bresnahan locomotor rating scale.

Article Snippet: To prepare cells for extracellular vesicle (EV) isolation, hMSCs were washed three times with phosphate-buffered saline (PBS, Thermo Fisher) to remove residual FCS-derived vesicles and cultured in serum-free DMEM for 48 h. The conditioned medium was then harvested and subjected to differential ultracentrifugation for small EV (sEV) isolation, following MISEV2023 recommendations [ ] and the protocol by Théry et al., 2006 [ ].

Techniques: Injection, Saline, Derivative Assay

Journal: Neurotherapeutics

Article Title: Continuous intravenous infusion of human mesenchymal stromal cell-derived small extracellular vesicles in spinal cord injured rat modulates extracellular matrix and has greater therapeutic efficacy than multiple single injections

doi: 10.1016/j.neurot.2026.e00892

Figure Lengend Snippet: Recovery of growth trajectory after SCI. (a): Representative images showing the gross appearance rats 70 days post- SCI, following 3-day treatment or 6-day treatment with PBS vehicle alone or hMSC-sEVs in PBS. Scale bar indicates 3 cm. (i–j): Line graphs of body weight in 3-day treatment (b) and 6-day treatment (c). Dashed lines at 300 and 350 g indicate approximate weights of 3-day PBS treated and 3 day hMSC-sEVs treated groups at 70 days post SCI and are included as references to compare 3 day and 6 day treatment conditions. (d, e): Bar graphs comparing the body weight (d) and head-body lengths (e) of PBS or hMSC-sEVs treated rats 70 days post-SCI. n = 15 for all hMSC-sEVs treatment groups and 7 or 8 for individual PBS treatment groups and n = 15 for combined daily injection and pump PBS groups with 3-day or 6-day treatment. Statistical significance: ◇p < 0.05, ◇◇p < 0.01, ◇◇◇p < 0.001 for comparisons between daily MSC-sEVs and PBS-treated groups; ◆p < 0.05, ◆◆p < 0.01, ◆◆◆p < 0.001 for comparisons between continuous MSC-sEVs and PBS-treated groups. Abbreviations: SCI: Spinal Cord Injury, PBS: Phosphate-Buffered Saline, hMSC-sEVs: Human Mesenchymal Stem Cell-Derived Small Extracellular Vesicles, n: Sample Number.

Article Snippet: To prepare cells for extracellular vesicle (EV) isolation, hMSCs were washed three times with phosphate-buffered saline (PBS, Thermo Fisher) to remove residual FCS-derived vesicles and cultured in serum-free DMEM for 48 h. The conditioned medium was then harvested and subjected to differential ultracentrifugation for small EV (sEV) isolation, following MISEV2023 recommendations [ ] and the protocol by Théry et al., 2006 [ ].

Techniques: Injection, Saline, Derivative Assay

Journal: Neurotherapeutics

Article Title: Continuous intravenous infusion of human mesenchymal stromal cell-derived small extracellular vesicles in spinal cord injured rat modulates extracellular matrix and has greater therapeutic efficacy than multiple single injections

doi: 10.1016/j.neurot.2026.e00892

Figure Lengend Snippet: Expression of Collagen and Fibronectin at the lesion site. (a) Fluorescence micrographs showing lesion sites in representative animals treated with PBS or hMSC-sEVs at 14 days post-SCI, stained with CD206 (red) and fibronectin (green). Enlarged views of boxed areas from a and b, highlighting the co-localization of CD206 and fibronectin. (b) Fluorescence micrographs showing lesion sites in representative animals treated with PBS or hMSC-sEVs at 70 days post-SCI, stained with CD206 (red), fibronectin (green), and collagen 1 (magenta). (c) Western blot analysis of fibronectin, collagen 1, and collagen 5 levels in spinal cord segments centered around the lesion site at 70 days post-SCI, comparing PBS and hMSC-sEVs treated animals. (d–f) Quantitative analysis of western blot data for fibronectin (d), collagen 1 (e), and collagen 5 (f) normalized to control levels, showing significant reductions in extracellular matrix protein deposition in hMSC-sEV-treated groups compared to PBS-treated groups. Scale bar in lower right a = 20 μm and applies to all other high magnification images. Scale bars in upper right a and lower right b = 100 μm and apply to all low magnification images. Boxed areas marked with an asterisk (∗) in the top row indicate the regions shown at higher magnification in the bottom rowStatistical significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ∗∗∗∗p < 0.0001. PBS: Phosphate-Buffered Saline, Abbreviations: hMSC-sEVs: Human Mesenchymal Stem Cell-Derived Small Extracellular Vesicles, SCI: Spinal Cord Injury, CD206: Cluster of Differentiation 206.

Article Snippet: To prepare cells for extracellular vesicle (EV) isolation, hMSCs were washed three times with phosphate-buffered saline (PBS, Thermo Fisher) to remove residual FCS-derived vesicles and cultured in serum-free DMEM for 48 h. The conditioned medium was then harvested and subjected to differential ultracentrifugation for small EV (sEV) isolation, following MISEV2023 recommendations [ ] and the protocol by Théry et al., 2006 [ ].

Techniques: Expressing, Fluorescence, Staining, Western Blot, Control, Saline, Derivative Assay

Journal: iScience

Article Title: Kinetics of immune responses elicited after three mRNA COVID-19 vaccine doses in predominantly antibody-deficient individuals

doi: 10.1016/j.isci.2022.105455

Figure Lengend Snippet:

Article Snippet: MACS BSA solution , Miltenyi Biotec , Cat# 130-091-376.

Techniques: Functional Assay, Virus, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Recombinant, Saline, Modification, Expressing, Transfection, Luciferase, Blocking Assay, Enzyme-linked Immunospot, Staining, Software

Journal: Oncotarget

Article Title: Inhibition of ERα/ERK/P62 cascades induces “autophagic switch” in the estrogen receptor-positive breast cancer cells exposed to gemcitabine

doi: 10.18632/oncotarget.10363

Figure Lengend Snippet: A. MCF-7 cells were treated with or without scramble siRNA or si-ERα, followed by gemcitabine treatment (4 μg/ml) for 48 h. Then total cell lysates were subjected to immunoblot analysis with indicated antibodies. The data represented a typical result and the experiments were conducted three times with similar results. B. The phosphorylated levels of ERK at Thr202/Tyr204 were detected by immunoblot after treatment with gemcitabine combined with PD98059 or DMSO (added before gemcitabine treatment for 1 h). C. MCF-7 cells were treated by gemcitabine (4 μg/ml) combined with PD98059 or DMSO for 48 h. The levels of LC3-I and LC3-II detected by Western blotting were quantified by densitometry using ImageJ software. The ratio of LC3-II to LC3-I was evaluated in three independent experiments. D. MCF-7 cells were treated with scramble siRNA or si-Atg5 for 48 h, followed by gemcitabine and PD98059 for another48 h. The percentage of living cells relative to the control group was detected by MTT assay. E. MCF-7 cells were treated with scramble siRNA or si-BECN1 for 48 h, followed bygemcitabine and PD98059 for another48 h. The percentage of living cells relative to the control group was detected by MTT assay. F. MCF-7 and MDA-MB-231 cells were treated by gemcitabine with different concentrations (MCF-7: 0, 2, 4, 8 μg/ml; MDA-MB-231: 0, 0.5, 1, 2 μg/ml), and the phosphorylation of ERα (ser167), ERK (Thr202/Tyr204) and expression of LC3-I and LC3-II were detected. All the results (mean ± SE) were from three independent experiments.**, P<0.01;***, P<0.001.

Article Snippet: The Annexin V Apoptosis Detection Kit FITC (88-8005) and Propidiumlodide Staining Solution (00-6990) was from eBioscience, Inc. PD98059 (s1177) was from Selleck.cn.

Techniques: Western Blot, Software, Control, MTT Assay, Phospho-proteomics, Expressing

Journal: Oncotarget

Article Title: Inhibition of ERα/ERK/P62 cascades induces “autophagic switch” in the estrogen receptor-positive breast cancer cells exposed to gemcitabine

doi: 10.18632/oncotarget.10363

Figure Lengend Snippet: A. MCF-7 cells that exposed to scramble siRNA or si-ERα for 48 h were treated by gemcitabine alone (4 μg/ml) or gemcitabine + CQ (2.5 μmol/L, added before gemcitabine treatment for 1 h) for another 48 h. Then the cells were treated with MDC (50 μmol/L) for 15 minutes and fixed. The samples were analyzed by confocal microscope. B. MCF-7 cells were treated by gemcitabine, gemcitabine+ PD98059 (30 μmol/L, added before gemcitabine treatment for 1 h) or DMSO for 48 h. Then total cell lysates were subjected to immunoblot analysis with indicated antibodies. The data represented a typical experiment conducted three times with similar results. C. MCF-7 cells were exposed to scramble siRNA or si-ERα for 48 h, and treated by gemcitabine or gemcitabine + CQ for another 48 h. The expression levels of LC3-I/II and P62 proteins were evaluated by Western blotting. β-actin was used as a loading control. D. MCF-7 cells were exposed to scramble siRNA or si-P62 for 48 h, and treated by gemcitabine or gemcitabine + CQ for another 48 h. The expression of LC3-I and LC3-II proteins was detected by immunoblot analysis and actin was used as a loading control. E. Working model for the “autophagic switch”. In ER negative MDA-MB-231, gemcitabine induced the protective autophagy. While in ER positive MCF-7 cells, the phosphorylation of ERα-ERK cascades induced by gemcitabine activated the P62-mediated autophagic degradation excessively, and as a result, it may lead to the collapse of cellular function and autophagic cell death. Inhibition of ERα-ERK cascades removed the excessive activation from the autophagic degradation process induced by gemcitabine, thus resulted in the autophagy that the degree was under a certain threshold, and was beneficial for the adaptation of cells in unfavorable conditions, thereby contributing to the cells survival.

Article Snippet: The Annexin V Apoptosis Detection Kit FITC (88-8005) and Propidiumlodide Staining Solution (00-6990) was from eBioscience, Inc. PD98059 (s1177) was from Selleck.cn.

Techniques: Microscopy, Western Blot, Expressing, Control, Phospho-proteomics, Cell Function Assay, Inhibition, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Pancreatic expression of CPT1A is essential for whole body glucose homeostasis by supporting glucose-stimulated insulin secretion

doi: 10.1016/j.jbc.2025.108187

Figure Lengend Snippet: CPT1 A activity is dispensable for maintenance of β-cell function and glucose tolerance in female mice. A , A glucose tolerance test in 16 to 28 week old female Cpt1a CON versus Cpt1a Pdx1−/− mice with respective AUC calculation ( B ). C , glucose- and KCl-stimulated insulin secretion in islets isolated from 30 week old female Cpt1a CON versus Cpt1a Pdx1−/− mice with respective AUC calculations for glucose- and KCl-stimulated time points ( D ), that is, time points highlighted in gray in panel C . E , insulin content in the same islets as shown in panel C . F , fasting serum insulin in 15 week old and 30 week old female Cpt1a CON versus Cpt1a Pdx1−/− mice. G , insulin-positive area and ( H ) islet fraction calculated from formalin-fixed paraffin-embedded pancreatic tissue in 30 week old female Cpt1a CON versus Cpt1a Pdx1−/− mice. I , Ins1, Ins2, Mafa, and Nkx6.1 mRNA levels in isolated islets from 30 week old female Cpt1a CON versus Cpt1a Pdx1−/− mice. n = 23 to 28 ( A and B ), n = 3 to 5 ( C – E ), n = 7 ( F – I ). ns = not significant, ∗ p < 0.05 and ∗∗ p < 0.01. CPT1A, carnitine palmitoyltransferase 1A; AUC, area under the curve.

Article Snippet: FFPE sections were dewaxed and subjected to heat-induced epitope retrieval (20 min at 100 °C, Biocare Medical Decloaking Chamber) with either a pH 6.0 citrate [ALDH1A3, glucagon, Ki-67] or pH 9.0 tris-based buffer [MafA, Nkx6.1] (H-3300, H-3301; Vector Laboratories).

Techniques: Activity Assay, Cell Function Assay, Isolation, Formalin-fixed Paraffin-Embedded

Journal: The Journal of Biological Chemistry

Article Title: Pancreatic expression of CPT1A is essential for whole body glucose homeostasis by supporting glucose-stimulated insulin secretion

doi: 10.1016/j.jbc.2025.108187

Figure Lengend Snippet: Markers of β-cell maturity are unchanged in male mice with a pancreatic reduction in CPT1A activity. A , Ins1, Ins2, Mafa, Nkx6.1, Pdx1, and Slc2a2 mRNA levels in isolated islets from 30 week male Cpt1a CON versus Cpt1a Pdx1−/− mice. B , immunofluorescence staining for insulin ( green ) costaining with MafA ( red ), Nkx6.1 ( red ; C ), and glucagon ( red ; F ) in formalin-fixed paraffin-embedded (FFPE) pancreatic tissue from 30 week old male Cpt1a CON versus Cpt1a Pdx1−/− mice. D , fasting serum insulin in 12 week old, 30 week old, and 52 week old, ( E ) fasting serum proinsulin:C-peptide ratio, and ( G ) fasting serum glucagon in 30 week old male Cpt1a CON versus Cpt1a Pdx1−/− mice. H , glucose-stimulated glucagon secretion, and ( I ) fold change in glucose-stimulated glucagon secretion, in islets isolated from 12 week old male Cpt1a CON versus Cpt1a Pdx1−/− mice. n = 7 to 8 ( A ), n = 5; the scale bar represents 100 μm ( B , C , and F ), n = 14 to 24 ( D ), n = 6 ( E ), n = 10 to 15 ( G ), and n = 4 ( H and I ). ns = not significant, ∗∗ p < 0.01, ∗∗∗ p < 0.001. CPT1A, carnitine palmitoyltransferase 1A.

Article Snippet: FFPE sections were dewaxed and subjected to heat-induced epitope retrieval (20 min at 100 °C, Biocare Medical Decloaking Chamber) with either a pH 6.0 citrate [ALDH1A3, glucagon, Ki-67] or pH 9.0 tris-based buffer [MafA, Nkx6.1] (H-3300, H-3301; Vector Laboratories).

Techniques: Activity Assay, Isolation, Immunofluorescence, Staining, Formalin-fixed Paraffin-Embedded

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chitinase 3-like 1-CD44 interaction promotes metastasis and epithelial-to-mesenchymal transition through β-catenin/Erk/Akt signaling in gastric cancer

doi: 10.1186/s13046-018-0876-2

Figure Lengend Snippet: Upregulation of CHI3L1 correlates with distant metastasis and poor prognosis of human GCs. a Western blot analysis of CHI3L1 protein expression in 5 pairs of gastric tissues (GC and adjacent non-cancerous tissues) after curative resection. Data represent mean ± SEM; * p < 0.05 (Student’s t test). b Immunohistochemical analysis of CHI3L1 protein levels in GC samples on tissue microarrays. Representative examples of CHI3L1 expression in adjacent non-cancerous gastric tissues, GC tissues and GC metastases tissues (lymph node and pancreas) are shown. Scale bars represent 100 μm. c - e Serum CHI3L1 levels in GC patients ( n = 100) and normal controls ( n = 50) ( c ); Serum CHI3L1 levels in GC patients with different tumor invasive depth ( d ) ( n = 17 [T1 + T2], n = 83 [T3 + T4]) and lymph node metastasis ( e ) ( n = 21 [N0 + N1], n = 79 [N1 + N2]). Mann-Whitney U test was used to assess p values; * p < 0.05. f - i The overall survival of patients with high or low CHI3L1 expression in GC tissues. Kaplan-Meier test was used to analyze p values

Article Snippet: Human serum CHI3L1 levels (R&D Systems; Cat. #DC3 L10) and total (R&D Systems; Cat. #MB100B) and active (Biolegend, San Diego, CA; Cat. #437707) mouse TGF-β1 levels in mouse broncho-alveolar lavage fluid were measured using ELISA kits as directed by the manufacturer.

Techniques: Western Blot, Expressing, Immunohistochemical staining, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chitinase 3-like 1-CD44 interaction promotes metastasis and epithelial-to-mesenchymal transition through β-catenin/Erk/Akt signaling in gastric cancer

doi: 10.1186/s13046-018-0876-2

Figure Lengend Snippet: Associations between CHI3L1 Expression and Clinical Pathological Characteristics in Patients with GC

Article Snippet: Human serum CHI3L1 levels (R&D Systems; Cat. #DC3 L10) and total (R&D Systems; Cat. #MB100B) and active (Biolegend, San Diego, CA; Cat. #437707) mouse TGF-β1 levels in mouse broncho-alveolar lavage fluid were measured using ELISA kits as directed by the manufacturer.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chitinase 3-like 1-CD44 interaction promotes metastasis and epithelial-to-mesenchymal transition through β-catenin/Erk/Akt signaling in gastric cancer

doi: 10.1186/s13046-018-0876-2

Figure Lengend Snippet: CHI3L1 binds to CD44 which also interacts with IL-13Rα2. a Western blot analysis of CHI3L1, CD44 and IL-13Rα2 protein expression in various gastric cancer cell lines. b - c Lysates from AGS and MGC803 cells were immunoprecipitated (IP) with control IgG and anti-CD44 or anti-CHI3L1 antibody, and then immunoblotted as indicated. Five percent of total cell lysates were used for the input. d Co-localization of CD44 (green) and CHI3L1 (red) in AGS (upper panel), MGC803 (middle panel) and GC tissues from patient #1 (lower panel) by immunofluorescent confocal microscopy (Magnification: 630×). Scale bars represent 10 μm. e - f Lysates from AGS and MGC803 cells were immunoprecipitated (IP) with IgG and anti-CD44 or anti-IL-13Rα2 antibody, and then immunoblotted as indicated. Five percent of total cell lysates were used for the input. g Co-localization of CD44 (green) and IL-13Rα2 (red) in AGS (upper panel), MGC803 (middle panel) and GC tissues from patient #2 (lower panel) by immunofluorescent confocal microscopy (Magnification: 630×). Scale bars represent 10 μm

Article Snippet: Human serum CHI3L1 levels (R&D Systems; Cat. #DC3 L10) and total (R&D Systems; Cat. #MB100B) and active (Biolegend, San Diego, CA; Cat. #437707) mouse TGF-β1 levels in mouse broncho-alveolar lavage fluid were measured using ELISA kits as directed by the manufacturer.

Techniques: Western Blot, Expressing, Immunoprecipitation, Control, Confocal Microscopy

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chitinase 3-like 1-CD44 interaction promotes metastasis and epithelial-to-mesenchymal transition through β-catenin/Erk/Akt signaling in gastric cancer

doi: 10.1186/s13046-018-0876-2

Figure Lengend Snippet: CHI3L1 triggers Erk and Akt signaling through CD44. a and b Western blot analysis of Erk and Akt activation in the AGS and MGC803 cells which were pre-exposed with the control antibody or functional CD44 blocking antibody and treated with rhCHI3L1 (500 ng/ml) for the indicated times. c and d Western blot analysis of CD44 and IL-13Rα2 knockdown efficacy in AGS ( c ) and MGC803 ( d ) transfected with scramble, CD44 or IL-13Rα2 shRNA. e and f Westen blot evaluation of the activation of Erk and Akt in AGS and MGC803 cells which stably expressing scrambled, CD44 or IL-13Rα2 shRNA and were cultured in the presence or absence of rhCHI3L1 (500 ng/ml). g Westen blot analysis of the activation of Erk and Akt in bone marrow-derived macrophages (BMDM) from WT and CD44 −/− mice which were incubated with the recombinant mouse CHI3L1 (500 ng/ml) for the noted periods of time

Article Snippet: Human serum CHI3L1 levels (R&D Systems; Cat. #DC3 L10) and total (R&D Systems; Cat. #MB100B) and active (Biolegend, San Diego, CA; Cat. #437707) mouse TGF-β1 levels in mouse broncho-alveolar lavage fluid were measured using ELISA kits as directed by the manufacturer.

Techniques: Western Blot, Activation Assay, Control, Functional Assay, Blocking Assay, Knockdown, Transfection, shRNA, Stable Transfection, Expressing, Cell Culture, Derivative Assay, Incubation, Recombinant

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chitinase 3-like 1-CD44 interaction promotes metastasis and epithelial-to-mesenchymal transition through β-catenin/Erk/Akt signaling in gastric cancer

doi: 10.1186/s13046-018-0876-2

Figure Lengend Snippet: CHI3L1 regulates β-Catenin signaling via CD44. a Western blot analyses were used to evaluate β-catenin phosphorylation (Ser675 or Ser552), and active-β-Catenin (ABC) after exposure to rhCHI3L1 (500 ng/ml) in the presence of control or CD44 neutralizing antibody (10 μg/ml) for the noted periods of time. b - d AGS cell were pretreated with control IgG or CD44 blocking antibody (10 μg/ml). Then, the immunofluorescence assays of p-β-catenin (Ser552 or Ser675) and active-β-catenin (ABC) (all in red) were performed in AGS cell treated with rhCHI3L1 (500 ng/ml). DAPI (blue) was used as a nuclear counterstain. The quantification of nuclear β-catenin positive staining in at least 200 counted cells was presented as percentage ± SEM. Magnification: 400×, Scale bars represent 20 μm. Results shown here are the representative of three independent experiments. Statistical significance was calculated using ANOVA ( b - d ). * p < 0.05

Article Snippet: Human serum CHI3L1 levels (R&D Systems; Cat. #DC3 L10) and total (R&D Systems; Cat. #MB100B) and active (Biolegend, San Diego, CA; Cat. #437707) mouse TGF-β1 levels in mouse broncho-alveolar lavage fluid were measured using ELISA kits as directed by the manufacturer.

Techniques: Western Blot, Phospho-proteomics, Control, Blocking Assay, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chitinase 3-like 1-CD44 interaction promotes metastasis and epithelial-to-mesenchymal transition through β-catenin/Erk/Akt signaling in gastric cancer

doi: 10.1186/s13046-018-0876-2

Figure Lengend Snippet: Roles of CHI3L1 in promoting GC growth and metastasis in vitro. a The effects of rhCHI3L1 on AGS and MGC803 cells proliferation were measured by CCK8 assay. Data are presented as mean ± SEM. b Colony formation assays were performed for AGS and SGC7901 cells. c and d Proliferation of AGS ( c ) and MGC803 ( d ) cells were evaluated by EdU incorporation assay. e The invasion assay of AGS and MGC803 cells. f AGS cells transfected with scramble control or shRNAs targeting CD44 were subjected to wound closure assays. g GC cells were incubated with rhCHI3L1for the noted periods of time. Protein levels of Vimentin and Snail were analyzed by Western blot. CHI3L1 (500 ng/ml) in combination with control or functional CD44 neutralizing antibody (10 μg/ml) were used in the experiments. Results shown here are the representative of three independent experiments. Scale bars represent 100 μm. Statistical significance was calculated using ANOVA ( a - f ). * p < 0.05

Article Snippet: Human serum CHI3L1 levels (R&D Systems; Cat. #DC3 L10) and total (R&D Systems; Cat. #MB100B) and active (Biolegend, San Diego, CA; Cat. #437707) mouse TGF-β1 levels in mouse broncho-alveolar lavage fluid were measured using ELISA kits as directed by the manufacturer.

Techniques: In Vitro, CCK-8 Assay, Invasion Assay, Transfection, Control, Incubation, Western Blot, Functional Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chitinase 3-like 1-CD44 interaction promotes metastasis and epithelial-to-mesenchymal transition through β-catenin/Erk/Akt signaling in gastric cancer

doi: 10.1186/s13046-018-0876-2

Figure Lengend Snippet: CD44v3 physically interacts with CHI3L1 and IL-13Rα2. a Illustration of CD44 gene and alternative spliced variants (e.g., CD44s, CD44v3, v6 and v9 isoforms) which contain external, transmembrane (TM) and intracellular domain. b The structure of CD44v3 transmembrane protein, which contains the hyaluronic acid (HA) binding sites at the external N-terminal region, a heparin sulfate (HS) assembly site in v3 domain, and the signaling regulator binding sites at the cytoplasmic region. CD44v3 domain amino acid sequences and v3 peptide used in the current study (in red) were listed. c - e Binding of CD44v3 extracellular domain (ECD) ( c ) or CD44v3 peptide ( d and e ) to rhCHI3L1 or rhIL-13Rα2 ECD. The binding affinity was evaluated by the absorbance at 450 nm in a direct ELISA. Results shown are representative of a minimum of three independent experiments. The values represent the mean ± SEM in triplicate; *p < 0.05. f and g Measurement of the binding affinity of CD44v3 peptide to rhCHI3L1 ( f ) or rhIL-13Rα2 ECD ( g ) by biolayer interferometry (BLI). Various concentrations of CD44v3 peptide were shown. All experiments were performed in triplicate. h Binding of CD44v6 peptide to rhCHI3L1 or rhIL-13Rα2 ECD evaluated by a direct ELISA as describe above. i - j Measurement of the binding affinity of CD44s ECD to rhCHI3L1 ( i ) or rhIL-13Rα2 ECD ( j ) by biolayer interferometry (BLI). CD44s ECD was immobilized and CHI3L1 (500 μM) or IL-13Rα2 (500 μM) was in the mobile phase

Article Snippet: Human serum CHI3L1 levels (R&D Systems; Cat. #DC3 L10) and total (R&D Systems; Cat. #MB100B) and active (Biolegend, San Diego, CA; Cat. #437707) mouse TGF-β1 levels in mouse broncho-alveolar lavage fluid were measured using ELISA kits as directed by the manufacturer.

Techniques: Binding Assay, Direct ELISA

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chitinase 3-like 1-CD44 interaction promotes metastasis and epithelial-to-mesenchymal transition through β-catenin/Erk/Akt signaling in gastric cancer

doi: 10.1186/s13046-018-0876-2

Figure Lengend Snippet: CHI3L1 mediated tumorigenesis through CD44 in vivo. a A representative image of tumor growth in nude mice subcutaneously inoculated with lenti-shCHI3L1- or lenti-shControl-transfected MGC803 cells. b The growth curve of subcutaneous tumor from MGC803 cells infected with lenti-shCHI3L1 or lenti-shControl in nude mice ( n = 10 animals per group). c Comparison of tumor weight from two groups at the end of the experiment. d Evaluation of cell proliferative activity by Ki-67 staining in subcutaneous xenografts from MGC803 cells infected with lenti-shControl or lenti-shCHI3L1. e and f H&E staining of the representative SGC7901 cells lung metastatic lesions in nude mice ( e ). The total area of invasive lesions on the lung slice section represents the invasive tumor volume in the lungs ( f ). Sizing bar indicates 100 μm. g Representative photograph of lungs from WT and CD44 −/− mice 2 weeks after challenge with B16-F10 melanoma cells. Comparison of the number of pleural melanoma colonies in lungs from tumor cells challenged WT and CD44 −/− mice ( n = 8 animals per group). h and i The levels of total ( h ) and active ( i ) TGF-β1 in broncho-alveolar lavage (BAL) fluids from WT mice and CD44 −/− mice. j Schematic representation of the CHI3L1/CD44-IL-13Rα2 signaling pathway in promoting GC cell metastasis. Data are presented as mean ± SEM, * p < 0.05 (Student’s t test)

Article Snippet: Human serum CHI3L1 levels (R&D Systems; Cat. #DC3 L10) and total (R&D Systems; Cat. #MB100B) and active (Biolegend, San Diego, CA; Cat. #437707) mouse TGF-β1 levels in mouse broncho-alveolar lavage fluid were measured using ELISA kits as directed by the manufacturer.

Techniques: In Vivo, Transfection, Infection, Comparison, Activity Assay, Staining

Journal: PLoS ONE

Article Title: Antibody-Free Magnetic Cell Sorting of Genetically Modified Primary Human CD4+ T Cells by One-Step Streptavidin Affinity Purification

doi: 10.1371/journal.pone.0111437

Figure Lengend Snippet: In Antibody-Free Magnetic Cell Sorting (A) transfected or transduced cells co-express a gene or shRNA of interest with a streptavidin-binding cell surface affinity tag. Cells are selected by incubation with streptavidin-conjugated beads then, after washing to remove unbound cells, released by incubation with excess biotin. SBP-ΔLNGFR comprises the 38 amino acid SBP fused to the N-terminus of the truncated LNGFR (B). Expression of SBP-ΔLNGFR at the cell surface was tested 48 hrs after transient transfection of 293Ts with pHRSIN-HA-SBP-ΔLNGFR by staining with streptavidin-APC (C). After a further 72 hrs, cells expressing SBP-ΔLNGFR were selected from the bulk population using magnetic streptavidin-conjugated beads: (i) Dynabeads Biotin Binder (Invitrogen) or (ii) Streptavidin MicroBeads (Miltenyi) (D). Purity of transfected cells before (black) and after (red) selection was assessed by staining with anti-LNGFR-PE. Background staining of cells transfected with a control vector is shown (grey).

Article Snippet: 293Ts were harvested with enzyme-free cell dissociation buffer and Dynabeads Human T-Activator CD3/CD28 beads were removed from primary human CD4+ T cells using a DynaMag-2 magnet (Invitrogen).

Techniques: FACS, Transfection, shRNA, Binding Assay, Incubation, Expressing, Staining, Selection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Antibody-Free Magnetic Cell Sorting of Genetically Modified Primary Human CD4+ T Cells by One-Step Streptavidin Affinity Purification

doi: 10.1371/journal.pone.0111437

Figure Lengend Snippet: 293Ts were transiently transfected or lentivirally transduced with pHRSIN-SE-PGK-SBP-ΔLNGFR-W (encoding EGFP and SBP-ΔLNGFR; A) or pHRSIREN/β2 m-PGK-SBP-ΔLNGFR-W (encoding shRNA to β2 m and SBP-ΔLNGFR; B) and stained with streptavidin-APC plus/minus anti-HLA-A2-PE. Transfected/transduced cells are either GFP+/streptavidin-APC+ or HLA-A2-low/streptavidin-APC+ (dashed circles). Primary human CD4+ T cells were lentivirally transduced with the same constructs then selected using Dynabeads Biotin Binder. Purity of cells before (green or blue) and after (red) selection was assessed by GFP fluorescence (C) or staining with anti-HLA-A2-PE (D). Transduced cells are either GFP+ or HLA-A2-low (dashed boxes). Background staining of untransfected/unstransduced controls is shown (grey).

Article Snippet: 293Ts were harvested with enzyme-free cell dissociation buffer and Dynabeads Human T-Activator CD3/CD28 beads were removed from primary human CD4+ T cells using a DynaMag-2 magnet (Invitrogen).

Techniques: Transfection, Transduction, shRNA, Staining, Construct, Selection, Fluorescence

Journal: PLoS ONE

Article Title: Antibody-Free Magnetic Cell Sorting of Genetically Modified Primary Human CD4+ T Cells by One-Step Streptavidin Affinity Purification

doi: 10.1371/journal.pone.0111437

Figure Lengend Snippet: 293Ts were transiently transfected with pSpCas9(BB)-P2A-SBP-ΔLNGFR (encoding gRNA to β2 m and Cas9-P2A-SBP-ΔLNGFR under the CBh promoter; A) and stained with anti-MHC-I-AF647 before (blue) or after (red) selection with Dynabeads Biotin Binder (B). Transfected cells with β2 m knockouts are MHC-I low (dashed boxes). Background staining of untransfected controls is shown (light grey).

Article Snippet: 293Ts were harvested with enzyme-free cell dissociation buffer and Dynabeads Human T-Activator CD3/CD28 beads were removed from primary human CD4+ T cells using a DynaMag-2 magnet (Invitrogen).

Techniques: Transfection, Staining, Selection

Journal: PLoS ONE

Article Title: Antibody-Free Magnetic Cell Sorting of Genetically Modified Primary Human CD4+ T Cells by One-Step Streptavidin Affinity Purification

doi: 10.1371/journal.pone.0111437

Figure Lengend Snippet: Primary human CD4+ T cells were lentivirally transduced with pHRSIREN/β2 m-PGK-SBP-ΔLNGFR-W (encoding shRNA to β2 m and SBP-ΔLNGFR under the PGK promoter) and either rested for 2 weeks (pale blue) or re-stimulated with CD3/CD28 Dynabeads 3 days prior to analysis (dark blue). Cells were co-stained with anti-HLA-A2-PE and anti-LNGFR-APC, and expression levels of SBP-ΔLNGFR compared in HLA-A2-low cells (A). Transduction with pHRSIN-SE-PGK-SBP-ΔLNGFR-W was then compared with pHRSIN-SE-P2A-SBP-ΔLNGFR-W (encoding GFP-P2A-SBP-ΔLNGFR under the SFFV promoter; B). Transduced cells are GFP+/LNGFR-APC+ (dashed circles). Background staining of untransfected/unstransduced controls is shown (grey). Finally, primary human CD4+ T cells were transduced with the optimised pHRSIREN-S-SBP-ΔLNGFR-W and pHRSIN-SE-P2A-SBP-ΔLNGFR-W lentivectors (C) encoding 2 different shRNAs and 2 different exogenous genes. Following selection with Dynabeads Biotin Binder, purity was assessed by staining with anti-LNGFR-PE (D). Each datapoint represents % LNGFR+ for a different construct (shRNA or exogenous gene) and means and SEMs are shown. Viability and functional activity of selected (expressing a control shRNA) and mock (unselected) cells were compared (E). Viability was measured 4 days after selection, and cells either rested or re-stimulated with CD3/CD28 Dynabeads. Resting and re-stimulated cells were stained with CD69-APC (day 2) and enumerated using CytoCount beads (days 1–3). CD69 expression by resting (grey) versus re-stimulated mock (pale blue) and selected (pink) cells is shown. Fold-increases in viable cell numbers following re-stimulation (proliferation) were calculated using day 1 as a baseline. Experiments were conducted in triplicate and means and SEMs are shown. cPPT – central polypurine tract; RRE – Rev response element; * – packaging signal; LTR – long terminal repeat; WPRE – Woodchuck Hepatitis Virus post-transcriptional regulatory element.

Article Snippet: 293Ts were harvested with enzyme-free cell dissociation buffer and Dynabeads Human T-Activator CD3/CD28 beads were removed from primary human CD4+ T cells using a DynaMag-2 magnet (Invitrogen).

Techniques: Transduction, shRNA, Staining, Expressing, Selection, Construct, Functional Assay, Activity Assay

Journal: Frontiers in Ecology and Evolution

Article Title: Chemosensory Receptors in the Larval Maxilla of Papilio hospiton

doi: 10.3389/fevo.2021.795994

Figure Lengend Snippet: FIGURE 6 | Screening of candidate ligands on OR1. Multiple traces of fluorescence variation associated with Ca++i increase in HEK293A cells transfected with CpomOrco + PmachOR1 (left) and CpomOrco + PhospOR1 (right). (A) Application of 500 µM doses of VUAA1, L-nicotine, caffeine, D-(-)-salicin, quercetin, and AITC. (B) Two consecutive applications of AITC (500 µM) to non-transfected HEK293A. A reversible effect on non-transfected cells was observed, excluding its relations with the human TRPA1 (Cattaneo et al., 2017b), which is in accordance with the documented absence of TRPA1-transcripts in HEK cells (BioGPS Cell Line Gene Expression Profiles—HEK293). N = 67 for all experiments; black bar: stimulus (10 s for VUAA1, 20 s for ligands).

Article Snippet: Human Embryonic Kidney (HEK293A) cells were grown to semi-confluence in 35-mm Petri dishes containing HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, United States), 2 mM L-glutamine, and 100 μg/ml penicillin/streptomycin (Invitrogen)] at 37◦C and 5% CO2.

Techniques: Transfection, Gene Expression

Journal: Frontiers in Ecology and Evolution

Article Title: Chemosensory Receptors in the Larval Maxilla of Papilio hospiton

doi: 10.3389/fevo.2021.795994

Figure Lengend Snippet: FIGURE 7 | Functional studies on TRP Painless. (A) Mean ± SE of fluorescence variation associated with PhospPain (N = 33) and PmachPain (N = 28) upon stimulation with 500 µM allyl-isothiocyanate (AITC, two consecutive applications). (B) Application of 500 µM doses of L-nicotine, caffeine, D-(-)-salicin, quercetin on HEK-cells transfected with PhospPain that were found responsive to AITC. Black bars: stimulus (20 s). (C) Mean ± SE of fluorescence variation associated with PhospPain (N = 94) and PmachPain (N = 107) upon stimulation with heat (T ∼42–44◦C). Right: positive control experiment stimulating HEK293A cells transfected with dTRPA1(B) (N = 101). Red line: time point for thermal experiments preceding an endogenous-effect observed in the phase of testing HEK-cells transfected for Papilio Painless. The demonstrated Ca++-dependency of this effect on HEK cells (Ong et al., 2015; Shalygin et al., 2015) may associate the activation of cation channels most probably belonging to the asset of thermal-gated channels selective for Calcium (Xiao et al., 2011).

Article Snippet: Human Embryonic Kidney (HEK293A) cells were grown to semi-confluence in 35-mm Petri dishes containing HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, United States), 2 mM L-glutamine, and 100 μg/ml penicillin/streptomycin (Invitrogen)] at 37◦C and 5% CO2.

Techniques: Functional Assay, Transfection, Positive Control, Activation Assay