Journal: Biomedicines

Article Title: Skin, Liver, and Kidney Interactions Contribute to Skin Dryness in Aging KK-Ay/Tajcl Mice

doi: 10.3390/biomedicines10102648

Figure Lengend Snippet: Effect of aging on ROS levels in the skin ( A ) and liver ( B ); and the expression of Iba1( C ) and CCR7 ( D ) in KK-Ay/TaJcl mice compared with the control. Western blot diagram of Iba1, CCR7, and β-actin with molecular weight markers ( E ). We measured the levels of ROS using a commercial kit and Western blotting for the analysis of Iba1 and CCR7. Values are expressed as the mean ± SD derived from five animals. * p < 0.05. Control: C57BL/6j mice.

Article Snippet: The membranes were washed with Tris-buffered saline–0.1% Tween 20 three times and were incubated at room temperature for 1 h with primary antibodies against Iba1 (marker of macrophage; 1:1000; Wako), chemokine receptor 7 (CCR7) (marker of M1 macrophage; 1:1000; Abcam, Cambridge, MA, USA), and β-actin as a loading control (1:5000; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Western Blot, Molecular Weight, Derivative Assay

Journal: Translational Neurodegeneration

Article Title: Dynamic changes of CX3CL1/CX3CR1 axis during microglial activation and motor neuron loss in the spinal cord of ALS mouse model

doi: 10.1186/s40035-018-0138-4

Figure Lengend Snippet: Primers for qPCR

Article Snippet: After blocking with 5% skim milk prepared in tris-buffered saline-Tween 20 (TBST) (20 mM Tris, pH 7.2, 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature, membranes were incubated for 14 h at 4 °C with primary antibodies against CX3CL1 (bs-0811R, 1: 1000, Bioss), CX3CR1 (AF5825, 1: 100, R&D Systems), β-tubulin (2128S, 1:2000, Cell Signaling Technology).

Techniques:

Journal: Translational Neurodegeneration

Article Title: Dynamic changes of CX3CL1/CX3CR1 axis during microglial activation and motor neuron loss in the spinal cord of ALS mouse model

doi: 10.1186/s40035-018-0138-4

Figure Lengend Snippet: CX3CL1 were mainly located in neurons and decreased during MNs loss in TG mice. a Double immunofluorescent staining of CX3CL1 (green) and NeuN (red) in the anterior horn of lumber spinal cord of WT and TG mice at the age of 40, 90 and 120 days. Scale bar = 50 μm. b Integrated density of CX3CL1 staining. The values are expressed as mean ± SD, * p < 0.05 versus age-matched WT mice, ** p < 0.001 versus age-matched WT mice, n = 5 in each group

Article Snippet: After blocking with 5% skim milk prepared in tris-buffered saline-Tween 20 (TBST) (20 mM Tris, pH 7.2, 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature, membranes were incubated for 14 h at 4 °C with primary antibodies against CX3CL1 (bs-0811R, 1: 1000, Bioss), CX3CR1 (AF5825, 1: 100, R&D Systems), β-tubulin (2128S, 1:2000, Cell Signaling Technology).

Techniques: Staining

Journal: Translational Neurodegeneration

Article Title: Dynamic changes of CX3CL1/CX3CR1 axis during microglial activation and motor neuron loss in the spinal cord of ALS mouse model

doi: 10.1186/s40035-018-0138-4

Figure Lengend Snippet: The mRNA and protein levels of CX3CL1 in the spinal cord anterior horns. a qPCR analysis of CX3CL1 mRNA level. b Western blot analysis of CX3CL1 protein level. c Quantitative analysis the CX3CL1 Protein level. The values are expressed as mean ± SD, * p < 0.05 versus age-matched WT mice, ** p < 0.001 versus age-matched WT mice, n = 5 in each group

Article Snippet: After blocking with 5% skim milk prepared in tris-buffered saline-Tween 20 (TBST) (20 mM Tris, pH 7.2, 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature, membranes were incubated for 14 h at 4 °C with primary antibodies against CX3CL1 (bs-0811R, 1: 1000, Bioss), CX3CR1 (AF5825, 1: 100, R&D Systems), β-tubulin (2128S, 1:2000, Cell Signaling Technology).

Techniques: Western Blot

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: ( A ) Representative images showing the spatial localization of endogenous MT1-MMP in perforations of aneural AChR clusters (arrows) in fixed Xenopus muscle cultures. ( B ) Representative images showing the extent of fluorescent gelatin degradation in area covered by MT1-MMP-mCherry-overexpressing (MT1-mCherry) muscle cells in the presence or absence of MMP inhibitors BB-94 or BB-2516. 8-bit pseudo-color images highlight the relative fluorescence intensity of AChR clusters in different conditions. ( C ) Quantification on the effects of MT1-MMP-mCherry overexpression on the formation of AChR top and bottom clusters in response to BB-94 or BB-2516 treatment. n = 163 (Control), 123 (MT1-mCherry), 119 (MT1-mCherry + BB-94), and 62 (MT1-mCherry + BB-2516) muscle cells from 4 independent experiments. ( D ) Quantification on the effects of MT1-MMP-mCherry overexpression on the extent of fluorescent gelatin degradation and the intensity of aneural AChR clusters in response to BB-94 or BB-2516 treatment. For gelatin intensity measurement: n = 30 (Control), 39 (MT1-mCherry), 33 (MT1-mCherry + BB-94), and 31 (MT1-mCherry + BB-2516) muscle cells from 3 independent experiments. For AChR intensity measurement: n = 14 (Control), 19 (MT1-mCherry), 21 (MT1-mCherry + BB-94), and 16 (MT1-mCherry + BB-2516) muscle cells from 3 independent experiments. Scale bars represent 10 μm. Data are represented as mean ± SEM. One-way ANOVA with Dunnett’s multiple comparisons test ( C ), one-way ANOVA with Turkey’s multiple comparisons test ( D ), *, **, **** represent p≤0.05, 0.01, and 0.0001 respectively.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Fluorescence, Over Expression

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: ( A ) Representative images showing the intracellular trafficking of MT1-MMP vesicles in a cultured muscle cell expressing both MT1-MMP-mCherry (MT1-mCherry) and EB1-GFP. The time-lapse series of two regions (i and ii) outlined in the merge image showed (i) the search-and-capture of MT1-mCherry vesicles (arrows) by an EB1-GFP comet (arrowheads); and (ii) the bidirectional movement of MT1-mCherry vesicles (arrows) along the microtubules (arrowheads). ( B ) Kymographs showing the spatiotemporal correlation between MT1-mCherry and EB1-GFP signals along K1 and K2 lines indicated in the merge image. The maximal projection of MT1-mCherry and EB1-GFP signals was constructed from 40 frames in a 40 s time-lapse series. Arrows indicate the examples of lateral displacement of initially stationary MT1-mCherry vesicles after EB1-GFP comets had passed through. ( C ) Representative TIRF-FRAP images showing the local capturing of MT1-mCherry vesicles at AChR clusters. After photobleaching, the recovery of MT1-mCherry signals and their trajectories in two regions of interest indicate (i) MT1-mCherry vesicles (arrows) were transported to and captured at the perforation of AChR clusters; and (ii) two groups of MT1-mCherry vesicles (red and green arrows) were transported to and captured at the same site of AChR cluster periphery over a period of 120 s. ( D ) Kymographs showing the spatiotemporal capture of MT1-mCherry vesicles at AChR clusters. Two kymographs were constructed from 120 time-lapse images along K3 and K4 lines, as indicated in the merge image (top panels in C). Arrowheads and arrows indicate the sites of MT1-mCherry capture at the perforated and peripheral regions of AChR clusters, respectively. Scale bars represent 10 μm, unless stated otherwise.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Cell Culture, Expressing, Construct

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: Representative images showing the localization of some endogenous MT1-MMP signals at the vesicular compartment (arrows), as visualized by vesicle-associated membrane protein 1 (VAMP1). For clarity, two regions were shown in the 2X magnified view. Scale bar represents 10 μm.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques:

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: ( A ) Representative images showing the effects on aneural AChR clustering in cultured muscle cells over-expressing different levels of MT1-MMP-pHluorin (MT1-pHluorin). Arrows indicate the spatial localization of MT1-pHluroin at perforated regions of AChR clusters. ( B ) Quantification on the effects on aneural AChR cluster formation in cultured muscle cells with different levels of MT1-pHluorin over-expression. Cultured muscle cells with average MT1-pHluorin intensity above the cutoff value of >500 (arbitrary unit) were classified as high expression. n = 150 (Control), 32 (Low MT1-pHluorin level), and 15 (High MT1-pHluorin level) muscle cells from 3 independent experiments. ( C ) Representative sets of images showing the change in MT1-pHluorin fluorescence intensity in response to a sequential switching of various culture media with different pH levels. Arrows indicate the spatial localization of MT1-pHluroin signals in the perforated region of AChR clusters. 8-bit pseudo-color images highlight the change in the relative intensity of MT1-pHluorin. ( D ) Representative sets of time-lapse images showing the extent of AChR cluster remodeling in muscle cells with different MT1-pHluorin expression levels. 8-bit pseudo-color images in the insets highlight the change in the relative intensity of AChR clusters. The percentage values indicate the relative fluorescence intensity of AChR clusters after 24 hr. ( E ) A scatter plot analysis showing the correlation between MT1-pHluorin intensity and the percentage change in AChR intensity over 24 hr in cells with different MT1-pHluorin expression levels. The black line indicates a linear correlation. R = −0.7188, p=0.0056. n = 13 from 3 independent experiments. ( F ) Representative TIRF-FRAP images showing the surface insertion of MT1-pHluorin at AChR clusters in cultured muscle cells. Arrows and arrowheads indicate the spatial insertion of MT1-pHluorin at the perforation and periphery of AChR clusters, respectively. ( G ) Kymographs showing the spatiotemporal insertion of MT1-pHluorin at AChR clusters. Two kymographs were constructed from 180 time-lapse images along the lines of K1 and K2, as indicated in the merge image (left panel). Arrows indicate some newly inserted MT1-pHluorin that were relatively stable, while arrowheads indicate that some were dispersed shortly after surface insertion. ( H ) Quantification on the number of events of MT1-pHluorin surface insertion per unit area (μm 2 ) of aneural AChR clusters over the entire time-lapse duration. n = 10 (Control) and 10 (CLASP-MO) muscle cells from 3 independent experiments. Scale bars represent 10 μm, unless otherwise specified. Data are represented as mean ± SEM. One-way ANOVA with Bonferroni’s multiple comparisons test (B), Student’s t-test ( H ), **, *** represent p≤0.01 and 0.001 respectively. n.s.: non-significant.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Cell Culture, Expressing, Over Expression, Fluorescence, Construct

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: ( A ) Representative images showing the increased signals of endogenous MT1-MMP at the perforated regions of aneural AChR clusters after 8 hr agrin stimulation. ( B ) Quantification on the time-dependent increase in the localization of MT1-MMP signals at aneural AChR clusters in cultured muscle cells upon agrin treatment. n = 67 (Control, 4 hr), 92 (Agrin, 4 hr), 118 (Control, 8 hr), and 93 (Agrin, 8 hr) muscle cells from 3 independent experiments. Scale bar represents 10 μm. Data are represented as mean ± SEM. Student’s t-test, **, *** represent p≤0.01 and 0.001 respectively.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Cell Culture

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: Western blot analysis showing the reduced endogenous MT1-MMP protein level in Xenopus embryos injected with MT1-MMP-MO. α-tubulin was used as a loading control.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Western Blot, Injection

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: ( A ) Representative images showing the localization of pre-existing and newly inserted AChR clusters at nerve-muscle contacts (arrows) in control co-cultures (WT (M)) and in the chimeric co-cultures of wild-type neurons and muscle cells with control morpholino (Control MO (M)), MT1-MMP-MO (MT1-MO (M)), or MT1-MMP-mCherry and MT1-MMP-MO (MT1-mCherry + MT1-MO (M)) muscles. Fluorescent dextran indicates the muscle cells derived from MO-injected embryos. 8-bit pseudo-color images highlight the relative fluorescence intensity of pre-existing and newly inserted AChR signals. ( B ) Quantification on the percentage of nerve-muscle contacts with AChR clusters in control nerve-muscle co-cultures and in different chimeric co-cultures. n = 127 (WT), 120 (Control MO), 146 (MT1-MO), or 34 (MT1-mCherry + MT1-MO) nerve-muscle pairs from 3 independent experiments. ( C ) Quantification on the intensity of pre-existing AChR signals per unit length of nerve-muscle contacts in control co-cultures and in different chimeric co-cultures. n = 29 (WT), 30 (Control MO), 32 (MT1-MO), or 23 (MT1-mCherry + MT1-MO) nerve-muscle pairs from 4 independent experiments. ( D ) Quantification on the intensity of newly inserted AChR signals per unit length of nerve-muscle contacts in control co-cultures and in different chimeric co-cultures. n = 24 (WT), 23 (Control MO), 25 (MT1-MO), or 19 (MT1-mCherry + MT1-MO) nerve-muscle pairs from 3 independent experiments. Scale bar represents 10 μm. Data are represented as mean ± SEM. One-way ANOVA with Sidak’s multiple comparisons test, *, **, *** represent p≤0.05, 0.01, and 0.001 respectively. n.s.: non-significant.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Derivative Assay, Injection, Fluorescence

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: ( A ) A schematic diagram illustrating the use of laser-based photobleaching approach to differentially identify the contribution of aneural AChR clusters and diffuse AChRs for the assembly of nerve-induced synaptic AChR clusters. ( B ) Representative images showing the differential contribution of aneural AChR clusters and diffuse AChRs to nerve-induced synaptic AChR clusters in control co-cultures (WT) and in the chimeric co-cultures of wild-type neurons and muscle cells with control MO or MT1-MMP MO. Yellow boxes (right panels) indicate the region of photobleaching. White boxes show a magnified view of nerve-muscle contacts in 1-d old co-cultures. Dotted lines highlight the periphery of muscle cells. Fluorescent dextran signals in the insets indicate the muscle cells with microinjected MO. 8-bit pseudo-color images highlight the relative fluorescence intensity of AChR signals. Arrows indicate synaptic AChR clusters at nerve-muscle contact sites. Arrowheads indicate the original location of aneural AChR clusters. ( C ) Quantification on the intensity of AChR signals at the nerve-muscle contacts in control co-cultures and in chimeric co-cultures either with or without photobleaching. The control groups without photobleaching indicate the contribution from aneural AChR clusters + diffuse AChRs, n = 15 (WT), 9 (Control MO), and 9 (MT1-MMP MO) nerve-muscle pairs from 3 independent experiments. The experimental groups with photobleaching of aneural AChR clusters indicate the contribution from diffuse AChRs only, n = 23 (WT), 13 (Control MO), and 8 (MT1-MMP MO) nerve-muscle pairs from 3 independent experiments. Scale bars represent 10 μm. Data are represented as mean ± SEM. Two-way ANOVA with Sidak’s multiple comparison test, *, ** represent p≤0.05 and 0.01 respectively.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Fluorescence

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: ( A ) Representative confocal images showing the localization of endogenous MT1-MMP at perforations of synaptic AChR clusters (arrows, top panels) in longitudinal cryosections of adult rat soleus muscles. The specificity of MT1-MMP antibody was verified by pre-incubating anti-MT1-MMP primary antibody with recombinant human MT1-MMP protein (rhMT1-MMP, bottom panels). Neurofilament (NF) was used as a neuronal marker. ( B ) Representative confocal images showing the postsynaptic localization of MT1-MMP at NMJs as demonstrated by the surgical denervation experiment. Staining of AChR, MT1-MMP, and presynaptic marker (NF and synaptophysin (Syn)) was performed using cross sections from adult rat soleus muscles either without (top panels) or with sciatic nerve cut after 4 days (bottom panels). ( C ) Representative confocal images showing aneural versus synaptic AChR clusters in whole-mount diaphragms from wild-type (WT) control and MT1-MMP -/- mice at E13.5 (left panels) and E18.5 (right panels). Whole-mount tissues were stained for AChR and NF. The superimposed 3D reconstruction images were generated by z-stack images using Imaris. Synaptic AChR clusters (yellow) were identified when signals of AChR and NF overlapped with each other, whereas other AChR signals (red) represent aneural AChR clusters. ( D–G ) Quantification on the density of aneural ( D ) versus synaptic ( E ) AChR clusters, the width of end-plate bands ( F ), and the length of axonal branches ( G ) in diaphragm muscles between wild-type and MT1-MMP -/- mouse embryos at E13.5 and E18.5. n = 4 (E13.5, WT) and 5 (E13.5, MT1-MMP -/- ), 5 (E18.5, WT), and 3 (E18.5, MT1-MMP -/- ) embryos from three independent experiments. Scale bars represent 10 μm ( A ) or 100 μm ( C ). Data are represented as mean ± SEM. Two-way ANOVA with Sidak’s multiple comparison test ( D and E ), Student’s t-test ( F and G ), *, **, *** represent p≤0.05, 0.01, and 0.001 respectively. n.s.: non-significant.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Recombinant, Marker, Staining, Generated

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: ( A ) Schematic diagram showing the proposed mechanisms underlying site-directed MT1-MMP trafficking and surface insertion for the regulation of focal ECM degradation and the recruitment of ECM-induced bottom aneural AChR clusters to nerve-induced synaptic clusters at developing NMJs. ( B ) Logical flow diagram proposing key events in the mechanistic role of ECM-induced PLS assembly in MT1-MMP trafficking and surface insertion, and the functional role of MT1-MMP-mediated ECM degradation in postsynaptic development. The relevant data presented in the main figures that support each of the individual proposed events are highlighted in red.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Functional Assay

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet:

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Recombinant, Sequencing, Cell Attachment Assay, Software, Microscopy, Imaging, Electron Microscopy

Journal: BMC complementary medicine and therapies

Article Title: A whole transcriptome profiling analysis for antidepressant mechanism of Xiaoyaosan mediated synapse loss via BDNF/trkB/PI3K signal axis in CUMS rats.

doi: 10.1186/s12906-023-04000-0

Figure Lengend Snippet: Fig. 9 Effects of XYS on BDNF/trkB/PI3K signal axis in hippocampal. A The protein level of BDNF. B The protein level of trkB. C The protein level of p-trkB. D The protein level of PI3K. E The protein level of Akt. F The protein level of p-Akt. G Band diagram of Western blotting. Data are expressed as means ± SEM, n = 3 in each group. *P < 0.05; ** P < 0.01 vs. the control group; # P < 0.05; ## P < 0.01 vs. the CUMS group; & P < 0.05; && P < 0.01 vs. the XYS group

Article Snippet: The PVDF membrane were blocked with skimmed milk(5%) in Tris-buffered saline containing Tween at room temperature for 1 h, then incubated with primary antibodies against BDNF (1:200, cat#:28205-1-AP, Proteintech, China), TrkB (1:600, cat#:13129-1-AP, Proteintech, China), p- TrkB (1:1000, cat#:PA5-36695, Thermo Fisher, United States), PI3K (1:1200, cat#:20584-1-AP, Proteintech, China), Akt(1:50,000, cat#:Ab154598, Abcam, USA), p- Akt (1:10,000, cat#:66444-1-lg, Proteintech, China), β-actin at a dilution of 1/1000 at 4 °C overnight.

Techniques: Western Blot, Control

Journal: The Journal of Neuroscience

Article Title: S-Nitrosylation of p62 Inhibits Autophagic Flux to Promote α-Synuclein Secretion and Spread in Parkinson's Disease and Lewy Body Dementia

doi: 10.1523/jneurosci.1508-21.2022

Figure Lengend Snippet: Figure 1. S-Nitrosylation of p62 in PD/LBD models. A, B, S-Nitrosylation of exogenous and endogenous p62 by the NO do- nor SNOC. HA-p62 transfected SH-SY5Y cells (A) or untransfected SH-SY5Y cells (B) were exposed to 100 mM freshly prepared SNOC or old SNOC (from which NO had been dissipated). After 20min, cell lysates were subjected to the biotin-switch assay. The “ascorbate minus” sample served as a negative control. SNO-p62 and total (input)-p62 detected by immunoblot with anti-p62 antibody. C, S-Nitrosylation of p62 by endogenously generated NO. SH-SY5Y cells exposed to 1 mM rotenone (Rot) in the presence of 1 mM L-arginine and subjected to biotin-switch assay. D, Ratio of SNO-p62/input p62 (lanes 1 and 3). Data are mean 6 SEM; n = 3. **p , 0.01, Student’s t test. E, S-Nitrosylation of p62 in transgenic PD/LBD mouse model. Brain lysates from 4-month-old control (WT) or human Thy1 promoter-driven a-syn-overexpressing mice subjected to biotin- switch assay. F, Ratio of SNO-p62/input p62 from WT or a-syn-overexpressing mice. Data are mean 6 SEM; n = 4 mice in each group. *p , 0.05, Student’s t test. G, S-Nitrosylation of p62 in hiPSC-DA neurons. A53T mutant a-syn or isogenic con- trol (corr) hiPSC-DA neurons in the presence or absence of 1 mM rotenone were subjected to the biotin-switch assay. The corr and A53T blots are derived from the same gel. H, Ratio of SNO-p62/input p62 (lanes 1, 3, and 5). For each histogram, data are mean 6 SEM; n = 3. **p , 0.01, ***p , 0.001, ANOVA with Tukey’s correction.

Article Snippet: Membranes were blocked with Odyssey blocking buffer (Li-Cor, 927-40 000) for 30min at room temperature and then probed with primary antibodies against p62 (1:3000, BD Transduction Laboratories, 610833; Proteintech, 18 420-1-AP; or Progen, GP62-C), LC3B (1:3000, Sigma, L7543), actin (1:5000, Millipore, MAB1501), or flotillin-1 (1:2000, BD Transduction Laboratories, 610820).

Techniques: Transfection, Biotin Switch Assay, Negative Control, Western Blot, Generated, Transgenic Assay, Control, Mutagenesis, Derivative Assay

Journal: The Journal of Neuroscience

Article Title: S-Nitrosylation of p62 Inhibits Autophagic Flux to Promote α-Synuclein Secretion and Spread in Parkinson's Disease and Lewy Body Dementia

doi: 10.1523/jneurosci.1508-21.2022

Figure Lengend Snippet: Figure 2. Non-nitrosylatable p62(C331A) mutant mimics SNO-p62 to increase LC3 binding. A, SH-SY5Y cells were transfected with HA-p62 and GFP-LC3. After 1 d, cells were exposed to 200 mM old or fresh SNOC, and 30min later, lysed and immunoprecipitated with anti-HA antibody. Total cell lysates (Input) and immunoprecipitates (IP) were probed with anti-GFP and anti-HA antibodies on immunoblots. “Heavy chain” shows presence of antibody. B, Coimmunoprecipitated GFP-LC3 levels normalized to immunoprecipitated HA-p62. Data are mean 6 SEM; n = 3. **p , 0.01, Student’s t test. C, SH-SY5Y cells were transfected with C-terminal HA-tagged WT p62 or mutant p62(C331A). Cells were exposed to 100 mM old or fresh SNOC and biotin-switch assay performed 30 min later. D, Ratio of SNO-p62/input p62 for C-terminal WT HA-p62 or mutant p62(C331A) transfected SH-SY5Y cells. Data are mean 6 SEM; n = 3. ***p , 0.001 by Student’s t test. E, SH-SY5Y cells were transfected with HA-tagged WT p62 or mutant p62(C331A). The next day, cells were exposed to 200 mM old or fresh SNOC; 30min later, lysates were prepared and immunoprecipitated with anti-HA antibody. Total cell lysates (Input) and immunoprecipitates (IP) were immunoblotted with anti-GFP and anti-HA antibodies. F, Coimmunoprecipitated GFP-LC3 levels normalized to immunoprecipitated HA-p62. Data are mean 6 SEM; n = 3. *p , 0.05, ANOVA with Tukey’s correction. G-I, Homology model of the p62- LC3 complex. G, Overall structure of the p62-LC3 complex. Homology modeling of the p62 fragment (magenta) was docked to the crystal structure of LC3 (gray [2ZJD]). A network of Asp-Arg interactions (shown as dotted lines with distance between the atoms shown in Å) is present around p62(Cys331); these interactions facilitate binding of p62 to LC3 (H) or p62(Ala331) (I). See Extended Data Figures 2-1 and 2-2.

Article Snippet: Membranes were blocked with Odyssey blocking buffer (Li-Cor, 927-40 000) for 30min at room temperature and then probed with primary antibodies against p62 (1:3000, BD Transduction Laboratories, 610833; Proteintech, 18 420-1-AP; or Progen, GP62-C), LC3B (1:3000, Sigma, L7543), actin (1:5000, Millipore, MAB1501), or flotillin-1 (1:2000, BD Transduction Laboratories, 610820).

Techniques: Mutagenesis, Binding Assay, Transfection, Immunoprecipitation, Western Blot, Biotin Switch Assay

Journal: The Journal of Neuroscience

Article Title: S-Nitrosylation of p62 Inhibits Autophagic Flux to Promote α-Synuclein Secretion and Spread in Parkinson's Disease and Lewy Body Dementia

doi: 10.1523/jneurosci.1508-21.2022

Figure Lengend Snippet: Figure 3. Knock-in p62(C331A) mutant inhibits autophagic flux. A, WT or mutant p62(C331A) knock-in cells exposed to 400 nM BafA1 for 4 h were lysed and immunoblotted with anti-p62, anti-LC3, and anti-actin antibodies. B, C, Immunoblot quantification of relative p62 expression levels normalized to actin (B), and LC3-II/LC3-I ratio (C). Data are mean 6 SEM; n = 5. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001, ANOVA with Tukey’s correction. D, Representative confocal images of WT or mutant p62(C331A) in knock-in cells taken 6 h after pre- staining with 1 mM DALGreen and 0.1 mM DAPRed dye for 30 min. Scale bar, 20mm. E, Quantification of DALGreen and DAPRed puncta. Data are mean 6 SEM; n = 3. *p , 0.05 (total puncta, WT vs C331A) by Student’s t test. **p , 0.01 (DAPRed puncta, WT vs C331A) by ANOVA with Tukey’s correction. F, Representative TEM images showing ultrastructure of autophagic vacuoles in WT (boxed areas; i, ii) or mutant p62(C331A) knock-in cells (boxed areas; iii, iv). Arrowhead indicates autolysosome. Arrows indicate autophagosomes. See Extended Data Figures 3-1, 3-2, and 3-3.

Article Snippet: Membranes were blocked with Odyssey blocking buffer (Li-Cor, 927-40 000) for 30min at room temperature and then probed with primary antibodies against p62 (1:3000, BD Transduction Laboratories, 610833; Proteintech, 18 420-1-AP; or Progen, GP62-C), LC3B (1:3000, Sigma, L7543), actin (1:5000, Millipore, MAB1501), or flotillin-1 (1:2000, BD Transduction Laboratories, 610820).

Techniques: Knock-In, Mutagenesis, Western Blot, Expressing, Staining

Journal: The Journal of Neuroscience

Article Title: S-Nitrosylation of p62 Inhibits Autophagic Flux to Promote α-Synuclein Secretion and Spread in Parkinson's Disease and Lewy Body Dementia

doi: 10.1523/jneurosci.1508-21.2022

Figure Lengend Snippet: Figure 4. Mutant p62(C331A) increases a-syn and p62/LC3 secretion. A, Immunoblots for p62, LC3, and Flotillin1 in supernatants or EVs obtained after ultracentrifugation from CM collected from V5-a-syn-transfected WT p62 or mutant p62(C331A) knock-in cells. LC3-I was the predominant form in supernatants, while LC3-II was the major form in EVs. B, C, V5-a-syn levels in EVs (B) and supernatants (C) measured by ELISA. Data are mean 6 SEM; n = 4. *p , 0.05, Student’s t test. D, Vesicle flow cytometry of CM (concentrated with 100 kDa cutoff filter) from mNeonGreen-tagged human a-syn (mNG-a-syn)-transfected WT p62 or mutant p62(C331A) knock-in cells. mNG-a-syn1 events were determined using a gate set on a bivariate plot of diam- eter versus mNG fluorescence for untransfected WT cell-derived EVs. E, Quantification of mNG-a-syn-positive EVs by flow cytometry. Inset, Immunoblot of CM used for flow cytometry. Data are mean 6 SEM; n = 3. *p , 0.05, Student’s t test. F, G, a-syn levels by ELISA in EVs (F) and supernatants (G) ultracentrifuge fractions of conditioned media from isogenic control or A53T hiPSC-DA neuronal cultures in the presence or absence of 1 mM L-NAME. Data are mean 6 SEM; n = 3. *p , 0.05, **p , 0.01, Sidak’s correction. See Extended Data Figures 4-1, 4-2, and 4-3.

Article Snippet: Membranes were blocked with Odyssey blocking buffer (Li-Cor, 927-40 000) for 30min at room temperature and then probed with primary antibodies against p62 (1:3000, BD Transduction Laboratories, 610833; Proteintech, 18 420-1-AP; or Progen, GP62-C), LC3B (1:3000, Sigma, L7543), actin (1:5000, Millipore, MAB1501), or flotillin-1 (1:2000, BD Transduction Laboratories, 610820).

Techniques: Mutagenesis, Western Blot, Transfection, Knock-In, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence, Derivative Assay, Control

Journal: The Journal of Neuroscience

Article Title: S-Nitrosylation of p62 Inhibits Autophagic Flux to Promote α-Synuclein Secretion and Spread in Parkinson's Disease and Lewy Body Dementia

doi: 10.1523/jneurosci.1508-21.2022

Figure Lengend Snippet: Figure 5. p62(C331A) mutant increases cell-to-cell transmission of a-syn. A, Schematic diagram of dual-cell BiFC sys- tem. In this assay, the hemi-Venus-a-syn (V1S) construct expresses a-syn conjugated to the N-terminal fragment of Venus, and a-syn-hemi-Venus (SV2) expresses a-syn conjugated to the C-terminal fragment of Venus. Upon a-syn dime- rization/aggregation, the combination of V1S and SV2 emits green fluorescence. B, Scheme of analysis of a-syn cell-to-cell spread using the BiFC system. V1S-transfected WT or mutant p62(C331A) knock-in cells were plated on transwell inserts (donor cells), and SV2-transfected WT cells were plated on coverslips (recipient cells). One day after transfection, the donor and recipient cells were incubated for 3 d. C, Representative BiFC images (top panels) plus Hoechst staining (bottom pan- els). Scale bar, 20mm. D, E, Quantification of green Venus-a-syn puncta (D) and relative intensity (E). Data are mean 6 SEM; n = 3. *p , 0.05, **p , 0.01, Student’s t test.

Article Snippet: Membranes were blocked with Odyssey blocking buffer (Li-Cor, 927-40 000) for 30min at room temperature and then probed with primary antibodies against p62 (1:3000, BD Transduction Laboratories, 610833; Proteintech, 18 420-1-AP; or Progen, GP62-C), LC3B (1:3000, Sigma, L7543), actin (1:5000, Millipore, MAB1501), or flotillin-1 (1:2000, BD Transduction Laboratories, 610820).

Techniques: Mutagenesis, Transmission Assay, Construct, Fluorescence, Transfection, Knock-In, Incubation, Staining

Journal: The Journal of Neuroscience

Article Title: S-Nitrosylation of p62 Inhibits Autophagic Flux to Promote α-Synuclein Secretion and Spread in Parkinson's Disease and Lewy Body Dementia

doi: 10.1523/jneurosci.1508-21.2022

Figure Lengend Snippet: Figure 6. S-Nitrosylation of p62 in human LBD brains and schema for a-syn cell-to-cell spread. A, S-Nitrosylation of human LBD and control brains. Human brain tissues with relatively short postmortem times (described in Extended Data Fig. 6-1) were subjected to biotin-switch assay. B, Ratio of SNO-p62/input p62 from control or LBD brains. Data are mean 6 SEM; n = 5 brains in each group. **p , 0.01, Student’s t test. C, Combined data for ratio of SNO-p62/Input p62 from Figures 1D, F, H and 6B (asterisks are from the original figures and retain their original meaning; vertical dotted line at “1” indicates the control for each condition from data shown in each of the other figures listed above). D, Schema of mechanism for S-nitrosylated p62 increas- ing cell-to-cell transmission of a-syn. Cytoplasmic misfolded a-syn can be directly released via nonclassical exocytosis or exophagy, yielding free a-syn in the extracellular medium (Ejlerskov et al., 2013). Alternatively, misfolded a-syn can be relegated to multivesicular bodies in the cell and then released into the extracellular space via enriched exosomes or EVs (Jang et al., 2010; Alvarez-Erviti et al., 2011; Danzer et al., 2012; Lee et al., 2013). SNO-p62 enhances the release of misfolded a-syn via both of these pathways by enhancing protein buildup because of block- ade of autophagy at the stage of autolysosome formation. Released a-syn is then internalized by neighboring cells, and can seed endogenous a-syn to form aggregates (Luk et al., 2009; Nonaka et al., 2010). EV-mediated uptake occurs through direct endocytosis of the vesicle (Lee et al., 2008), while EV-independent uptake of a-syn may be mediated by binding to a receptor on the cell surface of the cell with subsequent endocytosis and endosome formation of the complex (Mao et al., 2016). See Extended Data Figure 6-1.

Article Snippet: Membranes were blocked with Odyssey blocking buffer (Li-Cor, 927-40 000) for 30min at room temperature and then probed with primary antibodies against p62 (1:3000, BD Transduction Laboratories, 610833; Proteintech, 18 420-1-AP; or Progen, GP62-C), LC3B (1:3000, Sigma, L7543), actin (1:5000, Millipore, MAB1501), or flotillin-1 (1:2000, BD Transduction Laboratories, 610820).

Techniques: Control, Biotin Switch Assay, Transmission Assay, Blocking Assay, Binding Assay

Journal: Journal of Orthopaedic Surgery and Research

Article Title: Inhibition of NUCB2 suppresses the proliferation, migration, and invasion of rheumatoid arthritis synovial fibroblasts from patients with rheumatoid arthritis in vitro

doi: 10.1186/s13018-022-03453-2

Figure Lengend Snippet: NUCB2 mRNA expression in the synovium of patients with RA. A – C The expression level of NUCB2 mRNA in the synovium of patients with RA was higher than that in normal controls (GSE77298, GSE55235, and GSE1919); D – G High expression of NUCB2 mRNA was associated with the cell cycle, DNA replication, extracellular matrix (ECM)–receptor interaction, and focal adhesion in the GSE77298 dataset. All data are presented as the means ± SEMs. * P < 0.05, ** P < 0.01 vs. control group

Article Snippet: After blocking with 5% nonfat dried milk in Tris-buffered saline (TBS) containing 1% Tween-20 for 2 h at room temperature, the membranes were incubated overnight with specific primary antibodies against NUCB2/nesfatin-1 (R&D Systems, # MAB5949) and β-actin (Abcam, #ab8227) at 4 °C overnight.

Techniques: Expressing

Journal: Journal of Orthopaedic Surgery and Research

Article Title: Inhibition of NUCB2 suppresses the proliferation, migration, and invasion of rheumatoid arthritis synovial fibroblasts from patients with rheumatoid arthritis in vitro

doi: 10.1186/s13018-022-03453-2

Figure Lengend Snippet: Effect of NUCB2 silencing on RASF proliferation. A RASFs were stably transfected with sh-NUCB2 NC, sh-NUCB2#1, and sh-NUCB2#2, and the mRNA level of NUCB2 was analyzed using RT-qPCR. B , C The protein level of nesfatin-1 was analyzed using Western blotting. D The growth curves of cells were evaluated by CCK-8 assay after knocking down NUCB2 in RASFs. E , F An EdU assay was performed to evaluate cell proliferation. The samples were imaged at × 200 magnification. Three experiments were performed using RASFs isolated from three different RA patients, and a representative result is shown. ** P < 0.01 vs. sh-NUCB2 NC group

Article Snippet: After blocking with 5% nonfat dried milk in Tris-buffered saline (TBS) containing 1% Tween-20 for 2 h at room temperature, the membranes were incubated overnight with specific primary antibodies against NUCB2/nesfatin-1 (R&D Systems, # MAB5949) and β-actin (Abcam, #ab8227) at 4 °C overnight.

Techniques: Stable Transfection, Transfection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, EdU Assay, Isolation

Journal: Journal of Orthopaedic Surgery and Research

Article Title: Inhibition of NUCB2 suppresses the proliferation, migration, and invasion of rheumatoid arthritis synovial fibroblasts from patients with rheumatoid arthritis in vitro

doi: 10.1186/s13018-022-03453-2

Figure Lengend Snippet: Effect of NUCB2 silencing on the migration and invasion of RASFs. A , B , D Transwell assays were performed to assess the migration and invasion of RASFs. The samples were imaged at × 100 magnification. C , E Cell migration was assessed using a wound healing assay. The samples were imaged at × 100 magnification. Three experiments were performed using RASFs isolated from three different RA patients, and a representative result is shown. ** P < 0.01 vs. sh-NUCB2 NC group

Article Snippet: After blocking with 5% nonfat dried milk in Tris-buffered saline (TBS) containing 1% Tween-20 for 2 h at room temperature, the membranes were incubated overnight with specific primary antibodies against NUCB2/nesfatin-1 (R&D Systems, # MAB5949) and β-actin (Abcam, #ab8227) at 4 °C overnight.

Techniques: Migration, Wound Healing Assay, Isolation

Journal: Molecular Medicine Reports

Article Title: Long noncoding RNA STCAT16 suppresses cell growth and its expression predicts prognosis in patients with gastric cancer

doi: 10.3892/mmr.2019.10128

Figure Lengend Snippet: Expression level of STCAT16 affects GC cell proliferation and colony formation in vitro . (A) Proliferative ability of BGC-832 cells following STCAT16 knockdown. (B) Proliferative ability of AGS cells following STCAT16 overexpression. (C and D) Colony formation ability of BGC-832 cells following STCAT16 knockdown. (E and F) Colony formation ability of AGS cells following STCAT16 overexpression. Cells in the sh-STCAT16 group were transfected with STCAT16-shRNA-759. Magnification, ×100. *P<0.05 vs. corresponding control. STCAT16, stomach cancer-associated transcript 16; sh, short hairpin RNA; NC, negative control.

Article Snippet: Sections were incubated with 5% bovine serum albumin (cat. no. LLBB-1000-01, SurModics, Inc., Eden Prairie, USA) to block non-specific protein binding for 10 min at 37°C, followed by 1-h incubation at room temperature and overnight at 4°C with monoclonal antibody against Ki-67 (1:50 dilution; cat. no. ab16667; Abcam, Cambridge, UK).

Techniques: Expressing, In Vitro, Over Expression, Transfection, shRNA, Negative Control

Journal: Molecular Medicine Reports

Article Title: Long noncoding RNA STCAT16 suppresses cell growth and its expression predicts prognosis in patients with gastric cancer

doi: 10.3892/mmr.2019.10128

Figure Lengend Snippet: Effects of STCAT16 overexpression on GC cell growth and proliferation in vivo . (A) Tumour formation in nude mice at 6 weeks following injection with cells transfected with STCAT16-pcDNA3.1 or mock-NC vectors. (B) Growth curve of tumours derived from cells transfected with STCAT16-pcDNA3.1 or mock-NC vectors. Haematoxylin and eosin staining results in tumours derived from cells transfected with (C) STCAT16-pcDNA3.1 or (D) mock-NC vectors. MKI67 staining results in tumours derived from cells transfected with (E) STCAT16-pcDNA3.1 or (F) mock-NC vectors. Magnification, ×400. *P<0.05 vs. corresponding control. GC, gastric cancer; NC, negative control; STCAT16, stomach cancer-associated transcript 16.

Article Snippet: Sections were incubated with 5% bovine serum albumin (cat. no. LLBB-1000-01, SurModics, Inc., Eden Prairie, USA) to block non-specific protein binding for 10 min at 37°C, followed by 1-h incubation at room temperature and overnight at 4°C with monoclonal antibody against Ki-67 (1:50 dilution; cat. no. ab16667; Abcam, Cambridge, UK).

Techniques: Over Expression, In Vivo, Injection, Transfection, Derivative Assay, Staining, Negative Control

Journal: Frontiers in Microbiology

Article Title: The supernatant of Lactiplantibacillus plantarum 25 is more effective than extracellular vesicles in alleviating ulcerative colitis and improving intestinal barrier function

doi: 10.3389/fmicb.2025.1742486

Figure Lengend Snippet: Effects of LP25-derived extracellular vesicles and LP25 supernatant on the intestinal mucosal barrier in acute ulcerative colitis mice. (A,B) Western blot analysis of barrier-associated proteins ZO-1, Occludin, Claudin-1, and α-SMA in colonic tissues from mice treated with 3% DSS alone, low-dose LP25 EVs (1 mg/kg), high-dose LP25 EVs (2 mg/kg), low-dose LP25 supernatant (125 mg/kg), and high-dose LP25 supernatant (250 mg/kg). (C–F) Quantitative densitometric analysis of the respective protein bands. (G) Representative hematoxylin and eosin (H&E) staining of colonic tissues from each group (100×, scale bar = 50 μm; magnified views at 400×). Black arrows indicate inflammatory cell infiltration. Data are presented as mean ± SD ( n = 3). *** p < 0.001, **** p < 0.0001 indicate significant differences compared to the model group; # p < 0.05, significant difference between low-dose LP25 EVs and low-dose LP25 supernatant groups; && p < 0.01, &&& p < 0.001, significant differences between high-dose LP25 EVs and high-dose LP25 supernatant groups.

Article Snippet: Membranes were blocked with 5% non-fat milk in PBST for 2 h at room temperature and incubated overnight at 4 °C with rabbit primary antibodies against anti-ZO-1 (1: 1000), anti-Claudin-1 (1: 1000), anti-Occludin (1: 1000), anti-α-SMA (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-Villin (1: 2000) (Proteintech, Chicago, United States), anti-intestinal alkaline phosphatase (IAP) (1: 5000) (Abcam, UK), anti-CD86 (1: 1000), anti-Arg-1 (1: 1000), anti-TLR4 (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-NF-κB p65 (1: 2000) (Zenbio, Chengdu, China), anti-phospho-NF-κB p65 (1: 1000) (Abmart, Shanghai, China), anti- IκB-α (1: 5000), anti-phospho-IκB-α (1:2000) (Zenbio, Chengdu, China), and GAPDH (1:10000) (Affinity Biosciences, Cincinnati, United States).

Techniques: Derivative Assay, Western Blot, Staining

Journal: Frontiers in Microbiology

Article Title: The supernatant of Lactiplantibacillus plantarum 25 is more effective than extracellular vesicles in alleviating ulcerative colitis and improving intestinal barrier function

doi: 10.3389/fmicb.2025.1742486

Figure Lengend Snippet: Effects of LP25-derived extracellular vesicles and LP25 supernatant on intestinal mucosal barrier integrity in DSS-induced acute ulcerative colitis mice. (A–F) Immunofluorescence staining of colonic tissues showing tight junction proteins ZO-1, Occludin, and Claudin-1 in each treatment group (magnification 10×; scale bar = 100 μm; insets show magnified images at 20×), along with corresponding fluorescence intensity quantification. Data are presented as mean ± SD ( n = 3). * p < 0.05, *** p < 0.001, **** p < 0.0001 indicate significant differences compared to the model group; # p < 0.05, ## p < 0.01 indicate significant differences between the low-dose LP25 EVs group and the low-dose LP25 supernatant group; & p < 0.05, && p < 0.01 indicate significant differences between the high-dose LP25 EVs group and the high-dose LP25 supernatant group.

Article Snippet: Membranes were blocked with 5% non-fat milk in PBST for 2 h at room temperature and incubated overnight at 4 °C with rabbit primary antibodies against anti-ZO-1 (1: 1000), anti-Claudin-1 (1: 1000), anti-Occludin (1: 1000), anti-α-SMA (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-Villin (1: 2000) (Proteintech, Chicago, United States), anti-intestinal alkaline phosphatase (IAP) (1: 5000) (Abcam, UK), anti-CD86 (1: 1000), anti-Arg-1 (1: 1000), anti-TLR4 (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-NF-κB p65 (1: 2000) (Zenbio, Chengdu, China), anti-phospho-NF-κB p65 (1: 1000) (Abmart, Shanghai, China), anti- IκB-α (1: 5000), anti-phospho-IκB-α (1:2000) (Zenbio, Chengdu, China), and GAPDH (1:10000) (Affinity Biosciences, Cincinnati, United States).

Techniques: Derivative Assay, Immunofluorescence, Staining, Fluorescence

Journal: Frontiers in Microbiology

Article Title: The supernatant of Lactiplantibacillus plantarum 25 is more effective than extracellular vesicles in alleviating ulcerative colitis and improving intestinal barrier function

doi: 10.3389/fmicb.2025.1742486

Figure Lengend Snippet: Effects of LP25-derived EVs and LP25 supernatant on TJ proteins expression in LPS-stimulated Caco-2 cells. (A) Caco-2 cells were stimulated with lipopolysaccharide (LPS) at concentrations of 0, 1, 5, 10, and 50 μg/mL. TNF-α mRNA expression was measured by qPCR to determine the optimal concentration for inducing inflammation. A concentration of 10 μg/mL was selected for subsequent experiments. *** p < 0.001, **** p < 0.0001 indicate significant differences compared to the 0 g/mL group; # p < 0.05 between 5 g/mL and 10 g/mL. (B) Cell viability was assessed by CCK-8 assay following treatment with LP25 EVs at concentrations of 120, 240, 480, and 960 g/mL. * p < 0.05 compared to the 120 g/mL group. (C) Cell viability was assessed after treatment with LP25 supernatant at concentrations of 0.25, 2.5, 25, and 250 g/mL. Concentrations of 25 g/mL and 250 g/mL were selected for further experiments. ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. 250 g/mL group. (D,E) Western blot analysis of TJ proteins (ZO-1, Occludin and Claudin-1) in Caco-2 cells treated with 10 g/mL LPS alone or in combination with low-dose LP25 EVs (120 g/mL), high-dose LP25 EVs (240 g/mL), low-dose LP25 supernatant (25 g/mL), or high-dose LP25 supernatant (250 g/mL). (F–H) Densitometric quantification of TJ proteins levels. (I) Representative immunofluorescence staining of ZO-1 in each treatment group, with DAPI staining for nuclei (scale bar = 50 m). (J) Quantitative analysis of ZO-1 fluorescence intensity. Data are presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 indicate significant differences compared to the LPS group; # p < 0.05, ## p < 0.01 indicate significant differences between the low-dose LP25 EVs group and the low-dose LP25 supernatant group; & p < 0.05, && p < 0.01, &&& p < 0.001 indicate significant differences between the high-dose LP25 EVs group and the high-dose LP25 supernatant group.

Article Snippet: Membranes were blocked with 5% non-fat milk in PBST for 2 h at room temperature and incubated overnight at 4 °C with rabbit primary antibodies against anti-ZO-1 (1: 1000), anti-Claudin-1 (1: 1000), anti-Occludin (1: 1000), anti-α-SMA (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-Villin (1: 2000) (Proteintech, Chicago, United States), anti-intestinal alkaline phosphatase (IAP) (1: 5000) (Abcam, UK), anti-CD86 (1: 1000), anti-Arg-1 (1: 1000), anti-TLR4 (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-NF-κB p65 (1: 2000) (Zenbio, Chengdu, China), anti-phospho-NF-κB p65 (1: 1000) (Abmart, Shanghai, China), anti- IκB-α (1: 5000), anti-phospho-IκB-α (1:2000) (Zenbio, Chengdu, China), and GAPDH (1:10000) (Affinity Biosciences, Cincinnati, United States).

Techniques: Derivative Assay, Expressing, Concentration Assay, CCK-8 Assay, Western Blot, Immunofluorescence, Staining, Fluorescence